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Yeast

Yeast 2005; 22: 835894.


Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1249

Review

A history of research on yeasts 9: regulation of sugar


metabolism1
James A. Barnett* and Karl-Dieter Entian
School of Biological Sciences, University of East Anglia, Norwich NR4
Institut f
ur Mikrobiologie, Universitat Frankfurt, Marie-Curie-Strae 9,

*Correspondence to:
James A. Barnett, School of
Biological Sciences, University of
East Anglia, Norwich NR4
7TJ, UK.
E-mail: j.barnett@uea.ac.uk

7TJ, UK
D-60439 Frankfurt/Main, Germany

Keywords: history of yeast research; Pasteur effect; Kluyver effect; Custers effect;
Crabtree effect; glucose repression; glucose inactivation

Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Pasteur effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pasteurs observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Studies by Meyerhof, Warburg and others: 1920s and 1930s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6-Phosphofructokinase: Engelhardt and Sakov . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Saccharomyces cerevisiae and the Pasteur effect. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Custers effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Kluyver effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kluyvers observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Observations of Sims and Barnett . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experiments of Jack Pronk and his colleagues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kluyver effect mutants: fds and gal2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Crabtree effect (repression of respiration) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Glucose repression in yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Genetic analysis of glucose repression and identification of the genes involved . . . . . . . . . . . . . . . . . .
Nomenclature of genes and their synonyms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Zimmermanns selection system for mutants defective in glucose repression . . . . . . . . . . . . . . . . . .
Entians analysis of hexokinases and their role in glucose repression . . . . . . . . . . . . . . . . . . . . . . . . .
Carlsons analysis of sucrose-non-fermenting (snf ) mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Repressors and activators under regulatory control of the Snf/Cat kinase . . . . . . . . . . . . . . . . . . . . .
The current model of glucose repression: single and double control systems . . . . . . . . . . . . . . . . . . . .
Classification of glucose-repressible genes according to their regulation . . . . . . . . . . . . . . . . . . . . . .
Enzyme inactivation and the regulation of gluconeogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Holzers analyses of glucose inactivation (catabolite inactivation) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Genetic analysis of glucose inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Glucose inactivation: proteasomal versus vacuolar degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 Previous

articles in this series: [2126,28,30,31].

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Introduction
The present article continues the description of the history of research on induction and repression of
individual enzymes, begun in number 7 of this series [25]. Herein are described some quite recent findings,
which were made after molecular biological methods had become usual for studying these regulatory
processes in yeasts. Some account is also given below of the investigation of certain well-known, general
regulatory mechanisms that control sugar metabolism, and which involve enzyme induction, repression
and inactivation.
These mechanisms which regulate sugar metabolism have been called the Pasteur, Kluyver, Custers
and Crabtree effects (naming the scientists who first described the respective phenomena), glucose or
catabolite repression, and glucose or catabolite inactivation (Table 1). What has been called the Crabtree
effect in yeasts should, as will be discussed below, be called glucose repression. Such regulatory
effects involve enzyme synthesis and enzyme activity (Table 2). In describing the original findings
and the development of later research, an attempt is made to give clear definitions of the phenomena
described, as well as an exposition of their physiological roles in Saccharomyces cerevisiae and, as
far as is known, in other yeasts too. An account is also given of the history of research on the
inter-regulation of glycolysis and gluconeogenesis.2 The story of studying these processes, like many
aspects of microbiology, began with the work of Louis Pasteur3 who, in 1861, described how the
growth of yeast per gram of sugar consumed was much greater under aerobic than anaerobic conditions
[281].

The Pasteur effect


Many kinds of cell utilize exogenously-supplied sugar faster under anaerobic than under aerobic
conditions. This is the Pasteur effect. However, the term has been used variously and the effect has
been reported as occurring in many different organisms and tissues. There has been a great deal of
confused writing on the subject, as indicated in the three quotations from the 1930s which follow this
paragraph, and the large numbers of publications on this topic have been reviewed extensively (e.g.
[46,83,84,203,226,293,329,336]).
The intimate relations between the two processes [oxidation and fermentation] has occupied many biochemists since
Pasteur discovered their quantitative interdependence, now known as the Pasteur Reaction. Pasteur found that there
was some sort of equilibrium between oxidation and fermentation. If oxidation is suppressed by lack of oxygen,
fermentation begins. If we promote again oxidation, fermentation is set to rest. The mechanism of this relation has
been one of the most attractive puzzles of biochemistry ever since (Albert von Szent-Gyorgyi 1937 [365, p. 166]).
By far the great majority [of experts on the Pasteur effect] . . . belong to a class which, vaguely aware of the
Pasteur effect . . . rather accidentally obtain some sort of Pasteur effect, often with some special organism and set
of conditions, and announce boldly, not infrequently in Nature (or in the good old days, Naturwissenschaften), that
here is the explanation of the Pasteur effect. It is this human, indeed lovable, but mathematically-impossible-thatthey-could-all-be-right class that we must be wary of (Dean Burk4 1939 [46, p. 421]).
2 Glycolysis is the anaerobic breakdown of sugar to pyruvate; gluconeogenesis is the formation of D-glucose from compounds which
are not carbohydrates.
3 Some accounts of the following scientists, who are mentioned here, are given in earlier articles of this series: C. F. Cori [23], E. Fischer
[28], E. F. Gale [25], J. S. Haldane, D. Keilin, E. P. Kennedy [24], A. J. Kluyver [30], H. A. Krebs [23], A. L. Lehninger [31], F. A.
Lipmann [23], B. Magasanik [25], O. F. Meyerhof [23], J. Monod [25], P. Ostern [23], L. Pasteur [22], A. Sols [23], S. Spiegelman
[25], A. von Szent-Gyorgyi [24], O. Warburg [23].
4 Dean Burk (19041988), American biochemist, worked at University College London, the Kaiser Wilhelm Institute in Berlin and
Harvard and Cornell Universities. He became chief chemist at the National Cancer Institute, Bethesda [8].

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Table 1. Regulatory phenomena


Name

What happens

Underlying factors

Some key references

PASTEUR EFFECT

Sugar used faster


anaerobically than
aerobically (insignificant in
Saccharomyces cerevisiae)

Oxidized cytochrome inactivates


6-phosphofructokinase

Pasteur 1861 [282]; Meyerhof


1925 [258]; Warburg 1926 [367];
Lipmann 1933, 1934 [224,225];
Engelhardt and Sakov 1943 [95];
Lagunas and Gancedo [211]

CUSTERS EFFECT

Brettanomyces and
Dekkera spp. ferment
D-glucose to ethanol and
CO2 faster in aerobic than
in anaerobic conditions

Much acetic acid is produced via an


NAD+ -aldehyde dehydrogenase.
Consequently, anaerobically, the
high NADH : NAD+ ratio inhibits
glycolysis

Custers 1940 [73], Wiken and


colleagues 1961 [382], Scheffers
1961, 1966 [312,313]

KLUYVER EFFECT

Ability to use
oligosaccharide or
galactose aerobically, but
not anaerobically, although
glucose is fermented

Probably caused mainly by slower


uptake of sugar anaerobically

Kluyver and Custers 1940 [194],


Sims and Barnett 1978 [324],
Barnett and Sims 1982 [32],
Barnett 1992 [20], Weusthuis and
colleagues 1994 [378,379]

CRABTREE EFFECT

Adding glucose to tumour


cells lowers the respiration
rate

Decrease of ADP concentration in


mitochondria

Crabtree 1929 [71], Ibsen 1961


[175]

GLUCOSE

Repression of respiration

Repression of structural genes of


respiratory enzymes

Spiegelman and Reiner 1947 [331],


Magasanik 1961 [240], Bartley and
colleagues [287289],
Zimmermann and colleagues 1977
[398,399], Entian and Mecke 1982
[106], Nehlin and colleagues 1991
[269], DeVit and colleagues 1997
[82], Gancedo 1998 [129], Carlson
1999 [47]

Decrease of enzyme
activity within minutes
after adding glucose

Phosphorylation (rapidly
reversible) and proteolytic
degradation (irreversible) of
enzyme

Holzer and colleagues 1966 [390],


Gancedo 1971 [125], Lenz and
Holzer 1980 [220], Entian and
colleagues 1983 [102], Rose and
colleagues 1988 [306], Hammerle
and colleagues 1998 [152], Schule
and colleagues 2000 [318]

REPRESSION

(glucose effect,
carbon catabolite
repression)

GLUCOSE
INACTIVATION

(catabolite
inactivation)

As considerable confusion exists in the current literature as to the real nature of the Pasteur effect, it is necessary
to explain Pasteurs original conceptions and to describe his experimental results on the effect of oxygen on
carbohydrate catabolism (Kendal Dixon5 1937 [84, p. 432]).

Pasteurs observations
The first relevant publication was that of Pasteur himself, who in 1861 found the growth yield of brewers
yeast, per gram of sugar consumed, to be many times greater aerobically than anaerobically. Eventually,
this observation was shown to have very wide significance for understanding the biochemistry of many
kinds of cell which are capable of both aerobic and anaerobic metabolism. Pasteur put 100 cm3 of sugar
5 Kendal Cartwright Dixon (19111990), Irish biochemist and medical man, worked on carbohydrate and lipid metabolism in Cambridge
from 1933, where he became Professor of Cellular Pathology [9].

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J. A. Barnett and K.-D. Entian

Table 2. Major regulatory mechanisms in carbohydrate metabolism


Kind of regulation

Physiological observation

Examples

Mechanism

Mechanisms regulating enzymic activity


Allosteric activation
and inactivation

Immediate reversible gain or


loss of enzymic activity

6-Phosphofructokinase
pyruvate kinase

Activators or inhibitors change


substrate affinity

Interconversion by
covalent
modification

Reversible loss of enzymic


activity within minutes

Fructose-1,6-bisphosphatase

Usually phosphorylation of
enzyme

Inactivation

Irreversible loss of enzymic


activity

Fructose-1,6-bisphosphatase
and other mainly gluconeogenic
and glyoxylate cycle enzymes

Specific proteolysis of the


enzyme

Mechanisms regulating enzyme synthesis


Induction

Increase in enzymic activity in


response to presence of inducer
(substrate or structurally similar
compound)

GAL and MAL genes

Activation of transcription upon


binding of specific gene
activators

Repression

No further enzyme synthesis


due to a stop of transcription of
the encoding gene

Genes encoding
glucose-repressible enzymes

Inhibition of transcription upon


binding of specific gene
repressors

Derepression

Increase in specific activity of


enzyme after removing
repressing substrate

Genes encoding glucoserepressible enzymes

Release from repression due


to de-binding of gene
repressors

solution with a little protein into a 250 cm3 flask and boiled the solution to remove the oxygen. After
cooling, he introduced a very small amount of beer yeast and placed the drawn-out neck of the flask
under mercury (see [22]). The yeast grew only a little and the sugar was fermented: 60 to 80 parts of
sugar were consumed for 1 part of yeast formed. He wrote:
If the experiment is done in contact with the air and over a large surface area . . . much more yeast is produced for the
same quantity of sugar consumed. The air loses oxygen which is absorbed by the yeast. The latter grows vigorously,
but its characteristic capacity to ferment tends to disappear in these conditions. For one part of yeast formed, only
4 to 10 parts of sugar are transformed. The yeast nevertheless retains its capacity to cause fermentation. Indeed
fermentation appears greatly increased if the yeast is again cultured with sugar in the absence of free oxygen.6

Studies by Meyerhof, Warburg and others: 1920s and 1930s


As a sequel to Pasteurs observations, in the 1920s Otto Meyerhof and Otto Warburg examined differences
between the aerobic and anaerobic breakdown of sugar in yeast, muscle and other tissues. Various tissues,
such as muscle, were already known to form lactate from sugar in the absence of oxygen (see [23]).
Wanting to test whether oxygen uptake increases when cells begin to grow [366], Warburg compared
the respiration rates of certain rat cancer cells with those of normal rat cells [372,376]. He found the
cancer cells to have (a) the same rate of oxygen consumption as the normal cells but (b) a much higher
6 Si lexp
erience est faite au contact de lair et sur une grande surface. . .Pour la meme quantite de sucre disparu, il se fait beaucoup plus
de levure. Lair en contact c`ede de loxyg`ene qui est absorbe par la levure. Celle-ci se developpe e nergiquement, mais son caract`ere de
ferment tend a` disparatre dans ces conditions. On trouve en effet que pour 1 partie de levure formee, il ny aura que 4 a` 10 parties de
sucre transforme. Le role de ferment de cette levure subsiste neanmoins et se montre meme fort exalte si lon vient a` la faire agir sur le
sucre en dehors de linfluence du gaz oxyg`ene libre [282, p. 80].

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839

rate of lactate formation, even in the presence of oxygen. In addition, ethyl isocyanide, which inhibited
heavy-metal catalysis of certain oxidations, abolished the slowing down of glycolysis by oxygen. From
such observations, he concluded:
Respiration and fermentation are thus connected by a chemical reaction, which I call the Pasteur reaction after its
discoverer.7

Working with both yeast and muscle, it was Meyerhof who was the first to examine Pasteurs observations
of differences between the aerobic and anaerobic breakdown of carbohydrate. Meyerhof found that
glycogen was catabolized by frog muscle more slowly when in oxygen than in nitrogen [257]. Then,
working with several kinds of yeast, he showed indisputably that the rate of sugar breakdown by some
yeasts is greater in the absence than in the presence of air [258]. He measured oxygen uptake and carbon
dioxide output, using Warburg manometers,8 and estimated the quotients QO2 and QCO2 ,9 both with
washed yeast at 25 C in phosphate solution (0.1 M KH2 PO4 ) and with a high concentration of D-glucose
(0.28 M).
A brewers bottom yeast had about the same rate of oxygen uptake, whether in buffer alone or when
supplied with glucose, and the high rate of carbon dioxide production was similar (QCO2 > 200) in air
or under nitrogen (Table VA of [258]). This finding of Meyerhofs has since been reported many times
for strains of Saccharomyces cerevisiae (e.g. [342]): that is to say, with a high concentration of glucose,
sugar catabolism is entirely anaerobic, even in aerated cultures. Hence, for such a yeast, the Pasteur effect
cannot occur.
Warburg had already found that carbon monoxide inhibits the respiration of bakers yeast by combining
with a component of the respiratory system of the cell [369]. During a visit to Warburg in the winter
of 19271928, the English physiologist Archibald Hill10 told him about the light-sensitivity of the
carbon monoxidehaemoglobin complex discovered in 1896 by John Scott Haldane and James Smith11
[151], [204, p. 26]. Promptly investigating, Warburg found that the carbon monoxide compound of his
respiratory enzyme (Atmungsferment, see [24]), was also light-sensitive (Figure 1). So, by illuminating
his yeast suspensions with monochromatic light of different wavelengths and known intensities, he
measured the absorption spectrum of this Atmungsferment [373375]. Furthermore, measuring the
inhibition of respiration by his yeast in different mixtures of carbon monoxide and oxygen (replacing
carbon monoxide by nitrogen as a control), Warburg was able to calculate the relative affinity (K ) of his
Atmungsferment for oxygen and carbon monoxide as a partition constant:
K =

[CO]
n
.
(1 n) [O2 ]

where n is the ratio of the respiration rate in the presence of carbon monoxide to the rate in its absence.
Hans Krebs comments that:
. . . to devise and to carry out the experiments and to develop the mathematical analysis of the measurements required
very exceptional experimental and theoretical skill. First he [Warburg] had to find sources of monochromatic light
of sufficient intensity, then he needed methods for measuring the gas exchanges and light intensities, and finally he
had to elaborate the theory for the quantitative interpretation of the measurements . . . It was this work for which
Warburg was awarded the Nobel Prize for Medicine and Physiology in 1931 [204, p. 27].
7 Atmung und G
arung sind also durch eine chemische Reaktion verbunden, die ich nach ihrem Entdecker Pasteursche Reaktion nenne
[367, p. 435].
8 Warburg manometers are described in article 5 of this series [23, p. 516].
9Q
3
O2 and QCO2 were expressed as mm of O2 taken up or of CO2 produced, respectively, per mg dry weight of yeast per hour.
10 Archibald Vivian Hill (18861977), English physiologist, was professor first at Manchester University from 1923, then at University
College London from 1926. He shared the 1922 Nobel Prize for physiology or medicine with Otto Meyerhof for work on heat production
in muscle contraction [187].
11 James Lorrain Smith (18621931), Scottish physiologist, worked at Oxford with J. S. Haldane on air pollution caused by breathing.
He moved to Queens College, Belfast, in 1894, where he became professor in 1901. Subsequently he held chairs in Manchester and
Edinburgh [150].

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J. A. Barnett and K.-D. Entian

Figure 1. Results of one of Warburgs experiments on the action of light on the carbon monoxide inhibition of yeast
respiration. Reproduced with permission from [371, p. 81]. Dunkel = dark; hell = light

The Pasteur effect was studied further in many other kinds of cell. Using Warburgs methods to
investigate the effects of carbon monoxide on the aerobic metabolism of several animal tissues, Hans
Laser12 confirmed some of Warburgs observations, which are shown in Figure 1, and found the following:
(a) the rate of respiration was the same in oxygen + carbon monoxide as in oxygen + nitrogen mixtures;
(b) replacing nitrogen by carbon monoxide increased the rate of glycolysis to that in fully anaerobic
conditions; (c) the effect of carbon monoxide was reversed by light and he showed that, whereas
respiration was unaffected, aerobic glycolysis increased up to the level of anaerobic glycolysis [213].

6-Phosphofructokinase: Engelhardt and Sakov


In 1933, Fritz Lipmann suggested that the Pasteur effect might be a consequence of the oxidation
of a glycolytic enzyme by an electron carrier, such as a cytochrome [224]. As a development of
Lipmanns view, in 1943, Vladimir Engelhardt13 and Nikolai Sakov14 established the major role of
6-phosphofructokinase15 in producing the Pasteur effect [95]. Using fractionated muscle extract, they
investigated sensitivity to oxidation (by various redox dyes16 ) of certain enzymes of the glycolytic
12 Hans Laser (18991980), German biochemist, worked at the Kaiser Wilhelm Institute for Cell Physiology at Berlin, but came to
England as a refugee from the Nazi government in 1934. He worked for over 30 years at the Molteno Institute, Cambridge, where his
research included a study of lysis of cells in patients with malaria and the study of neoplastic cells [7].
13 Vladimir Alexandrovich Engelhardt (18941984) was a great and much-liked Russian biochemist, who discovered oxidative
phosphorylation and the functioning of myosin as an ATPase. He was professor of biochemistry at Kazan University from 1929 and
from 1935 at the Institute of Biochemistry of the Academy of Sciences of the USSR in Moscow whence, in the early 1940s when the
war was approaching Moscow, he was evacuated to Kazakhstan in Central Asia [94,192,326].
14 Nikolai E. Sakov died in the battle for Stalingrad in 1942, his joint work with Engelhardt having been completed in 1941 [94,192].
15 6-Phosphofructokinase was discovered in 1936 by Pawel Ostern and his colleagues [275], see [23].
16 Redox dyes are mostly coloured when oxidized and colourless when reduced. Engelhardt and Sakov found inhibition by dyes with
E  0 > +0.05 V, such as 2,6-dibromophenolindophenol or 2,6-dichlorophenolindophenol. E  0 is the approximate electrode potential, when
there are equal concentrations of both oxidized and reduced forms at pH 7. Relations of the oxidationreduction (redox) potential,
electromotive force and ionic concentration had been worked out in 1889 by Hermann Walther Nernst (18641941) [272].

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841

pathway and found only one of them to be sensitive, namely, 6-phosphofructokinase (PFK), which
catalyses the following reaction:
D-fructose

6-phosphate + ATP D-fructose 1,6-bisphosphate

They also showed that oxidized cytochrome inactivated PFK, as Engelhardt relates:
Evidently, the effect of these agents, completely alien to the normal catalytic system of the cell, even if highly
suggestive, was only of an indirect kind. But an impressive proof of the validity of the findings was obtained when
an exactly similar effect was found using the major physiological oxidizing system, cytochrome and its oxidase.
In the presence of a suitable intermediate carrier, oxidized cytochrome by itself taken in stoichiometric amount,
inhibited the phosphofructokinase. But, most important, the inhibition could be obtained with minute, catalytic
amounts of cytochrome in the presence of cytochrome oxidase. In air, almost complete inhibition is observed,
whereas in nitrogen no inhibition occurs. This experiment can well be regarded as the closest modelling of the
Pasteur effect under the most simplified conditions [94, pp. 910].

Since this work was finished during the middle of World War II, it was impracticable for the Russian
authors to send their script abroad, so it was published in Russian in the journal Biokhimia. Consequently,
as it was not widely known, this work was not cited in the 1950s and early 1960s by the various authors
who presented evidence that changes in PFK activity underlie the Pasteur effect (e.g. [279,309]).
At the same time as these Russian experiments, two American workers were obtaining results consistent
with those of Engelhardt and Sakov. First, Carl Cori was suggesting that PFK has a regulatory role in
muscle glycolysis, writing:
. . . hexosemonophosphate . . . a normal constituent of muscle . . . can increase considerably under certain
experimental conditions without any increase in the formation of lactic acid. This indicates that the reaction between
fructose-6-phosphate and adenosinetriphosphate in intact muscle is a limiting factor as regards the rate at which
lactic acid is formed and carbohydrate is oxidized [70, p. 183].

Second, Joseph Melnicks17 findings, published in 1941 and 1942, accorded with the suggestion that the
Pasteur effect could be brought about by the action of cytochrome and cytochrome oxidase on PFK.
The photochemical absorption spectra, obtained with bakers yeast, indicated that the three proteins,
known as (a) Pasteur enzyme, (b) Atmungsferment or (c) cytochrome oxidase, were all the same enzyme
[253,254,333]. As David Keilin wrote:
It is, therefore, reasonable to assume that cytochrome oxidase is the component showing the light-sensitive inhibition
by carbon monoxide and the photochemical absorption spectrum of the catalytic system involved in the Pasteur
reaction [188, p. 268].

The allosteric effectors of 6-phosphofructokinase have been identified relatively recently, and the
effect of their inhibition is different for various organisms. Many allosteric inhibitors (more than 20,
including cytochrome) for 6-phosphofructokinases have been found in vitro. However, in vivo, the major
allosteric inhibitor of 6-phosphofructokinase is ATP and the major allosteric activators are fructose
2,6-bisphosphate and AMP. The extent of activation and inhibition by these effectors differs between
organisms. Fructose 2,6-bisphosphate, first discovered in mammalian cells [357], is the main activator of
6-phosphofructokinase18 in S. cerevisiae (see Figure 19, below) [191], (for review see [40]).
17 Joseph Lewis Melnick (19142001), American medical microbiologist, worked especially on enteroviruses at Yale University. He
became Professor of epidemiology in 1954 (Historical Register of Yale University, 19511968, p. 523) and moved to a chair at Baylor
College of Medicine, Houston, in 1958 [38,255,364].
18 S. cerevisiae has two enzymes that phosphorylate D-fructose 6-phosphate. The best known glycolytic enzyme, named 6phosphofructokinase-1, is a heterooctamer with 4 - and 4 -subunits [195], which are encoded by genes PFK1 (-subunit) and
PFK2 (-subunit) [67,159]. 6-Phosphofructokinase-1 phosphorylates D-fructose 6-phosphate to the glycolytic intermediate fructose 1,6bisphosphate, whereas 6-phosphofructokinase-2 (encoded by PFK26 and PFK27 ) phosphorylates D-fructose 6-phosphate to D-fructose
2,6-bisphosphate.

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J. A. Barnett and K.-D. Entian

Table 3. The Pasteur effect: chronology of some findings


Date

Author

Findings

1861

Pasteur [281,282]

Brewers yeast growth yield per gram of sugar used is 20 times greater aerobically
than anaerobically

1892

Brown [42]

With high concentration of brewers top yeast (so growth was insignificant),
fermentation found to be independent of oxygen supply

1920

Meyerhof [257]

Rate of glycogen breakdown in muscle greater anaerobically than aerobically

1925

Meyerhof [258]

Rate of sugar breakdown by some yeasts greater anaerobically than aerobically

1926

Warburg [367]

1. The term Pasteursche Reaktion used for a hypothetical chemical reaction linking
respiration and fermentation.
2. CO inhibits yeast respiration.

1928

Warburg and Negelein


[373375]

CO + respiratory enzyme complex cleaved by light and absorption spectrum of


enzyme determined

1933

Lipmann [224]

Oxidizing agents stop glycolysis in yeast and muscle: O2 itself inhibits glycolysis

1937

Laser [213]

In several animal tissues, CO increases glycolysis but does not alter respiration
rate

1941

Stern and Melnick


[253,333]

Light reverses formation of CO-enzyme complex in yeast and retina

1943

Engelhardt and Sakov [95]

Cytochrome + cytochrome oxidase inactivated 6-phosphofructokinase

1962

Passoneau and Lowry


[279]

Rediscovery of Engelhardt and Sakovs findings

1980

Van Schaftingen, Hue and


Hers [357]

Fructose 2,6-bisphosphate stimulates 6-phosphofructokinase

Table 3 shows the chronological sequence of some of the work on the Pasteur effect.

Saccharomyces cerevisiae and the Pasteur effect


In 1966 R. H. De Deken19 recorded differences between a number of yeast species in their rates of
oxidative respiration and of non-oxidative fermentation, when growing aerobically on D-glucose [78].
The yeasts varied from those that are completely oxidative under these conditions, such as Candida
utilis, to others that are completely fermentative, such as Schizosaccharomyces pombe (Table 4). The
figures given in the table are simply illustrative, since considerable variations occur between strains of
the same species and under differing experimental conditions. However, De Dekens results indicate
clearly that C. utilis [227] and Kluyveromyces lactis [308] are both likely to be better yeasts for studying
the Pasteur effect than Saccharomyces cerevisiae. The occurrence of the Pasteur effect has also been
described in Saccharomyces bayanus (uvarum), Schizosaccharomyces pombe [227] and Schwanniomyces
occidentalis [286].
But there are special problems in interpreting much of the work published on the Pasteur effect in
yeasts. Because Pasteurs original observations were on yeast, and to biochemists yeast usually means
Saccharomyces cerevisiae, most of the work has been done with that species. Now, as indicated by
19 R. H. De Deken (1927?) worked on yeast biochemistry and biochemical cytology at the Institut de Recherches du Centre
dEnseignement et de Recherches des Industries Alimentaires et Chimiques (Brussels) in the 1950s and 1960s.

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Table 4. Rates of oxidative respiration and non-oxidative fermentation by several


yeasts growing aerobically in 17 mM-D-glucose at pH 6.5 (temperature and growth phase
unspecified). Rates are in l of gas per 107 yeast cells per 10 min at atmospheric pressure
(results of De Deken 1966 [78]). Names in parentheses are those given by De Deken but
are not in use currently
Yeast

Oxygen
uptake

Carbon dioxide
evolved by fermentation

Saccharomyces cerevisiae (italicus)

0.0

94.5

Kluyveromyces thermotolerans (Torulopsis dattila)

0.0

52.0

Schizosaccharomyces pombe

0.0

40.6

Dekkera bruxellensis (Brettanomyces lambicus)

1.2

9.3

Torulaspora delbrueckii (Torulopsis colliculosa)

10.7

30.2

Kluyveromyces lactis (Torulopsis sphaerica)

25.7

3.5

Candida tropicalis

27.7

0.9

Pichia (Hansenula) anomala

24.1

0.0

Candida utilis

30.0

0.0

De Dekens observations, as well as by some of Meyerhofs results described above, D-glucose almost
completely represses the aerobic metabolism of many strains of S. cerevisiae, even when oxygen is present.
Accordingly, such yeasts in the presence of glucose cannot show the Pasteur effect. Indeed, Rosario
Lagunas, studying two strains, found the Pasteur effect to be insignificant during growth on glucose,
galactose or maltose and very low during ammonia starvation [208]. Furthermore, Walter Bartley20
(Figure 2) and his colleagues stated that cells of S. cerevisiae grown on glucose (at 50 mM or more) do
not form mitochondria [289], the enzymes of the tricarboxylic acid cycle being repressed [287].
However, detecting mitochondria21 in anaerobically grown or glucose-repressed S. cerevisiae requires
special techniques for fixing and staining [72]. Since the 1960s, it has been accepted that this yeast when
metabolizing anaerobically does have mitochondria in a smaller, somewhat elusive form [74] and these
have sometimes been called promitochondria [285,311]. In the 1970s, Barbara Stevens, by means of
a remarkable electron micrographic study of serial thin sections and computer-aided three-dimensional
reconstructions, showed the volume of the promitochondria to occupy about 3% of the cell volume in
glucose-repressed cells, and as much as 1012% in derepressed respiring cells [334].
Nonetheless, Lagunas and her colleagues have observed the Pasteur effect in Saccharomyces cerevisiae
in resting (non-growing) cells [210,211], the resting condition being obtained by depriving the yeast of a
source of nitrogen. When growing, the cells respired only 320% of the sugar they catabolized; whereas
resting cells respired as much as 25100%. Accordingly, it became practicable to detect the Pasteur effect
in such resting cells. Lagunas and her colleagues attributed this reduced rate of fermentation (>10% of
that in growing cells) to inactivation of the transport system by which the sugar enters the cells [210].
Furthermore, they pointed out that previous studies of the Pasteur effect in Saccharomyces cerevisiae
[169,237,258,309] had indeed been done with resting cells. Lagunas and Carlos Gancedo found, even
20 Walter Bartley (19161994), English biochemist, worked in Hans Krebs laboratory, first in Sheffield and then in Oxford as a
technician and later as a research student. Bartley became deputy director of Krebs Medical Research Council Unit for Cell Metabolism
at Oxford and returned to Sheffield in 1963 as professor of biochemistry [10,13,15].
21 Mitochondria, the sites in eukaryotes of tricarboxylic acid cycle reactions and oxidative phosphorylation (see [24]).

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Figure 2. Walter Bartley. Photo courtesy of Joan Brown

for resting cells, that the magnitude of the Pasteur effect is very small in S. cerevisiae [211], Lagunas
commenting further:
S. cerevisiae shows physiological characteristics very different from those often reported even in good textbooks
of microbiology and biochemistry. The fact that the yeast obtains a small benefit from aerobiosis and that [the]
Pasteur effect is neither important nor was discovered in this microorganism should not be ignored any longer [209,
p. 227].

To sum up, Pasteurs finding is undoubtedly correct, namely, that the increase in cell mass anaerobically
is much smaller than aerobically. However, what is now called the Pasteur effect the generalization
that the presence of oxygen decreases the rate of sugar breakdown does not occur in all yeasts, let
alone all other organisms. Indeed, the Pasteur effect is insignificant in his own experimental organism,
which was likely to have been Saccharomyces cerevisiae or S. pastorianus. Two characteristics of these
particular yeasts may explain his findings.
First, the lower growth yield anaerobically was probably because these yeasts are unable to synthesize
ergosterol and unsaturated fatty acids in the absence of oxygen, as Arthur Andreasen22 and Theodore
Stier23 found in the 1950s [4,5]. Second, the biphasic (or diauxic) growth of S. cerevisiae on glucose
22 Arthur A. Andreasen, who worked with Stier at Bloomington, was with Lynferd Wickerham in the early 1940s at the University of
Illinois, Urbana, working on preserving yeasts by freeze-drying for the degree of Master of Science [380].
23 Theodore James Blanchard Stier (19031991), American cellular physiologist, was professor of physiology at Indiana University
from 1947 (information kindly supplied by Kristen Walker of Indiana University Archives).

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Figure 3. Typical biphasic (diauxic) growth of Saccharomyces cerevisiae on D-glucose in aerobic batch culture. The first
phase (about 06 h) is characterized by production of ethanol which, after the disappearance of glucose, is used as the
carbon source for growth (from [186]). Reprinted from Advances in Applied Microbiology 28, G. Kappeli, Regulation of
carbon metabolism in Saccharomyces cerevisiae and related yeasts: 181209, copyright 1986, with permission from Elsevier

(Figure 3) may be the underlying reason for the higher yield of biomass when oxygen is present. In phase
1, glucose is fermented to ethanol; and in phase 2, the ethanol is respired.
The change in free energy for the anaerobic conversion of D-glucose into ethanol, given by:
C6 H12 O6 2EtOH + 2CO2 ,

G  = 235 kJ[205]

is much less than that for the aerobic oxidation of D-glucose, given by:
C6 H12 O6 + 6O2 6CO2 + 6H2 O,

G  = 2873 kJ[205]

so, when there is a change from anaerobic to aerobic conditions, less glucose is consumed.
For S. cerevisiae and other fermentative yeasts, the rapid fermentative catabolism of glucose to
ethanol, accompanied by secretion of acids, such as succinate (as Pasteur found in 1860 [280]) and
acetate (reviewed in [277]), generates an environment in which yeasts have an advantage, as they are
generally more acid- and ethanol-tolerant than most bacteria. Hence, where there are high concentrations
of sugar, such as in rotting figs or grapes, these relatively slow-growing eukaryotic microbes can compete
successfully with most (fast-growing) prokaryotes.

The Custers effect


In 1940, when working in Albert Kluyvers (Figure 4) laboratory in Delft, Mathieu Custers24 studied
yeasts of the genera Dekkera and Brettanomyces, which are important in the brewing of the rather acid
Belgian lambic beer [147]. In contrast to the Pasteur effect, Custers described how these yeasts ferment
D-glucose to ethanol faster under aerobic conditions than anaerobically [73]. He also reported that they
produce considerable amounts of acetic acid in addition to the ethanol. Custers called this behaviour of
Brettanomyces the negative Pasteur effect (see [382]). Lex Scheffers and his colleagues confirmed the
24 Mathieu Theodoor Jozef Custers, Dutch microbiologist, defended his doctors thesis on 3 May 1940, 1 week before the German army
invaded The Netherlands. He became a school teacher in Amsterdam and died before 1970 (W. A. Scheffers, personal communication).

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J. A. Barnett and K.-D. Entian

Figure 4. Albert Jan Kluyver. Photo courtesy of C. T. Kluyver

existence of this effect in a number of strains of Brettanomyces and Dekkera [382] and renamed it the
Custers effect in 1966 [313].
Measuring respiratory exchanges with Warburg manometers, Scheffers found a marked Custers effect in
Dekkera anomala (Brettanomyces claussenii ), which was harvested from shaken aerobic cultures [312].
He also reported the stimulation in D. anomala of anaerobic fermentation by various additions to the
suspensions of this yeast. These additives included acetone, ether, acetaldehyde, acetone, pyruvic acid,
formaldehyde, 3-hydroxy-2-butanone (acetoin25 ), 1,3-dihydroxyacetone, butanone (methyl ethyl ketone)
and -oxoglutaric acid. He wrote:
The results suggest an action of the carbonyl compounds as H-acceptors in enzymatic dehydrogenation . . . Oxidized
coenzyme I (DPN) [NAD+ ] enhances anaerobic fermentation to an extent depending on its concentration . . . it is
tentatively suggested that the inhibition of the start of fermentation in Br. claussenii under anaerobic conditions is,
at least in part, due to a shortage of DPN. This inhibition is abolished on addition of O2 or of other substances able
to oxidize DPNH enzymatically [312, p. 41].

Later, Scheffers described how, on adding exogenously the hydrogen-acceptor, 3-hydroxy-2-butanone,


the rate of fermentation by Dekkera bruxellensis (Brettanomyces intermedius) is increased when under
anaerobic conditions (Figure 5) [313]. He and his colleagues published additional evidence that glycolysis
is slowed by lowering the concentration of NAD+ (Figure 6) [50]. This is because production of acetic
acid involves reduction26 of NAD+ . The NAD+ is restored by any system which re-oxidizes NADH,
such as NADH dehydrogenase, an electron carrier of the respiratory chain.
25 Acetoin (3-hydroxy-2-butanone) may be reduced to butane-2,3-diol by the action of butanediol dehydrogenase:
CH3 CO CH(OH) CH3 + NADH + H+ CH3 CH(OH) CH(OH) CH3 + NAD+ [79].
26 As described for bacterial acetate production, such as by Pseudomonas fluorescens [178].

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847

Figure 5. Fermentation of D-glucose by Dekkera bruxellensis (CBS 1943). Results of Scheffers, published in 1966.
Reproduced from [313], courtesy W. A. Scheffers and by permission of Nature Publishing Group. Symbols: , in
aerobic conditions + or exogenous 103 M 3-hydroxy-2-butanone (acetoin); , in anaerobic conditions; , in anaerobic
conditions + 103 M 3-hydroxy-2-butanone; , with 0.12% oxygen

Figure 6. Custers effect: reduction of NAD(P)+ by formation of acetate from acetaldehyde lowers the concentration of
NAD+ , which is necessary for oxidizing glyceraldehyde 3-phosphate in glycolysis

The Kluyver effect


Kluyvers observations
In 1940, Kluyver and Custers reported that although Candida (Torulopsis) utilis can ferment D-glucose
anaerobically to ethanol and carbon dioxide, this yeast can (unlike Saccharomyces cerevisiae) utilize
maltose aerobically only. Thus they confirmed earlier reports that certain yeasts were able to use the
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J. A. Barnett and K.-D. Entian

Table 5. Abilities of Candida utilis and Saccharomyces cerevisiae to utilize


D-glucose and maltose

aerobic growth

D-glucose

fermentation

aerobic growth

maltose

fermentation

Candida utilis

Saccharomyces
cerevisiae

component hexoses of certain disaccharides anaerobically, yet could use those disaccharides aerobically
only [194]. Thirty-eight years later, this phenomenon was named the Kluyver effect [324].
The problem of the Kluyver effect can be seen from Table 5. Given that the first step in maltose
catabolism is:
-glucosidase
maltose + H2 O 2 D-glucose
why doesnt Candida utilis ferment maltose? Kluyver and Custers reasoned that the organism is able
to synthesize its numerous different cell compounds from the unsplit disaccharide . . . seems utterly
absurd [194, p. 132]. Their view was consistent with the findings of Emil Fischer who, at the end of the
nineteenth century, had firmly established for yeasts that oligosaccharides are always hydrolysed before
they are fermented27 ( [117], and see [28]). Hence, the inability of C. utilis to ferment glucose was not
easy to interpret.
Indeed, Kluyver and Custers found no lack of -glucosidase activity in a strain of Kluyveromyces
thermotolerans (Torulopsis dattila), which gave the Kluyver effect with maltose. Working in the late
1930s, they suggested that the effect was caused by anaerobic conditions reversibly inactivating some
glycoside hydrolases, such as -glucosidase [194, p. 159]. On 10 May 1940, the German army invaded
Holland, so that Kluyvers research was severely interrupted for several years [185] and it was not until
the 1950s that an alternative explanation became available; namely, inactivation of the mechanism of
transport across the plasma membrane. Such an explanation became feasible after Jacques Monod and
his colleagues had characterized selective permeation systems, which are responsible for the entry of
metabolites into microbial cells (e.g. [300], see [25]).
Results of investigating the same problem for maltose utilization by Mucor rouxii in 1969 were
interpreted to mean that a functional respiratory chain is required for maltose penetration into the cell
[119], as had been suggested the previous year for yeasts [16, pp. 566567]. Furthermore, in other
contexts, there were reports that certain yeasts required oxygen for the transport of sugars into their
cells. For example, (a) a non-fermenting yeast, Rhodosporidium toruloides, was found to transport Dglucose actively under aerobic conditions, but not to take up that sugar anaerobically [202], and (b) a
27 In 1922, Richard Willst
atter (18721942) and Gertrud Oppenheimer (18931948) had disputed Fischers view [386]. They found that
certain yeasts ferment lactose more rapidly than they ferment D-glucose, D-galactose or an equimolar mixture of the two and, hence,
concluded that the first metabolic step is not necessarily hydrolytic. Their evidence for direct fermentation of oligosaccharides remained
a matter of dispute (e.g. [217,218]) until 1949, when Alfred Gottschalk pointed out that the rate of entry of a sugar across the plasma
membrane might limit the rate of catabolism of that sugar [143].

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849

respiratory-deficient mutant of Saccharomyces pastorianus was shown to have a much reduced rate of
maltose uptake compared with the wild-type [310].

Observations of Sims and Barnett


However, it was not until the late 1970s that Tony Sims28 and Barnett began investigating the physiology
of the Kluyver effect in yeasts [324]. Basing their information on a survey by taxonomists [231], they listed
the responses of 100 species which appeared to show the effect for at least one of nine oligosaccharides,
commenting: This effect is widespread and possibly at least as common amongst yeasts as the Pasteur
effect. Table 6 gives examples from these authors list and illustrates the finding that there was no
obvious pattern of occurrence of the Kluyver effect; on the contrary, there was striking individuality
among yeasts in their response to each substrate.
Sims and Barnett extended the notion of the Kluyver effect to the utilization of D-galactose. The route
by which D-galactose is transformed to D-glucose 6-phosphate (see [25]), itself an intermediate of the
glycolytic pathway (Figure 7), involves no net oxidation. Hence, there seemed to be no reason for the
catabolism of D-galactose to differ from that of D-glucose in its oxygen requirements.
These workers studied yeasts which gave this effect with maltose, cellobiose and D-galactose, using a
carbon dioxide electrode to measure CO2 output under both aerobic and anaerobic conditions. A ninefold increase in the rate of CO2 output occurred only a few seconds after admitting air into an anaerobic
suspension of maltose-grown Candida utilis and was immediately linear (Figure 8). The rapidity of the
Table 6. Nine yeast species which show the Kluyver effect with more than one sugar: their utilization of certain
glycosides and D-galactose (after Sims and Barnett [324])
Species

SUC

MAL

CEL

TRE

LAC

MEL

MLZ

MeG

RAF

Gal

Candida chilensis

Candida ergatensis

Candida haemulonii

Candida utilis

Debaryomyces castellii

Debaryomyces
polymorphus

Metschnikowia lunata

Pichia heimii

Pichia naganishii

Pichia strasburgensis

K, Kluyver effect, i.e. fermentation negative and aerobic growth positive; +, fermentation and aerobic growth both positive; , fermentation
and aerobic growth both negative; ?, doubt as to how results should be interpreted; SUC, sucrose; MAL, maltose; CEL, cellobiose; TRE,
,-trehalose; LAC, lactose; MEL, melibiose; MLZ, melezitose; MeG, methyl -D-glucopyranoside; RAF, raffinose; Gal, D-galactose.
Notes: (i) All these yeasts ferment D-glucose to give ethanol and carbon dioxide. (ii) The tests used to provide this information were crude and
unquantitative [17]. Those results given as + or should be repeatable; those in doubt are listed as ?. However, growth rates of, for example,
0.5 or 0.05 generations h1 might both be registered as +. The results come from [29].
28 Anthony

Peter Sims (19331990), English biochemist, worked at the University of East Anglia, Norwich on the regulation of
metabolism in Candida utilis, other fungi and green plants [19].
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Figure 7. Routes of galactose and glucose catabolism (Leloir pathway simplified)

Figure 8. Representation of recorder traces for aerobic and anaerobic carbon dioxide output by maltose-grown Candida
utilis (NCYC 737). A suspension (0.4 mg dry wt/ml) of C. utilis was carbon-starved aerobically for 2 hours in Difco yeast
nitrogen base. 10 ml was transferred to a CO2 electrode chamber and made anaerobic by bubbling with nitrogen. Traces:
(i) Negative control: the rate of endogenous CO2 by the yeast was recorded for about 2 min; air was then admitted for
about 5 s () and the recording was continued; (ii) endogenous anaerobic CO2 was recorded; 5 mol maltose (MAL )
were added at about 2 minutes and air was admitted at about 3 minutes (O2 ), the yeast then again made anaerobic and
further recordings were made of anaerobic and aerobic CO2 output. Reproduced from [324]

changes was suggestive of some form of activation and deactivation, rather than the slower processes
involving induction or derepression, for which enzymic (or carrier) synthesis is essential (see [101]).
Moreover, with C. utilis, which shows the Kluyver effect for the -glucoside, cellobiose,29 there was no
loss of -glucosidase activity associated with a change from aerobic to anaerobic conditions [324].
29 Whereas maltose (4-O--D-glucopyranosyl-D-glucopyranose) is an -linked glucose-glucose disaccharide, cellobiose (4-O--Dglucopyranosyl-D-glucopyranose) is the same, but -linked:

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851

Since inactivity of the hydrolases did not appear to explain the Kluyver effect, it seemed worth
investigating whether the carriers which take sugars into the cells might be deactivated, as had been
suggested previously [16,119]. The crude results from tests by taxonomists also indicated that transport
might well be an important factor. For those oligosaccharides which are mostly hydrolysed in the
cytosol30 (Table 7), 70100% of the yeasts showed the Kluyver effect. On the other hand, for those
usually hydrolysed outside the plasma membrane, the corresponding figure was <35% [324] (Table 8).
For sucrose, however, the figure was about 40%: probably this is because sucrose (-D-fructofuranosyl
-D-glucopyranoside) is a double glycoside, that is, both a -fructoside and an -glucoside. Although
most yeasts hydrolyse sucrose with an external invertase, which is a -fructosidase, many do so with
an internal (cytosolic) -glucosidase (Figure 9; for review, see [18]). Accordingly, Sims and Barnett
measured the rates of entry of sugars into the cells.
Results of experiments with Kluyvers Kluyveromyces thermotolerans (Torulopsis dattila) were
consistent with the transport of D-[1-3 H]fucose by the D-galactose carrier (Figure 10); and this transport
was about four times faster aerobically than anaerobically (Table 9). No such effect was observed with
2-deoxy-D-glucose (2-deoxy-D-arabino-hexose), illustrating the important fact that the Kluyver effect
occurs only with certain transport systems in any given yeast.

Figure 9. External hydrolysis of sucrose by invertase and internal hydrolysis by -glucosidase

Figure 10. Structures of D-galactose and D-fucose


30 Generally hydrolysed in the cytosol : maltose, cellobiose, lactose, melezitose and methyl -D-glucopyranoside. Generally hydrolysed
outside the plasma membrane: raffinose and melibiose [18].

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Table 7. Location of hydrolysis of oligosaccharides in most yeasts [18]

Table 8. Percentage of species apparently showing the Kluyver effect.


Oligosaccharides tested include those usually hydrolysed in the cytosol
and others hydrolysed externally to the plasma membrane. Results of a
taxonomic survey; the list includes only those yeasts for which all strains
are able to (a) grow aerobically on the specified oligosaccharide and
(b) ferment D-glucose. Data from [29]
Oligosaccharides

Percentage of species
showing Kluyver effect

Usually hydrolysed in cytosol


melezitose
lactose
methyl -D-glucopyranoside
cellobiose
maltose
,-trehalose

98
92
87
86
73
54

Usually hydrolysed externally


sucrose
raffinose
melibiose

33
24
14

Entry of maltose into Candida utilis, too, was much slower anaerobically than aerobically. In further
work, on the unregulated maltose uptake of a mutant31 of Saccharomyces cerevisiae, Barnett and Sims
found that the active transport of exogenous maltose ceases on switching from aerobic to anaerobic
31 The

mutant was defective in glucose repression and had uncontrolled uptake of maltose [98].

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853

Table 9. Rates of uptake of D-[1-3 H]fucose by galactose-grown Kluyveromyces thermotolerans under aerobic
and anaerobic conditions. Note: the inhibition of uptake
by D-galactose is consistent with the entry of D-fucose by
the D-galactose carrier. (From [324])
Condition

Inhibitor

Rate of uptake
nmol min1 (mg dry wt)1

Aerobic

none

20.7

Anaerobic

none

4.8

Aerobic

D-galactose

1.28

Anaerobic

D-galactose

0.84

Figure 11. The ability of a mutant strain of Saccharomyces cerevisiae, to concentrate exogenously-supplied maltose.
, Aerobic uptake; , anaerobic uptake; - - - - , equilibrium conditions, when exogenous and endogenous maltose
concentrations are the same. Reproduced from Barnett and Sims 1982 [32]. The mutant, which was defective in glucose
repression, had uncontrolled uptake of maltose [98]

conditions so that, consequently, the yeast did not concentrate maltose anaerobically (Figure 11) [32].
They extended their study of the requirement of oxygen for the active transport of sugars into other
yeasts, using strains of Kluyveromyces marxianus and Debaryomyces polymorphus. Experiments with
the non-metabolizable analogue of lactose, TMG32 (methyl 1-thio--D-galactopyranoside), showed that
these yeasts, too, required an oxygen supply for the active transport of lactose, which Barbara Schulz
and Milan Hofer later confirmed for D. polymorphus [321].
Although Barnett and Sims found that active transport ceases under anaerobic conditions, facilitated
diffusion,33 by which the glycosides can also enter the cells, seemed to be unaffected. Hence, they
concluded that the control mechanism underlying the Kluyver effect (a) probably also acts at a later
stage of catabolism, such as in the pathway from pyruvate to ethanol (Figure 12), and (b) is not mediated
by the slower processes involving induction or repression [32].
32 TMG (methyl 1-thio--D-galactopyranoside) was used by Adam Kepes for studying the kinetics of -galactoside transport into
Escherichia coli in the 1950s [190] (see also [25]).
33 Facilitated diffusion is carrier-mediated movement across a membrane which, unlike active transport, depends on a concentration
gradient and not on expenditure of metabolic energy (for review see [91]).

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Figure 12. Anaerobic and aerobic pathways of sugar catabolism in yeasts

Hendrik van Urk and his colleagues found the levels of pyruvate decarboxylase (see Figure 12) in
Saccharomyces cerevisiae and Candida utilis to be associated with the rate of catabolic flux in the
anaerobic utilization (fermentation) of D-glucose [358]. Observations on six species of yeast by Sims and
Barnett were consistent with these findings [325]. Five of these yeasts utilized one or more disaccharides
aerobically, but not anaerobically, although all used D-glucose anaerobically, that is, all five showed the
Kluyver effect; but the sixth yeast, S. cerevisiae, did not do so. When grown on a glycoside with which
it showed the Kluyver effect, each yeast had much less pyruvate decarboxylase activity than when grown
on a glycoside with which it did not give the effect (exemplified in Table 10). There was no consistent
corresponding lowering of activity of either alcohol dehydrogenase or of the relevant glycosidase.
Hence, they concluded, pyruvate decarboxylase may have a role in producing the Kluyver effect
[325, p. 295] and the chain of events might be as follows:
(a) Glycolytic flux may be low as a result of a combination of:
(i) The change from active transport to facilitated diffusion, which leads to a low concentration of
glycoside in the cytosol and;
(ii) The low affinity of the glycosidase for its substrate.34
(b) The consequent diminution of the rate of glycolysis leads to the rapid deactivation of pyruvate
decarboxylase, as described later for Kluyveromyces lactis [37], the enzyme being activated by its
substrate, pyruvate [39,174,314,324,335].
(c) While switching to anaerobic conditions activates pyruvate decarboxylase, transport is greatly slowed
down by a reduction in the supply of ATP, so pyruvate decarboxylase activation fails because of
reduced glycolytic flux.

Experiments of Jack Pronk and his colleagues


Although some later work on maltose catabolism by Candida utilis, published by Jack Pronk and his
colleagues at Delft in 1994, gave support to the notion that transport limitation is a factor in the Kluyver
effect, their findings with pyruvate decarboxylase conflicted with the idea that inactivation of that enzyme
was also a factor [378]. They found that pyruvate decarboxylase activities of C. utilis grown on maltose in
oxygen-limited culture had a higher flux even than in Saccharomyces cerevisiae under the same conditions.
The authors suggest that the Kluyver effect is caused by feedback inhibition of sugar transport by ethanol
[379].
34 Two

-glucosidases of Debaryomyces polymorphus have Km = 22 mM and 40 mM-cellobiose, respectively [361].

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855

Table 10. Specific activities of three enzymes in two yeasts grown on different sugars as sole source of
carbon: expressed as nmol substrate catalysed min1 (mg protein)1 . Results of Sims and Barnett [325]
Yeast

Carbon source for


growth

Pyruvate
decarboxylase

Alcohol
dehydrogenase

Glycosidasea

Candida viswanathii

D-glucose

0.13

1.04

maltose

0.11

0.32

0.33

cellobioseb

0.039

0.23

0.33

D-glucose

1.62

0.57

maltose

0.40

0.64

1.01

Saccharomyces
cerevisiae

a Yeasts grown on maltose were tested for -glucosidase activity. C. viswanathii grown on cellobiose was tested for
-glucosidase activity.
b C. viswanathii gave the Kluyver effect with cellobiose.

In order to test the hypothesis that yeasts, which show the Kluyver effect for sucrose, hydrolyse
it intracellularly [18,324], Pronk and his colleagues investigated sucrose uptake and metabolism by
Debaryomyces yamadae [184]. And, indeed, they concluded:
The results indicate that the Kluyver effect for sucrose in D. yamadae . . . is effected by rapid down-regulation of
the capacity of the sucrose carrier under oxygen-limited conditions [184, p. 1567].

Kluyver effect mutants: fds and gal2


In 1978, working in Norwich with Barnett, Entian attempted to isolate mutants of Kluyveromyces lactis
which did not show the Kluyver effect from strains that already did so. Although 40 000 colonies of
mutagenized cells grown aerobically on lactose plates were replica-plated onto maltose, cellobiose or
,-trehalose (all substrates giving the Kluyver effect with these yeasts), none of the colonies was able
to grow anaerobically on these sugars [100].
However, certain mutants failed to grow with glycerol. These fds mutants were totally aerobic and
depended entirely on anaerobic fermentation. However, they were not respiratory-deficient and, hence,
were similar phenotypically to the glucose derepression mutant snf1 of Saccharomyces cerevisiae (see
below). When these mutants were tested against substrates that gave the Kluyver effect, none was utilized
aerobically. Poisoning respiration with KCN immediately prevented uptake of these substrates and led
to an instant decrease in the concentration of D-glucose 6-phosphate. Adding glucose to these poisoned
cells promptly restored fermentation, which showed that glycolysis was still functioning. From these
observations and the genetical data, Entian and Barnett concluded:
All these results were consistent with the requirement of an energy supply for the transport of maltose, alpha,alphatrehalose or cellobiose, that involved the cytochrome system. [100, p. 325].

In the context of the Kluyver effect, Hiroshi Fukuhara has recently drawn attention to the failure of
gal2 mutants of Saccharomyces cerevisiae to use D-galactose anaerobically, although they will grow
on it aerobically [123]. (The GAL2 gene encodes the main galactose carrier [2,66,86,87].) Furthermore,
introducing a wild-type GAL2 gene into yeast with a gal2 mutant restores the ability to use galactose
anaerobically.
Results of some genetic experiments with Kluyveromyces lactis also give credence to the theory that
loss of the supply of metabolic energy, necessary for transport, has a role in producing the Kluyver effect.
Paola Goffrini and her colleagues in Parma have been studying the curious case of the Kluyver effect with
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Figure 13. Raff inose

the trisaccharide, raffinose (O--D-galactopyranosyl-(1 6)--D-glucopyranosyl -D-fructofuranoside,


Figure 13). This case is curious, because raffinose is usually hydrolysed outside the plasma membrane
by invertase (cf. Figure 9) to produce melibiose and D-fructose. Now K. lactis does not utilize melibiose
[29] and the fructose might be expected to be transported into the cells by a hexose carrier, as described
for Saccharomyces cerevisiae [65,201]. Hence, given that failure of transport across the membrane is
critical for producing the Kluyver effect, raffinose utilization would not be expected to be subject to the
effect. However, Goffrini and her colleagues report overcoming the effect in this yeast by introducing
sugar carrier genes from S. cerevisiae and they conclude:
These results strongly suggest that the sugar uptake step is the major bottleneck in the fermentative assimilation of
certain sugars in K. lactis and probably in many other yeasts [139, p. 427].

It is uncertain whether the tight coupling of concentrative monosaccharide transport to aerobic


metabolism described for Rhodosporidium toruloides [202], mentioned above, can be compared to
mechanisms underlying the Kluyver effect. In any case, a study of both phenomena could well assist
the progress towards an understanding of the transport of sugars into yeasts. Furthermore, Pronk and his
colleagues have suggested a potential industrial use for the Kluyver effect:
Because the use of yeast strains exhibiting a Kluyver effect obviates the need for controlled substrate-feeding
strategies to avoid oxygen limitation, such strains should be excellently suited for the production of biomass and
growth-related products from low-cost disaccharide-containing feedstocks [51, p. 621].

The Crabtree effect (repression of respiration)


Despite glucose repression in yeasts often being called the Crabtree effect, there are major differences
between these two phenomena, and so some explanation is given here of this effect and its history. In the
1920s, following up Warburgs findings that certain tumour tissues have a higher rate of glycolysis
than normal cells [368], Herbert Crabtree35 studied the respiration of tumour cells and found that
35 Herbert

Grace Crabtree (18921966), English biochemist, was with the Imperial Cancer Research Fund in London for 43 years [6].

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857

adding glucose decreased the respiration rate [71,175]. Unlike glucose repression in yeasts, the Crabtree
effect in tumour cells is commonly explained in terms of a decrease in ADP within the mitochondria
[55,292] because ADP is imported into the mitochondria by an exchange with cytoplasmic ATP. If
efficient glucose fermentation produces a high concentration of ATP in the cytoplasm, importing of
ADP into the mitochondria is prevented, and the consequent depletion of ADP leads to a lower rate of
respiration.
This, however, does not explain why 2-deoxy-D-glucose (2DG) produces a Crabtree effect [396]. In
1958, Kenneth Ibsen and his colleagues [176] showed that the level of ATP decreases almost immediately
after adding 2DG, and the Crabtree effect could be measured within 20 seconds after adding glucose.
From these observations, and also because 2DG gives a Crabtree effect too, these authors concluded
that the level of cytoplasmic ATP is overcome by a disproportionate reaction in the mitochondria of
2ADP ATP + ADP, ADP being exported into the cytoplasm. This export decreases the concentration
of ADP within the mitochondria.
In 1961, Benno Hess and Britton Chance carefully studied the kinetics of the Crabtree effect in tumour
cells [163], distinguishing between a short-term Crabtree effect, occurring within 2 minutes of adding
glucose, and a long-term Crabtree effect, which occurred after 2030 minutes. The short-term effect was
explained by an excess of ATP within the mitochondria and the long-term effect by reduced import of
ADP into the mitochondria. Both result in depleted mitochondrial ADP.

Glucose repression in yeasts


The eccentric behaviour of Saccharomyces cerevisiae, when supplied with D-glucose, has already been
mentioned in this series [31]: even in air, most of the pyruvate formed by glycolysis is channelled to
ethanol, rather than into the tricarboxylic acid cycle (Figure 14) and, accordingly, the yeasts respiratory
activity is decreased. When in 1966 De Deken described the catabolism of glucose by a number of
yeast species (Table 4), he named this decrease in respiration, produced by glucose, the Crabtree
effect after Crabtrees findings [78].
However, the physiological reasons for the lower rate of respiration after adding glucose are completely
different in yeast and tumour cells. Whereas, as described above, the respiratory decrease in tumour
cells depends solely on metabolic changes (ADP depletion), the corresponding respiratory decrease in
yeast cells is caused by the repression of the structural genes responsible for synthesizing respiratory
enzymes [128]. Hence, the term Crabtree effect is a misnomer for glucose repression in yeasts
[101].
Glucose repression was first reported for Escherichia coli by Helen Epps and Ernest Gale, who termed
it glucose effect [111]. Later, Boris Magasanik used the term catabolite repression instead [240]: in
1961, he wrote:
. . . considerations led us . . . to formulate the concept that catabolites which are formed rapidly from glucose
accumulate in the cell and repress the formation of enzymes . . . It is this interpretation of the glucose effect which
suggests catabolite repression as an appropriate name for this phenomenon [240, p. 251].

In 1998, Juana Maria Gancedo explained:


When [glucose or fructose] is present, the enzymes required for the utilization of alternative carbon sources are
synthesized at low rates or not at all. This phenomenon is known as carbon catabolite repression, or simply catabolite
repression, and since no catabolite derived from glucose and involved in the repression has been yet identified,
the term glucose repression has been proposed . . . I still use the term catabolite repression as well as glucose
repression, to stress that other sugars, such as galactose or maltose, are able to affect the synthesis of enzymes
repressed by glucose [129, p. 334].

In 1942, Epps and Gale had described their glucose effect as follows: the presence of glucose in
the medium during the growth of Escherichia coli suppresses the formation of certain enzymes [111].
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(A)

Figure 14. Diagram of aspects of metabolism of D-glucose and ethanol by Saccharomyces cerevisiae in (A) derepressed and
(B) glucose-repressed cells (after Ronne [304])

Solomon Spiegelman and John Reiner in 1947, and Wilbur Swanson36 and Charles Clifton37 in 1948,
reported a similar finding for Saccharomyces cerevisiae (or S. pastorianus) [331,342]. In their excellent
paper, Spiegelman and Reiner carefully examined the galactose metabolizing pathway, which they referred
to as galactozymase (see [25]). They observed that a yeast pre-grown with galactose, and thereafter
transferred to a glucose-containing medium, lost its galactozymase activity; but this loss was prevented
by adding azide38 (Figure 15). Two years later, azide was shown to inhibit phosphorylation [232].
Studies of glucose repression, described below, have shown that the presence of glucose in the growth
medium stops the transcription of glucose-repressible genes. As a consequence, after adding glucose:
(a) the total amount of certain enzymes remains constant; (b) however, the specific activity (enzyme
activity per mg protein) decreases, because the number of cells that do not transcribe increases [96].
In 1948, Swanson and Clifton gave an account of the effects of glucose repression (although they
did not use this expression) in Saccharomyces cerevisiae. When the yeast grew aerobically in batch
36 Wilbur H. Swanson (1903?) worked with Charles Clifton at the Department of Bacteriology and Experimental Pathology, School
of Medicine, Stanford University, California in the 1940s, moving to San Jose State College in 1948.
37 Charles Egolf Clifton (19041976), American microbial biochemist, worked at the Department of Bacteriology and Experimental
Pathology, School of Medicine, Stanford University from 1929, becoming professor of bacteriology (information kindly supplied by
Patricia A. French of Lane Medical Library, Archives and Special Collection Department, Stanford University, School of Medicine).
38 Sodium azide (NaN ) prevents the coupling of ADP phosphorylation to aerobic respiration [232]; in 1949, Eugene Kennedy and
3
Albert Lehninger found that isolated mitochondria catalyse oxidative phosphorylation, which is coupled to the oxidation of intermediates
of the tricarboxylic acid cycle [189].

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859

(B)

Figure 14. Continued

Figure 15. Loss of galactozymase activity by Saccharomyces sp. on adding D-glucose. The loss was prevented by adding
, activity after adding NaN3 ;
loss of activity in control
NaN3 . Arrows () indicate when NaN3 was added.
suspension, without NaN3 . Reproduced from Spiegelman and Reiner (1947) [331]; The Journal of General Physiology, 1947,
vol. 31, p. 183, by copyright permission of The Rockefeller University Press

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Figure 16. The tricarboxylic acid and glyoxylate cycles. Reproduced from [197], courtesy of H. L. Kornberg and by
permission of Elsevier

culture on 56 mM D-glucose, alcoholic fermentation predominated until the glucose disappeared from the
medium [342]. Sixteen years later, Walter Bartley and his colleagues published three key papers (in 1964
and 1965) which described a major step towards understanding glucose repression in Saccharomyces
cerevisiae. Sugars in the medium for growing this yeast aerobically caused an anaerobic type of
metabolism as measured by ethanol production, D-glucose being much more effective in this respect
than was D-galactose. This glucose repression affected enzymes of the tricarboxylic acid cycle, both
glucose and galactose repressing the key enzymes of the glyoxylate cycle39 (Figure 16) almost completely.
Furthermore, glutamate dehydrogenase was more than 50 times more active in S. cerevisiae when it was
grown on pyruvate than on D-glucose [287]. In addition, Bartley and his colleagues found no mitochondrial
structures in yeast grown aerobically on glucose but, with the removal of glucose, mitochondria reappeared
as the yeasts ability to respire acetate returned [288,289]. And in 1971, Alberto Sols and his colleagues
wrote:
There is considerable uncertainty as to whether the impairment of respiration caused by glucose is: (i) a case of the
catabolite repression that affects the synthesis of many catabolic enzymes (Polakis et al., 1965 [289]; DeDeken,
1966 [78]; C. P. M. Gorts, 1967 [141]); (ii) related to the disassembly of normal mitochondrial structures; or
(iii) involves a combination of factors. The mechanism(s) of catabolite repression in general is far from clear, and
is currently under study in several laboratories [330, p. 301].

Many kinds of enzyme in yeasts have been found to be subject to glucose repression. These
include respiratory enzymes [110,337], glyoxylate cycle enzymes [287,389], gluconeogenic enzymes
[126,130,148], disaccharide hydrolysing enzymes [85,120,212,341,359,360] and many others (Table 11).
39 Glyoxylate

cycle (a modification of the tricarboxylic acid cycle) by which two molecules of acetate form one molecule of C4 dicarboxylic acid, occurs not only in yeasts, but also in bacteria (e.g. [200]), filamentous fungi (e.g. [68,196]) and green plants (e.g.
[198]).

Acetate
Citrate
Oxaloacetate

isoCitrate
Malate

Acetate

Glyoxylate

Succinate

The glyoxylate cycle was first described by Kornberg, Krebs and Madsen in 1957 [199,205]. In 1960, Barnett and Kornberg published
evidence of its occurrence in the yeasts, Kluyveromyces lactis, Saccharomyces cerevisiae and Zygosaccharomyces bailii [27]. However,
Schizosaccharomyces pombe is said to lack two key enzymes of the cycle [112], which may explain its reported inability to utilize
acetate as sole carbon source for growth [207, p. 345].
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861

By the mid-1980s it was clear that the underlying regulation of glucose repression in Escherichia
coli differed from that in yeasts. Research on E. coli had shown D-glucose to lower the levels of
cAMP40 , which nucleotide is necessary for the transcription of genes sensitive to carbon catabolite
repression [177,353]. However, evidence was accumulating that this was not true of yeasts [241]. Adding
exogenous cAMP to strains of Saccharomyces cerevisiae that were permeable to it did not prevent
the repression of galactokinase [246] and levels of cAMP were at least twice as high in repressed
S. cerevisiae, Schizosaccharomyces pombe or Kluyveromyces marxianus as in the non-repressed yeasts
[112].
Today, it is clear that the molecular mechanism of glucose repression in E. coli differs completely
from that in S. cerevisiae. In E. coli, binding of cAMP to the cAMP receptor protein (CRP or CAP41 ) is
necessary for the transcription of glucose repressible enzymes [93,323,400]. By contrast, there is no CRP
homologous protein in S. cerevisiae and, unlike E. coli, there is a transient increase in cAMP concentration
in S. cerevisiae within 2 minutes of adding glucose [248,291,356]. Indeed, there is no evidence that the
level of cAMP in yeasts is associated with the glucose repression of synthesis of enzymes, such as
invertase [261].

Genetic analysis of glucose repression and identification of the genes involved


This historical review takes into account the following steps in the analysis of gene function:
(a) identifying the gene loci involved, by means of isolating mutants; (b) isolating the respective genes,
in most cases by plasmid complementation of the respective mutants; (c) sequencing the genes;42 and
(d) characterizing the biochemical function of the proteins encoded by each gene.

Nomenclature of genes and their synonyms


There are many synonyms for the genes involved in regulating glucose repression and it is difficult to
decide which name should be used for each gene. Genetic convention is to prefer the name used in the
first description of a mutant. However, many mutants which proved to be synonymous were isolated
independently. Often their allelism was demonstrated much later than their original description, in many
cases only after their respective wild-types had been isolated and sequenced. Accordingly, the chosen
name of each gene could be that used when it was first sequenced.
Mark Johnston has suggested that, like the nomenclature of mitochondrial proteins, the glucoserepression community should decide on new names, each of which would refer to the genes function.
Although this idea is attractive, this historical survey is no place to generate further confusion with yet
more names. Accordingly, in order to help follow the complexities of the molecular-genetic control of
glucose repression, the large number of pleiotropic genes involved and their synonyms are summarized
in Table 12 (see below).43
In the early 1970s, several repression mutants were described. One of these, flk1, was highly pleiotropic:
for this mutant, invertase, -glucosidase and flocculent growth were each non-repressible [310]. Earlier,
Oliver Lampen and his colleagues had described a mutant with a similar phenotype, but had only
characterized it biochemically [137,261]. Mutant flk1 was found later to be allelic to tup1, whose function
is described below in Table 13.
40 cAMP, cyclic AMP, adenosine 3 , 5 -cyclic monophosphate, is formed from ATP in a reaction catalysed by adenylate cyclase and has
regulatory functions in many kinds of organism. cAMP was first reported in a yeast in 1966 [56].
41 The cAMP receptor protein is also named CAP (for catabolite gene activator protein).
42 Understanding the molecular basis of glucose repression became possible in the 1970s with the development of methods of gene
isolation and sequencing. The first yeast gene was probably cloned in 1976 [338] and yeast transformations were reported in 1978 [35,166].
Accordingly, in the 1980s, many genes corresponding to glucose-repression mutants were isolated and their sequences determined.
43 For ease of reading, gene synonyms are given in brackets where original findings are reported and, thereafter, preference is given to
the gene name which has been first sequenced.

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Table 11. Repression by D-glucose of some enzymes in yeasts


Enzyme repressed

Yeast

Repression (%)

ACONITATE HYDRATASE

S. cerevisiae

51

Comparing growth in 50 mM D-glucose with


that in 44 mM pyruvate [287]

P. anomala
S. cerevisiae

39
91

Grown in 167 mM D-glucose; then incubated


for 4 h in (a) 56 mM D-glucose or (b) 61 mM
NaAc; (a) compared with (b) [88]

87

Comparing growth in 167 mM D-glucose with


that in 0.43 M ethanol [384]

K. marxianus

>99

Comparing growth in 111 mM D-glucose with


that in 11 mM D-glucose [75]

S. cerevisiae

>99

Comparing growth in different concentrations


of D-glucose, from 250 mM to 3 M [85]

91

Comparing growth in 111 mM D-glucose with


that in 17 mM D-glucose [310]

(aconitase)
ALCOHOL DEHYDROGENASE

-FRUCTOFURANOSIDASE
(invertase or inulinase)

S. pastorianus

Conditions

FRUCTOSE BISPHOSPHATASE

C. salmanticensis
D. carsonii
D. hansenii
P. anomala
P. membranifaciens
Rh. glutinis
Rh. minuta
Rh. mucilaginosa
Rhs. toruloides
Sp. pararoseus
S. cerevisiae

>98
69
62
82
90
86
52
89
76
95
>98

Comparing growth in 111 mM D-glucose with


that in 0.43 M ethanol [130]

-GALACTOSIDASE

K. marxianus

>99

Comparing growth in 111 mM D-glucose with


that in 11 mM D-glucose [76]

-GLUCOSIDASE

K. marxianus
K. dobzhanskii

90

Comparing growth in 1 mM D-glucose with


that in 0.1 mM D-glucose [239]

GLUTAMATE DEHYDROGENASE

S. cerevisiae

50

Comparing growth in 50 mM D-glucose with


that in 44 mM pyruvate [287]

ISOCITRATE DEHYDROGENASE
(NAD+ )

S. cerevisiae

59

Comparing growth in 50 mM D-glucose with


that in 44 mM pyruvate [287]

30

Grown in 167 mM D-glucose; then incubated


for 4 h in (a) 56 mM D-glucose or (b) 61 mM
NaAc; (a) compared with (b) [88]

67

Comparing growth in 50 mM D-glucose with


that in 44 mM pyruvate [287]

ISOCITRATE DEHYDROGENASE
(NADP+ )

S. cerevisiae

In 1975 Michael Ciriacy44 (Figure 17) devised an electrophoretic system by which he could distinguish
three isoenzymes of alcohol dehydrogenase: alcohol dehydrogenase I (Adh1p45 ), alcohol dehydrogenase
II (Adh2p) and mitochondrial alcohol dehydrogenase (Adh3p). He showed that Adh1p is mainly present
44 Michael

Ciriacy (19471996), German geneticist, studied the regulation of alcohol dehydrogenase isoenzymes, showed the first
Ty1 retrotransposon integration to be responsible for constitutive adh2 expression, and characterized glucose carriers of Saccharomyces
cerevisiae genetically. He was in Fritz Zimmermanns laboratory at Darmstadt from 1977 to 1981, when he became a professor at the
Institute of Microbiology at the University of Dusseldorf [160].
45 Abbreviations used for proteins, for which each gene is responsible, are written as the abbreviation of genes name, printed in roman
type, with the first letter a capital, e.g. Adh1. This may also be written Adh1p: the p is added (for protein) to prevent misunderstanding.
This convention differs from that used for the genes; e.g. the wild-type structural gene of alcohol dehydrogenase I is written ADH1 (in
italic capitals) and a mutant is adh1 (italic lower case).
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863

Table 11. Continued


ISOCITRATE LYASE

MALATE DEHYDROGENASE

MALATE SYNTHASE

OLIGO-1,6-GLUCOSIDASE

P. anomala
Rh. glutinis
S. cerevisiae

94
99
99

Grown in 167 mM D-glucose; then incubated


for 4 h in (a) 56 mM D-glucose or (b) 61 mM
NaAc; (a) compared with (b) [88]

99

Comparing growth in 83 mM D-glucose with


that in 61 mM NaAc [389]

P. anomala

Grown in 167 mM D-glucose; then incubated


for 4 h in (a) 56 mM D-glucose or (b) 61 mM
NaAc; (a) compared with (b) [88]

S. cerevisiae

44
87

P. anomala
Rh. glutinis
S. cerevisiae

S. pastorianus

94
99
95

Grown in 167 mM D-glucose; then incubated


for 4 h in (a) 56 mM D-glucose or (b) 61 mM
NaAc; (a) compared with (b) [88]

73

Comparing growth in 50 mM D-glucose with


44 mM pyruvate [287]

99

Comparing growth in 83 mM D-glucose with


that in 61 mM NaAc [389]

87

Comparing growth in 111 mM D-glucose with


that in 17 mM D-glucose [310]

67
55
50
5
10
>65
975
63
79

Comparing growth in 111 mM D-glucose with


that in 0.43 M ethanol [127]

(isomaltase)
PHOSPHOENOLPYRUVATE
CARBOXYKINASE

C. pelliculosa
Cr. humicola
P. anomala
Rh. glutinis
Rh. mucilaginosa
S. cerevisiae
S. pastorianus
Z. fermentati
Z. rouxii

Comparing growth in 83 mM D-glucose with


that in 61 mM NaAc [389]

C, Candida; Cr, Cryptococcus; D, Debaryomyces; K, Kluyveromyces; P, Pichia; Rh, Rhodotorula; Rhs, Rhodosporidium; S, Saccharomyces; Sp, Sporidiobolus;
Z, Zygosaccharomyces

during growth with glucose and is the major enzyme involved in ethanol production, whereas Adh2p
is subject to glucose repression [60,61]. He also described a mutation of the ADH2 promoter46 which
made alcohol dehydrogenase II47 insensitive to glucose repression [62]. Molecular analysis showed that
this insensitivity was caused by a promoter insertion of the yeast transposon Ty1 [384].
The first pleiotropic48 mutants of glucose repression, cat1 and cat2, were isolated by Fritz Zimmermann
in 1977 [398] by screening for mutants that could grow on glucose, but not on ethanol, as the
carbon source. The cat1 mutants failed to derepress various enzymes: -glucosidase, invertase, and
also gluconeogenic and respiratory enzymes; hence, these mutants did not grow with ethanol, maltose or
sucrose as a sole source of carbon.
That same year, Ciriacy, who later joined Zimmermanns laboratory, established a system for selecting
mutants in which there was no glucose repression. Ciriacy used haploid mutants lacking a constitutive
alcohol dehydrogenase49 and, from these haploid mutant strains, he selected new mutants which could not
46 A

promoter is a DNA region upstream to the coding sequence of a gene, which binds RNA polymerase.
dehydrogenase II (Adh2p), encoded by the gene ADH2, catalyses the first step of gluconeogenesis from ethanol. Adh2p is
cytoplasmic, necessary for alcohol degradation and is repressed by glucose several hundred-fold [236].
48 A pleiotropic mutation has more than one phenotypic effect.
49 Alcohol dehydrogenase I (Adh1p), unlike Adh2p, is the enzyme responsible for the formation of ethanol in alcoholic fermentation.
47 Alcohol

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Figure 17. Michael Ciriacy

Figure 18. Connexion between the glyoxylate and tricarboxylic acid cycles (modified from a diagram by Kornberg and
Madsen, published in 1957 [199])

grow on glycerol or ethanol [63]. These new mutants included ccr1, in which there was no derepression
of (a) enzymes of gluconeogenesis, (b) isocitrate lyase (of the glyoxylate cycle; see Figures 16 and 18)
or (c) fructose bisphosphatase50 (Figure 19), that is, the strains carrying ccr1 could not synthesize these
enzymes, whether or not glucose was present. Ciriacys ccr1 mutant was allelic to Zimmermanns cat1
50 Fructose bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) is often written fructose-1,6-bisphosphatase
in order to distinguish it clearly from fructose-2,6-bisphosphate 2-phosphatase, EC 3.1.3.46, (also written fructose-2,6-bisphosphatase).
Fructose bisphosphatase catalyses D-fructose 1,6-bisphosphate + H2 O D-fructose 6-bisphosphate + ortho-phosphate. Fructose bisphosphatase was first prepared from kidney and liver in 1943 by George Gomori [140] and its specificity for fructose 1,6-bisphosphate was
reported in 1955 [250].

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865

Figure 19. The regulation of glycolysis by activators and deactivators

(Ciriacy, personal communication) as well as to the snf1 mutant (Schuller and Entian, unpublished),
isolated by Marian Carlson [48] (see Table 12).
The cat1 mutation affects all the glucose-repressible enzymes and, as described below, later biochemical
analysis has shown that cat1 encodes the most central element in the regulatory circuit of glucose
repression, a protein kinase, named Snf1-4-kinase (see below).

Zimmermanns selection system for mutants defective in glucose repression


A further advance in the genetic analysis of glucose repression was Zimmermanns development in
1977 of a powerful system for selecting mutants which resisted glucose repression [399] (described in
an earlier article in this series [25]). Working with Saccharomyces cerevisiae growing exponentially on
glucose as carbon source, he plated this yeast on medium containing low concentrations (0.61.8 mM)
of 2-deoxy-D-glucose (2DG) plus raffinose.
The selection of mutants depended on certain properties of 2DG:
1. S. cerevisiae (and other yeasts), although using D-glucose, does not use 2DG for growth; 2DG is
toxic, as it is phosphorylated and incorporated into the cell wall, which becomes severely damaged
[162,180,181].
2. S. cerevisiae hydrolyses the raffinose by means of invertase [387,388], to give melibiose and D-fructose,
of which only fructose is utilized.
3. S. cerevisiae takes up D-glucose, D-fructose and 2DG by the same carriers [65,201].
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Table 12. Genetic and biochemical characterization of genes involved in glucose repression
description
Gene

Mutant isolation

Mutant phenotypes

Gene
sequence

Asterisk indicates first

of wild-type protein
Physiological role

Subunits of the central Snf/Cat complex


CAT1

Zimmermann and
colleagues 1977
[398]

CCR1 Ciriacy 1977 [63]


SNF1 Carlson and
colleagues 1981
[48]
CAT3

Entian and
Zimmermann
1982 [108]

SNF4 Neigeborn and


Carlson 1987
[271]
SIP1

No growth on non-fermentable
Celenza and
carbon sources; no derepression of Carlson
-glucosidase, invertase,
1986 [52]
gluconeogenic or glyoxylate cycle
enzymes
Zimmermann and colleagues 1977
[398]

Catalytic subunit of a Ser/Thr-specific


protein kinase complex, phosphorylates
activators and repressors involved in
glucose repression.
Celenza and Carlson 1986 and 1989
[52,53]

No growth on non-fermentable
Schuller and
carbon sources; no derepression of Entian 1988
-glucosidase, invertase,
[319]
gluconeogenic or glyoxylate cycle
enzymes
Entian and Zimmermann 1982 [108]
Celenza and
colleagues
1989 [54]

-Subunit of the Ser/Thr-specific


Snf1/Cat1 kinase
Celenza and colleagues 1989 [54]
Partial nuclear localization
Schuller and Entian 1988 [319]

Yeast two-hybrid interaction with


Snf1-kinase
Yang and colleagues 1992 [394]

-Subunits (scaffold proteins) of the


Ser/Thr-specific Snf1/Cat1 kinase
Yang and colleagues 1994 [395]

Yang and
colleagues 1992
[394]

SIP2

Yang and
colleagues 1992
[394]

GAL83

Yang and
colleagues 1992
[394]

Gene repressors under control of Snf/Cat complex


MIG1

Nehlin and
Ronne 1990
[270]
CAT4 Schuller and
Entian 1991 [320]
SSN1 Vallier and
Carlson 1994
[355]

Multicopy inhibitor of GAL gene


induction.
Nehlin and Ronne 1990 [270].
No repression of invertase or
-glucosidase; increased
hexokinase P11 activity
Schuller and Entian 1991 [320]

Nehlin and
Ronne 1990
[270]

C2 H2 Zinc-finger protein binds as


repressor to most glucose-repressible
genes
Nehlin and Ronne 1990 [270]
Released from the nucleus upon Snf/Catcatalysed phosphorylation
DeVit and colleagues 1997 [82]

Gene repressors not under control of Snf/Cat complex


MIG2

Lutfiyya and
Johnston 1996
[235]

Overexpression of MIG2 represses Lutfiyya and


SUC2
Johnston 1996
[235]

C2 H2 Zinc-finger protein binds to


Mig1p binding site, not a target of
Snf/Cat kinase
Lutfiyya and colleagues 1998 [234]

Zimmermann found that glucose-grown wild-type cells take up the toxic 2DG but, because the invertase
is glucose-repressed, do not hydrolyse the raffinose. Spontaneous mutants occur in all populations; those
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Table 12. Continued


Gene activators under control of Snf/Cat complex
CAT8

DIL1

SIP4

Hedges and
colleagues 1995
[158]
Rahner and
colleagues 1996
[295]
Lesage and
colleagues 1996
[221]

No derepression of gluconeogenic
or glyoxylate cycle enzymes. Fails
to grow with ethanol as carbon
source.
No derepression of isocitrate lyase

Interacts with Snf1 in the yeast


two-hybrid system

Hedges and
colleagues 1995
[158]
Rahner and
colleagues 1996
[295]
Lesage and
colleagues 1996
[221]

Binuclear zinc cluster (Zn2 Cys6 ) gene


activator binds to CSRE elements
Hedges and colleagues 1995 [158],
Rahner and colleagues 1999 [294]
Activation through Snf/Cat
phosphorylation
Randez-Gil and colleagues 1997
[296]
Binuclear zinc cluster (Zn2 Cys6 ) gene
activator binds to CSRE elements.
Activated through Snf/Cat
phosphorylation.
Vincent and Carlson 1998 [362]

Subunits of the Glc7 phosphatase


CID1

GLC7

HEX2

REG1

Neigeborn and
Carlson 1987 [271]

Constitutive invertase synthesis

Peng and
colleagues 1990
[284]
Entian and
Zimmermann
1980 [107]

Does not accumulate glycogen

Matsumoto and
colleagues 1983
[246]

No repression of invertase or
-glucosidase; partial
derepression of respiratory
enzymes; increased hexokinase
PII activity, inhibited by maltose
No repression of galactokinase

Glc7 type 1 protein phosphatase


Tu and Carlson 1994 [351].
Dephosphorylates Snf1 at Thr-210
McCartney and Schmidt 2001 [249]
Feng and
colleagues 1991
[114]
Niederacher and
Entian 1991
[273]

Subunit of Glc7
Tu and Carlson 1995 [352]

Other proteins involved in the signalling of glucose repression


HEX1

HXK2

CAT80

GRR1

Entian and
colleagues 1977
[109]

Lobo and Maitra


1977 [229]

Entian and
colleagues 1977
[109]

No repression of invertase or
-glucosidase; partial
derepression of respiratory
enzymes; decreased hexokinase
activity
Reduced glucose phosphorylation

Frohlich
and
colleagues 1985
[121]

No repression of invertase or
-glucosidase; abnormal cell
shape

Bailey and
Woodword 1984
[14]

Flick and
Johnston 1991
[118]

Hexokinase PII gene. Possibly


important in signalling glucose
repression
Entian 1980 [97],
Entian and Mecke 1982 [106].
Phosphorylated through Snf/Cat
complex
Randez-Gil and colleagues 1998
[297,298]
Possibly indirect effect on
glucose-repression; part of a
ubiquitin-conjugating enzyme
complex which regulates Rgt1p, a
regulator of certain hexose
transporters (HXT-genes)
Li and Johnston 1997 [222]

which have non-repressible invertase can hydrolyse the raffinose to give melibiose and D-fructose. So
such mutants become supplied with an excess of exogenous fructose molecules, which competitively
prevent the uptake of 2DG (Entian, unpublished). Hence, those cells that grew in a medium containing
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2DG + raffinose were derepressed mutants which had high invertase activity, even when glucose was the
carbon source.
Table 12 lists genes and their mutants which are involved in glucose repression and derepression and
summarizes the phenotypic effects of the mutations. The mutants, hex1 [399], hex2 and cat80 [109,399],
were isolated using Zimmermanns selection system. These mutants all had pleiotropic effects on glucose
repression, and their functional analysis (described below) showed all three genes, HEX1, HEX2 and
CAT80, to be central components of the regulatory circuit of glucose repression. Each of these three
mutants affected the glucose repression of invertase, -glucosidase and respiratory enzymes, but not the
repression of gluconeogenic enzymes. Later, in the 1980s, various other methods led to the isolation of
mutants which were allelic to those mutants that Entian and Zimmermann had obtained in 1977 and 1980
[107,399]. Mutant glr1 (allelic to hex1 ) was selected by using glucosamine instead of 2-deoxy-D-glucose
[259]; reg1 (allelic to hex2 ) was isolated using a selection system for non-repressible galactokinase [246];
and grr1 (allelic to cat80 ) [14] was selected on medium containing 0.1 M D-galactose and 0.6 mM 2DG.

in glucose repression
Entians analysis of hexokinases and their role
When analysing the way these non-repressible mutants functioned, Entian appreciated that the pattern
of derepression strongly resembled that of wild-type cells during growth on galactose, which had been
carefully examined by Polakis and Bartley in 1965 [287]. Hexokinase is the only glycolytic enzyme that
is by-passed during growth on galactose (see Figure 12 of article 7 in this series [25]) and the regulatory
role of the genes was underlined, when enzymic analysis revealed that hexokinase activity was much
decreased in hex1 and significantly increased in hex2 mutants [107,109,206,278]. The original cat80
mutant showed normal hexokinase activity but, with a different genetic background, the grr1 mutation
(which was allelic to cat80 ) gave a three-fold higher hexokinase activity [14].
Also in the 1970s, Pabitra Maitra51 and Zita Lobo52 carried out a careful genetic analysis of glucose
phosphorylation in Saccharomyces cerevisiae. They identified the loci of the structural genes for the
hexokinase isoenzymes PI and PII and their respective mutants, hxk1 and hxk2, as well as the glucokinase
mutant, glk1 [243]. Their system differed from that of Zimmermann: they selected mutants with an
increasing resistance to higher concentrations of 2-deoxy-D-glucose than he had used, the cells being
resistant to 2DG because of their failure to phosphorylate this sugar [228230,242].
Saccharomyces cerevisiae, it should be explained, possesses three hexose-ATP-kinases, namely (a) two
hexokinases (both EC 2.7.1.1), PI and PII53 [69], which phosphorylate both D-glucose and D-fructose
and (b) the D-glucose-specific glucokinase (EC 2.7.1.12) [242,244]. The relative rates of activity of PI
and PII differ: the rate of PI with D-fructose is about three times that with D-glucose, whereas PII gives
nearly the same rates with both substrates [215,393].
By the 1980s, the biochemistry of yeast hexokinases had been studied extensively (see [99] for review)
and, using the mutants of Lobo and Maitra, Entian was able to show that (a) the hex1 mutant corresponded
to the hexokinase PII structural gene hxk2 [97,106] (Table 12) and (b) HXK2 was markedly overexpressed in hex2 mutants. These findings indicated a regulatory as well as a catalytic function for
hexokinase PII, which was found to be important for triggering glucose repression [103,106].
After identifying hexokinase PII as a key enzyme in glucose repression, Entian wrote:
As shown previously . . . the lowered hexokinase activity [in hex1-mutants] was not associated with reduced
metabolite levels. This agrees with the similar catalytic activities of mutant and wild-type hexokinases at low
substrate concentrations . . . We hypothesize that, in addition to its catalytic activity, hexokinase PII also has a
regulatory component. This of course requires that the enzyme changes considerably, depending on the availability
of hexoses or their catabolic derivatives [97 p. 637].
51 Pabitra K. Maitra (b. 1932), Indian biochemist and geneticist, worked at the Tata Institute of Fundamental Research in Bombay
(Mumbai). Together with Zita Lobo, he made major contributions to the genetics and biochemistry of yeast carbohydrate metabolism.
52 Zita Lobo (?2000), Indian biochemist, was married to P. K. Maitra, with whom she published most of her 23 papers on carbohydrate
metabolism and genetics of yeasts [303].
53 In the 1960s, Saccharomyces cerevisiae was found to have two distinct hexokinase isoenzymes, PI and PII [135,215,322].

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It is still not known how hexokinase PII acts on the regulatory system. The original hypothesis
of an enzyme with a dual function, both catalytic and regulatory [97,106], has been neither proved
nor disproved. Entian and his colleagues have isolated hexokinase PII mutants with defective glucose
repression which, nonetheless, maintain their catalytic activity [103,104] but these mutants have not
been characterized at a molecular level. Furthermore, overexpression of the structural gene for yeast
glucokinase, GLK1 (which is only 40% homologous with the structural genes for yeast hexokinases
I and II), did not restore glucose repression. Hence the hexokinases, themselves, do have a specific
regulatory role. On the other hand, David Botstein and his colleagues obtained mutants which gave
evidence of a marked association between hexokinase catalytic activity and glucose repression [238] and
similar results were obtained after domain swapping experiments between hexokinase isoenzymes PI and
PII [305]. So, thus far, the question remains unresolved.

Carlsons analysis of sucrose-non-fermenting (snf ) mutants


The pleiotropic hex2 mutants were sensitive to maltose, so that adding maltose to a culture of such
mutant strains inhibited their growth, glycolysis and protein synthesis [98]. This effect of maltose on
these mutants was the consequence of its uncontrolled uptake and hydrolysis, which produced a high
intracellular concentration of glucose [98,105]. Entian exploited this maltose toxicity, as it provided
a convenient system for the selective isolation of mutants which are specifically required for maltose
utilization, as well as others involved in general carbon catabolite repression.
The cat3 mutant, reported in 1982 [108], was almost identical phenotypically to the cat1 mutant
described in 1977 [398]. Later, CAT1 and CAT3 were shown to encode major components of a multimeric
protein kinase complex and the epistasis of hex2 (= reg1) is explained, as this gene encodes a subunit
of the counteracting phosphatase Glc7p [351,352].
Also during the 1980s, Carlson and Botstein began another systematic investigation into glucose
repression in Saccharomyces cerevisiae. They and their colleagues isolated a large number of mutants
which failed to utilize sucrose and were named snf mutants (for sucrose non fermenter) [48]. Two of
these mutants, snf1 and snf4, proved to be of major importance towards furthering the understanding of
the molecular mechanism which underlies glucose repression. The snf1 mutants were shown to be allelic
to cat1 and the snf4 mutants to cat3 (Table 12 gives the synonyms of some of these alleles).
Various findings were important for further augmenting the understanding of the molecular control of
glucose repression:
1.
2.
3.
4.

Carlsons showing that SNF1 encodes a protein kinase [52,53] was particularly important.
Sequencing established that the CAT3 [319] and SNF4 genes [54] are identical.
The Cat3p protein was located in the nucleus [54,319].
Co-immunoprecipitation experiments showed that SNF1 and SNF4 form a common protein complex
(Snf1pSnf4p)54 [54].
5. Epistasis was observed of cat1 (= snf1 ) and cat3 (= snf4 ) mutants over the hex2 (= reg1 ) mutation
[108].
6. It was found that reg1 (= hex2 ), a subunit of the GCL7, encodes protein phosphatase 155 [351,352].
Further major advances became practicable with the establishment of the yeast two-hybrid genetic
system for studying interactions between proteins (Figure 20), described in 1989 by Stan Fields and
54 The physical interaction of SNF1 and SNF4 was used to establish the yeast two-hybrid system [116], which is even today an effective
method for obtaining first indications of physical proteinprotein interactions and is used in functional genome analyses for many species.
55 GCL7 is an essential gene. Originally, a mutant with non glucose-repressible invertase was isolated and called cid1 (for constitutive
invertase derepression) [271]. Later analysis of this mutant showed it to be viable (T152K) within the GLC7 gene [351].

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Figure 20. Diagram of the two-hybrid system of Fields and Song (based on their Figure 1) [116]. The GAL4 protein
is a transcriptional activator, which expresses genes encoding enzymes of the galactose pathway. (1) The GAL4 protein
consists of two separable domains, which do not function when separated: (a) Gal4-BD is a domain which binds specific
DNA sequences (UASG ); and (b) Gal4-AD is a domain which activates gene transcription. (2) Fields and Song separated
these two domains as two genes and (3) fused Gal4-BD to protein X and Gal4-AD to protein Y. (4) If X and Y interact to
form a dimer, the two domains are brought together and transcription is activated

Ok-kyu Song [116].56 This system has become widely used for selecting proteins that interact with a
known protein. By means of yeast two-hybrid screenings, Carlsons group used Snf1p to identify three
other proteins, Sip1, Sip2 and Gal83, which interact with the Snf/Cat kinase [53,394,395], and domain
interaction analyses, using the same system, indicated that these three (Sip1p, Sip2p and Gal83p) act
as alternative scaffold proteins.57 Under conditions of glucose repression, these scaffold proteins build
bridges; Gal83p, for example, is the bridge between Snf1p and Snf4p and, under derepressing (low
glucose) conditions, this bridge may support the direct interaction between the now phosphorylated
Snf1p and its positively acting regulatory protein Snf4p (Figure 21) [179].58 The scaffold subunits are
also responsible for the intracellular location of the Snf/Cat complex [363]. The major scaffold proteins
for glucose repression seem to be Gal83p, because it directs the Snf/Cat kinase complex with its nuclear
localizing sequence (NLS) to the nucleus [363], and Sip2, because it is N -myristoylated [12]59 and
therefore retains the Snf/Cat complex in the cytoplasm due to its fixation at the plasma membrane
[223].
56 Fields and Song made use of the characteristics of the transcriptional activator protein GAL4p of Saccharomyces cerevisiae [116]. This
activator has functional domains for DNA binding (Gal4p-BD) and for gene activation (Gal4p-AD), which Fields and Song separated
as two genes. Gal4p-BD is fused to one protein, X, and Gal4p-AD to protein Y. If X and Y interact to form a dimer, this dimerization
brings the Gal4p-AD and Gal4p-BD together. As a result, transcription of genes regulated by GAL4p DNA-binding sites is activated
and the activation can be detected. This system has made it practicable to identify and clone genes, the products of which interact with
a known protein of special interest [59]. The known protein is fused to Gal4p-BD and expressed in a gal4 deletion strain. Libraries of
these fusions are screened for clones which activate a GAL4-regulated promoter.
57 The S. cerevisiae Snf/Cat kinase is a homologue of the highly conserved AMP-activated serine/threonine kinases, which are found
in plants, Drosophila, Caenorhabditis elegans, mammals and fungi (for review, see [154]). The Snf/Cat kinase contains a catalytic
-subunit encoded by SNF1, a regulatory subunit encoded by CAT3 (SNF4 ), and the -subunits, which act as scaffold proteins,
having structural roles as temporary structural frameworks but no catalytic properties.
58 This interpretation of the genetic results of yeast two-hybrid analysis was recently confirmed biochemically, using tandem-affinitypurification and MALDITOFMS (mass spectrometry) to analyse the protein composition of protein complexes [134]. The N-terminal
glycine residue of Sip2 is modified by myristoylation [12].
59 Protein N-myristoylation promotes weak and reversible proteinmembrane interactions [283].

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871

Figure 21. Diagram of protein interactions involved in regulating glucose repression in Saccharomyces cerevisiae (after
Carlson [47]). Events in low glucose concentration: (1) scaffold proteins bring Snf1p kinase and Snf4p protein together;
(2) Snf4p protein activates Snf1p kinase; (3) Snf1 kinase permits transcription of glucose-repressed genes

Repressors and activators under regulatory control of the Snf/Cat kinase


The major repressor, Mig1p, that binds to glucose-repressible proteins, was isolated and characterized in
the early 1990s by Hans Ronne and his colleagues [269,270]. They found that the mig1 mutants reversed
glucose repression for certain genes, including SUC2 (encoding invertase) as well as the GAL1 and
GAL4 genes (encoding galactokinase and the GAL gene specific activator Gal4p).
At about the same time, the cat4 and the ssn1 mutants were isolated as suppressors of the expression
of (a) -glucosidase (maltase) in cat1 and cat3 mutant strains [320] and (b) invertase [355]. Finding
these mutants to be allelic to mig1 (H.-J. Schuller, personal communication) provided the first indication
that Mig1p and the Snf/Cat kinase are interrelated.
Although binding sites for the Mig1p repressor were detected in nearly all glucose-repressible genes,
those genes concerned with gluconeogenesis were unaffected. This difference in response to mig1 mutants
was explained after Entian and his colleagues had identified the Cat8p gene activator (zinc cluster60 )
protein [158,294], which was also found as DIL1 when Hans-Joachim Schuller and his colleagues
screened for mutants that failed to derepress the gluconeogenic enzyme, isocitrate lyase, encoded by
ICL1 [295]. Cat8p is necessary specifically for the transcription of gluconeogenic genes and, hence,
is essential for growing yeast on non-fermentable carbon sources. Recent use of microarray analyses
has confirmed this highly specific function of Cat8p with about 30 target genes, of which 12 are
strongly regulated by Cat8p. These include all the structural genes of the enzymes of gluconeogenesis
[156,290].
The CAT8 gene, itself, is under the regulatory control of Mig1p and, hence, its expression
is repressed on glucose. However, expression of CAT8 is not sufficient for the transcription of
gluconeogenic genes, because the Cat8 protein needs post-translational activation via Snf/Cat kinasemediated phosphorylation [296]. The existence of this dual control system for the expression of
gluconeogenic genes explains why all attempts, even with highly stringent selection systems, have failed
[252].
60 Zinc cluster proteins: some regulators of transcription contain a zinc cluster (or zinc finger) which enables them to bind to specific
DNA sequences. For example, X-ray crystallography has shown that Gal4p binds to certain DNA sequences, CGG N11 CCG, with each
zinc cluster recognizing a CGG triplet. Many DNA-binding proteins have a zinc cluster, which is a polypeptide chain bound to a zinc
atom.

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Figure 22. Regulation of Class I glucose-repressible genes, such as the structural genes for SUC2 and CAT8. If glucose is
present in the medium, the Mig1 repressor binds to the respective URS sites (upstream repressing sequences) of Class I
genes and prevents their transcription. Under these conditions, Snf/Cat kinase is inactive. If glucose is exhausted in the
medium, the Snf/Cat kinase is activated and phosphorylates the Mig1 repressor (probably with the nuclear Snf/CatGal83
complex kinase). Consequently, the phosphorylated Mig1 repressor is exported to the cytoplasm. It is not known whether
Mig1 phosphorylation is a prerequisite for the dissociation of Mig1 from the URS of the respective structural genes

Another important step towards understanding the physiology of glucose repression has been the finding
by Johnston and his colleagues that the Mig1p repressor is exported from the nucleus, within minutes,
when cells are derepressed by depriving them of exogenous glucose [82].
A series of observations, summarized below in this paragraph, have provided the basis for understanding
the roles of the Snf/Cat kinase and Mig1p in glucose repression (see Figures 22 and 23). As mentioned
above, mig1 and its allelic isolates, cat4 [320] and ssn1 [355], partly suppressed the snf1 mutant,
indicating that the Snf1p kinase deactivates Mig1p. Snf/Cat kinase has been shown to phosphorylate
Mig1p at least in vitro [276,327,348]. Furthermore, Johnston and his fellow authors have given convincing
evidence that Mig1p phosphorylation brings about nuclear exclusion of Mig1p by means of binding the
nuclear exportin Msn5p [81], which had been previously identified genetically as a multi-copy suppressor
of Snf1p [113]. In order to exert repression, Mig1p requires the additional interaction with the repressors
Cyc8p (= Ssn6p) and Tup1p; hence cyc8 (= ssn6 ) and tup1 mutants fail to repress respiratory enzymes
and/or invertase [49,307,349]. Mutations within both these genes have pleiotropic effects and, having first
been identified by virtue of such different effects, these mutants have been given several synonymous
designations, which are listed in Table 13. Cyc8p and Tup1p, physically associated within a large nuclear
protein complex [383], interfere with chromatin structure [92] and both of them act as mediator proteins
for other regulatory proteins, which explains their pleiotropic effects (for review, see [328]).
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Figure 23. Regulation of Class II glucose-repressible genes, such as the structural genes for gluconeogenesis and the
glyoxylate cycle. If glucose is exhausted in the medium, the Snf/Cat kinase is activated and has a dual function. First, Snf/Cat
kinase phosphorylates the Mig1 repressor (probably catalysed by the nuclear Snf/CatGal83 complex). Consequently,
the phosphorylated Mig1 repressor is exported to the cytoplasm. Second, Snf/Cat kinase phosphorylates the Cat8 gene
activator (probably catalysed by the plasma membrane-bound Snf/CatSip2 complex). NB: the CAT8 structural gene is also
a Class I glucose-repressible gene under Mig1 control. In order to function, the CAT8 gene must be released from glucose
repression; its post-translational activation is via the Snf/Cat kinase

The current model of glucose repression: single and double61 control systems
To sum up, the mechanism of glucose repression is now quite well understood and may be described
simply as follows. The Snf/Cat kinase is the central element for glucose repression and regulates the
activity of the respective gene repressors and activators. Under conditions of glucose limitation, the
Snf/Cat kinase complex is activated when it is phosphorylated. The catalytically active Snf/Cat kinase
complex then has a dual function:
1. It phosphorylates the Mig1p repressor, so inhibiting its action, and enabling it to be exported from
the nucleus.
2. By means of phosphorylation, it activates specific gene activators such as Cat8p.
61 Double

control system: two systems, and if one fails the other one will take over.

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Table 13. Pleiotropic effects of mutants of the genes CYC8 and TUP1
Mutant

cyc8

tup1

Allelic
designations

Phenotype

cyc8

Over-expression of iso-2-cytochrome c [307]


Producing non-repressible invertase [349]

ssn6

Extragenic suppressor of a snf1 (cat1) mutation [49]

tup1

Ability to take up thymidine [381]


Constitutive invertase synthesis on glucose [349]

a ARS,

flk1

Flocculent growth; the first genetically characterized mutant with non-repressible


invertase, -glucosidase and galactose utilization [310,332]

umr7

Resistant to UV-induced CAN1 mutations [219]

amm1

Stabilizing ARSa -defective plasmids [345]

cyc9

Over-expression of iso-2-cytochrome c [307]

aar1

-Specific mating type defect [153,262]

aer2

Increased expression of CYC1, CYC7 (iso-2-cytochrome c) and GAL1, [397]

sfl2

Flocculation phenotype [122]

autonomous replicating sequence necessary for chromosome and plasmid replication.

Transcriptional
Repressor

Glucose

Glucose-repressible
Structural Genes

Snf/C at kinase
complex

Transcriptional
Activator

Figure 24. Simplified diagram of the regulation of glucose repression. The Snf/Cat kinase is inactive if glucose is available in
the medium. Hence, the Snf/Cat complex has a dual function in regulating glucose repression. If activated, when no glucose
is available, Snf/Cat kinase (a) inactivates the transcriptional repressor and (b) activates the transcriptional activator; so that
finally the transcriptional repressor dissociates from the glucose-repressible structural genes and the activator binds to the
structural gene

Figure 24 gives a simplified picture of these events.


Three kinases, Pak1p, Tos3p and Elm1, can activate the Snf/Cat kinase [170,265,340] from which
the Pak1 kinase seems to derive its major physiological function, as Pak1-dependent phosphorylation
of Snf/Catp is important for the nuclear localization of the Snf/CatGal83 complex [157]. It is still not
known how the kinases that activate the Snf/Cat kinase complex are, themselves, activated when glucose
is depleted, or how hexokinase PII interferes with the system.
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Three findings in the late 1990s have provided further evidence for a regulatory role of hexokinase PII
in glucose repression. First, hexokinase PII is present in the nucleus as well as in the cytoplasm [297];
second, there is an Snf1p-dependent phosphorylation of hexokinase PII [296]; and third, hexokinase has
been identified as a target for Hex2p-dependent phosphorylation [3]. These three findings are evidence
of the interrelation of hexokinase PII and the mechanism of glucose repression. However, it is still
unclear whether the effects observed with hexokinase PII are responsible for, or just the consequence of,
glucose-dependent regulation.

Classification of glucose-repressible genes according to their regulation


Three classes of glucose-repressible genes have been recognized: (I) genes whose expression is under
a single glucose control mechanism; (II) genes whose expression is under a single glucose control
mechanism, but which also require induction; and (III) genes which are under additional glucose control
mechanisms.
The expression of Class I genes mainly depends on the release of the Mig1p repressor from the
respective promoters and can be described as follows. After its Snf/Cat1-dependent phosphorylation,
Mig1p is then exported from the nucleus and derepresses several genes, which include the invertase
structural (SUC ) genes and gene activators such as CAT8 (Figure 22).
Expression of Class II genes requires the release of Mig1p from the promoter and the additional binding
of an inducible gene activator. This class (II) of genes includes those concerned with catabolizing maltose
(MAL genes) and galactose (GAL genes).
Class III genes are strictly controlled by glucose. Their expression depends on (a) the release of Mig1p
from the promoter and (b) the binding of specific gene activators. Class III genes are mainly those
belonging to crucial metabolic pathways for the utilization of non-fermentable carbon sources, such as
those catabolized via gluconeogenesis and the glyoxylate cycle. The specific gene activator is CAT8, a
class I gene. However, transcription of CAT8 is, of itself, insufficient for transcribing the genes concerned
with gluconeogenesis. Furthermore, the Cat8 protein is activated by phosphorylation, which is catalysed
by Snf/Cat kinase. In other words, the dual control of class III genes prevents failure of glucose repression
of the gluconeogenic enzymes. And such failure would, of course, be disastrous for the cell. After its
activation, Cat8p also binds to its functional homologue Sip4p [221], which then enforces derepression
of gluconeogenic genes (Figure 23).

Enzyme inactivation and the regulation of gluconeogenesis


A yeast growing on a non-fermentable source of carbon, such as acetate, ethanol, glycerol or lactate,
requires high activities of enzymes of both the gluconeogenic pathway (Figure 19) and the glyoxylate
cycle (Figures 16 and 18). Adding D-glucose to such a yeast leads to the onset of glycolysis and to high
activity of fructose bisphosphatase. Without regulation of the enzyme activity of the two reciprocal
pathways (glycolysis and gluconeogenesis), an energy-wasting futile cycle would ensue, between
phosphofructokinase and fructose bisphosphatase (see Figure 19).
Accordingly, certain enzymes are strictly regulated by several biochemical and genetic systems, which
are dependent on the nature of the available carbon source. These enzymes include both the two key
enzymes of gluconeogenesis (phosphoenolpyruvate carboxykinase and fructose bisphosphatase, encoded
by genes PCK1 and FBP1 ) and the enzymes of the glyoxylate cycle (isocitrate lyase, malate synthase and
cytoplasmic malate dehydrogenase, encoded by genes ICL1, MLS1 and MDH2, respectively). In addition
to allosteric inhibition of fructose bisphosphatase by both AMP [126] and D-fructose 2,6-bisphosphate62
[132], the transcription of all genes which encode the gluconeogenic and glyoxylate cycle enzymes are
62 D-fructose

2,6-bisphosphate was demonstrated in Saccharomyces cerevisiae in 1981 [216].

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J. A. Barnett and K.-D. Entian

subject to glucose repression (see above). Furthermore, in 1965 Helmut Holzer63 (Figure 25) and his
fellow-workers had found that malate dehydrogenase activity rapidly disappears on adding D-glucose to
the medium [390], and this crucial finding drew attention to another important mechanism that regulates
the amount of enzyme in the cell, that is, the specific proteolysis of enzymes64 (see also above, Table 2).
This specific proteolysis of enzymes, which occurs when glucose is added to yeast cells, has been studied
extensively for many years and is called glucose (or catabolite) inactivation (Figure 26) [168].
As long ago as 1947, Spiegelman and Reiner had found that adding D-glucose to cells of
Saccharomyces species grown on D-galactose not only repressed but also rapidly inactivated the synthesis
of galactozymase (the galactose catabolizing pathway) [331]. This inactivation occurred within 4 hours
of adding glucose. A similar result was reported in the 1950s by J. J. Robertson and Harlyn Halvorson,
who found that the ability of maltose-grown S. cerevisiae to ferment maltose to ethanol and carbon dioxide
was stopped about 3 hours after adding D-glucose, although enzymic activity and glucose fermentation
were not destroyed [302].
Confirming their conclusion that it was the maltose uptake system which was inactivated, Coen Gorts65
found that adding maltose did not prevent this inactivation [142]; hence, it was the presence of glucose and
not the absence of maltose which caused the inactivation. Recovery of maltose uptake after about 1 hour in
glucose-free, maltose-containing medium was inhibited by adding cycloheximide, which prevented protein

Figure 25. Helmut Holzer. Photo courtesy of Karl Decker


63 Helmut

Holzer (19211997), German biochemist, was a pioneer in the study of enzyme regulation. When he was aged 18, World
War II began, and he was obliged to undertake labour (Arbeitdienst) constructing fortifications along the Rhine. He was in the German
army in France and then was wounded in Russia. Holzer joined Feodor Lynens group at Munich in 1945 to work on the metabolism
of butanol in yeast cells, moving to Hamburg in 1953 and thence to become professor of biochemistry at Freiburg-im-Breisgau in 1956
[77].
64 Several of these mechanisms, particularly induction, were considered in article number 7 of this series.
65 Coen P. M. G
orts (b. 1935), Dutch microbial biochemist, worked in the Botanical Laboratory, State University, Utrecht, The
Netherlands, from 1961 to 1972, when he moved to the education department. (C. P. M. Gorts, personal communication).
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877

Figure 26. Catabolite inactivation, as illustrated by Helmut Holzer in 1976 [168]. Reprinted from Trends in Biochemical
Sciences 1: 178181, copyright 1976, with permission from Elsevier

synthesis [354]. Spiegelman and Reiner had observed earlier that the galactose pathway (galactozymase)
was inactivated only when cells were suspended in buffer; not when they were growing [331]. Later,
Holzer and his colleagues showed that this inactivation was due to a reduced substrate affinity of the
galactose carrier [245].

Holzers analyses of glucose inactivation (catabolite inactivation)


A different kind of inactivation was found for malate dehydrogenase [390]; its cytoplasmic isoenzyme
was rapidly inactivated within 3060 minutes after adding glucose, even when the cells were growing
[90,115]. Similar findings were obtained for various other enzymes, namely, isocitrate lyase [89], fructose
bisphosphatase [125], phosphoenolpyruvate carboxykinase [127,148] and 2-isopropylmalate synthase (of
the leucine biosynthetic pathway) [45]. Adding cycloheximide66 did not prevent the inactivation of
fructose bisphosphatase [125] or phosphoenolpyruvate carboxykinase [127]; hence, glucose inactivation
seemed to be independent of synthesis of the enzymes de novo. However, cycloheximide did prevent
inactivation of cytoplasmic malate dehydrogenase [115] but did not prevent this same inactivation in a
tryptophan-auxotrophic mutant [90], either in starved cells [268] or in a temperature-sensitive mutant at
elevated temperature [267]. These observations provided evidence that protein synthesis de novo is not
needed for glucose inactivation of cytoplasmic malate dehydrogenase. Holzer called this phenomenon
catabolite inactivation but, since it was shown that no glycolytic catabolite occurring after D-glucose
6-phosphate is necessary, the term glucose inactivation seemed more appropriate [102].
Cycloheximide, however, prevented the recovery of all these enzymes, which suggested that they were
irreversibly degraded proteolytically, and Dieter Mecke and his colleagues provided direct evidence for
this interpretation: they showed immunologically that, on adding glucose, the amount of cytoplasmic
malate dehydrogenase decreased in proportion to the enzymic activity [149,266]. This finding was
also confirmed for fructose bisphosphatase [124] and phosphoenolpyruvate carboxykinase [264]. Further
evidence of a proteolytic degradation came from the observation that phenylmethanesulphonyl fluoride
66 Cycloheximide

is often used as an inhibitor of protein synthesis, but it is reported to inhibit glycolysis markedly [145].

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J. A. Barnett and K.-D. Entian

(PMSF), an inhibitor of serine-proteases,67 prevented the inactivation of -isopropylmalate synthase in


permeabilized cells [45] and also prevented the inactivation of isocitrate lyase and fructose bisphosphatase
in normal (non-permeabilized) cells [146].
What happens in glucose inactivation seemed even odder: Holzer explained that, when studying
inactivation of fructose bisphosphatase, the results of some of their control experiments were inconsistent
with there being an irreversible degradation of the enzyme. He and his colleagues showed that adding
glucose to cells in their stationary phase (after growth) caused a very rapid loss of 50% of enzyme
activity within 1 or 2 minutes. Astonishingly, this rapid inactivation appeared reversible for more than 15
minutes (after adding glucose) as, during that time, the activity could be recovered, even in the presence
of cycloheximide (Figure 27) [220]; moreover, experiments with antibodies showed that the enzyme was
still cross-reacting during that period [346]. This rapid reversible inactivation is brought about by an
enzyme conversion, in which fructose bisphosphatase is phosphorylated [263,247] at serine residue 11
[301]. In vitro, the phosphorylation could be catalysed with a cAMP-dependent protein kinase and the
phosphorylation was faster when fructose-2,6-bisphosphatase was present [133]. In vivo, a marked cAMP
increase occurred within 2 minutes of adding glucose [347], while the concentration of D-fructose 1,6bisphosphate increased greatly [216]. Proton ionophores also triggered the phosphorylation of fructose
bisphosphatase, showing that the membrane potential is important for the increase in cAMP [248].
The finding that fructose bisphosphatase was phosphorylated before its irreversible degradation was, at
first, thought to indicate that this conversion triggered the proteolysis of this enzyme. However, Entian
and Matthias Rose showed that a mutated form of fructose bisphosphatase, where Ser [11] was replaced
by Ala [11], no longer underwent rapidly reversible inactivation, but was still susceptible to irreversible
glucose inactivation [306]. Commenting on this work, the Gancedos wrote:
It can be concluded that phosphorylation of FbPase is not required for the irreversible inactivation and could be an
independent mechanism of regulation, as phosphorylation increases the sensitivity of FbPase towards the inhibitors
fructose-2,6-bisphosphate and AMP [131, p. 367].

Isocitrate lyase was also shown to decrease in activity, reversibly, after phosphorylation [233].

Figure 27. Reactivation of fructose bisphosphatase activity after inactivation by glucose. At zero time, 3 and 30 minutes
after adding glucose, cells were washed and resuspended in 0.1 M potassium phosphate buffer (pH 6.0) with or without
cycloheximide. Reproduced from [220] by permission of Elsevier
67 Proteases (or proteinases), a term originally used in 1928 by Wolfgang Grassmann (18981978) and Hanns Dyckerhoff (19041965)
[144], are orthodoxly called peptidases or peptide hydrolases; this term applies to any enzymes that hydrolyse peptide bonds [274].

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879

Genetic analysis of glucose inactivation


Although the rapid reversible inactivation of some gluconeogenic enzymes after phosphorylation is
physiologically significant, the findings described above do not explain how glucose inactivation occurs, or
how it is catalysed. Using mutants with blocked glycolysis [64], rapid reversible and irreversible glucose
inactivation were found generally in glucose-6-phosphate isomerase (pgi ), triose-phosphate isomerase
(tpi ) and phosphoglycerate kinase (pgk ) mutants [102]. This clearly showed that no metabolites occurring
after glucose 6-phosphate in the glycolytic pathway are needed to trigger both types of inactivation.
Unexpectedly, mutants of phosphoglycerate mutase (pgm) and pyruvate kinase (pgk ) showed a rapid
reversible inactivation only, but there was no irreversible inactivation. This was explained in terms of
interference of the triosephosphates with the proteolytic machinery. Under anaerobic conditions, rapid
reversible inactivation was normal in all glycolytic block mutants, but irreversible inactivation did not
occur without the expenditure of respiratory energy. Irreversible inactivation was prevented both in a
pyruvate kinase (pyk ) mutant under all conditions and in glycolytic block mutants anaerobically; this
rapid inactivation remained reversible, even for as long as 2 hours after adding glucose [102].
These findings provide striking evidence for a proteolytic degradation pathway and research has been
focused on identifying the proteases which were responsible for this degradation. Proteases in yeast
were first identified and characterized biochemically; and protease A68 was one of the first enzymes
to be described in yeast [136,80,385]. In the early 1980s all known yeast proteases were located
inside the vacuole; an exception being aminopeptidase B, which was within the vacuolar membrane.
Of these, protease A is of major importance, because it is necessary for the maturation69 of protease
B, carboxypeptidase Y and aminopeptidase I.70 Mutation within the structural gene of each of these
three proteases only eliminated the activity of the respective protease itself. Accordingly, such mutants
were used to test whether or not these proteases are involved in glucose inactivation of gluconeogenic
enzymes; however, such tests gave conflicting results.
Hui-Ling Chiang and Randy Schekman reported the importing of fructose bisphosphatase into so-called
Vid vesicles (diameter 3040 nm) and the subsequent vacuolar inactivation of this enzyme [57]. They
also isolated vid mutants, defective in vacuolar inactivation; the inactivation depended on protease A and
other proteins, such as Vid22p, Cpr1p and Vid24p (Figure 28) [43,44,58].
By contrast to the above findings, Dieter Wolf and his colleagues have reported that, in mutants
of protease A and protease B, catabolite inactivation of fructose bisphosphatase is independent of
vacuolar proteolysis [251,344,392]; and they have provided clear evidence that inactivation of fructose
bisphosphatase requires polyubiquitinylation71 at the cytosolic 26 S proteasome72 [315317].
Although isolating mutants defective in glucose inactivation has proved exceedingly difficult because
there were no appropriate selection systems, Entian and his colleagues partly solved this problem in the
1990s. They found that the N-terminal fragment of fructose bisphosphatase, which is composed of 291
N-terminal amino acid residues, is necessary for glucose inactivation and that a fusion of this fragment
with E. coli -galactosidase makes the -galactosidase susceptible to glucose inactivation. By means of
68 Protease

A was purified and described in 1980 [256]; originally designated EC 3.4.23.6, later 3.4.23.25, and named saccharopepsin,
yeast endopeptidase A or Saccharomyces aspartic proteinase [377,391].
69 These proteases are primarily translated as larger proteins from which the active proteases are matured after proteolytic cleavage.
70 This became clear after isolating mutants defective for (a) protease A (pep4 ) [155], (b) protease B (prb1 ) [161,183],
(c) carboxypeptidase Y (prc1 ) and (d) carboxypeptidase S (cps1 ); for review see [34,164,165]; for activities of several proteases (protease
A, protease B, and carboxypeptidase Y), see [182,350].
71 Ubiquitin is a small protein (8.5 kDa) which attaches to proteins as a preliminary to their destruction in proteasomes. Ubiquitin has
been described as the cellular equivalent of the black spot of Robert Louis Stevensons Treasure Island: the signal for death [36, p.
635].
72 Proteasomes, which occur in the yeast cytoplasm and nucleus, are nanocompartments, where proteolysis is confined. The term was
first used in 1988 for particles in HeLa cells [11], and described as very large peptidases with several non-identical subunits and, later,
as 20 S cylindrical particles in various eukaryotes [193,343]. In order to move proteins to a proteasome, they are polyubiqutinylated by
means of an enzyme cascade, consisting of an ubiquitin-activating enzyme, ubiquitin-conjugating enzymes and ubiquitinprotein ligases
which mark these proteins for degradation [34,164,165].
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Figure 28. The concept of fructose bisphosphatase (FBPase) inactivation in the vacuole, by means of Vid vesicles (adapted
from [43]). When starved cells of Saccharomyces cerevisiae are given D-glucose, FBPase is taken into Vid vesicles and then
degraded in the vacuole. The first step involves at least two cytosolic proteins, Ssa2p and Cpr1p, the level of the latter being
regulated by the plasma membrane protein Vid22p. Formation of Vid vesicles is thought to be regulated by the ubiquitin
conjunction enzyme Ubc1p. Delivery of FBPase by Vid vesicles to the vacuole depends on Vid24p. Vacuolar proteinase
degrades the FBPase

this FBPaselacZ fusion, these authors established a screening system for isolating mutants which had
a defect in glucose inactivation, so that it became practicable to isolate three independent gid-mutants
(glucose inactivation deficient, gid1, gid2 and gid3 ).73
All gid-mutants were defective in the glucose inactivation of several enzymes, namely, cytoplasmic
malate dehydrogenase, isocitrate lyase and phosphoenolpyruvate decarboxylase, but other functions of the
proteasome were unaffected [152]. Of particular interest was finding the N -terminal proline residue to be
essential for inactivating fructose bisphosphatase, and a replacement with any other amino acid residue
made this enzyme resistant to glucose inactivation. Hence, the N -terminal proline residue is essential
for polyubiquitinylation and proteasomal degradation [152]. That cytoplasmic malate dehydrogenase and
isocitrate lyase also share a conserved proline residue at the N -terminus provides further evidence for its
importance for glucose inactivation. A major advance in the understanding of glucose inactivation was
the cloning of the GID3 gene and finding that it was identical with UBC8, which encodes a ubiquitinconjugating enzyme. Like fructose bisphosphatase, Ubc8p is cytoplasmic, and vesicle isolation and
proteinase degradation experiments also showed fructose bisphosphatase to be degraded in the cytoplasm
under these inactivation conditions [318].

Glucose inactivation: proteasomal versus vacuolar degradation


Currently, two distinguished research groups are publishing controversial observations on the molecular
mechanism of the enzyme degradation which is triggered by glucose. Either the enzymes are degraded in
the vacuole (Chiang and his colleagues) or they are degraded in proteosomes (Wolf and his colleagues).
Both groups publish biochemical and genetic observations which support their own respective hypotheses.
These apparently conflicting results might conceivably be explained by the existence of more than one
pathway for degrading those enzymes which are subject to glucose inactivation. Indeed, there is evidence
that the degradation of the galactose carrier (Gal2p) differs from that of fructose bisphosphatase [171].
73 The

gid mutants were analysed further by Michael Thumm, Dieter Wolf and their colleagues; see also [33].

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881

For galactose, the events may be described as follows. The galactose carrier is first ubiquitinated,
then undergoes endocytosis and, finally, is degraded in the vacuole [172,173]. As already mentioned in
Spiegelmans original paper of 1947, inactivation of the galactose carrier occurs only in resting cells, not in
growing cells [331]. On the other hand, glucose inactivation of gluconeogenic enzymes occurs in growing
cells only. There is strong biochemical and genetic evidence that gluconeogenic enzymes are degraded in
the proteasomes of growing cells: in several mutants of the proteosomal pathway, gluconeogenic enzymes
were not inactivated. This is so, in particular, in the case of the gid3-mutant of the ubiquitin-conjugating
enzyme Ubc8p and has been found more recently with the gid6-mutant of the de-ubiquitinating enzyme
Ubp14p, which prevents the inhibition of proteasomal function [299].
Systematic screening of the mutant deletion collection, EUROSCARF,74 led unexpectedly to finding
several mutants which affect the vacuolar degradation of fructose bisphosphatase [299]. These mutants
include Vid24p, a peripheral membrane protein at vesicles (Vid vesicles). These vesicles are thought
to transport fructose bisphosphatase to the vacuole (Figure 28) [58]. The involvement of Vid24p in the
proteasomal and the vacuolar pathways does not seem to be consistent with the glucose inactivation
of deficient phenotype vidp24 mutants. However, this dual function in the two pathways is not
a general feature of vid mutants, as other vid mutants, such as vid22 and vid27, which are involved
in vacuolar fructose bisphosphatase degradation [43,44], show a typical proteasomal inactivation of this
enzyme [299]. Future research will show whether there are, indeed, two alternative pathways for glucoseinduced degradation of gluconeogenic enzymes. Current work suggests that proteasomal degradation is
the major pathway when Saccharomyces cerevisiae is growing, whereas the vacuolar degradation pathway
may apply when the cells are starved.
Although at first an esoteric subject, the specific proteolysis of proteins became a major field of research
for understanding cellular regulation. In 1996 Wolfgang Hilt and Dieter Wolf wrote:
It is obvious that the few substrates of proteasomes discovered to date represent only the tip of the iceberg and
it will be a great challenge to uncover all the cellular processes that proteasomes are involved in, as well as the
detailed mechanisms underlying these selective processes [164, p. 101].

This kind of work is likely to have major impacts outside yeast research. An obvious example is that
of proteasome inhibitors, which are undergoing clinical trials for the chemotherapy of some cancers
[1,138,260], since proteasomes occur in a wide range of organisms, including man [34].

Conclusion
The Pasteur, Kluyver and Custers effects are responses by yeasts to changes in the amount or character
of the sugars available to them. Enzymic regulation, induction, repression and inactivation bring about
these effects and make possible other adaptations to alterations in the supplies of nutrients.
The early research on microbial adaptations, from 1900 onwards, was physiological and particularly
concerned with enzyme induction, which depends on which particular sugar is accessible to the microbe.
However, with the development of microbial genetics and molecular biology in the second half of the
twentieth century, it became possible to examine the molecular genetics underlying these regulatory
phenomena. The first major molecular analysis of a microbial adaptation, carried out during the 1960s to
1980s, was that of the induction and repression of enzymes of the galactose pathway in Saccharomyces
cerevisiae. The complex interactions of proteins produced by the various GAL genes, such as activation
by Gal4p or repression by Gal80p, were elucidated by the 1990s and this work is discussed in article
number 7 of the present series [25].
Towards the end of twentieth century, further complexities of the molecular control of sugar metabolism
have been unravelled. Such work has depended on both the development of DNA transformation
74 EUROSCARF, the European S accharomyces cerevisiae Archive for Functional Analysis, contains deletion mutants of all yeast genes
(http://www.srd-biotec.de/euroscarf or http://web.uni-frankfurt.de/fb15/mikro/euroscarf/).

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systems in yeasts in the late 1970s, first achieved by Jean Beggs [35] and Gerry Fink [166], and
also on recombinant DNA technology generally, such as by the creation of yeast vector systems,
initially by David Botstein [41] and Kevin Struhl [339]. Many other techniques have contributed to
the spectacular advances described above. Immunological methods, such as immunoblotting [167] and
immunofluorescence microscopy [57] in the 1990s, have made it practicable to follow the movements of
regulatory proteins across various membrane barriers in the cell.
One of the main mechanisms by which microbes adapt to changes is by regulating gene expression,
and, as understanding what genes do is an essential part of molecular biology, this subject has made
many biochemical phenomena in general, and microbial adaptation in particular, more comprehensible.
The roles of certain proteases, kinases and phosphorylations in regulating enzymic activities provide some
examples. Because these processes are complex, involve a number of interacting genes and proteins and
are in many cases compartmented, the processes are often difficult for non-molecular biologists to follow.
Research on the molecular biology of cellular regulation is very active today and will undoubtedly
bring to light even greater complexities. Since the entire genome of Saccharomyces cerevisiae has
been sequenced (the first eukaryote for which this was done), deletion mutants are available for all
approximately 6000 yeast genes [138a]. Consequently, there is enormous progress in understanding the
molecular biology of yeast as a eukaryotic model system. New techniques, such as transcriptome analysis
[214], the genome-wide yeast two-hybrid analysis [176a] and the use of MALDI MS for proteome analysis
of the analysis of TAP-purified protein [134], all generate huge amounts of information which can only
be handled by modern bioinformatic methods.

Acknowledgements
The authors thank the following most warmly for their help: Joan Brown, Evelyne Dubois, Dylan Edwards, Robert Hauer,
Reginald Hems, C. T. Kluyver, Peter Kotter, Matthias Rose and W. A. Scheffers. In addition, they thank L. K. Barnett
for all her work towards greatly improving both the writing and illustrations. J. A. B. also thanks the Royal Society for a
research grant.

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