Molecular characterization of
MRG19
of
Saccharomyces cerevisiae
Implication in the regulation of galactose and nonfermentable carbon source
utilization
Firdous A. Khanday*, Maitreyi Saha and Paike Jayadeva Bhat
Laboratory of Molecular Genetics, Biotechnology Center, Indian Institute of Technology, Powai, Mumbai, India
We have reported previously that multiple copies of MRG19
suppress GAL genes in a wild-type but not in a gal80 strain of
Saccharomyces cerevisiae. In this report we show that dis-
ruption of MRG19 leads to a decrease in GAL induction
when S. cerevisiae is induced with 0.02% but not with 2.0%
galactose. Disruption of MRG19 in a gal3 background (this
strain shows long-term adaptation phenotype) further delays
the GAL induction, supporting the notion that its function is
important only under low inducing signals. As a corollary,
disruption of MRG19 in a gal80 strain did not decrease the
constitutive expression of GAL genes. These results suggest
that MRG19 has aroleinGAL regulation only when the
induction signal is weak. Unlike the effect on GAL gene
expression, disruption of MRG19 leads to de-repression of
CYC1-driven b-galactosidase activity. MRG19 disruptant
also showed a twofold increase in the rate of oxygen uptake
as compared with the wild-type strain. ADH2, CTA1,
DLD1,andCYC7 promoters that are active during non-
fermentative growth did not show any de-repression of
b-galactosidase activity in the MRG19 disruptant. Western
blot analysis indicated that MRG19 is a glucose repressible
gene and is expressed in galactose and glycerol plus lactate.
Experiments using green fluorescent protein fusion con-

network of gene interaction leading to exquisite co-ordina-
tion between different cellular processes [10].
Gal4p, a DNA binding transcriptional activator, acti-
vates the GAL genes in response to galactose. Although
Gal4p remains bound to the upstream activating sequences
of GAL genes in noninducing conditions, Gal80p inhibits
transcriptional activation. This is due to a physical interac-
tion between Gal4p and Gal80p [11]. In response to
galactose, Gal3p interacts with Gal80p, thereby allowing
Gal4p to cause rapid transcription of GAL genes
[1,2,4,12,13]. The long-term adaptation phenotype exhibited
by a gal3 strain [14], is due to Gal1p, which has Gal3p-like
signal transduction activity in addition to galactokinase
activity [15]. Recent experiments have demonstrated that
Gal3p directly interacts with Gal80p in the presence of
galactose and ATP [16–19]. It has also been demonstrated
that a tripartite complex is formed between Gal3p-Gal80p-
Gal4p in response to galactose and ATP [3]. The current
view is that the interaction of Gal3p with Gal80p allows the
transcription-activating domain of Gal4p to interact with
the general transcription factors, thereby causing transcrip-
tion activation of GAL genes [20,21]. It has been suggested
that the interaction of Gal3p with Gal80p may not result in
the dissociation of Gal80p from Gal4p [22] but may cause
Gal80p to shift to a second site on Gal4p [19]. Based on the
results that Gal3p is cytoplasmic and Gal80p is distributed
in both the nucleus and the cytoplasm, it has been suggested
Correspondence to P. J. Bhat, Laboratory of Molecular Genetics,
Biotechnology Center, Indian Institute of Technology, Powai,
Mumbai 400 076, India.

high induction signal [30]. Results presented in this
communication indicate that Mrg19p is a regulator of
GAL and CYC1 expression. We present evidence that
Mrg19p is an integral component required for the maximal
induction of GAL when the induction signal is weak.
Results indicate that Mrg19p is a canonical repressor of
CYC1. Based on the above, we propose that Mrg19p
regulates fermentation and aerobic oxidation.
MATERIALS AND METHODS
Strains, media and growth conditions
Table 1 provides the details of yeast strains used in this
study. Yeast strains were grown at 30 °Cinrichyeast
extract peptone (YEP) or defined synthetic drop-out or
synthetic complete media as described [31]. Carbon sources
were added to YEP, synthetic drop-out or synthetic
complete media to a final concentration of 2% w/v glucose,
2% or 0.02% galactose and/or 3% glycerol plus 2%
potassium lactate (v/v) pH 5.7. Yeast transformations were
carried out as described [32]. Escherichia coli strain XL1-
Blue was used for plasmid construction and amplification.
Bacterial transformation was carried out as described [33].
E. coli strain BL21 (DE3) was used for expression of fusion
protein from pET32(a). E. coli XL1-Blue and BL21 (DE3)
strains were grown at 37 °C in Luria–Bertani broth with
ampicillin at a final concentration of 75 lgÆmL
)1
wherever
required for plasmid maintenance [34]. For the induction of
fusion protein in BL21 (DE3), isopropyl thio-b-
D

introduced in p19C-KX by digesting with SalI and filling in
with dNTPs and the resulting plasmid was named p19C-S.
This construct was expected to induce a protein of 49 kDa.
To determine subcellular localizations of Mrg19p, two
in-frame fusion constructs with GFP were made. A 2.9-kb
SmaI–SalIfragmentofMRG19 was subcloned into SmaI–
SalI digested pGFP-N-FUS [38] and the resulting plasmid
was named pGFP-N-19FUS. pGFP-N-19FUS was further
digested by SmaI–HindIII to remove the nuclear localization
signal (NLS) and the resulting plasmid was named pGFP-
N-NLSFUS.
Strain constructions
A derivative of ScPJB644 with LEU2 was constructed as
follows. ScPJB644 was transformed to leucine prototrophy
with a 5.4-kb genomic fragment containing LEU2 gene,
which was isolated by digesting YEp13 with PstI. The
Table 1. List of strains.
Name Genotype Source
Sc289-1 MATa ura3-52 trp1-289 gal7Dgal1D Laboratory stock
Sc285 MATa ura3-52 leu2-3, 112 gal80 J.E. Hopper
Sc285-19D MATa ura3-52 leu2-3, 112 gal80 mrg19:: LEU2 This study
ScPJB644-L MATa ura3-52 leu2:: LEU2 trp1 This study
ScPJB644-19D MATa ura3-52 leu2-3112 trp1, mrg19::LEU2 Laboratory stock
ScPJB644-19D MATa ura3-52 leu2-3, 112 trp1 mrg19::LEU2 This study
Sc385 MATa ura3-52 leu2-3, 112 ade1 ile, MEL1 GAL3::LEU2 J.E.Hopper
Sc385-19D MATa ura3-52 leu2-3, 112 ade1 ile MEL1, GAL3::LEU2, mrg19::LEU2 This study
H190 MATa SUC2 ade2-1 can1-100 his3–11,15, leu2-3112 trp1-1 ura3-1 mig1-
€
aa2::LEU2 H. Ronne
W303-1D MATa SUC2 ade2-1 can1-100 his3-11,15, leu2-3112 trp1-1 ura3-1 H. Ronne

SDS/PAGE. E. coli strain bearing parent vector (pET32a)
or the plasmid construct (p19C-S) with and without IPTG,
respectively, served as the controls. As expected, a protein of
molecular weight 49 kDa was induced from transformant
bearing the p19C-S in the presence, but not in the absence,
of IPTG. For immunization, a protein of molecular mass
49 kDa was isolated using preparative SDS/PAGE fol-
lowed by electro-elution and then precipitated by acetone.
After collecting blood (to obtain preimmune serum), 100 lg
protein along with Freund’s complete adjuvant was injected
subcutaneously at more than one spot into albino rabbits.
Two weeks after the primary injection, three booster doses
of 100 lg protein were given in incomplete Freund’s
adjuvant. One week after the last booster dose, rabbit was
bled through the marginal vein. Serum was collected after
allowing clot formation at room temperature for 1 h
followed by centrifugation.
Western blot analyses
Cells were harvested by centrifuging at 5000 g for 5 min and
washed once with cold autoclaved double distilled water.
Whole cell extracts were prepared in the presence of
protease inhibitor cocktail and phenylmethanesulfonyl
fluoride as described. Supernatant obtained from the whole
cell extract was treated with polyethyleneimine to a final
concentration of 0.03% and then centrifuged at 4 °Cat
10 000 g for 2 min. Protein was estimated as described [41].
Supernatant obtained from the above step was kept in a
boiling water bath with gel loading buffer for 5 min and was
subjected to SDS/PAGE on a 7.5% gel. An equal amount
of protein was loaded in all lanes. Proteins were transferred

independent transformants and the result of four different
experiments is presented. Protein was estimated by the
Bradford method. Specific activities are represented as nmol
product formedÆmin
)1
Æmg protein
)1
.
Analysis of O
2
consumption
Cells grown on glycerol plus lactate as carbon source were
harvested either in the log phase or in the stationary phase.
The cells were washed three times with ice-cold distilled
water; the wet weight of the pellets was determined and
resuspended in oxygraph buffer [1% yeast extract, 0.1%
K
2
HPO
4
,0.12%(NH
4
)
2
SO
4
(pH 4.5)] at 100 mg cellÆmL
)1
.
Oxygen consumption rates were measured using a Clark-

sion in response to galactose. Recently, it was shown that
the activity of wild-type GAL4 is not different whether 0.02%
or 2.0% galactose is used for induction. However,
GAL4S699 A is defective in GAL gene induction at 0.02%
but not 2% galactose, indicating a difference in the galactose
signalling mechanism [27]. As MRG19 was isolated as a
multicopy suppressor of galactose toxicity at low galactose
5842 F. A. Khanday et al. (Eur. J. Biochem. 269) Ó FEBS 2002
concentration [30], we surmised that disruption of MRG19
might effect GAL induction only at low galactose concen-
trations. This hypothesis was tested by monitoring galac-
tokinase induction as a function of time in the wild-type and
in the MRG19 disruptant when cells were induced by either
0.02% or 2.0% galactose. It is clear from the results that
galactokinase activity is reduced by  50% in the disruptant
as compared with the wild-type, only when the cells were
induced by 0.02% galactose (Fig. 1). These results indicate
that MRG19 is required for maximal GAL gene induction
under conditions when the induction signal is weak.
Disruption of MRG19 in a gal3 background leads to a
delay in long-term adaptation phenotype. To further
investigate the idea that MRG19 function is necessary for
maximum expression of GAL genes only under conditions
when the induction signal is weak, GAL gene expression was
monitored in both a gal3 and a gal3mrg19 strain. The
delayed induction of GAL genes in a gal3 strain is due to the
weak induction signal transduced by the GAL1 gene [15].
Therefore, it was expected that disruption of MRG19 in a
gal3 strain (i.e. gal3mrg19) would not show any change in
the GAL gene expression if the two lie in the same induction

600
of 0.5 in synthetic me-
dium containing glycerol plus lactate and galactose was added to the
culture to a final concentration of 0.02% or 2.0%. After galactose
addition, cells were allowed to grow for 20, 60 and 140 min Galacto-
kinase activity was determined as described in Materials and methods.
Specific activity is represented as nanomoles of [
14
C]galactose phos-
phorylatedÆmin
)1
Æmg protein
)1
.
Fig. 2. Delayed long-term adaptation phenotype of the mrg19gal3
strain. Wild-type, gal3, mrg19 (in duplicate) and six independent
segregants of genotype gal3mrg19 obtained from three tetrads, were
grown on synthetic complete medium containing 2% glucose and
replica plated onto synthetic complete media containing 2% galactose.
Cells in (A), (B) and (C) were allowed to grow on synthetic complete
media containing 2% galactose for 20, 35 and 50 h, respectively.
Ó FEBS 2002 MRG19 as a bi-functional regulator (Eur. J. Biochem. 269) 5843
MRG19
as a regulator of iso-1-cytochrome C
Disruption of MRG19 results in the de-repression of the
CYC1 promoter. We reported previously that multiple
copies of MRG19 suppress CYC1 driven galactokinase [30].
If MRG19 is a canonical repressor of CYC1,thenitis
expected that disruption of MRG19 would result in the
de-repression of CYC1 promoter. To determine this, we

density.
To corroborate the above conclusion, we monitored the
rate of oxygen uptake in log and stationary phase cultures
of wild-type and MRG19 disruptant cells. The rate of
oxygen uptake was increased in wild-type and MRG19
disruptant cells in response to exogenously added ethanol
indicating that the cells are able to metabolize the carbon
source (Fig. 6, Compare 1 and 2 or 3 and 4) However, an
increase of 50% in the rate of oxygen uptake was
observed in the MRG19 disruptant as compared with the
wild-type in the absence of exogenously added ethanol
(Fig. 6, compare 1 and 3). A similar pattern was observed
even in the presence of exogenously added ethanol (Fig. 6,
compare 2 and 4). The rate of oxygen uptake in wild-type
and MRG19 disruptant cells obtained from log phase
cultures was indistinguishable either in absence or in the
presence of exogenously added ethanol (data not shown).
The above result is consistent with the observation that
CYC1 is de-repressed in mrg19 disruptant cells only at
stationary phase.
Effect of disruption of MRG19 on b-galactosidase activity
driven by promoters, which are active in a nonfermentable
carbon source. Since disruption of MRG19 de-represses the
CYC1 promoter, we expected that it might also de-repress
promoters that are active in the presence of a nonferment-
Fig. 3. Galactokinase activity in gal80MRG19 and gal80mrg19 strains.
Cells were grown to D
600
of 0.5 in synthetic complete medium con-
taining glycerol plus lactate and galactokinase activity was determined

Hap1p [46,47] and therefore it is not surprising that MRG19
affects the CYC1 promoter but not CYC7.Therefore,we
tested whether MRG19 also de-represses DLD1, which has
similar regulatory features to those of CYC1 [48]. Results
indicate that there was no difference in DLD1 promoter
driven b-galactosidase activity in wild-type and MRG19
disruptant at lower and higher cell density (Fig. 5A). Based
on the above studies, we conclude that MRG19 is a specific
repressor of the CYC1 promoter.
Expression and localization of Mrg19p
Expression of Mrg19p is carbon source dependent. Poly-
clonal antiserum was raised against a portion of Mrg19p
corresponding to residues 700–984. To detect Mrg19p, we
carried out Western blot analysis of cell-free protein
extracts, obtained from cells grown under different experi-
mental conditions. We could not detect Mrg19p in extracts
obtained from wild-type cells grown in glucose (Fig. 7, lane
2). A band corresponding to an expected molecular mass of
Fig. 6. Oxygen uptake in wild-type and MRG19 disrupted strains. Rate
of oxygen uptake in the presence (shaded bar) and absence (open bar)
of exogenously added ethanol in cells obtained from stationary phase
culture grown in glycerol plus lactate were monitored in wild-type
(1 and 2) and MRG19 disruptant cells (3 and 4).
Fig. 5. b-galactosidase activity in wild-type and MRG19 disrupted
strains bearing the indicated fusion constructs. Transformants were
grown to D
600
of 0.5 and 1.5, in minimal synthetic medium containing
glycerol plus lactate. All values are the means of duplicates from five
independent transformants. Specific activity is represented as nmol

detected in wild-type cells grown in glucose at low as well as
high cell density (Fig. 8, lane 4 and 5). We surmised that the
absence of Mrg19p from glucose-grown wild-type cells, at
both low and high cell density, is due to the low level of
expression. To test this possibility, we monitored Mrg19p
expression in a wild-type strain transformed with multiple
copies of MRG19 grown in glucose. It is clear from the results
that Mrg19p was absent from cells obtained at low cell
density but is present at high cell density (Fig. 8, lane 2 and 3).
To decipher whether the expression of Mrg19p during
diauxic shift is due either to withdrawal of glucose
repression or to other signals that emanate during diauxic
shift wild-type, as well as multicopy MRG19 transformants,
were grown in glycerol plus lactate and expression of
Mrg19p was monitored in response to glucose. It was
observed that Mrg19p expression decreased within 45 min
of glucose addition in both the wild-type strain and the
transformants (Fig. 9). We wanted to determine whether
the glucose repression of MRG19 is mediated by MIG1.
Mrg19p expression was monitored in MIG1 disruption
(Fig. 10, lane 1 and 2) and wild-type (Fig. 10, lane 3 and 4)
strains bearing multiple copies of MRG19 and grown in the
presence of glucose (Fig. 10, lane 1 and 3) and glycerol
(Fig. 10, lane 2 and 4). It is clear from the results that
Fig. 9. Mrg19p expression in response to the addition of glucose.
Extracts obtained from wild-type (lanes 4, 5 and 6) and wild-type strain
transformed with multiple copies of MRG19 (lanes 1, 2 and 3) grown in
complete synthetic medium containing either glycerol plus lactate
alone (lanes 3 and 6), or glycerol plus lactate containing glucose for
different periods of time (lanes 1, 2, 4 and 5). As an internal control, the

Mig1p binding site in the promoter of MRG19 (http://
cgsigma.cshl.org/jian/). However, our results do not exclude
the possibility of glucose inactivation of Mrg19p in addition
to glucose repression.
Subcellular localization of Mrg19p. We expected Mrg19p
to be localized in the nucleus based on its effect on
expression of GAL and CYC1 promoters. Mrg19p has been
predicted to be nuclear localized with a probability of 0.890
(). Database search showed that amino
acid sequence from 432A to 450T of Mrg19p is similar to
the NLS, present in many of the nuclear localized proteins
of yeast [49,50]. Since MRG19::GFP fusion protein could
not be detected when expressed from its own promoter
(data not shown), we constructed two MRG19::GFP
plasmids, wherein the fusion protein is expressed from
MET25 promoter. One of them retained the putative NLS
while the other did not (Materials and methods). Wild-type
cells transformed with the above constructs grown in the
absence of methionine were observed using confocal
microscopy. It is clear that MRG19::GFP fusion protein
with NLS is localized both in the cytoplasm as well as in the
nucleus (Fig. 11A). On the other hand, MRG19::GFP
fusion without NLS was not colocalized with DAPI
(Fig. 11B) indicating that it is not localized in the nucleus.
The presence of MRG19::GFP fusion with NLS in the
cytoplasm could be due to over-expression. As a control,
MRG19::GFP fusion protein could not be detected in cells
grown in the presence of methionine (data not shown).
However, the above results do not exclude the possibility
that Mrg19p may also be a cytoplasmic protein. Localiza-

regulatory network to direct galactose through fermentative
and oxidative pathways and we suggest that Mrg19p is a
component of this network.
If MRG19 is required for efficient induction of GAL
genes when the induction signal is weak, what could be its
physiological significance? It is conceivable that MRG19
may play a vital role in maintaining the GAL gene induction
at optimal levels to ensure near complete utilization of
galactose when the concentration of galactose is decreasing
in the medium. To explain the mechanism of MRG19 in
facilitating GAL gene induction, under low induction signal,
we consider the following possibility. Mrg19p might be
required to stabilize the active Gal4p–Gal80p complex,
which is formed less frequently under low induction signal
(such as low galactose concentration, or in a gal3 mutant),
and accordingly, disruption of MRG19 impairs GAL gene
induction. This observation is consistent with: (a) that
disruption of MRG19 does not interfere in the constitutive
expression of GAL genes in a gal80 strain (see Results); and
(b) that disruption of MRG19 does not affect the GAL gene
induction in a wild-type strain induced with 2% galactose
(100 times more than that required to activate the GAL
genes maximally). According to this view, the strong
induction signal (2% galactose) might be adequate to
stabilize the active Gal4p–Gal80p complex and therefore
disruption of MRG19 may not have any effect.
Recently it has been suggested that SRB10 dependent
S699 phosphorylation of Gal4p is required for stabilizing
the transiently induced active Gal4p–Gal80p complex in a
strain defective in GAL gene induction [27]. An srb10gal3

not shown). It has been well documented that over-
expression of transcription factors or activators interfere
with the normal transcription by a phenomenon commonly
referredtoasÔsquelchingÕ [53]. In light of this, it is also
possible that over-expression of Mrg19p could sequester
factors necessary for GAL1 transcription when the cells are
induced with galactose. It is possible that under these
conditions (recall that over-expression of Mrg19p in a
constitutive strain does not suppress galactokinase expres-
sion) the affect of squelching may not manifest due to the
strong activation function provided by unencumbered
Gal4p.
Based on: (a) the suppression of CYC1 promoter upon
over-expression of MRG19 [30]; (b) de-repression of the
CYC1 promoter upon disruption of MRG19 but not other
promoters such as ADH2, DLD1, CTA1 and CYC7;and
(c) the 50% increase in oxygen uptake in MRG19 disrupted
strain, we suggest that MRG19 is a specific repressor of
CYC1. Under in vitro conditions Hap1p (one of the major
regulators of CYC1) forms a large complex with other as yet
unidentified cellular proteins [47]. It has been shown that
upon interaction with hemin, the large complex is converted
to a smaller complex which led to the proposal that hemin
might mask the binding site of the repressor. Therefore, the
possibility that Mrg19p could be a member of Hap1p
complex remains to be tested.
If MRG19 were a repressor of CYC1 involved in the
regulation of carbon flow through mitochondria (when the
cells are growing in galactose or glycerol), one would expect
CYC1 to get de-repressed in a MRG19 disruptant even

tion by being an auxiliary member of the RNA
polymerase II holoenzyme complex. This view is sup-
ported by the observation that the function of MRG19 is
reminiscent of Gal11p in many respects, which has been
shown to be a component of the RNA polymerase II
holoenzyme [55]. Experiments to test the above possibil-
ity are underway.
ACKNOWLEDGEMENTS
This work was supported by Department of Science and Technology
(India) (SP/SO/D-55/99). We thank J.E. Hopper, H. Ronne, J.
Verdiere, T. Lodi, H. Ruis, K.M. Dombek, A. Kabir and Gurumurthy
for providing plasmids and yeast strains. We are grateful to V.G.
Daftari of Bharat Serums and Vaccine Ltd. Mumbai, for providing the
facility to raise antibodies against Mrg19p. We thank K. Sastry and
A. Atre for helping us to carry out laser scanning confocal microscopy
for GFP studies. We thank P. Phale for the use of the oxygraph. We
thank P.V. Balaji and S. Kumar for useful suggestions in the
preparation of this manuscript.
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