FEMS Microbiology Reviews 32 (1986) 179-187 179

Published by Elsevier

FER 00012

Catabolite repression mutants of yeast *

(Catabolite repression; Saccharomyces cerevisiae; yeast mutants)

Juana M. Gancedo and Carlos Gancedo

Instituto de Investigaciones Biorr~dicas del C.S.I.C., Facultad de Medicina de la U.A.M., ,4rzobispo Morcillo 4, 28029 Madri~ Spain

Received 18 November 1985

Accepted 10 December 1985

1. SUMMARY lizable carbon sources [1]. The old 'glucose effect'

The mecharlism of catabolite repression in yeast
is not well understood, although it has been estab-
lished that cAMP does not play a role similar to
that found in Escherichia coli. To identify the
elements implicated in catabolite repression in
yeast, a variety of mutants affected in this process
have been isolated by different research groups. A
systematic review of the results reported in the
literature is presented. The conclusion that can be
drawn is that the mechanism of catabolite repres-
sion is a complex one, with no single gene control-
ling all the genes subject to repression. The ex-
pression of a given gene or set of genes is con-
trolled by several regulatory genes, but it is not yet
known whether these genes act cooperatively or
sequentially.
2. INTRODUCTION
Catabolite repression, 'a new name for an old
phenomenon' proposed by Magasanik in 1961,
designates the inhibition of the synthesis of cer-
tain enzymes by glucose or other rapidly metabo-
* This paper is dedicated to our teacher of chemical engineer-
ing Professor E. Costa, for his stimulating lectures and
warm support
has p,771ed generations of biochemically oriented
microbiologists, and the elucidation of its mecha-
nism has been a long-standing problem in biology.
In the last 10 years, a model to explain the mecha-
nism of catabolite repression in E. coli has been
worked out [2,3]. Briefly, it was found that glucose
produces a decrease in the levels of cAMP, and
that cAMP is necessary for the transcription of
genes sensitive to catabolite repression. Thus, in
the presence of glucose, transcription of these
genes is impaired and the enzymes coded by them
are not synthesized. Although this model is prob-
ably a simplification which does not take into
account the possibility of a negative control by
specific metabolites [4] it is the first step in the
understanding of catabolite repression in any
organism. The useful concept of the unity of bio-
chemistry led to the assumption that the mecha-
nism operating in E. coil would also be functional
in other microorganisms [5]. However, in the last 4
years, evidence has accumulated showing that the
mechanism of catabolite repression in yeast may
not be similar to that found in E. coli. In particu-
lar the role of cAMP has been challenged as a
consequence of results obtained in genetic and
biochemical experiments [6,7].
To identify the different elements implicated in
the mechanism of catabolite repression in yeast, a
convenient approach is the isolation and char-

0168-6445/86/$03.50 © 1986 Federation of European Microbiological Societies

180
acterization of mutants affected in the process. A
number of laboratories have tried this approach
and a variety of mutants has been described. With
the aim of facilitating the integration of the exist-
ing data, we have examined the available literature
on this topic and have analyzed the results in a
systematic way. We present here a review of the
state of the field and propose a preliminary model
of the interaction between the different elements
implicated in the process of catabolite repression
in yeast.
2.1 Strains used
A variety of strains, some of them poorly de-
fined from a genetic point of view, have been
used. In some cases this makes the genetic char-
acterization of the mutants difficult and hinders
comparative studies. Ideally, a good strain for
studying catabolite repression should have the fol-
lowing characteristics: (a) it should grow on a
variety of carbon sources that require for their
metabolism enzymes subject to catabolite repres-
sion; (b) it should be well defined from a genetic
point of view; (c) it should be easily transformed;
(d) it should have a selectable marker to detect
transformants easily, e.g., ura3, leu2; (e) an iso-
genic strain of opposite mating type should be
available; (f) the diploid should be easily amena-
ble to genetic analysis. Perhaps it could be useful
to construct a small number of such 'ideal' (or
almost ideal) strains and adopt them as standard
organisms in laboratories working in this area.
This would allow an easy comparison of the re-
suits from different laboratories.
2.2. Some considerations about nomenclature
The addition of glucose to a yeast culture causes
inhibition of the synthesis of certain proteinL This
we shall term repression. When glucose is ex-
hausted the synthesis of some of these proteins
may start and this process is called derepression.
However, for the synthesis of some other enzymes
to occur, depletion of glucose is not enough, and
the presence of an inducer is required. Therefore,
these enzymes are both repressible and inducible.
Repression by glucose and inducibility are con-
trolled by different mechanisms, and it is possible
for an enzyme to become constitutive (i.e., with its
synthesis independent of inducer) while remaining
sensitive to repression by glucose.
3. M E T H O D O L O G Y FOR OBTAINING
M U T A N T S A F F E C T E D IN CATABOLITE RE-
PRESSION
Several approaches have been used to obtain
mutants which are resistant to catabolite repres-
sion (non-repressible) or that do not derepress
sensitive enzymes even in the absence of glucose
(non-derepressible).
3.1. Non-repressible mutants
One of the most general methods to obtain
mutants with non-repressible enzymes has been
the use of non-metabolizable glucose analogues
that act as gratuitous repressors, like'glucosamine
or 2-deoxyglucose. The yeast is plated in the pres-
ence of the analogue on a medium whose utiliza-
tion requires the expression of enzymes sensitive
to catabolite repression, e.g., a medium with sac-
charose or maltose as carbon source. Colonies
growing in these conditions are selected as puta-
tive mutants [8-13]. In some cases diploids were
used as starting material to obtain dominant
mutants [14,15].
Another approach is based on the existence of
different isoenzymes of alcohol dehydrogenase
with different regulatory properties. A D H I is the
enzyme involved in fermentation of glucose and is
present in high levels when the yeast grows on this
sugar. A D H II is the isoenzyme implicated in the
utilization of ethanol and is repressed by glucose.
Strains possessing only A D H II grow poorly on
glucose. Good growth on glucose of these strains
could occur if A D H II fulfilled the role of the
fermentative enzyme. This in turn would imply
that the enzyme was no longer repressible by
glucose. With this rationale mutants have been
obtained possessing only A D H II, by selection of
large colonies on 10% glucose [16] or of cells able
to grow on glucose in the presence of an inhibitor
of respiration, like antimycin [17]. These ap-

proaches allow the obtention of mutants affected
not only in the control of ADH II but also in the
regulation of other repressible enzymes.
Mutants affected in gal7 (which codes for
galactose-l-phosphate uridyl transferase) have also
been used as starting material. Mutants affected in
gal7 do not grow on a medium with ethanol and
galactose, probably due to the accumulation of
galactose-l-phosphate but grow on glucose plus
galactose, since glucose represses galactokinase
synthesis. Mutants that have lost catabolite repres-
sion of galactokinase can be selected as non-
growers on glucose plus galactose [18,19].
Direct methods have also been used occasion-
ally. The yeast is plated in conditions where only
deregulated mutants will produce enzymes whose
products may be detected by a colour reaction
[20,21] or even in the case of catalase by the
formation of oxygen bubbles after incubation with
n202 [22].
Empirical approaches with no immediate theo-
retical explanation have also been employed. In
some backgrounds, the loss of respiratory com-
petence causes an inability to grow on maltose.
Revertants able to grow on maltose in the rho-
state can be isolated and show resistance to
catabolite repression of several enzymes [23]. Re-
vertants of non-derepressible mutants have also
yielded organisms with loss of catabolite repres-
sion [24].
3.2. Non-derepressible mutants
The phenotype of non-derepressible mutants is
lack of growth on carbon sources that require
derepression of some enzyme(s) fc~r their utiliza-
tion. Non-derepressible mutants have therefore
been isolated as non-growers on glycerol, and on
maltose [25] or sucrose [26]. Since petites are also
unable to grow on carbon sources that can be used
only via respiration, it is important to discard
them at this stage.
For the obtention of non-derepressible mutants,
strains with altered alcohol dehydrogenase have
also been used. Starting with a mutant possessing
only ADH II, small colonies on ethanol plus 0.4%
glucose were selected. These organisms form small
colonies because they are unable to derepress ADH
181
II (and other enzymes) even when the glucose has
been exhausted [16].
Finally, mutants with non-derepressible en-
zymes have also been obtained as revertants of
non-repressible mutants [17].
4. CHARACTERISTICS OF THE MUTANTS
Once mutants potentially affected in catabolite
repression have been isolated, it is necessary to
test whether the phenotype observed is actually
due to a mutation controlling repression or dere-
pression. This is done by testing the activities of a
number of enzymes sensitive to catabolite repres-
sion under adequate conditions. In cases where
resistance to analogues is used as a selection pro-
cedure, mutants will frequently be obtained which
are resistant to the toxic effect of the analogue but
still sensitive to repression by glucose ([10,12] this
laboratory, unpublished).
In the mutants obtained so far not all enzymes
tested responded similarly, i.e., while some had
lost the control by catabolite repression others
conserved it. This result suggests strongly that
there are several circuits implicated in catabolite
repression and that there is apparently no 'master
gene' governing the whole process.
In Table 1, different mutations are shown that
block the derepression of several enzymes. Genes
identified as acting on the derepression process
are listed in Table 2. Although CA TI and CAT3
have the same characteristics, they have been
shown to belong to different complementation
groups. It was originally thought that CCRI was
allelic with CAT1 [16] but this now seems un-
likely, since CCR1 is aUelic with SNF1 [17] and
SNFI has different properties from CA TI. Genes
CCR2, CCR3 and CCR4 appear similar, but CCR2
and CCR3 belong to different complementation
groups. It could be, however, that CCR4 is identi-
cal to CCR2 or CCR3. From the data available,
no consistent pattern of action emerges, since even
the derepression of hydrolytic enzymes like in-
vertase and maltase is not controlled by the same
set of genes.
Other genes required for derepression are
HAP2, TYE2 and TYE4. They have not yet been
182

"Fable 1
Characteristics of mutants unable to derepress enzymes subject to catabolite repression
Abbreviations: M A L , m a h a s e ; F B P , fructose 1,6-bisphosphatase; I C E isocitrate l y a s e ; S U C , i n v e r t a s e ; S D H , succinate dehydro-
genase; M D H , malate dehydrogenase; A D H II, alcohol dehydrogenase I1; M S , malate synthase.

Selection procedure Enzymes affected Name of mutation Reference

Lack of growth on glycerol and MAL, FBP, ICL carl [251
on maltose
Lack of growth on glycerol and SUC snfl a [26,531
on sucrose
Small colonies on ethanol + 0.4% FBP, ICL, SDH, MDH ccrl, ccr2, [16]
glucose, starting with an a d h l - strain ccr3
Reversion of a strain hex2 to MAL, FBP, ICL carl, cat3 [241
growth on maltose + glucose
Reversion of a mutant with ADH II, ICL, MDH, MS ccr4 [171
non-repressible A D H II

a This mutant does not ferment galactose or maltose and exhibits poor growth on ethanol or lactate.

thoroughly characterized and were identified as
follows. A fl-galactosidase gene was placed under
control of a D N A sequence that makes the
synthesis of iso-l-cytochrome c sensitive to
catabolite repression, and then introduced into a
wild-type yeast. The transformed yeast was
mutagenized, and mutants producing lower than
basal levels of fl-galactosidase in the presence of
glucose were selected. These mutants, named hap2
did not grow on non-fermentable carbon sources.
It was suggested that HAP2 may encode an
activator for the synthesis of one or more cyto-
chrome genes [27]. TYE2 and TYE4 have been
identified as necessary for the constitutive expres-
sion of A D H II in strains that carry a Ty insertion
in the ADR3 gene [28]. These genes are also
necessary for full expression of A D H II and iso-
citrate lyase in the wild type. In the mutants tye2
and tye4 the derepressed levels of these enzymes
are about 30% of those in the parental strain.
Mutants ras2, which lack a protein activating
adenylate cyclase [29] fail to grow on gluconeo-
genic carbon sources like glycerol or ethanol, while
still growing on sucrose or raffinose [30,31]. Since
malate dehydrogenase, phosphoenolpyruvate
carboxykinase and fructose-l,6-bisphosphatase are
derepressed in ras2 mutants, it could be thought
that the gene RAS2 controls the synthesis of re-
spiratory enzymes, although other causes may be
responsible for this lack of growth.
Table 3 shows a list of yeast mutants with a
number of enzymes that have lost the repression
by glucose. In some cases mutants have been
isolated as resistant to glucose analogues but have

Table 2
Genes that control the derepression of different enzymes sensitive to catabolite repression
Abbreviations for enzymes as in Table 1. + , Affects expression of the corresponding gene; - , without effect; + , partial effect; nt, not
tested.

Gene Enzymes affected

MAL SUC ADH II FBP ICL MS MDH SDH Respiratory enzymes
CATI + - nt + + nt =h nt -
CAT3 + - nt + + nt =t= nt -
C C R I ~- S N F ! + + + + + nt + + nt
CCR2 - nt nt + + nt + + :t:
CCR3 - nt + + + + + + +
C C R 4 ( =- C C R 2 o r - nt + + + + + nt +
CCR3?)
 183

not been further characterised [8,11]. These
mutants have not been included in Table 3 since
resistance to the analogue may be caused by a
mechanism not related to catabolite repression
(see above). Some of the mutants presented in the
Table 3 appear to affect the repression of only one
enzyme e.g., ADR3, GAL82, cgrl, while most of
them have a pleiotropic effect. Genes affecting the
expression of several enzymes are listed in Table
4. Although very similar in their properties, HEXI,
HEX2 and CAT80 belong to different comple-
mentation groups. Mutants hexl have decreased
levels of hexokinase (see 4.3) while mutants hex2
show an increased concentration of hexokinase II
[32]. In addition hex2 mutants cannot grow in the
presence of maltose, a fact apparently related to
uncontrollable and excessive maltose uptake [33].
GRRI is not allelic to HEXI or HEX2 [15] and
probably also not to CAT80, since grrl mutants
have elevated levels of hexokinase activity, a fea-
ture not shared by cat80 mutants. The known
properties of mutants FLKI and FH4C make it
possible that they are allelic. Little information is
available on dgr2, 1710 and regl, although the
last two are similar and differ in their properties
from other mutants described.
Finally, the mutations in genes CCR91 (equal
to CCR96) and CCR80 present the particularity
of being dominant. Although CCR91 and CCRS0
are in some respect similar they are probably not
allelic since glucose fermentation is severely im-
paired in CCR80 but not in CCR91 mutants.

Table 3
Characteristics of mutants that do not repress enzymes subject to catabolite repression
Abbreviations for enzymes as in other tables, GAK, Galactokinase; CYT, cytochrome oxidase; ISO, isomaltase.

Selection procedure Enzymes affected Name of mutation Reference

Growth on galactose + 2-deoxyglucose MAL, SUC, MDH [9]
Growth on galactose + 2-deoxyglucose a MAL, SUC, GAK [14]
Growth on galactose + 2-deoxyglucose MAL, SUC, GAK, CYT grrl [15]
Growth on raffinose + 2-deoxyglucose MAL, SUC, MDH, SDH hexl b, hex2 ¢ [9,34,44]
cat80
Growth on raffinose + 2-deoxyglucose MAL, SUC dgr2, dgr3 [45]
Growth on starch + 2-deoxyglucose d MAL, SUC, ISO [13]
Growth on maltose + glucosamine MAL, SUC, GAK, CYT girl [10]
Growth on lactate + glucosamine Respiratory enzymes garl, gar2, gar3 [12]
Big colonies on 10% glucose from a ADH II ADR3 [47]
strain with only ADH II
Rapid growth on 85[ glucose from • MDH, SDH, ICL, MS CCR80 [48]
strains lacking ADH I
Growth on maltose of a strain f MAL flkl [49,23]
unable to grow on maltose in the
respiratory deficient state
Growth on lactate of a mutant defi- iso-2-cytochrome c cyc9 [50]
cient in iso-1--cytochrome c
No growth on glucose + galactose of SUC, G A K regl [19]
mutants gal7 GALSI
No growth on glucose + galactose of GAK GAL82, GAL83 [18]
mutants gal7 GALSI
Colonies grown on 10% fructose and r MAL, SUC FH4C, 1710 [20]
producing invertase
Colonies grown on 8~ glucose and s SDH CCR91, CCR96 [21]
stained red with a tetrazolium salt
Colonies grown on 10~ glucose and Catalase T cgrl, cgr2, [22,51]
releasing O 2 when incubated with H202 cgr4 h, casl h
" The mutation is dominant; b decregsed hexokinase; c increased hexokinase and respiration not repressed by glucose, d mutation in
Schwanniomyces occidentalis; • poor fermentative capacity, f mutation produces flocculence; s cytochromes not repressed; h cyto-
chromes aa3 and b not repressed. Catalas¢ A and c type cytochromes repressed.
184

M u t a n t s garl, gar2 and gar3 a p p e a r to b e l o n g g r o u p presented nonrepressibility of invertase and

to a m o r e c o m p l e x system since only the d o u b l e
m u t a n t s garl gar2 or garl gar 3 are resistant to
repression.
4.2. Epistatic relations between the different muta-
tions affecting catabolite repression
T h e effects o f a gene at o n e locus on the
expression of a n o t h e r gene at a second locus
(epistasis) are n o t always s t r a i g h tf o r w a r d in the
case of c a t a b o l i t e repression. T h e simplest cases
are the d o u b l e m u t a n t s hex2 cat80, hex2 catl or
hex2 cat3 that b e h a v e like the respective cat
m u t a n t s except that they are u n a b l e to grow on
m a l t o s e [24]. M o r e c o m p l e x appears the relation-
ship be t ween cat1 or cat3 and h e x l . W h e n these
cat m u t a n t s are c o m b i n e d with h e x l , maltase,
invertase an d m a l a t e d e h y d r o g e n a s e are not sub-
j e c t to c a t a b o l i t e repression (as in h e x l ) while
fructose 1,6-bisphosphatase and isocitrate lyase
can not be derepressed (as in cat1 or cat3) A gene
epistatic to CA T I is CAT2. This last gene can
m u t a t e to cat2, a form that causes early derepres-
sion of e n z y m e s in glucose g r o w n cells. D o u b l e
m u t a n t s cat1 cat2 b e h a v e as cat2 [25].
maltase and a partial decrease in glucose phos-
p h o r y l a t i n g activity [34]. Closer e x a m i n a t i o n re-
vealed that this m u t a n t ( h e x l ) lacked the
hexokinase i so en zy m e P-II [35]. T r a n s f o r m a t i o n of
yeasts lacking hexokinase activity with the P-II
gene resulted in restoration of catabolite repres-
sion [36]. T h u s the hypothesis was put forward
that hexokinase P-II had a dual role in S.cerevisiae,
acting b o t h as a p h o s p h o r y l a t i n g e n z y m e and as a
regulatory protein. S u p p o r t for this idea w o u l d be
g a i n e d if o n e co u l d o b t a i n a m u t a n t in the gene
c o d i n g for hexokinase P-II that m a i n t a i n s its cata-
lytic activity and has lost the regulatory function.
This goal was achieved by E n t i a n an d FriShlich
[37] w h o o b t a i n e d a m u t a n t with these characteris-
tics. This m u t a n t was called h e x l R . However, up
to now, there is no hint as to how hexokinase P-II
acts in its role as regulatory protein.
5. I N T E R A C T I O N B E T W E E N T H E D I F F E R -
ENT ELEMENTS IMPLICATED IN CATA-
BOLITE REPRESSION: A PRELIMINARY
MODEL

4.3. The participation o f hexokinase P - I I in the A l t h o u g h o u r k n o w l e d g e is still fragmentary,

catabolite repression o f some enzymes the f u n c t i o n i n g of a gene subject to c a t a b o l i t e
\ repression could be c o n t e m p l a t e d as follows (Fig.
A m o n g the several m u t a n t s i s o l a t e d by 1). T h e expression of the gene is c o n t r o l l e d im-
Z i m m e r m a n and coworkers, a c o m p l e m e n t a t i o n m e d i a t e l y by two types of cis-acting sequences,

Table 4
Genes that control the repression of different enzymes subject to catabolite repression
Abbreviations as in other Tables. Data from Table 3.
Gene Enzymes affected
GAK MAL SUC FBP ICL MS MDH SDH Cytochromes
HEXI =--GLR1 =1: + + - - - ± + +
HEX2 nt + + - _ _ + + +a
CA T80 nt + + - - - + - nt
GRRI + + + nt nt nt nt nt +
FLKI =-CYC9 nt + + nt nt nt nt + +
FH4C nt + + nt nt nt nt nt nt
DGR2 =-DGR3 nt + + nt nt nt nt nt nt
1710 nt - + nt nt nt nt nt nt
REGI + - + nt nt nt nt nt nt
CCR91 =--CCR96 nt - nt nt nt - nt + +
CCR80 nt nt 5: :t: :t: 5: 5: + nt
a Cytochromes have not been measured but in hex2 mutants glucose does not repress respiration.

GENES ACTING IN TRANS
Acting p l e i o t r o p i c a l l y ~
/ / ~ ~ Acting
p-~lei oi i i : i ~ i i : s ~
Required for repression / ~
/
l Specific
Fig. 1. A possibleschemeof the functioningof a genesubjectto cataboliterepression.The metabolitesderivedfromthe utilizationof
the sugar could regulatethe expressionof the genesactingin trans, modulatethe interactionof theirproductswith the genesacting
in cis, or both. e, Activation,O, Inhibition.
enhancers or silencers, whose function is evident
from their names. Different sets of genes acting in
trans determine the activity of the enhancers and
silencers. These genes may act pleiotropically, af-
fecting the expression of several genes, or specifi-
cally, affecting only a particular one. The expres-
sion of the genes acting in trans and the interac-
tion of their products with the genes acting in cis
may in turn be controlled by metabolites whose
levels vary between repressed and derepressed
conditions. Examples of the aforementioned com-
ponents follow.
A typical enhancer is the UAS2 fragment up-
stream of the gene CYC1 [27], that allows at least
a 100-fold increase in the expression of iso-l-cyto-
chrome c. A silencer [38] could be exemplified by
ADR3, a fragment upstream of the gene A D H I I
which puts this gene under glucose control [39].
When ADR3 is deleted [39] or a Ty element
inserted [40], A D H I I is no longer sensitive to
glucose. However, in the case of the Ty insertion,
the constitutive expression of A D H I I requires the
function of at least 4 trans-acting genes, and is
therefore not due to a simple disruption of the
wild-type controlling site [28]. Examples of genes
necessary for derepression are CAT1 and CCRI,
which affect expression of several enzymes (pleio-
tropic effect), and ADR1 which specifically af-
fects A D H I I [41]. Mutations in these genes are
185
GENES ACTING IN CIS STRUCTURAL GENES
~ Expression
Si).encers
usually recessive, with the exception of mutants
like C A T 1 - 2 d [25] or A D R I c [41] which cause a
less tight repression. Genes necessary for repres-
sion can also act pleiotropicaUy, like HEXI,
CAT80 or CCR91, or specifically, like GAL82.
Mutations in these genes may be recessive (hexl,
cat80) or dominant (CCR91, GAL82). A recently
documented case of interplay between elements
like those of Fig. 1 is the interaction of the protein
GAL 4 with the DNA sequence UASG which is
necessary for derepression of the genes GAL 1 and
GAL I0. This interaction is blocked in the pres-
ence of glucose [52]. Metabolites whose levels vary
depending on the metabolic situation i.e., dere-
pressed vs. repressed, may play a role in catabolite
repression, although this has not yet been proved.
Likely candidates are glucose 6-phosphate, fruc-
tose 6-phosphate, fructose 1,6-bisphosphate, fruc-
tose 2,6-bisphosphate or cAMP, whose concentra-
tions increase after glucose addition [7,42,43] Pre-
liminary results (Gancedo, J.M., unpublished re-
sults) suggest that a sugar has to be phosphory-
lated to exert repression.
ACKNOWLEDGEMENTS
The critical reading of the manuscript by Drs.
J. Conde (La Cruz del Campo, Seville) P. Erasco,
186
C.F. Heredia, R. Lagunas and F. Portillo is greatly
appreciated. Work in the laboratory of the authors
has been supported by grants of the Comisi6n
Asesora de lnvestigaci6n Ciendfica y T6cnica.
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