Escolar Documentos
Profissional Documentos
Cultura Documentos
Review
*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
836
836
837
838
840
842
845
847
847
849
854
855
856
857
861
861
865
868
869
871
873
875
875
877
879
880
881
882
836
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].
837
What happens
Underlying factors
PASTEUR EFFECT
CUSTERS EFFECT
Brettanomyces and
Dekkera spp. ferment
D-glucose to ethanol and
CO2 faster in aerobic than
in anaerobic conditions
KLUYVER EFFECT
Ability to use
oligosaccharide or
galactose aerobically, but
not anaerobically, although
glucose is fermented
CRABTREE EFFECT
GLUCOSE
Repression of respiration
Decrease of enzyme
activity within minutes
after adding glucose
Phosphorylation (rapidly
reversible) and proteolytic
degradation (irreversible) of
enzyme
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].
838
Physiological observation
Examples
Mechanism
6-Phosphofructokinase
pyruvate kinase
Interconversion by
covalent
modification
Fructose-1,6-bisphosphatase
Usually phosphorylation of
enzyme
Inactivation
Fructose-1,6-bisphosphatase
and other mainly gluconeogenic
and glyoxylate cycle enzymes
Repression
Genes encoding
glucose-repressible enzymes
Derepression
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
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].
840
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].
841
pathway and found only one of them to be sensitive, namely, 6-phosphofructokinase (PFK), which
catalyses the following reaction:
D-fructose
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.
842
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]
1925
Meyerhof [258]
1926
Warburg [367]
1. The term Pasteursche Reaktion used for a hypothetical chemical reaction linking
respiration and fermentation.
2. CO inhibits yeast respiration.
1928
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
1943
1962
1980
Table 3 shows the chronological sequence of some of the work on the Pasteur effect.
843
Oxygen
uptake
Carbon dioxide
evolved by fermentation
0.0
94.5
0.0
52.0
Schizosaccharomyces pombe
0.0
40.6
1.2
9.3
10.7
30.2
25.7
3.5
Candida tropicalis
27.7
0.9
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]).
844
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).
845
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.
846
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].
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
848
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].
849
respiratory-deficient mutant of Saccharomyces pastorianus was shown to have a much reduced rate of
maltose uptake compared with the wild-type [310].
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].
Copyright 2005 John Wiley & Sons, Ltd.
850
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:
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.
852
Percentage of species
showing Kluyver effect
98
92
87
86
73
54
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].
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]).
854
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.
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
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].
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
Copyright 2005 John Wiley & Sons, Ltd.
856
Grace Crabtree (18921966), English biochemist, was with the Imperial Cancer Research Fund in London for 43 years [6].
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.
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].
Copyright 2005 John Wiley & Sons, Ltd.
858
(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].
859
(B)
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
860
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].
Copyright 2005 John Wiley & Sons, Ltd.
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].
862
Yeast
Repression (%)
ACONITATE HYDRATASE
S. cerevisiae
51
P. anomala
S. cerevisiae
39
91
87
K. marxianus
>99
S. cerevisiae
>99
91
(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
-GALACTOSIDASE
K. marxianus
>99
-GLUCOSIDASE
K. marxianus
K. dobzhanskii
90
GLUTAMATE DEHYDROGENASE
S. cerevisiae
50
ISOCITRATE DEHYDROGENASE
(NAD+ )
S. cerevisiae
59
30
67
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).
Copyright 2005 John Wiley & Sons, Ltd.
863
MALATE DEHYDROGENASE
MALATE SYNTHASE
OLIGO-1,6-GLUCOSIDASE
P. anomala
Rh. glutinis
S. cerevisiae
94
99
99
99
P. anomala
S. cerevisiae
44
87
P. anomala
Rh. glutinis
S. cerevisiae
S. pastorianus
94
99
95
73
99
87
67
55
50
5
10
>65
975
63
79
(isomaltase)
PHOSPHOENOLPYRUVATE
CARBOXYKINASE
C. pelliculosa
Cr. humicola
P. anomala
Rh. glutinis
Rh. mucilaginosa
S. cerevisiae
S. pastorianus
Z. fermentati
Z. rouxii
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
864
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].
865
(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).
866
Table 12. Genetic and biochemical characterization of genes involved in glucose repression
description
Gene
Mutant isolation
Mutant phenotypes
Gene
sequence
of wild-type protein
Physiological role
Zimmermann and
colleagues 1977
[398]
Entian and
Zimmermann
1982 [108]
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]
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]
Yang and
colleagues 1992
[394]
SIP2
Yang and
colleagues 1992
[394]
GAL83
Yang and
colleagues 1992
[394]
Nehlin and
Ronne 1990
[270]
CAT4 Schuller and
Entian 1991 [320]
SSN1 Vallier and
Carlson 1994
[355]
Nehlin and
Ronne 1990
[270]
Lutfiyya and
Johnston 1996
[235]
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
Copyright 2005 John Wiley & Sons, Ltd.
867
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
Hedges and
colleagues 1995
[158]
Rahner and
colleagues 1996
[295]
Lesage and
colleagues 1996
[221]
GLC7
HEX2
REG1
Neigeborn and
Carlson 1987 [271]
Peng and
colleagues 1990
[284]
Entian and
Zimmermann
1980 [107]
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
Subunit of Glc7
Tu and Carlson 1995 [352]
HXK2
CAT80
GRR1
Entian and
colleagues 1977
[109]
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]
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
Copyright 2005 John Wiley & Sons, Ltd.
868
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].
869
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 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].
870
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].
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
872
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]).
Copyright 2005 John Wiley & Sons, Ltd.
873
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.
874
Table 13. Pleiotropic effects of mutants of the genes CYC8 and TUP1
Mutant
cyc8
tup1
Allelic
designations
Phenotype
cyc8
ssn6
tup1
a ARS,
flk1
umr7
amm1
cyc9
aar1
aer2
sfl2
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
875
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.
876
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
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).
Copyright 2005 John Wiley & Sons, Ltd.
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].
is often used as an inhibitor of protein synthesis, but it is reported to inhibit glycolysis markedly [145].
878
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].
879
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].
Copyright 2005 John Wiley & Sons, Ltd.
880
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].
gid mutants were analysed further by Michael Thumm, Dieter Wolf and their colleagues; see also [33].
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/).
882
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.
References
1. Adams J. 2003. The development of proteasome inhibitors as anticancer drugs. Cancer Cell 5: 417421.
2. Algeri AA, Bianchi L, Viola AM, Puglisi PP, Marmiroli N. 1981. IMP1 /imp1 : a gene involved in the nucleo-mitochondrial control
of galactose fermentation in Saccharomyces cerevisiae. Genetics 97: 2744.
3. Alms GR, Sanz P, Carlson M, Haystead TAJ. 1999. Reg1p targets protein phosphatase 1 to dephosphorylate hexokinase II in
Saccharomyces cerevisiae characterizing the effects of a phosphatase subunit on the yeast proteome. EMBO Journal 18: 41574168.
4. Andreasen AA, Stier TJB. 1953. Anaerobic nutrition of Saccharomyces cerevisiae I. Ergosterol requirement for growth in a defined
medium. Journal of Cellular and Comparative Physiology 41: 2336.
5. Andreasen AA, Stier TJB. 1954. Anaerobic nutrition of Saccharomyces cerevisiae II. Unsaturated fatty acid requirement for growth
in a defined medium. Journal of Cellular and Comparative Physiology 43: 271281.
6. Anon 1967. Crabtree, Herbert Grace. Chemistry in Britain 3: 314.
7. Anon 1980. Dr Hans Laser. Lancet (vol. I for 1980): 270.
8. Anon 1988. Dean Burk, 84, chemist for Cancer Institute. New York Times 10 October: 8.
9. Anon 1992. Kendal Cartwright Dixon. Kings College Cambridge Annual Report; 2731.
10. Anon 1994. Professor Walter Bartley. The Times 2 September: 19.
11. Arrigo A-P, Tanaka K, Goldberg AL, Welch WJ. 1988. Identity of the 19S prosome particle with the large multifunctional protease
complex of mammalian cells (the proteasome). Nature 331: 192194.
12. Ashrafi K, Farazi TA, Gordon JI. 1998. A role for Saccharomyces cerevisiae fatty acid activation protein 4 in regulating protein
N -myristoylation during entry into stationary phase. Journal of Biological Chemistry 273: 2586425874.
13. Bailey E, Banks P. 1994. Obituary Emeritus Professor Walter Bartley. University of Sheffield Newsletter 7 October; 10.
14. Bailey RB, Woodword A. 1984. Isolation and characterization of a pleiotropic glucose repression resistant mutant of Saccharomyces
cerevisiae. Molecular and General Genetics 193: 507512.
15. Banks P. 1995. Walter Bartley 19161994. The Biochemist Feb/Mar: 4344.
16. Barnett JA. 1968. The biochemical differentiation of taxa, with special reference to the yeasts. In The Fungi, An Advanced Treatise,
vol 3, Ainsworth GC, Sussman AS (eds). Academic Press: New York; 557595.
17. Barnett JA. 1977. The nutritional tests in yeast systematics. Journal of General Microbiology 99: 183190.
Copyright 2005 John Wiley & Sons, Ltd.
883
18. Barnett JA. 1981. The utilization of disaccharides and some other sugars by yeasts. Advances in Carbohydrate Chemistry and
Biochemistry 39: 347404.
19. Barnett JA. 1990. A. P. Sims. The Independent 3 August.
20. Barnett JA. 1992. Some controls on oligosaccharide utilization by yeasts: the physiological basis of the Kluyver effect. FEMS
Microbiology Letters 100: 371378.
21. Barnett JA. 1998. A history of research on yeasts 1: work by chemists and biologists 17891850. Yeast 14: 14391451.
22. Barnett JA. 2000. A history of research on yeasts 2: Louis Pasteur and his contemporaries, 18501880. Yeast 16: 755771.
23. Barnett JA. 2003. A history of research on yeasts 5: the fermentation pathway. Yeast 20: 509543.
24. Barnett JA. 2003. A history of research on yeasts 6: the main respiratory pathway. Yeast 20: 10151044.
25. Barnett JA. 2004. A history of research on yeasts 7: enzymic adaptation and regulation. Yeast 21: 703746.
26. Barnett JA. 2004. A history of research on yeasts 8: taxonomy. Yeast 21: 11411193.
27. Barnett JA, Kornberg HL. 1960. The utilization by yeasts of acids of the tricarboxylic acid cycle. Journal of General Microbiology
23: 6582.
28. Barnett JA, Lichtenthaler FW. 2001. A history of research on yeasts 3: Emil Fischer, Eduard Buchner and their contemporaries,
18801900. Yeast 18: 363388.
29. Barnett JA, Payne RW, Yarrow D. 2000. Yeasts: Characteristics and Identification, 3rd edn. Cambridge University Press:
Cambridge.
30. Barnett JA, Robinow CF. 2002. A history of research on yeasts 4: cytology part I, 18901950. Yeast 19: 151182.
31. Barnett JA, Robinow CF. 2002. A history of research on yeasts 4: cytology part II, 19501990. Yeast 19: 745772.
32. Barnett JA, Sims AP. 1982. The requirement of oxygen for the active transport of sugars into yeasts. Journal of General
Microbiology 128: 23032312.
33. Bauer J. 1994. Isolation and characterization of Saccharomyces cerevisiae mutants with a defect in catabolite-inactivation. Thesis,
University of Frankfurt.
34. Baumeister W, Walz J, Zuhl F, Seemuller E. 1998. The proteasome: paradigm of a self-compartmentalizing protease. Cell 92:
367380.
35. Beggs JD. 1978. Transformation of yeast by a replicating hybrid plasmid. Nature 275: 104109.
36. Berg JM, Tymoczko JL, Stryer L. 2002. Biochemistry, 5th edn. Freeman: New York.
37. Bianchi MM, Tizzani L, Destruelle M, Frontali L, Wesolowski-Louvel M. 1996. The petite-negative yeast Kluyveromyces lactis
has a single gene expressing pyruvate decarboxylase activity. Molecular Microbiology 19: 2736.
38. Black FL, Hsiung GD, Phillips CA, Plummer G, Rapp F. 1975. A tribute to Joseph L. Melnick. Progress in Medical Virology 21:
VIIIX.
39. Boiteux A, Hess B. 1970. Allosteric properties of yeast pyruvate decarboxylase. FEBS Letters 9: 293296.
40. Boles E, Zimmermann FK, Thevelein JM. 1997. Metabolic signals. In Yeast Sugar Metabolism, Zimmermann FK, Entian K-D
(eds). Technomic: Lancaster, Penn; 379407.
41. Botstein D, Falco SC, Stewart SE, Brennan M, Scherer S, Stinchcomb DT, Struhl K, Davis RW. 1979. Sterile host yeasts (SHY):
a eukaryotic system of biological containment for recombinant DNA experiments. Gene 8: 1724.
42. Brown AJ. 1892. Influence of oxygen and concentration on alcoholic fermentation. Journal of the Chemical Society 61: 369385.
43. Brown CR, Cui D-Y, Hung GG-C, Chiang H-L. 2001. Cyclophilin A mediates Vid22p function in the import of fructose-1,6bisphosphatase into Vid vesicles. Journal of Biological Chemistry 276: 4801748026.
44. Brown CR, McCann JA, Hung GG-C, Elco CP, Chiang H-L. 2002. Vid22p, a novel plasma membrane protein, is required for the
fructose-1,6-bisphosphatase degradation pathway. Journal of Cell Science 115: 655666.
45. Brown HD, Satyanarayana T, Umbarger HE. 1975. Biosynthesis of branched-chain amino acids in yeast: effect of carbon source
on leucine biosynthetic enzymes. Journal of Bacteriology 121: 959969.
46. Burk D. 1939. A colloquial consideration of the Pasteur and neo-Pasteur effects. Cold Spring Harbor Symposia on Quantitative
Biology 7: 420459.
47. Carlson M. 1999. Glucose repression in yeast. Current Opinion in Microbiology 2: 202207.
48. Carlson M, Osmond BC, Botstein D. 1981. Mutants of yeast defective in sucrose utilization. Genetics 98: 2540.
49. Carlson M, Osmond BC, Neigeborn L, Botstein D. 1984. A suppressor of SNF1 mutations causes constitutive high-level invertase
synthesis in yeast. Genetics 107: 1932.
50. Carrascosa JM, Viguera MD, Nun ez de Castro I, Scheffers WA. 1981. Metabolism of acetaldehyde and Custers effect in the yeast
Brettanomyces abstinens. Antonie van Leeuwenhoek 47: 209215.
51. Castrillo JI, Kaliterna J, Weusthuis RA, van Dijken JP, Pronk JT. 1996. High-cell-density cultivation of yeasts on disaccharides in
oxygen-limited batch cultures. Biotechnology and Bioengineering 49: 621628.
52. Celenza JL, Carlson M. 1986. A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science
233: 11751180.
53. Celenza JL, Carlson M. 1989. Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional
interaction with the SNF4 protein. Molecular and Cellular Biology 9: 50345044.
54. Celenza JL, Eng FJ, Carlson M. 1989. Molecular analysis of the SNF4 gene of Saccharomyces cerevisiae: evidence for physical
association of the SNF4 protein with the SNF1 protein kinase. Molecular and Cellular Biology 9: 50455054.
55. Chance B, Hess B. 1956. On the control of metabolism in ascites tumor cell suspensions. Annals of the New York Academy of
Sciences 63: 10081016.
Copyright 2005 John Wiley & Sons, Ltd.
884
56. Cheung WY. 1966. Adenosine 3 , 5 -phosphate and oscillations of DPNH in a cell-free extract of Saccharomyces carlsbergensis.
Biochimica et Biophysica Acta 115: 235239.
57. Chiang H-L, Schekman R. 1991. Regulated import and degradation of a cytosolic protein in the yeast vacuole. Nature 350: 313318.
58. Chiang MC, Chiang H-L. 1998. Vid24p, a novel protein localized to the fructose-1,6-bisphosphatase-containing vesicles, regulates
targeting of fructose-1,6-bisphosphatase from the vesicles to the vacuole for degradation. Journal of Cell Biology 140: 13471356.
59. Chien C, Bartel PL, Sternglanz R, Fields S. 1991. The two-hybrid system: a method to identify and clone genes for proteins that
interact with a protein of interest. Proceedings of the National Academy of Sciences of the USA 88: 95789582.
60. Ciriacy M. 1975. Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae I. Isolation and genetic analysis of adh mutants.
Mutation Research 29: 315326.
61. Ciriacy M. 1975. Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae II. Two loci controlling synthesis of the glucoserepressible ADH II. Molecular and General Genetics 138: 157164.
62. Ciriacy M. 1976. cis-Dominant regulatory mutations affecting the formation of glucose-repressible alcohol dehydrogenase (ADHII)
in Saccharomyces cerevisiae. Molecular and General Genetics 145: 327333.
63. Ciriacy M. 1977. Isolation and characterization of yeast mutants defective in intermediary carbon metabolism and in carbon
catabolite derepression. Molecular and General Genetics 154: 213220.
64. Ciriacy M, Breitenbach I. 1979. Physiological effects of seven different blocks in glycolysis in Saccharomyces cerevisiae. Journal
of Bacteriology 139: 152160.
65. Cirillo VP. 1968. Relationship between sugar structure and competition for the sugar transport system in bakers yeast. Journal of
Bacteriology 95: 603611.
66. Cirillo VP. 1968. Galactose transport in Saccharomyces cerevisiae I. Nonmetabolized sugars as substrates and inducers of the
galactose transport system. Journal of Bacteriology 95: 17271731.
67. Clifton D, Fraenkel DG. 1982. Mutant studies of yeast phosphofructokinase. Biochemistry 21: 19351942.
68. Collins JF, Kornberg HL. 1960. The metabolism of C2 compounds in micro-organisms 4. Synthesis of cell materials from acetate
by Aspergillus niger. Biochemical Journal 77: 430438.
69. Colowick SP. 1973. The hexokinases. In The Enzymes, 3rd edn, vol. 9, Boyer PD (ed.). Academic Press: New York; 148.
70. Cori CF. 1942. Phosphorylation of carbohydrates. In A Symposium on Respiratory Enzymes. University of Wisconsin Press: Madison,
WI; 175189.
71. Crabtree HG. 1929. Observations on the carbohydrate metabolism of tumours. Biochemical Journal 23: 536545.
72. Criddle RS, Schatz G. 1968. Promitochondria of anaerobically grown yeast. I. Isolation and biochemical properties. Biochemistry
8: 322334.
73. Custers MTJ. 1940. Onderzoekingen over het gistgeslacht Brettanomyces. Thesis, De Technische Hoogeschool te Delft.
74. Damsky CH, Nelson WM, Claude A. 1969. Mitochondria in anaerobically-grown, lipid-limited brewers yeast. Journal of Cell
Biology 43: 174179.
75. Davies A. 1956. Invertase formation in Saccharomyces fragilis. Journal of General Microbiology 14: 109121.
76. Davies A. 1956. Some factors affecting lactase formation and activity in Saccharomyces fragilis. Journal of General Microbiology
14: 425439.
77. Decker K. 2000. A life-long quest for biochemical regulation (Helmut Holzer, 19211997). Comprehensive Biochemistry 41:
531561.
78. De Deken RH. 1966. The Crabtree effect: a regulatory system in yeast. Journal of General Microbiology 44: 140156.
79. DeMoss RD, Bard RC, Gunsalus IC. 1951. The mechanism of the heterolactic fermentation: a new route of ethanol formation.
Journal of Bacteriology 62: 499511.
80. Dernby KG. 1917. Studien u ber die proteolytischen Enzyme der Hefe und ihre Beziehung zu der Autolyze. Biochemische Zeitschrift
81: 107208.
81. DeVit MJ, Johnston M. 1999. The nuclear exportin Msn5 is required for nuclear export of the Mig1 glucose repressor of
Saccharomyces cerevisiae. Current Biology 9: 12311241.
82. DeVit MJ, Waddle JA, Johnston M. 1997. Regulated nuclear translocation of the Mig1 glucose repressor. Molecular Biology of the
Cell 8: 16031618.
83. Dickens F. 1951. Anaerobic glycolysis, respiration, and the Pasteur effect. In The Enzymes. Chemistry and Mechanism of Action,
vol. 2, Sumner JB, Myrback K (eds). Academic Press: New York; 624683.
84. Dixon KC. 1937. The Pasteur effect and its mechanism. Biological Reviews 12: 431460.
85. Dodyk F, Rothstein A. 1964. Factors influencing the appearance of invertase in Saccharomyces cerevisiae. Archives of Biochemistry
and Biophysics 104: 478486.
86. Donnini C, Lodi T, Ferrero I, Algeri A, Puglisi PP. 1992. Allelism of IMP1 and GAL2 genes of Saccharomyces cerevisiae. Journal
of Bacteriology 174: 34113415.
87. Douglas HC, Condie F. 1954. The genetic control of galactose utilization in Saccharomyces. Journal of Bacteriology 68: 662670.
88. Duntze W, Atzpodien W, Holzer H. 1967. Glucose-dependent enzyme activities in different yeast species. Archiv fur Mikrobiologie
58: 296301.
89. Duntze W, Neumann D, Gancedo JM, Atzpodien W, Holzer H. 1969. Studies on the regulation and localization of the glyoxylate
cycle enzymes in Saccharomyces cerevisiae. European Journal of Biochemistry 10: 8389.
90. Duntze W, Neumann D, Holzer H. 1968. Glucose induced inactivation of malate dehydrogenase in intact yeast cells. European
Journal of Biochemistry 3: 326331.
Copyright 2005 John Wiley & Sons, Ltd.
885
91. Eddy AA. 1982. Mechanisms of solute transport in selected eukaryotic microorganisms. Advances in Microbial Physiology 23:
178.
92. Edmondson DG, Smith MM, Roth SY.1996. Repression domain of the yeast global repressor Tup1 interacts directly with histones
H3 and H4. Genes and Development 10: 12471259.
93. Emmer M, de Crombrugghe B, Pastan I, Perlman R. 1970. Cyclic AMP receptor protein of E. coli : its role in the synthesis of
inducible enzymes. Proceedings of the National Academy of Sciences of the USA 66: 480487.
94. Engelhardt V [W]A. 1982. Life and science. Annual Review of Biochemistry 51: 119.
95. Engelhardt VA, Sakov NE [|NGELXGARDT wa, sAKOW ne]. 1943. o MEHANIZME PASTEROWSKOGO \FFEKTA. [On the mechanism of the
Pasteur effect.] buoxuMuQ [Biokhimiya] 8: 936.
96. Entian K-D. 1978. Genetik des Kohlenhydratstoffwechsels von Saccharomyces cerevisiae. Thesis, Technische Universitat Darmstadt.
97. Entian K-D. 1980. Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression
in yeast. Molecular and General Genetics 178: 633637.
98. Entian K-D. 1980. A defect in carbon catabolite repression associated with uncontrollable and excessive maltose uptake. Molecular
and General Genetics 179: 169175.
99. Entian K-D. 1997. Sugar phosphorylation in yeast. In Yeast Sugar Metabolism. Zimmermann FK, Entian K-D (eds). Technomic:
Lancaster, Penn; 6779.
100. Entian K-D, Barnett JA. 1983. Some genetical and biochemical attempts to elucidate the energetics of sugar uptake and explain
the Kluyver effect in the yeast, Kluyveromyces lactis. Current Genetics 7: 323325.
101. Entian K-D, Barnett JA. 1992. Regulation of sugar utilization by Saccharomyces cerevisiae. Trends in Biochemical Sciences 17:
506510.
102. Entian K-D, Droll L, Mecke D. 1983. Studies on rapid reversible and non-reversible inactivation of fructose-1,6-bisphosphatase
and malate dehydrogenase in wild-type and glycolytic block mutants of Saccharomyces cerevisiae. Archives of Microbiology 134:
187192.
103. Entian K-D, Frohlich K-U. 1984. Saccharomyces cerevisiae mutants provide evidence of hexokinase PII as a bifunctional enzyme
with catalytic and regulatory domains for triggering carbon catabolite repression. Journal of Bacteriology 158: 2935.
104. Entian K-D, Hilberg F, Opitz H, Mecke D. 1985. Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants
with regulatory mutations responsible for glucose repression. Molecular and Cellular Biology 5: 30353040.
105. Entian K-D, Loureiro-Dias MC. 1990. Misregulation of maltose uptake in a glucose repression defective mutant of Saccharomyces
cerevisiae leads to glucose poisoning. Journal of General Microbiology 136: 855860.
106. Entian K-D, Mecke D. 1982. Genetic evidence for a role of hexokinase isozyme PII in carbon catabolite repression in Saccharomyces
cerevisiae. Journal of Biological Chemistry 257: 870874.
107. Entian K-D, Zimmermann FK. 1980. Glycolytic enzymes and intermediates in carbon catabolite repression mutants of
Saccharomyces cerevisiae. Molecular and General Genetics 177: 345350.
108. Entian K-D, Zimmermann FK. 1982. New genes involved in carbon catabolite repression and derepression in the yeast
Saccharomyces cerevisiae. Journal of Bacteriology 151: 11231128.
109. Entian K-D, Zimmermann FK, Scheel I. 1977. A partial defect in carbon catabolite repression in mutants of Saccharomyces
cerevisiae with reduced hexose phosphorylation. Molecular and General Genetics 156: 99105.
110. Ephrussi B, Slonimski PP, Yotsuyanagi Y, Tavlitzki J. 1956. Variations physiologiques et cytologiques de la levure au cours du
cycle de la croissance aerobie. Comptes Rendus des Travaux du Laboratoire Carlsberg, Serie Physiologique 26: 87102.
111. Epps HMR, Gale EF. 1942. The influence of the presence of glucose during growth on the enzymic activities of Escherichia coli :
comparison of the effect with that produced by fermentation acids. Biochemical Journal 36: 619623.
112. Eraso P, Gancedo JM. 1984. Catabolite repression in yeasts is not associated with low levels of cAMP. European Journal of
Biochemistry 141: 195198.
113. Estruch F, Carlson M. 1990. Increased dosage of the MSN1 gene restores invertase expression in yeast mutants defective in the
SNF1 protein kinase. Nucleic Acids Research 18: 69596964.
114. Feng Z, Wilson SE, Peng Z-Y, Schlender KK, Reimann EM, Trumbly RJ. 1991. The yeast GLC7 gene required for glycogen
accumulation encodes a type 1 protein phosphatase. Journal of Biological Chemistry 266: 2379623801.
115. Ferguson JJ, Boll M, Holzer H. 1967. Yeast malate dehydrogenase: enzyme inactivation in catabolite repression. European Journal
of Biochemistry 1: 2125.
116. Fields S, Song OK. 1989. A novel genetic system to detect proteinprotein interactions. Nature 340: 245246.
117. Fischer E, Lindner P. 1895. Ueber die Enzyme einiger Hefen. Berichte der Deutschen Chemischen Gesellschaft 28: 30343039.
118. Flick JS, Johnston M. 1991. GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with
leucine-rich repeats. Molecular and Cellular Biology 11: 51015112.
119. Flores-Carreon A, Reyes E, Ruz-Herrera J. 1969. Influence of oxygen on maltose metabolism by Mucor rouxii . Journal of General
Microbiology 59: 1319.
120. Friis J, Ottolenghi P. 1959. Localization of invertase in a strain of yeast. Comptes Rendus des Travaux du Laboratoire Carlsberg
31: 259271.
121. Frohlich K-U, Entian K-D, Mecke D. 1985. The primary structure of the yeast hexokinase PII gene (HXK2 ) which is responsible
for glucose repression. Gene 36: 105111.
122. Fujita A, Matsumoto S, Kuhara S, Misumi Y, Kobayashi H. 1990. Cloning of the yeast SFL2 gene: its disruption results in
pleiotropic phenotypes characteristic for tup1 mutants. Gene 89: 9399.
Copyright 2005 John Wiley & Sons, Ltd.
886
123. Fukuhara H. 2003. The Kluyver effect revisited. FEMS Yeast Research 3: 327331.
124. Funayama S, Gancedo JM, Gancedo C. 1980. Turnover of yeast fructose-bisphosphatase in different metabolic conditions. European
Journal of Biochemistry 109: 6166.
125. Gancedo C. 1971. Inactivation of fructose-1,6-diphosphatase by glucose in yeast. Journal of Bacteriology 107: 401405.
126. Gancedo C, Salas ML, Giner A, Sols A. 1965. Reciprocal effects of carbon sources on the levels of the AMP-sensitive fructose1,6-diphosphatase and phosphofructokinase in yeast. Biochemical and Biophysical Research Communications 20: 1520.
127. Gancedo C, Schwerzmann K. 1976. Inactivation by glucose of phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae.
Archives of Microbiology 109: 221225.
128. Gancedo JM. 1992. Carbon catabolite repression in yeast. European Journal of Biochemistry 206: 297313.
129. Gancedo JM. 1998. Yeast carbon catabolite repression. Microbiology and Molecular Biology Reviews 62: 334361.
130. Gancedo JM, Gancedo C. 1971. Fructose-1,6-diphosphatase, phosphofructokinase and glucose-6-phosphate dehydrogenase from
fermenting and non-fermenting yeasts. Archives of Microbiology 76: 132138.
131. Gancedo JM, Gancedo C. 1997. Gluconeogenesis and catabolite inactivation. In Yeast Sugar Metabolism, Zimmermann FK,
Entian K-D (eds). Technomic: Lancaster, Penn; 359377.
132. Gancedo JM, Mazon MJ, Gancedo C. 1982. Kinetic differences between two interconvertible forms of fructose-1,6-bisphosphatase
from Saccharomyces cerevisiae. Archives of Biochemistry and Biophysics 218: 478482.
133. Gancedo JM, Mazon MJ, Gancedo C. 1983. Fructose 2,6-bisphosphate activates the cAMP-dependent phosphorylation of yeast
fructose-1,6-bisphosphatase in vitro. Journal of Biological Chemistry 258: 59985999.
134. Gavin AC and 37 others. 2002. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature
415: 141147.
135. Gazith J, Schulze IT, Gooding RH, Womack FC, Colowick SP. 1968. Multiple forms and subunits of yeast hexokinase. Annals of
the New York Academy of Sciences 151: 307331.
136. Geret L, Hahn M. 1898. Zum Nachweis des im Hefepresssaft enthaltenen proteolytischen Enzyms. Berichte der Deutschen
Chemischen Gesellschaft 31: 202205.
137. Ghosh BK, Montenecourt B, Lampen JO. 1973. Abnormal cell envelope ultrastructure of a Saccharomyces mutant with invertase
formation resistant to hexoses. Journal of Bacteriology 116: 14121420.
138. Gillessen S, Groettrup M, Cerny T. 2002. The proteasome, a new target for cancer therapy. Onkologie 25: 534539.
138a. Goffeau A and 15 others. 1996. Life with 6000 genes. Science 274: 546567.
139. Goffrini P, Ferrero I, Donnini C. 2002. Respiration-dependent utilization of sugars in yeasts: a determinant role for sugar transporters.
Journal of Bacteriology 184: 427432.
140. Gomori G. 1943. Hexosediphosphatase. Journal of Biological Chemistry 148: 139149.
141. Gorts CPM. 1967. Effect of different carbon sources on the regulation of carbohydrate metabolism in Saccharomyces cerevisiae.
Antonie van Leeuwenhoek 33: 451463.
142. Gorts CPM. 1969. Effect of glucose on the activity and the kinetics of the maltose-uptake system and of -glucosidase in
Saccharomyces cerevisiae. Biochimica et Biophysica Acta 184: 299305.
143. Gottschalk A. 1949. Direct fermentation of disaccharides by yeast. Wallerstein Laboratory Communications 12: 5569.
887
156. Haurie V, Perrot M, Mini T, Jeno P, Sagliocco F. 2001. The transcriptional activator Cat8p provides a major contribution to the
reprogramming of carbon metabolism during the diauxic shift in Saccharomyces cerevisiae. Journal of Biological Chemistry 276:
7685.
157. Hedbacker K, Hong S-P, Carlson M. 2004. Pak1 protein kinase regulates activation and nuclear localization of Snf1-Gal83 protein
kinase. Molecular and Cellular Biology 24: 82558263.
158. Hedges D, Proft M, Entian K-D. 1995. CAT8, a new zinc cluster-encoding gene necessary for derepression of gluconeogenic
enzymes in the yeast Saccharomyces cerevisiae. Molecular and Cellular Biology 15: 19151922.
159. Heinisch J. 1986. Isolation and characterization of the two structural genes coding for phosphofructokinase in yeast. Molecular and
General Genetics 202: 7582.
160. Heinisch J, Hollenberg CP, Zimmermann FK. 1997. In memory of Michael Ciriacy. Yeast 13: 881882.
161. Hemmings BA, Zubenko GS, Hasilik A, Jones EW. 1981. Mutant defective in processing of an enzyme located in the lysosome-like
vacuole of Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the USA 78: 435439.
162. Heredia CF, De La Fuente G, Sols A. 1964. Metabolic studies with 2-deoxyhexoses I. Mechanisms of inhibition of growth and
fermentation in bakers yeast. Biochimica et Biophysica Acta 86: 216223.
163. Hess B, Chance B. 1961. Metabolic control mechanisms VI. Chemical events after glucose addition to ascites tumour cells. Journal
of Biological Chemistry 236: 239246.
164. Hilt W, Wolf DH. 1996. Proteasomes: destruction as a programme. Trends in Biochemical Sciences 21: 96102.
165. Hilt W, Wolf DH. 2000. Proteasomes. The World of Regulatory Proteolysis. Landes Bioscience: Georgetown, IL.
166. Hinnen A, Hicks JB, Fink GR. 1978. Transformation of yeast. Proceedings of the National Academy of Sciences of the USA 75:
19291933.
167. Hoffman M, Chiang H-L. 1996. Isolation of degradation-deficient mutants defective in the targeting of fructose-1,6-bisphosphatase
into the vacuole for degradation in Saccharomyces cerevisiae. Genetics 143: 15551566.
168. Holzer H. 1976. Catabolite inactivation in yeast. Trends in Biochemical Sciences 1: 178181.
169. Holzer H, Holzer E, Schultz G. 1955. Zusammenhang zwischen Wachstum und aerober Garung. I. Versuche mit Hefezellen.
Biochemische Zeitschrift 326: 385404.
170. Hong S-P, Leiper FC, Woods A, Carling D, Carlson M. 2003. Activation of Snf1 and mammalian AMP-activated protein kinase
by upstream kinases. Proceedings of the National Academy of Sciences of the USA 100: 88398843.
171. Horak J, Regelmann J, Wolf DH. 2002. Two distinct proteolytic systems responsible for glucose-induced degradation of fructose1,6-bisphosphatase and the Gal2p transporter in the yeast Saccharomyces cerevisiae share the same protein components of the
glucose signaling pathway. Journal of Biological Chemistry 277: 82488254.
172. Horak J, Wolf DH. 1997. Catabolite inactivation of the galactose transporter in the yeast Saccharomyces cerevisiae: ubiquitination,
endocytosis, and degradation in the vacuole. Journal of Bacteriology 179: 15411549.
173. Horak J, Wolf DH. 2001. Glucose-induced monoubiquitination of the Saccharomyces cerevisiae galactose transporter is sufficient
to signal its internalization. Journal of Bacteriology 183: 30833088.
174. Hubner G, Weidhase R, Schellenberger A. 1978. The mechanism of substrate activation of pyruvate decarboxylase: a first approach.
European Journal of Biochemistry 92: 175181.
175. Ibsen KH. 1961. The Crabtree effect: a review. Cancer Research 21: 829841.
176. Ibsen KH, Coe EL, McKee RW. 1958. Interrelationships of metabolic pathways in the Ehrlich ascites carcinoma cells I. Glycolysis
and respiration (Crabtree effect). Biochimica et Biophysica Acta 30: 384400.
176a. Ito T, Ota K, Kubota H, Yamaguchi Y, Chiba T, Sakuraba K, Yoshida M. 2002. Roles for the two-hybrid system in exploration of
the yeast protein interactome. Molecular and Cellular Proteomics 1: 561566.
177. Jacquet M, Kepes A. 1969. The step sensitive to catabolite repression and its reversal by 3 5 cyclic AMP during induced synthesis
of -galactosidase in E. coli. Biochemical and Biophysical Research Communications 36: 8492.
178. Jakoby WB. 1958. Aldehyde oxidation I. Dehydrogenase from Pseudomonas fluorescens. Journal of Biological Chemistry 232:
7587.
179. Jiang R, Carlson M. 1996. Glucose regulates protein interactions within the yeast SNF1 protein kinase complex. Genes and
Development 10: 31053115.
180. Johnson BF 1968. Dissolution of yeast glucan induced by 2-deoxyglucose. Experimental Cell Research 50: 692694.
181. Johnson BF 1968. Lysis of yeast cell walls induced by 2-deoxyglucose at their sites of glucan synthesis. Journal of Bacteriology
95: 11691172.
182. Jones EW. 1977. Proteinase mutants of Saccharomyces cerevisiae. Genetics 85: 2333.
183. Jones EW, Zubenko GS, Parker RR. 1982. PEP4 gene function is required for expression of several vacuolar hydrolases in
Saccharomyces cerevisiae. Genetics 102: 665677.
184. Kaliterna J, Weusthuis RA, Castrillo JI, van Dijken JP, Pronk JT. 1995. Coordination of sucrose uptake and respiration in the yeast
Debaryomyces yamadae. Microbiology 141: 15671574.
185. Kamp AF, La Rivi`ere JWM, Verhoeven W. 1959. Albert Jan Kluyver. His Life and Work. North-Holland: Amsterdam.
186. Kappeli G. 1986. Regulation of carbon metabolism in Saccharomyces cerevisiae and related yeasts. Advances in Microbial
Physiology 28: 181209.
187. Katz B. 1978. Archibald Vivian Hill. Biographical Memoirs of Fellows of the Royal Society 24: 71149.
188. Keilin D. 1966. The History of Cell Respiration and Cytochrome. Cambridge University Press: Cambridge.
Copyright 2005 John Wiley & Sons, Ltd.
888
189. Kennedy EP, Lehninger AL. 1949. Oxidation of fatty acids and tricarboxylic acid cycle intermediates by isolated rat liver
mitochondria. Journal of Biological Chemistry 179: 957972.
889
890
260. Mitchell BS. 2003. The proteasome an emerging therapeutic target in cancer. New England Journal of Medicine 348: 25972598.
261. Montenecourt BS, Kuo S-C, Lampen JO. 1973. Saccharomyces mutants with invertase formation resistant to repression by hexoses.
Journal of Bacteriology 114: 233238.
262. Mukai Y, Harashima S, Oshima Y. 1991. AAR1/TUP1 protein, with a structure similar to that of the subunit of G proteins,
is required for a1-2 and 2 repression in cell type control of Saccharomyces cerevisiae. Molecular and Cellular Biology 11:
37733779.
263. Muller D, Holzer H. 1981. Regulation of fructose-1,6-bisphosphatase in yeast by phosphorylation/dephosphorylation. Biochemical
and Biophysical Research Communications 103: 926933.
264. Muller M, Muller H, Holzer H. 1981. Immunochemical studies on catabolite inactivation of phosphoenolpyruvate carboxykinase
in Saccharomyces cerevisiae. Journal of Biological Chemistry 256: 723727.
265. Nath N, McCartney RR, Schmidt MC. 2003. Yeast Pak1 kinase associates with and activates Snf1. Molecular and Cellular Biology
23: 39093917.
266. Neeff J, Hagele E, Nauhaus J, Heer U, Mecke D. 1978. Evidence for catabolite degradation in the glucose-dependent inactivation
of yeast cytoplasmic malate dehydrogenase. European Journal of Biochemistry 87: 489495.
267. Neeff J, Heer U. 1978. Catabolite inactivation of yeast cytoplasmic malate dehydrogenase. A process independent of protein
synthesis. FEBS Letters 85: 233236.
268. Neeff J, Mecke D. 1977. In vivo and in vitro studies on the glucose-dependent inactivation of yeast cytoplasmic malate
dehydrogenase. Archives of Microbiology 115: 5560.
269. Nehlin JO, Carlberg M, Ronne H. 1991. Control of yeast GAL genes by MIG1 repressor: a transcriptional cascade in the glucose
response. EMBO Journal 10: 33733377.
270. Nehlin JO, Ronne H. 1990. Yeast MIG1 repressor is related to the mammalian early growth response and Wilms tumour finger
proteins. EMBO Journal 9: 28912898.
271. Neigeborn L, Carlson M. 1987. Mutations causing constitutive invertase synthesis in yeast: genetic interactions with snf mutations.
Genetics 115: 247253.
272. Nernst W. 1889. Die elektromotorische Wirksamkeit der Jonen. Zeitschrift fur Physikalische Chemie 4: 129181.
273. Niederacher D, Entian K-D. 1991. Characterization of the Hex2 protein, a negative regulatory element necessary for glucose
repression in yeast. European Journal of Biochemistry 200: 311319.
274. Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. 2005. Enzyme Nomenclature;
http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/intro (accessed 3 February 2005).
276. Ostling
J, Ronne H. 1998. Negative control of the Migp repressor by Snf1p-dependent phosphorylation in the absence of glucose.
European Journal of Biochemistry 252: 162168.
277. Oura E. 1977. Reaction products of yeast fermentations. Process Biochemistry 12: , 35 1921.
278. Parlebas N, Chevalier MR. 1976. Biochemical studies on the cytosine permease of Saccharomyces cerevisiae. FEBS Letters 65:
327334.
279. Passoneau JV, Lowry OH. 1962. Phosphofructokinase and the Pasteur effect. Biochemical and Biophysical Research
Communications 7: 1015.
280. Pasteur L. 1860. Memoire sur la fermentation alcoolique. Annales de Chimie et de Physique 58: 323426.
281. Pasteur L. 1861. Experiences et vues nouvelles sur la nature des fermentations. Comptes Rendus Hebdomadaires des Seances de
lAcademie des Sciences, Paris 53: 12601264.
282. Pasteur [L]. 1861. Influence de loxyg`ene sur le developpement de la levure et la fermentation alcoolique. Bulletin de la Societe de
Paris (Resume de Seance du 28 juin 1861); 7980.
283. Peitzsch RM, McLaughlin S. 1993. Binding of acylated peptides and fatty acids to phospolipid vesicles: pertinence to myristoylated
proteins. Biochemistry 32: 1043610443.
284. Peng Z-Y, Trumbly RJ, Reimann EM. 1990. Purification and characterization of glycogen synthase from a glycogen-deficient strain
of Saccharomyces cerevisiae. Journal of Biological Chemistry 265: 1387113877.
285. Plattner H, Schatz G. 1969. Promitochondria of anaerobically grown yeast. III. Morphology. Biochemistry 8: 339343.
286. Poinsot C, Moulin G, Claisse M, Galzy P. 1987. Isolation and characterization of a mutant of Schwanniomyces castellii with altered
respiration. Antonie van Leeuwenhoek 53: 6575.
287. Polakis ES, Bartley W. 1965. Changes in the enzyme activities of Saccharomyces cerevisiae during aerobic growth on different
carbon sources. Biochemical Journal 97: 284297.
288. Polakis ES, Bartley W, Meek GA. 1964. Changes in the structure and enzyme activity of Saccharomyces cerevisiae in response to
changes in the environment. Biochemical Journal 90: 369374.
289. Polakis ES, Bartley W, Meek GA. 1965. Changes in the activities of respiratory enzymes during the aerobic growth of yeast on
different carbon sources. Biochemical Journal 97: 298302.
290. Proft M, Kotter P, Hedges D, Bojunga N, Entian K-D. 1995. CAT5, a new gene necessary for derepression of gluconeogenic
enzymes in Saccharomyces cerevisiae. EMBO Journal 14: 61166126.
291. Purwin C, Leidig F, Holzer H. 1982. Cyclic AMP-dependent phosphorylation of frucose-1,6-bisphosphatase in yeast. Biochemical
and Biophysical Research Communications 107: 14821489.
292. Racker E. 1956. Carbohydrate metabolism in ascites tumour cells. Annals of the New York Academy of Sciences 63: 10171021.
Copyright 2005 John Wiley & Sons, Ltd.
891
293. Racker E. 1974. History of the Pasteur effect and its pathobiology. Molecular and Cellular Biochemistry 5: 1723.
294. Rahner A, Hiesinger M, Schuller H-J. 1999. Deregulation of gluconeogenic structural genes by variants of the transcriptional
activator Cat8p of the yeast Saccharomyces cerevisiae. Molecular Microbiology 34: 146156.
295. Rahner A, Scholer A, Martens E, Gollwitzer B, Schuller H-J. 1996. Dual influence of the yeast Cat1p (Snf1p) protein kinase on
carbon source-dependent transcriptional activation of gluconeogenic genes by the regulatory gene CAT8 . Nucleic Acids Research
24: 23312337.
296. Randez-Gil F, Bojunga N, Proft M, Entian K-D. 1997. Glucose derepression of gluconeogenic enzymes in Saccharomyces cerevisiae
correlates with phosphorylation of the gene activator Cat8P. Molecular and Cellular Biology 17: 25022510.
297. Randez-Gil F, Herrero P, Sanz P, Prieto JA, Moreno F. 1998. Hexokinase PII has a double cytosolicnuclear localisation in
Saccharomyces cerevisiae. FEBS Letters 425: 475478.
298. Randez-Gil F, Sanz P, Entian K-D, Prieto JA. 1998. Carbon source-dependent phosphorylation of hexokinase PII and its role in
the glucose-signaling response in yeast. Molecular and Cellular Biology 18: 29402948.
299. Regelmann J, Schule T, Josupeit FS, Horak J, Rose M, Entian K-D, Thumm M, Wolf DH. 2003. Catabolite degradation of fructose1,6-bisphosphatase in the yeast Saccharomyces cerevisiae: a genome-wide screen identifies eight novel GID genes and indicates
the existence of two degradation pathways. Molecular Biology of the Cell 14: 16521663.
300. Rickenberg HV, Cohen GN, Buttin G, Monod J. 1956. La galactoside-permease dEscherichia coli . Annales de lInstitut Pasteur
91: 829857.
301. Rittenhouse J, Moberly L, Marcus F. 1987. Phosphorylation in vivo of yeast (Saccharomyces cerevisiae) fructose-1,6bisphosphatase at the cyclic AMP-dependent site. Journal of Biological Chemistry 262: 1011410119.
302. Robertson JJ, Halvorson HO. 1957. The components of maltozymase in yeast, and their behavior during deadapation. Journal of
Bacteriology 73: 186198.
303. Rodrigues V, VijayRaghavan K. 2000. Zita Lobo an obituary. Current Science 79: 13951396.
304. Ronne H. 1995. Glucose repression in fungi. Trends in Genetics 11: 1217.
305. Rose M, Albig W, Entian K-D. 1991. Glucose repression in Saccharomyces cerevisiae is directly associated with hexose
phosphorylation by hexokinases PI and PII. European Journal of Biochemistry 199: 511518.
306. Rose M, Entian K-D, Hofmann L, Vogel RF, Mecke D. 1988. Irreversible inactivation of Saccharomyces cerevisiae fructose-1,6bisphosphatase independent of protein phosphorylation at Ser [11]. FEBS Letters 241: 5559.
307. Rothstein RJ, Sherman F. 1980. Genes affecting the expression of cytochrome c in yeast: genetic mapping and genetic interactions.
Genetics 94: 871889.
308. Royt PW, MacQuillan AM. 1979. The Pasteur effect and catabolite repression in an oxidative yeast, Kluyveromyces lactis. Antonie
van Leeuwenhoek 45: 241252.
309. Salas ML, Vinuela E, Salas M, Sols A. 1965. Citrate inhibition of phosphofructokinase and the Pasteur effect. Biochemical and
Biophysical Research Communications 19: 371376.
310. Schamhart DHJ, Ten Berge AMA, van de Poll KW. 1975. Isolation of a catabolite repression mutant of yeast as a revertant strain
that is maltose negative in the respiratory-deficient state. Journal of Bacteriology 121: 747752.
311. Schatz G. 1965. Subcellular particles carrying mitochondrial enzymes in anaerobically grown cells of Saccharomyces cerevisiae.
Biochimica et Biophysica Acta 96: 342345.
312. Scheffers WA. 1961. On the inhibition of alcoholic fermentation in Brettanomyces yeasts under anaerobic conditions. Experientia
17: 4042.
313. Scheffers WA. 1966. Stimulation of fermentation in yeasts by acetoin and oxygen. Nature 210: 533534.
314. Schellenberger A, Hubner G. 1968. Binding of the substrate in yeast pyruvate decarboxylase. Angewandte Chemie International
Edition in English 7: 6869.
315. Schork SM, Bee G, Thumm M, Wolf DH. 1994. Site of catabolite inactivation. Nature 369: 283284.
316. Schork SM, Bee G, Thumm M, Wolf DH. 1994. Catabolite inactivation of fructose-1,6-bisphosphatase in yeast is mediated by the
proteasome. FEBS Letters 349: 270274.
317. Schork SM, Thumm M, Wolf DH. 1995. Catabolite inactivation of fructose-1,6-bisphosphatase of Saccharomyces cerevisiae.
Journal of Biological Chemistry 270: 2644626450.
318. Schule T, Rose M, Entian K-D, Thumm M, Wolf DH. 2000. Ubc8p functions in catabolite degradation of fructose-1,6bisphosphatase in yeast. EMBO Journal 19: 21612167.
319. Schuller H-J, Entian K-D. 1988. Molecular characterization of yeast regulatory gene CAT3 necessary for glucose derepression and
nuclear localization of its product. Gene 67: 247257.
320. Schuller H-J, Entian K-D. 1991. Extragenic suppressors of yeast glucose derepression mutants leading to constitutive synthesis of
several glucose-repressible enzymes. Journal of Bacteriology 173: 20452052.
321. Schulz B, Hofer M. 1986. Utilization of lactose in non-respiring cells of the yeast Debaryomyces polymorphus. Archives of
Microbiology 145: 367371.
322. Schulze IT, Colowick SP. 1969. The modification of yeast hexokinases by proteases and its relationship to the dissociation of
hexokinase into subunits. Journal of Biological Chemistry 244: 23062316.
323. Schwartz D, Beckwith JR. 1970. Mutants missing a factor necessary for the expression of catabolite-sensitive operons in E. coli .
In The Lactose Operon, Beckwith JR, Zipser D (eds). Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY; 417422.
324. Sims AP, Barnett JA. 1978. The requirement of oxygen for the utilization of maltose, cellobiose and D-galactose by certain
anaerobically-fermenting yeasts (Kluyver effect). Journal of General Microbiology 106: 277288.
Copyright 2005 John Wiley & Sons, Ltd.
892
325. Sims AP, Barnett JA. 1991. Levels of activity of enzymes involved in anaerobic utilization of sugars by six yeast species:
observations towards understanding the Kluyver effect. FEMS Microbiology Letters 77: 295298.
326. Slater EC. 1984. Vladimir Alexandrovich Engelhardt 18941984. Trends in Biochemical Sciences 9: 504505.
327. Smith FC, Davies SP, Wilson WA, Carling D, Hardie DG. 1999. The SNF1 kinase complex from Saccharomyces cerevisiae
phosphorylates the transcriptional repressor protein Mig1p in vitro at four sites within or near regulatory domain 1. FEBS Letters
453: 219223.
328. Smith RL, Johnson AD. 2000. Turning genes off by Ssn6-Tup1: a conserved system of transcriptional repression in eukaryotes.
Trends in Biochemical Sciences 25: 325330.
329. Sols A. 1976. The Pasteur effect in the allosteric era. In Reflections on Biochemistry, Kornberg A (ed.). Pergamon: Oxford; 199206.
330. Sols A, Gancedo C, DelaFuente G. 1971. Energy-yielding metabolism in yeasts. In The Yeasts, vol. 2, Rose AH, Harrison JS (eds).
Academic Press: London; 271307.
331. Spiegelman S, Reiner JM. 1947. The formation and stabilization of an adaptive enzyme in the absence of its substrate. Journal of
General Physiology 31: 175193.
332. Stark HC, Fugit D, Mowshowitz DB. 1980. Pleiotropic properties of a yeast mutant insensitive to catabolite repression. Genetics
94: 921928.
333. Stern KG, Melnick JL. 1941. The photochemical spectrum of the Pasteur enzyme in retina. Journal of Biological Chemistry 139:
301323.
334. Stevens BJ. 1977. Variation in number and volume of the mitochondria in yeast according to growth conditions. A study based on
serial sectioning and computer graphics reconstruction. Biologie Cellulaire 28: 3756.
335. Stoppani AOM, Actis AS, Deferrari JO, Gonzalez EL. 1952. Essential role of thiol groups in carboxylase. Nature 170: 842843.
336. Storey KB. 1985. A re-evaluation of the Pasteur effect: new mechanisms in anaerobic metabolism. Molecular Physiology 8:
439461.
337. Strittmatter CF. 1957. Adaptive variation in the level of oxidative activity in Saccharomyces cerevisiae. Journal of General
Microbiology 16: 169183.
338. Struhl K, Cameron JR, Davis RW. 1976. Functional genetic expression of eukaryotic DNA in Escherichia coli . Proceedings of the
National Academy of Sciences of the USA 73: 14711475.
339. Struhl K, Stinchcomb DT, Scherer S, Davis RW. 1979. High-frequency transformation of yeast: autonomous replication of hybrid
DNA molecules. Proceedings of the National Academy of Sciences of the USA 76: 10351039.
340. Sutherland CM, Hawley SA, McCartney RR, Leech A, Stark MJ, Schmidt MC, Hardie DG. 2003. Elm1p is one of three upstream
kinases for the Saccharomyces cerevisiae SNF1 complex. Current Biology 13: 12991305.
341. Sutton DD, Lampen JO. 1962. Localization of sucrose and maltose fermenting systems in Saccharomyces cerevisiae. Biochimica
et Biophysica Acta 56: 303312.
342. Swanson WH, Clifton CE. 1948. Growth and assimilation in cultures of Saccharomyces cerevisiae. Journal of Bacteriology 56:
115124.
343. Tanaka K,Yoshimura T, Kumatori A, Ichihara A, Ikai A, Nishigai M, Kameyama K, Takagi T. 1988. Proteasomes (multi-protease
complexes) as 20 S ring-shaped particles in a variety of eukaryotic cells. Journal of Biological Chemistry 263: 1620916217.
344. Teichert U, Mechler B, Muller H, Wolf DH. 1989. Lysosomal (vacuolar) proteinases of yeast are essential catalysts for protein
degradation, differentiation, and cell survival. Journal of Biological Chemistry 264: 1603716045.
345. Thrash-Bingham C, Fangman WL. 1989. A yeast mutation that stabilizes a plasmid bearing a mutated ARS1 element. Molecular
and Cellular Biology 9: 809816.
346. Tortora P, Birtel M, Lenz A-G, Holzer H. 1981. Glucose-dependent metabolic interconversion of fructose-1,6-bisphosphatase in
yeast. Biochemical and Biophysical Research Communications 100: 688695.
347. Tortora P, Burlini N, Hanozet GM, Guerritore A. 1982. Effect of caffeine on glucose-induced inactivation of gluconeogenic enzymes
in Saccharomyces cerevisiae: possible role of cyclic AMP. European Journal of Biochemistry 126: 617622.
348. Treitel MA, Kuchin S, Carlson M. 1998. Snf1 protein kinase regulates phosphorylation of the Mig1 repressor in Saccharomyces
cerevisiae. Molecular and Cellular Biology 18: 62736280.
349. Trumbly RJ. 1986. Isolation of Saccharomyces cerevisiae mutants constitutive for invertase synthesis. Journal of Bacteriology 166:
11231127.
350. Trumbly RJ, Bradley G. 1983. Isolation and characerization of aminopeptidase mutants of Saccharomyces cerevisiae. Journal of
Bacteriology 156: 3648.
351. Tu J, Carlson M. 1994. The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae.
Molecular and Cellular Biology 14: 67896796.
352. Tu J, Carlson M. 1995. REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae.
EMBO Journal 14: 59395946.
353. Ullmann A. 1985. Catabolite repression 1985. Biochemie 67: 2934.
354. Valentine RC, Green NM. 1965. Electron microscopy of an antibodyhapten complex. Journal of Molecular Biology 27: 615617.
355. Vallier LG, Carlson M. 1994. Synergistic release from glucose repression by mig1 and ssn mutations in Saccharomyces cerevisiae.
Genetics 137: 4954.
356. van der Plaat JB. 1974. Cyclic 3 , 5 -adenosine monophosphate stimulates trehalose degradation in bakers yeast. Biochemical and
Biophysical Research Communications 56: 580587.
Copyright 2005 John Wiley & Sons, Ltd.
893
357. van Schaftingen E, Hue L, Hers H-G. 1980. Fructose 2,6-bisphosphate, the probable structure of the glucose- and glucagon-sensitive
stimulator of phosphofructokinase. Biochemical Journal 192: 897901.
358. van Urk H, Schipper D, Breedveld GV, Mak PR, Scheffers WA, van Dijken JP. 1989. Localization and kinetics of pyruvatemetabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS
621. Biochimica et Biophysica Acta 992: 7886.
359. van Wijk R, Ouwehand J, van den Bos T, Koningsberger VV. 1969. Induction and catabolite repression of -glucosidase synthesis
in protoplasts of Saccharomyces carlsbergensis. Biochimica et Biophysica Acta 186: 178191.
360. van Wijk R, van de Poll KW, Speziali GAG. 1970. Catabolite repression of -glucosidase synthesis in yeast during nitrogen
limitation. Proceedings Koninklijke Nederlandsche Akademie van Wetenschappen, Series B 73: 357371.
361. Villa TG, Notario V, Villanueva JR. 1979. -Glucosidases in the yeast Pichia polymorpha. FEMS Microbiology Letters 6: 9194.
362. Vincent O, Carlson M. 1998. Sip4, a Snf1 kinase-dependent transcriptional activator, binds to the carbon source-responsive element
of gluconeogenic genes. EMBO Journal 17: 70027008.
363. Vincent O, Townley R, Kuchin S, Carlson M. 2001. Subcellular localization of the Snf1 kinase is regulated by specific beta subunits
and a novel glucose signaling mechanism. Genes and Development 15: 11041114.
364. Vonka V. 2001. Professor Joseph Louis Melnick Dr. Med. honoris causa Univerzity Karlovy (19142001). Sbornk Lekaosky 102:
547554.
365. von Szent-Gyorgyi A. 1937. Oxidation and fermentation. In Perspectives in Biochemistry, Needham J, Green DE (eds). Cambridge
University Press: Cambridge; 165174.
366. Warburg O. 1923. Versuche an u berlebendem Carcinomgewebe. Biochemische Zeitschrift 142: 317333.
894
393. Womack FC, Welch MK, Nielsen J, Colowick SP. 1973. Purification and serological comparison of the yeast hexokinases P-I and
P-II. Archives of Biochemistry and Biophysics 158: 451457.
394. Yang X, Hubbard EJA, Carlson M. 1992. A protein kinase substrate identified by the two-hybrid system. Science 257: 680682.
395. Yang X, Jiang R, Carlson M. 1994. A family of proteins containing a conserved domain that mediates interaction with the yeast
SNF1 protein kinase complex. EMBO Journal 13: 58785886.
396. Yushok WD, Batt WG. 1957. Influence of D-glucosone and 2-deoxy-D-glucose on respiration and the Crabtree effect. Abstracts of
the American Chemical Society 132nd Meeting; 23C24C.
397. Zhang M, Rosenblum-Vos LS, Lowry CV, Boakye KA, Zitomer RS. 1991. A yeast protein with homology to the -subunit of G
proteins is involved in control of heme-regulated and catabolite-repressed genes. Gene 97: 153161.
398. Zimmermann FK, Kaufmann I, Rasenberger H, Haumann P. 1977. Genetics of carbon catabolite repression in Saccharomyces
cerevisiae: genes involved in the derepression process. Molecular and General Genetics 151: 95103.
399. Zimmermann FK, Scheel I. 1977. Mutants of Saccharomyces cerevisiae resistant to carbon catabolite repression. Molecular and
General Genetics 154: 7582.
400. Zubay G, Schwartz D, Beckwith J. 1970. Mechanism of activation of catabolite-sensitive genes: a positive control system.
Proceedings of the National Academy of Sciences of the USA 66: 104110.