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 moni
Trang 1Molecular 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 a role in GAL 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, and CYC7 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-structs indicate that Mrg19p is localized in the nucleus consistent with the presence of a consensus nuclear locali-zation signal sequence Based on the above results, we pro-pose that Mrg19p is a regulator of galactose and nonfermentable carbon utilization
Keywords: carbon metabolism; CYC1 repressor; GAL genes; glucose repression; induction signal; transcriptional regulator
The reprogramming of molecular machinery mainly
brought about by transcriptional regulation, co-ordinates
different cellular processes as cells move from one
physio-logical state to another Since this is the key for the
evolutionary success of any organism, it is not surprising
that significant fraction of their genetic endowment is
dedicated to regulatory functions When yeast shifts from
the most preferred carbon source glucose to galactose, a
large increase in the synthesis of GAL gene products occurs,
without affecting its fermentative life style [1–4] Obviously,
during this transition, yeast has to make compensatory
changes in the pattern of gene expression to co-ordinate
galactose metabolism with various other cellular processes,
especially energy metabolism One of the obvious changes is
the de-repression of many glucose-repressed functions,
especially mitochondrial biogenesis [5–8] Recently,
genome-wide analysis has identified genes which previously
were not suspected to be induced in presence of galactose,
emphasizing the importance of the need for multiple pathways to integrate various cellular functions [9] Study
of utilization of galactose by Saccharomyces cerevisiae provides a convenient experimental system to probe into the 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 activatranscrip-tion 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.
Fax: + 91 22 572 3480, Tel.: + 91 22 576 7772,
E-mail: jayadeva@btc.iitb.ac.in
Abbreviation: IPTG, isopropyl thio-b- D -galactoside; YEP, yeast
extract peptone.
*Present address: School of Medicine, John Hopkins University,
Baltimore, MD 21205, USA.
(Received 15 July 2002, revised 27 September 2002,
accepted 10 October 2002)
Trang 2that the dynamics of their distribution is intrinsic to GAL
gene regulation [23] Recent studies have indicated that the
Gal80p–Gal80p interaction is required for complete
repres-sion of GAL genes [24]
Circumstantial evidence suggests that the energy status of
the cell is an important determinant of GAL gene induction
[15,25–27] This suggests that the availability of metabolic
energy is a rate-limiting step in the synthesis of GAL
enzymes that constitute 5% of the total soluble proteins
when the cell grows on galactose as sole carbon source
[28,29] Phosphorylation of S699 of Gal4p has been shown
to be important for activating transcription of GAL genes
when the induction signal is weak and has been suggested to
be a link between energy status and the GAL genetic switch
[27] Although, the importance of mitochondrial function in
galactose metabolism has been well recognized, the
molecular basis for the same has largely remained
unex-plored We had reported the isolation of MRG19 as a
multicopy suppressor of galactose toxicity at low but not at
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
M A T E R I A L S A N D M E T H O D S
Strains, media and growth conditions
Table 1 provides the details of yeast strains used in this
study Yeast strains were grown at 30C in rich yeast
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 colistrain BL21 (DE3)was used for expression of fusion
protein from pET32(a) E coli XL1-Blue and BL21 (DE3)
strains were grown at 37C in Luria–Bertani broth with
ampicillin at a final concentration of 75 lgÆmL)1wherever
required for plasmid maintenance [34] For the induction of fusion protein in BL21 (DE3), isopropyl thio-b-D -galacto-side (IPTG)was added to a final concentration of 1 mMat
an OD600of 0.5 and growth was allowed to continue for a further 2 h
Plasmids
A 4.7kb HindIII–SalI fragment obtained from YIp24 ADH2-lacZ(+)[35], containing ADH2::lacZ cassette, was subcloned into HindIII–SalI digested YCplac33 [36] and the resulting plasmid was named as YCfADH2::lacZ A 4-kb XbaI fragment was obtained from plasmid pAB2654 (unpublished data)containing the CYC7::lacZ cassette and was subcloned into XbaI digested YCplac33; the resulting plasmid was named YCfCYC7::lacZ A 5.6-kb PstI–SalI fragment obtained from YIpCTA1-lacZ [37], containing the CTA1::lacZ cassette, was subcloned into PstI–SalI digested YCplac33 and the resulting plasmid was named pYCfCTA 1::lacZ A portion of MRG19 was amplified by PCR using primers PJB102 (5¢-GACCGTAGGTACCATGTTGGCT TCAG-3¢)and PJB103 (5¢-CGGGCCCCTC GAGGCCCA TCATCTAA-3¢)carrying KpnI and XhoI sites, respectively After digesting the PCR product with KpnI and XhoI, it was cloned into KpnI–XhoI digested pET32a and the resulting plasmid was named p19C-KX The protein product obtained from the above construct upon induction with IPTG was found to be 67 kDa as expected As the induction of this truncated protein was low, a frame-shift mutation was 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–SalI fragment of MRG19 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.
Sc289-1 MATa ura3-52 trp1-289 gal7Dgal1D Laboratory stock
Sc285-19D MATa ura3-52 leu2-3, 112 gal80 mrg19:: LEU2 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-€ a a2::LEU2 H Ronne W303-1D MATa SUC2 ade2-1 can1-100 his3-11,15, leu2-3112 trp1-1 ura3-1 H Ronne
Trang 3mating type of ScPJB644–19D MATa was changed to
MATa by transforming with HO plasmid as described [15]
Sc285–19D was constructed by crossing Sc285 with
ScPJB644–19D of the opposite mating type The diploids
selected on synthetic complete glucose medium lacking
leucine and tryptophan were then sporulated [39] After
digesting the asci with cell wall degrading enzyme, random
spores were screened on synthetic complete glycerol plus
lactate medium lacking leucine to identify disrupted
MRG19locus, but containing 2-deoxygalactose to identify
gal80 allele [40] Sc385–19D was constructed by mating
Sc385 with ScPJB644–19D of the opposite mating type The
diploids selected in synthetic complete galactose medium
lacking tryptophan were sporulated Leu+ segregants
which are gal3mrg19 were isolated from tetrads of the
constitution 2+:2–::LEU+:LEU–by tetrad dissection
Expression of truncated Mrg19p and generation of
polyclonal antibodies
Antibodies against Mrg19p were raised as described [15]
The cells obtained after induction were treated with SDS gel
loading buffer, boiled for 5 min and subjected to analytical
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 4C at
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
onto nitrocellulose membrane and blocked with buffer
containing 1% milk powder for 1 h The blot was then
probed with 1 : 2000 diluted antiserum or preimmune
serum and incubated for 1 h Membrane was washed with
buffer four times for 5 min each The immunoblot was
probed with 1 : 2500 diluted secondary antibody
conjugat-ed with alkaline phosphatase All the experiments were
repeated at least three times
Galactokinase assay Cells were washed and extracts were prepared by the glass bead cell disruption method [4] Galactokinase activity was assayed as described [28] 14C-galactose (58 mCiÆmmol)1) was from Amersham The original stock of14C-galactose was diluted with cold galactose to achieve a final specific activity of 1 lCi per 4.7 lmol DE81 ion exchange paper was from Whatman International Ltd The radioactivity was counted in an LKB liquid scintillation counter using OCS liquid scintillant (Amersham) Each value is an average of four independent colonies and the assays were performed in triplicate
b-Galactosidase assay b-Galactosidase activity was assayed in cell extracts as described [39] Duplicate samples were taken for each determination Experiments were performed with five 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)1Æmg protein)1
Analysis of O2consumption 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%
K2HPO4, 0.12% (NH4)2SO4(pH 4.5)] at 100 mg cellÆmL)1 Oxygen consumption rates were measured using a Clark-type oxygen electrode, with 0.1 mM ethanol as substrate The rates were measured from the slope of a plot of O2 concentration vs time and expressed as nmol O2consumed per min per 10 mg wet weight of cells [42]
GFP fluorescence microscopy Wild-type cells were transformed with pGFP-N-19FUS and pGFP-N-NLSFUS plasmids Cells were grown to D600of 1.5 in methionine and uracil double drop-out glycerol (3%) lactate (2%)media [38] Cells were allowed to grow with 4¢,6-diamidino-2-phenylindole (DAPI)at a concentration
2 lgÆmL)1 for 1 h before microscopic observation Cells were harvested in the cold and green fluorescent protein (GFP)/DAPI fluorescence was monitored using a Zeiss LSM510 Scanning Confocal Microscope Images were recorded and processed inADOBE PHOTOSHOP6.0
R E S U L T S
MRG19 as a regulator of galactose utilization MRG19 disruptant is defective in galactokinase expres-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
Trang 4concentration [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
gal3strain (i.e gal3mrg19)would not show any change in
the GAL gene expression if the two lie in the same induction
pathway Alternatively, if they lie in different induction
pathway, gal3mrg19 would exhibit a further delay or may
not induce the GAL gene expression at all The growth
pattern of wild-type, gal3, mrg19 and gal3mrg19 strains was
monitored as a function of time on complete medium
containing galactose as a carbon source Wild-type and
mrg19strains grew on galactose plates within 12 h while
gal3 strain showed the characteristic delay in growth on
galactose Interestingly, the gal3mrg19 strain showed a
further delay in growth on galactose plate as compared with
the gal3 strain (Fig 2)
Disruption of MRG19 in a gal80 background does not affect constitutive GAL gene expression The results described above indicated that disruption of MRG19 affects expression of GAL genes only when the induction signal is weak This implied that loss of MRG19 function might not affect the constitutive GAL gene expression observed in a gal80 strain (strong induction signal) To determine whether this is true or not, galactokinase activity was determined in gal80MRG19 and gal80mrg19 strain grown in glycerol plus lactate As expected, disruption of MRG19 in a gal80 strain (gal80mrg19 strain)did not cause a discernible difference in galacto-kinase activity (Fig 3)in comparison with a gal80 strain (gal80MRG19 strain) This suggested that in a wild-type strain it is only when the induction signal is weak that the function of MRG19 is necessary for maximal GAL gene expression
Fig 1 Specific activity of galactokinase in wild-type cells and the
MRG19 disruptant Cells were grown to D 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)1Æmg 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.
Trang 5MRG19 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, then it is
expected that disruption of MRG19 would result in the
de-repression of CYC1 promoter To determine this, we
used a plate assay which is based on the observation that
2-deoxygalactose is toxic to wild-type yeast strains that
constitutively express galactokinase, due to the
accumula-tion of 2-deoxygalactose-1-phosphate [40] Growth of a
wild-type strain bearing the CYC1::GAL1 construct which
expresses galactokinase at a basal level on glycerol
plus lactate was marginally reduced in the presence of
2-deoxygalactose as compared to its vector control (Fig 4)
due to 2-deoxygalactose toxicity If MRG19 disruption
leads to a de-repression of the CYC1 promoter, we would
expect mrg19 transformed with CYC1::GAL1 to show a
diminished growth as compared with the wild-type control
Growth of an MRG19 disruptant bearing the
CYC1::GAL1construct was lower than that of the
wild-type transformed with same construct (Fig 4) Moreover,
growth of the MRG19 disruptant bearing CYC1::GAL1
was lower than that of the vector control These results
indicated that the disruption of MRG19 de-repressed the
CYC1promoter
Genome-wide expression analysis showed that MRG19
transcript levels increased fourfold during diauxic growth
[43], indicating that its function may be important at
higher cell density Therefore, in the MRG19 disruptant,
one would expect the CYC1 promoter to be de-repressed
to a greater extent at higher cell density than at a lower cell density To test the above prediction, CYC1 driven b-galactosidase activity was monitored in wild-type and the MRG19 disrupted strain b-galactosidase activity in the MRG19 disruptant was twofold higher than that in the wild-type strain (Fig 5A)only at a higher cell 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 onb-galactosidase activity driven by promoters, which are active in a nonfermentable carbon source Since disruption of MRG19 de-represses the CYC1promoter, 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
as described in Materials and methods.
Fig 4 Expression of galactokinase driven by the CYC1 promoter in the wild-type strain and the MRG19 disruptant Transformants (in tripli-cates)of wild-type and MRG19 disruptant bearing either vector (control)or CYC1::GAL1 construct were grown in Trp drop-out synthetic minimal medium containing glucose and were replica plated
on to Trp drop-out synthetic minimal medium containing glycerol plus lactate supplemented with 0.03% of 2-deoxygalatcose.
Trang 6able carbon source We tested this possibility by measuring
b-galactosidase activity driven by ADH2 and CTA1
promoters in wild-type and MRG19 disruptant at low and
high cell density Both of these promoters are under
the control of the Adr1p transcriptional activator
[44] b-galactosidase activity was the same in wild-type
and the MRG19 disruptant regardless of the cell density
(Fig 5) Although CYC1, ADH2 and CTA1 promoters are
active when cells are grown in ethanol, it is not surprising
that MRG19 disruption effects CYC1 but not ADH2 and
CTA1, as CYC1 is regulated through a pathway [45,46]
different from that of ADH2/CTA1 [44]
CYC1/CYC7form a duplicated pair of genes, which are functionally related but differentially regulated through Hap1p [47] We wanted to test whether CYC7 expression is also dependent on MRG19 Results indicate that b-galac-tosidase activity driven by CYC7 promoter is the same in the wild-type and the MRG19 disruptant (Fig 5B), indica-ting that suppression by MRG19 is specific to the CYC1 promoter Although Hap1p activates these two genes, CYC1 requires the Hap2/3/4/5 complex in addition to 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
product formedÆmin)1Æmg protein)1 (A)and (B)represent different
scales of b-galactosidase activity.
Fig 7 Detection of Mrg19p Extract obtained from transformants with multiple copies of MRG19, grown in Ura drop-out medium containing glycerol plus lactate (lane 1); wild-type strain grown in glucose (lane 2)and galactose (lane 3)and MRG19 disruptant grown in galactose (lane 4)were subjected to Western blot analysis as described
in materials and methods.
Trang 7125 kDa was detected in protein extracts obtained from
wild-type cells grown in galactose (Fig 7, lane 3) However,
this band was absent from extracts obtained from the
MRG19disrupted strain grown in galactose (Fig 7, lane 4)
We could detect an intense band in the control lane
corresponding to protein extract obtained from wild-type
transformed with multiple copies of MRG19 grown in
glycerol plus lactate (Fig 7, lane 1) Western blot analysis
carried out against the same protein extracts using
preim-mune serum did not detect the corresponding band (data
not shown) Based on the above, we conclude that the
antiserum specifically recognizes Mrg19p and it is expressed
in galactose but not glucose (Fig 7)
Genome-wide expression analysis indicated that the
MRG19transcript levels increase fourfold during the diauxic
shift [43] We wanted to determine whether the Mrg19p
profile also follows the same pattern Mrg19p could not be
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 above extracts were subjected to Western blot analysis using glucose-6-phosphate dehydrogenase (Zwf1p)antiserum as described in Mate-rials and methods.
Fig 8 Expression of Mrg19p as a function of cell density Wild-type
cells transformed with multiple copies of MRG19 (lane 2 and 3)and
wild-type cells (lanes 4 and 5)were allowed to grow to an OD 600 of 0.5
and 1.5, either in uracil drop-out minimal medium containing glucose
(lanes 2 and 3)or in synthetic complete medium containing glucose
(lanes 4 and 5) Extracts were subjected to Western blot analysis as
described in Materials and methods Lane 1 represents the extract
obtained from glycerol-grown wild-type strain transformed with
multiple copies of MRG19.
Fig 10 Glucose repression of MRG19 is MIG1 dependent Extracts obtained from wild-type (lane 3 and 4)and mig1 disrupted strains (lane
1 and 2)transformed with multiple copies of MRG19 grown in pres-ence of glucose (lane 1 and 3)or glycerol (lane 2 and 4)to D 600 of 0.5, were subjected to Western blot analysis As an internal control, the above extracts were subjected to Western blot analysis using glucose-6-phosphate dehydrogenase (Zwf1p)antiserum as described in Materials and methods Lane 5 contains extract from an mrg19 disrupted strain grown in the presence of glycerol.
Trang 8repression of Mrg19p is dependent on MIG1 function In
view of the results obtained from genome-wide analysis and
the results presented here, we suggest that Mrg19p is glucose
repressed This is consistent with the presence of a putative
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
(http://www.yale.edu) 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
MET25promoter 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::GFPfusion 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-tion of Mrg19p in the nucleus could not be observed in
genome-wide subcellular localization studies probably
because of detectability limitations (http://ygac.med.yale
edu/triples/cidlstqry.asp)
D I S C U S S I O N
Results presented in this study demonstrate that MRG19
plays a vital role in the regulation of GAL gene induction
and CYC1 expression Based on the expression of Mrg19p
in galactose, ethanol and glycerol plus lactate but not glucose, we suggest that its function is crucial when yeast grows in carbon sources that are metabolically inferior to glucose Although galactose is a fermentable carbon source similar to glucose, efficient utilization of galactose is dependent on mitochondrial function [8,15,25,26,51], implicating that the metabolic energy derived from fermen-tation alone may not be adequate for optimal growth The need for mitochondrial function is strengthened by the observation that: (a)galactose is also metabolized through mitochondrial respiration to a greater extent than sugars such as glucose, fructose and mannose; and (b)the generation time of respiratory incompetent cells is twice as long as that of respiratory sufficient cells when grown on galactose [51] It has been shown that the levels of glucose-6-phosphate and fructose-6-glucose-6-phosphate as well as those of ATP are significantly lower when cells utilize galactose as compared to glucose [52] Moreover, it has been demon-strated that within the first minutes after a galactose pulse, almost all of the galactose consumed is directed towards the TCA cycle [52] These studies clearly indicate that for the optimal utilization of galactose, the cell has designed a 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 strain does not activate the GAL genes due to a defect in phosphorylation of S699 of Gal4p [27] Based on the above observation, it has been suggested that Gal4p activity is controlled by two independent signal transduction path-ways: a Gal3p–galactose pathway and the holoenzyme associated cyclin dependent Srb10p kinase pathway The observation that disruption of MRG19 in a gal3 strain further delays long-term adaptation shows that, Mrg19p does not lie in the Gal3p–galactose pathway As the effect of MRG19 disruption is also observed under low induction
Fig 11 Subcellular localization of Mrg19p::GFP using confocal
microscopy Wild-type cells transformed with pGFP-19FUS (A)and
pGFP-NLSFUS lacking the nuclear localization signal (B)were
visualized by laser scanning confocal microscopy Co-localization was
monitored by carrying out DAPI staining of green fluorescing cells.
Arrows indicate the position of nucleus.
Trang 9signal, we suggest that MRG19 is also required for the
sensitive response
If Mrg19p is required for efficient transcription from the
GAL1 promoter under low induction signal, why should
over-expression lead to a decrease in galactokinase activity
in a wild-type strain but not in a strain constitutive for GAL
gene induction? It is conceivable that over-expression of
Mrg19p could sequester Gal80p–Gal4p complex thereby
decreasing galactokinase expression Consistent with this
idea, over-expression of MRG19 does not suppress the
constitutive expression in a gal80 [30] or GAL4cstrain (data
not shown) It has been well documented that
over-expression of transcription factors or activators interfere
with the normal transcription by a phenomenon commonly
referred to as squelching [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
CYC1promoter 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
during exponential growth However, we observed that
de-repression of CYC1 occurs only at higher cell density
This is consistent with our previous observation that that
MRG19disruptant strain attains a higher cell density during
stationary phase To rule out the possibility that the
previous results could reflect a difference due to auxotrophic
marker (LEU–vs LEU+)[54] rather than the difference at
MRG19locus, the stationary phase cell density of wild-type
and MRG19 disruptant in LEU+background was
moni-tored It was observed that the cell density was twofold
higher in the MRG19 disruptant than in the wild-type Our
inability to observe the de-repression of CYC1 during
exponential phase in MRG19 disruptant could be due to
other redundant pathways that regulate CYC1
Alternat-ively, Mrg19p expression might increase as a function of cell
density and therefore its effect might manifest only at a
higher cell density However, we were not able to detect any
such increase in Mrg19p expression using Western blot
analysis (data not shown)
Our results clearly show that MRG19 plays a
regula-tory role in the expression of genes driven by GAL1 and
CYC1 promoters However, the exact mechanism by
which Mrg19p regulates this process is yet to be determined We suggest that Mrg19p regulates transcrip-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
A C K N O W L E D G E M E N T S
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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