1)

Catalase is an enzyme found in almost all organisms that grow in oxygen and catalyzes the
following reaction:

2H2O2

2H2O + O2

Catalase Units/mg !

Absorption at 280 nm!

In order to understand the biochemical function of this enzyme, you purify it from mammalian
tissue culture cells. As part of these studies, you subject the protein to gel filtration and measure
the UV absorption and catalase activity in each fraction. You also analyze the gel filtration
fractions by SDS-PAGE and stain the gel with Coomasie Blue. The results of these experiments are
shown below.

Gel Filtration

240 kDa
60 kDa

10!
16!

9!

14!
8!
7!
6!
5!
4!
3!
2!
1!
0!

12!
10!
8!
6!
4!
2!
0!

0!

5!

10!

15!

20!

0!

Fraction Number!

5!

10!

15!

Fraction Number !

SDS-PAGE

20!

A) What is the molecular weight of the catalase protein?

B) What are the oligomeric forms of catalase?



C) Which oligomeric form of catalase is the enzymatically active form?



D) Why does higher molecular weight catalase flow through the gel filtration column faster than
lower molecular weight catalase?



Beta-oxidation of fatty acids can generate H2O2 as byproduct. Since H2O2 is highly reactive and can
damage proteins and nucleic acids, it must be inactivated. You reason that catalase should be
found in the organelles where beta-oxidation occurs.

E) Describe how you would use equilibrium density gradient ultracentrifugation to determine
which subcellular organelle(s) co-fractionate with catalase. For these experiments assume that
you have antibodies against proteins that are unique markers of each organelle.





F) Propose one way you could analyze your equilibrium density gradients, if you didnt have
access to any antibodies to marker proteins.




G) Your analysis reveals that catalase activity co-fractionates with mitochondria and with a novel
organelle you call the peroxisome. You think that catalase is present inside of each organelle, but
your PI thinks that it is just aggregating and running at the same position as mitochondria and the
peroxisome. Assuming that you have an antibody to catalase, propose an experiment that would
test whether or not catalase is inside or outside of mitochondria/peroxisomes.

2) You find it odd that the same enzymatic activity is present in two different organelles, so you
decide to purify both mitochondrial and peroxisome associated catalase. You analyze both
enzymes and get the following results:


















A) What is the Km and Vmax for Peroxisome Catalase (P-Catalase)?


B) What is the Km and Vmax for Mitochondrial Catalase (M-Catalase)?


C) If the concentration of H2O2 is 2mM, at what percent of their maximal rate are P catalase and
M-Catalase functioning?




D) Based on the difference in enzymatic activity, you propose that P-Catalase and M-Catalase are
different proteins. Devise a set of experiments to test your hypothesis.





E) Your experiments reveal that P-Catalase and M-Catalase are the same protein. Propose a
hypothesis to explain how the same protein can have two different levels of activity when purified
from different organelles (assume that the protein isnt denatured by the purification). Explain
how you would test your hypothesis.

3) In the budding yeast, Saccharomyces cerevisiae, catalase is only found in the peroxisome. You
reason that since beta-oxidation is restricted to the peroxisome in S. cerevisiae, that a screen to
identify mutants that cant grow on carbon sources that require beta-oxidation for their use will
identify mutants that are defective in catalase targeting to the peroxisome and peroxisome
biogenesis. You decide to screen for mutants that grow slowly on media that has oleic acid
(oleate), an 18-carbon, cis-monounsaturated fatty acid, as the sole carbon source.

A) Do you want to perform your screen with haploid or diploid Saccharomyces cerevisiae? Explain
your answer.


B) Describe how you would screen for mutants that grow slowly on oleic acid.



C) Would this screen identify genes required to sort catalase to the peroxisome if beta-oxidation
(and catalase) occurred in both the mitochondria and the peroxisome in yeast? Explain your
reasoning.




D) Your screen identified 7 mutants that grow slowly on oleic acid. Explain how you would
determine how many distinct genes are mutated in your collection of 7 mutants.



E) While you are most interested in the genes required to target catalase to the peroxisome , what
other processes will cause a slow growth phenotype on oleic acid when disrupted (i.e what other
types of mutants might you expect to get from this screen). For this question, you might find it
helpful to think about everything that must happen for oleic acid in the media to undergo beta-
oxidation in the peroxisome.


F) Describe a set of experiments to test which of your mutants, if any, are defective for targeting
catalase to peroxisomes.


G) Describe a set of experiments to determine which of your mutants , if any, are defective for
peroxisome formation.


H) Describe a set of experiments to determine which of your mutants is defective for the other
processes from part E that you proposed cause a slow growth phenotype on oleic acid when
disrupted.

4) Yeasts are able to use a wide range of carbon and energy sources. When glucose is present,
genes encoding enzymes required for the breakdown of non-fermentable carbon sources (i.e
glycerol, oleate) are normally turned off through a complex mechanism of gene regulation termed
glucose repression. -oxidation genes are repressed by glucose and become derepressed on non-
fermentable carbon sources. In addition, -oxidation genes are induced several fold in the
presence of oleate. This oleate induction is accompanied by a drastic expansion of the
peroxisomal compartment. In order to study this phenomenon more completely, you construct a
yeast strain where the URA3 gene is under the control of a peroxisome gene promoter. You
observe the following results when your strain is grown on different carbon sources:

Carbon Source

Glucose


Oleic Acid


Glycerol


Glucose + Oleic Acid
Glycerol + Oleic Acid

URA3 Expression

OFF

ON

ON

OFF

ON

Carbon Source

Glucose


Oleic Acid
Glycerol


Glucose + Oleic Acid
Glycerol + Oleic Acid

Growth on Ura- media

A) The URA3 gene is required for yeast to grow on uracil-deficient media. Assume the only copy of
the URA3 gene is your peroxisome promoter-URA3 construct. Predict whether the strain will grow
on Uracil-deficiemt media containing the following carbon sources.

B) Describe how you would use the peroxisome promoter-URA3 reporter in a genetic screen to
identify genes required for glucose repression.





C) The drug 5-FOA kills cells that express URA3, but does not affect cells that dont express URA3.
Describe how you would use the peroxisome promoter-URA3 reporter together with 5-FOA in a
genetic screen to identify genes required oleate induction.

D) Your genetic screen identifies several genes involved in glucose repression of the peroxisome
promoter: PXS1, PXS2, PXS3. Describe how you would test whether your alleles are dominant or
recessive.





In order to analyze PXS1-3, you generate null alleles of all three genes and dominant alleles in
PXS2 and PXS3. You analyze a series of double mutant combinations for their ability to cause
glucose repression of the reporter and get the following results:

Genotype
wild type

pxs1-
pxs2-

pxs3-


pxs2D
pxs3D

pxs1-,pxs2-
pxs1-,pxs3-
pxs2-,pxs3-
pxs2-, pxs3D
pxs2D, pxs3-

Glucose Ura- media

no growth

growth


no growth

no growth


growth


growth


growth


growth

no growth


growth

no growth

Glucose- Ura- media

growth

growth

no growth

no growth

growth

growth

growth

growth

no growth

growth

no growth

E) Is PXS1 required for glucose repression (keeping genes off in the presence of glucose) or
derepression (allowing genes to be transcribed in the absence of glucose)?

F) Is PXS2 required for glucose repression (keeping genes off in the presence of glucose) or
derepression (allowing genes to be transcribed in the absence of glucose)?

G) Is PXS3 required for glucose repression (keeping genes off in the presence of glucose) or
derepression (allowing genes to be transcribed in the absence of glucose)?

H) Diagram the glucose repression pathway for PXS1-3 including whether each gene is an
activator or repressor of the next step in the pathway.

5) You have decided to visually screen a collection of yeast strains where individual genes have
been tagged with green fluorescent protein (GFP). Your screen identifies 9 proteins that form
novel filaments shown below:



















A) Describe two experiments that you could use to determine which proteins are in the same
filament and which are not. Be sure to include negative controls where appropriate.




B) Gcd2p, Gcd6p, Gcd7p, Gcn3p, and Sui2p all co-localize to the same filament. Propose an
experiment to test whether they form a biochemical complex together.




C) Your experiment reveals that all 5 proteins are in a biochemical complex together. Propose two
experiments to find out which subunits make direct contact with each other.




D) You isolate deletions in GCD2, GCD6, GCD7, GCN3, and SUI2. Assume that each of these deletions
is viable. Propose a set of experiments to define the assembly pathway for these filaments.