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Originally published In Press as doi:10.1074/jbc.M400433200 on June 25, 2004

J. Biol. Chem., Vol. 279, Issue 37, 39165-39174, September 10, 2004
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The Reg1-interacting Proteins, Bmh1, Bmh2, Ssb1, and Ssb2, Have Roles in Maintaining Glucose Repression in Saccharomyces cerevisiae*

Kenneth M. Dombek{ddagger}, Nataly Kacherovsky, and Elton T. Young

From the Department of Biochemistry, University of Washington, Seattle, Washington 98195-7350

Received for publication, January 14, 2004 , and in revised form, June 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In Saccharomyces cerevisiae, a type 1 protein phosphatase complex composed of the Glc7 catalytic subunit and the Reg1 regulatory subunit represses expression of many glucose-regulated genes. Here we show that the Reg1-interacting proteins Bmh1, Bmh2, Ssb1, and Ssb2 have roles in glucose repression. Deleting both BMH genes causes partially constitutive ADH2 expression without significantly increasing the level of Adr1 protein, the major activator of ADH2 expression. Adr1 and Bcy1, the regulatory subunit of cAMP-dependent protein kinase, are both required for this effect indicating that constitutive expression in {Delta}bmh1{Delta}bmh2 cells uses the same activation pathway that operates in {Delta}reg1 cells. Deletion of both BMH genes and REG1 causes a synergistic relief from repression, suggesting that Bmh proteins also act independently of Reg1 during glucose repression. A two-hybrid interaction with the Bmh proteins was mapped to amino acids 187-232, a region of Reg1 that is conserved in different classes of fungi. Deleting this region partially releases SUC2 from glucose repression. This indicates a role for the Reg1-Bmh interaction in glucose repression and also suggests a broad role for Bmh proteins in this process. An in vivo Reg1-Bmh interaction was confirmed by copurification of Bmh proteins with HA3-TAP-tagged Reg1. The nonconventional heat shock proteins Ssb1 and Ssb2 are also copurified with HA3-TAP-tagged Reg1. Deletion of both SSB genes modestly decreases repression of ADH2 expression in the presence of glucose, suggesting that Ssb proteins, perhaps through their interaction with Reg1, play a minor role in glucose repression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In order to survive without glucose, their preferred carbon and energy source, yeast cells adapt their metabolic pathways to utilize alternative carbon sources (1, 2). When glucose is abundant, these adaptive responses are kept in check at both the transcriptional and post-transcriptional levels (3-5). The signaling pathway primarily responsible for the transcriptional control of this process is referred to as glucose repression (5-7). One component of this pathway is a type I protein phosphatase complex composed of the PP1 catalytic subunit, Glc7, and its associated regulatory subunit, Reg1 (8). Many regulatory subunits interact with PP1c1 utilizing an RVXF motif (9, 10). This motif has a general consensus sequence of (R/K)X0-1(V/I)(P)(F/W), where X represents any amino acid and (P) is any amino acid except proline (11). The RHIHF sequence, present at amino acids 464-468 of Reg1, matches this general consensus sequence and is required for binding to Glc7 and for maintaining glucose repression (12, 13).

One function of PP1c regulatory subunits is to direct the catalytic subunit to appropriate targets for dephosphorylation (14). For Reg1, these targets appear to be proteins involved in glucose repression (8), including Snf1, the yeast AMP-activated protein kinase required for release of most glucose-repressible genes from repression (2, 5-7, 15). Phosphorylation of Thr210 in the activation loop of the Snf1 kinase by the Pak1, Elm1, or Tos3 kinases is required for activating kinase activity (15-17). Reg1 interacts with the kinase domain of activated Snf1, directing Glc7 phosphatase activity to the kinase activation loop (18). When glucose in the growth medium becomes abundant, the Glc7 phosphatase is activated by an unknown mechanism, and Thr210 is dephosphorylated, causing Snf1 to assume an inactive closed conformation (15).

Little is known about how the activity of Reg1-Glc7 might be regulated. Neither expression nor subcellular localization of Reg1 is regulated by a carbon source (1, 13, 19). There is also no direct evidence indicating that the interaction of Reg1 with Glc7 is significantly regulated by a carbon source (8). The activity of Reg1-Glc7 might be regulated by post-translational phosphorylation of Reg1. Reg1 is phosphorylated in a carbon source-dependent manner, and this phosphorylation is Snf1-dependent (20). Reg1 is not phosphorylated in cells lacking Hxk2, the isozyme of hexokinase implicated in glucose repression (21), and its interaction with Snf1 is weakened (20). This suggests that Hxk2-dependent phosphorylation of Reg1 may regulate the interaction with and, presumably, the activity of Snf1. A major caveat to this conclusion is that phosphorylation was assessed using proteins expressed from the strong ADH1 promoter and primarily utilized LexA-Reg1 fusions that were targeted to the nucleus, a cellular compartment from which Reg1 appears to be excluded (13). This conclusion also suggests that expression of genes controlled by Snf1 and Reg1 should be co-regulated by Hxk2. Repression of ADH2, the gene encoding the glucose repressible alcohol dehydrogenase in the yeast Saccharomyces cerevisiae, is not relieved in cells expressing an allele of HXK2 that is defective in the repression of other genes by glucose (22). Repression is partially relieved, however, in cells lacking Reg1, and this requires Snf1 (22, 23). Therefore, the role of glucose-regulated phosphorylation in the control of Reg1-Glc7 activity remains unclear.

In order to expand our understanding of how the Reg1-Glc7 phosphatase is regulated and to identify new targets or processes controlled by this complex, we and others have performed yeast genomic two-hybrid library screens looking for proteins that interact with Reg1 (24).2 These screens identified the yeast homologs of mammalian 14-3-3 proteins, Bmh1 and Bmh2, and the nonconventional heat shock proteins, Ssb1 and Ssb2, as putative Reg1-binding proteins. Since 14-3-3 proteins play roles in other signal transduction pathways (25) and the role of Ssb proteins in signal transduction is uncertain, the Reg1-Bmh interaction and the role of Bmh proteins in glucose repression were further characterized (24). The interaction with Bmh2 was confirmed by glutathione S-transferase pull-down assays using highly expressed tagged proteins and was shown to require the N-terminal 313 amino acids of Reg1. However, no role for Bmh protein in glucose repression could be demonstrated. The interaction with Reg1 was not regulated by glucose availability, and glucose-repressible SUC2 expression was not released from repression in {Delta}bmh1{Delta}bmh2 cells.

In this study, we show that Bmh proteins do play a role in glucose repression in S. cerevisiae and that for SUC2 expression the interaction with Reg1 is required to maintain complete repression. The region of Reg1 that interacts with Bmh protein was mapped to amino acids 187-232 of the N terminus, a region that is evolutionarily conserved among fungi. Copurification of Bmh proteins with HA3-TAP-tagged Reg1 protein expressed at physiological levels from the chromosomal locus confirmed that Reg1 and Bmh proteins interact in vivo. Ssb proteins also copurified with HA3-TAP-tagged Reg1. Deletion of SSB1 and SSB2 caused a modest relief of ADH2 expression from glucose repression, providing evidence that Ssb proteins play a minor role in this process.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Media and Growth Conditions—Media and culture conditions were essentially as described by Sherman (26). Yeast cells were grown at 30 °C in YEP medium as described previously (22). Cells having URA3-selectable plasmids were grown in synthetic medium lacking amino acids and uracil and containing 0.4% (w/v) casamino acids and 40 µg/ml tryptophan. Cells having URA3- and LEU2-selectable plasmids were grown in the same medium with amino acid dropout solution lacking leucine instead of casamino acids and tryptophan. Repressing liquid medium contained 5% (w/v) glucose as the carbon source (YEPD or SD), and derepressing medium contained either 3% (v/v) ethanol in YEP broth (YEPE) or 0.05% glucose and 3% ethanol in synthetic medium broth. Agar plates were prepared using the same recipes as for liquid medium except that the glucose concentration was reduced to 2% (w/v). Selection for G418 or clonNat resistance was performed by plating cells on YEPD agar plates containing either 300 µg/ml G418 (Calbiochem) or 100 µg/ml clonNat (Werner BioAgents, JENA-Cospeda, Germany), respectively.

Yeast Strains and Their Construction—Yeast strains used in this study are listed in Table I. Oligonucleotides used in strain constructions are listed in Table II. Newly created strains were constructed using standard genetic methods (30). Yeast strains were transformed using the lithium acetate transformation method as described by Gietz and Woods (31). The adr1::LEU2 and bcy1::URA3 deletions were introduced into strains 10560-4d and RRY1216 using the one-step gene disruption technique as described previously (13). DNA fragments used for gene tagging or gene deletion with kanMX (32) or natMX (33) were generated by polymerase chain reactions using the Expand Long Template PCR system from Roche Applied Science following the recommendations in the product manual.


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  		TABLE I
Yeast strains used in this study

 


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  		TABLE II
Oligonucleotides used for diagnostic PCR, gene tagging and deletion, and plasmid constructions

 
A yeast strain of the 10560-4d genetic background having the chromosomal REG1 locus deleted was created by introducing the {Delta}reg1::LEU2 deletion into a 10560-4d diploid, sporulating the diploid, and then screening for random segregants with the genotype of KDY126. A yeast strain in which the BMH1, BMH2, and REG1 genes are deleted was created by mating a MAT{alpha} {Delta}reg1::LEU2 random segregant obtained during the construction of KDY126 with RRY1216, sporulating the diploid, and then screening for random segregants having all three deletions. Small colonies that grew on SD plates lacking leucine and having colony morphologies similar to that of RRY1216 were isolated and tested by diagnostic PCR for the presence of the {Delta}bmh1, {Delta}bmh2, and {Delta}reg1 deletions. Primers BMH1-A and BMH1-B were used to test for the wild type BMH1 gene, and primers BMH2-A and BMH2-B were used to test for the wild type BMH2 gene. The absence of PCR products with these two primer pairs along with the observation that these segregants had the same colony morphology as RRY1216 was taken as evidence that both BMH genes had been deleted. The presence of the {Delta}reg1::LEU2 deletion in these segregants was confirmed by PCR as described previously (22).

The REG1 chromosomal locus of KDY121 was tagged at the 3'-end of its open reading frame with the HA3-TAP coding sequence by integrative transformation with a DNA fragment created by PCR. This fragment was generated by amplifying DNA sequence from plasmid pYM12-HA3-TAP using primers REG1-T5 and REG1-T3. Expression of HA3-TAP-tagged Reg1 protein in G418-resistant transformants was confirmed by Western blot analysis using anti-HA monoclonal antibody 12CA5 (Roche Applied Science). Three independent transformants expressing the tagged protein were then tested for expression of an integrated ADH2-lacZ reporter plasmid by assaying {beta}-galactosidase activity in permeabilized whole cells after growth in repressing YEPD broth and derepressing YEPE broth. All three transformants had levels of {beta}-galactosidase equal to that of the untagged starting strain, KDY80. This indicated that the HA3-TAP-tagged Reg1 protein was fully functional and behaved identically to the untagged protein in repressing ADH2 expression.

The yeast strain KDY157, which is of the BY4741 genetic background and has deletions of the SSB1 and SSB2 genes, was created by first introducing a {Delta}ssb2::natMX deletion into BY4742 using integrative transformation. The DNA fragment used in the transformation was created by PCR using primers SSB2_del-5' and SSB2_del-3' and pAG25 as the template (33). Then one of the clonNat-resistant transformants was mated with BY4741{Delta}ssb1. The resulting diploid was sporulated, and random segregants were screened for the presence of both G418 and clonNat resistances. The absence of wild type SSB1 and SSB2 genes in these segregants was confirmed by diagnostic PCR with the primer pair SSB1-A and SSB1-B and the pair SSB2-A and SSB2-B, respectively. These segregants exhibited the slow growth phenotype reported for cells of the DS10 genetic background with the {Delta}ssb1{Delta}ssb2 deletions (34).

Plasmids Used and Their Construction—The plasmids used in this study are listed in Table III, and the oligonucleotides used in their construction are listed in Table II. Plasmid pKD134 was constructed by first amplifying by PCR the BMH1 open reading frame from plasmid p3.29.21 using Pfu polymerase (Stratagene, La Jolla, CA) with primers BMH1-A and BMH1-B. p3.29.21 is a pGAD-C3-based two-hybrid library plasmid having the GAD sequence fused in frame with codons -17 to 267 of BMH1, which had been cloned in a two-hybrid library screen using LexA-Reg1 as bait.2 Then the resulting 0.8-kb PCR product was digested with EcoRI and XhoI and ligated to EcoRI-SalI-digested pGAD-C1.


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  		TABLE III
Plasmids used in this study

 
A pRS316-based CEN plasmid carrying the reg1{Delta}187-232 allele was constructed by ligating PCR fragments amplified from both the 5' and 3' ends of the REG1 gene with a vector fragment of pKD89. The region of the REG1 gene 5' to the deletion was amplified from pKD89 using Turbo-Pfu polymerase (Stratagene, La Jolla, Ca) with primers REG1-BK1-R and pRS-5' (13) and then digested with BglII and EcoRI to yield a 1.5-kb fragment. The region 3' to the deletion was similarly amplified using primers REG1-BK1-F and pRS-3' (13) and digested with BglII and XhoI to yield a 2.7-kb fragment. These fragments were simultaneously ligated to the EcoRI-XhoI vector fragment from pKD89, yielding plasmid pKD143.

Five new lexA-REG1 two-hybrid plasmids employed in mapping the Bmh1 interaction site were constructed by exchanging the wild type 3.3-kb ClaI-SalI fragment of the REG1 open reading frame in pKD123 with a ClaI-SalI fragment from the pRS316-based CEN plasmid carrying the desired allele of REG1. The CEN plasmid source for each constructed plasmid was as follows: pKD92 for pKD129, pKD94 for pKD130, pKD96 for pKD131, pKD97 for pKD132, and pKD143 for pKD144. Except for pKD143, which is described above, the source plasmids have been described previously (13). A sixth new plasmid, pKD133, which has the lexA-REG1 fusion containing a deletion of codons 2-198, had to be constructed differently because the ClaI site fell within the first 198 codons of the REG1 open reading frame. A 2.9-kb fragment containing the 3'-end of the REG1 open reading frame beginning at codon 199 was amplified by PCR with Pfu polymerase using primers 123-1-3' and pRS-3' (13) and pKD123 as template. This PCR product was digested with EcoRI and XhoI and then ligated to the vector fragment of EcoRI-XhoI-digested pKD123. Finally, a lexA-REG1 two-hybrid plasmid having lexA fused in frame to codons 86-247 of the REG1 gene was constructed by ligating together the following DNA fragments: a 6.9-kb NotI-BglII vector fragment from plasmid pKD132, a 2-kb EcoRI-NotI fragment from pKD132 carrying the ADH1 promoter and lexA sequences, and a 0.5-kb EcoRI-BglII fragment carrying codons 86-247 of REG1. The EcoRI-BglII fragment was amplified from plasmid pKD123 sequence by PCR using primers N1.5-3' and N1.5-5'.

Amino Acid Sequence Alignment of REG1 Homologs—The amino acid sequences of Reg1 homologs were obtained from the following sources: S. cerevisiae, Saccharomyces Genome Database (available on the World Wide Web at www.yeastgenome.org/); Aspergillus nidulans, Neurospora crassa, and Ustillago maydis, The Fungal Genome Initiative at The Broad Institute, Center for Genome Research at the Whit-head Institute (available on the World Wide Web at www.broad.mit.edu/annotation/fungi/fgi/); Schizosaccharomyces pombe, The Wellcome Trust Sanger Institute (available on the World Wide Web at www.genedb.org/genedb/pombe/index.jsp). Blocks of sequence conserved among the Reg1 homologs were identified using the segment pair overlap alignment method of the MACAW program (38) and the Block Maker program of the Blocks-based Tools at the Fred Hutchinson Cancer Research Center (available on the World Wide Web at blocks.fhcrc.org/blocks/) (39).

Purification of TAP-tagged Reg1 Protein—Yeast cells were grown, and extracts were prepared using a combination of the Seraphin Lab TAP protocol (available on the World Wide Web at www-db.embl-heidelberg.de/jss/servlet/de.embl.bk.wwwTools.GroupLeftEMBL/ExternalInfo/seraphin/TAP.html) and the liquid nitrogen method for preparing yeast cell extracts described by Kaplan and Sorger (40). Yeast strains KDY121 (REG1-HA3-TAP) and KDY80 (REG1) were grown in YEPD broth to an A600 of ~1.5. One-half of each culture was harvested, and the cells were washed with a solution of 10% (w/v) glycerol and 2% (w/v) glucose at 4 °C to maintain the cells in a repressed state during processing. These repressed cells were frozen on powdered dry ice and stored at -80 °C. The other half of each culture was harvested, the cells were washed once with cold YEP, and then the cells were suspended in a volume of room temperature YEPE broth equal to the volume of culture harvested. After incubating at 30 °C for 3 h, these derepressed cultures were harvested, and cell pellets were prepared as described above except that glucose was not added to the glycerol washing solution. Proteins were extracted from each frozen pellet by grinding the cells in liquid nitrogen and then suspending the resulting fine powder in 10 ml of TAP extraction buffer (10 mM K-HEPES, pH 7.9, 200 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml aprotinin). For extract prepared from repressed cells, 2% (w/v) glucose was also included in the TAP extraction buffer. Each suspension was held on a reciprocating platform at 4 °C for 20 min. Unbroken cells and large cell debris were pelleted by centrifugation at 2300 x g at 4 °C for 10 min. The supernatant was then clarified by sequential spins of 20,500 rpm at 4 °C for 30 min and 33,500 rpm at 4 °C for 1 h 24 min in a Beckman ultracentrifuge using a 50Ti rotor (Beckman). The supernatant was dialyzed against TAP dialysis buffer (20 mM K-HEPES, 50 mM KCl, 0.2 mM EDTA, 20% (w/v) glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml aprotinin) at 4 °C for 3 h. To each ml of extract recovered from the dialysis was added 1 µl of 1 M phenylmethylsulfonyl fluoride, and then the extract was frozen and stored at -80 °C. Reg1-HA3-TAP protein was purified from the dialyzed extracts as described by Rigaut et al. (41).

Mass Spectrometry of TAP-purified Proteins—Trypsin digestion of TAP-purified proteins and HPLC-coupled ion trap mass spectrometry of the resulting peptide mixtures were performed by the Proteomics Resource Center of the Fred Hutchinson Cancer Research Center (Seattle, WA). This analysis identified 19-60 peptides for each of the most abundant proteins covering 43-50% of their amino acid sequence. All other proteins identified yielded five or fewer peptides.

Enzyme Assays—{beta}-galactosidase activities were determined in either permeabilized yeast cells or whole cell extracts as described by Guarente (42). Invertase activity was measured in whole cells essentially as described by Celenza and Carlson (43). ADH enzyme activity was analyzed in yeast extracts using an in-gel ADH activity assay. Proteins were separated on a nondenaturing polyacrylamide gel and visualized by in-gel chromogenic staining for ADH activity as described by Williamson et al. (44). The protein concentration of cell extracts was determined using the Bio-Rad protein assay reagent (Bio-Rad).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Bmh Protein Is Required to Maintain the Glucose-repressed State of ADH2 Expression—To determine whether Bmh proteins play a role in glucose repression, ADH2 expression was examined in cells lacking functional Bmh1 and Bmh2. ADH2 encodes the alcohol dehydrogenase isozyme responsible for ethanol assimilation in the absence of a fermentable carbon source. Its expression is tightly repressed in the presence of glucose, and Reg1 is required to maintain this repression (22). Deletion of the REG1 gene causes a low level of constitutive ADH2 expression during growth on glucose. Because deletion of both BMH1 and BMH2 is lethal in most laboratory strain genetic backgrounds (35), a congenic set of deletion strains having the {sum}1278b genetic background was utilized for these experiments. For an unknown reason, BMH1 and BMH2 are not essential for viability in this genetic background (27). Similar to what was previously seen for {Delta}reg1 cells (22), deletion of both BMH1 and BMH2 allowed constitutive ADH2 expression in glucose-repressed cells (Fig. 1). However, expression was substantially higher in derepressed cells, indicating that repression had not been completely abolished. Deletion of either BMH1 or BMH2 alone had no effect on the normal regulation of ADH2 expression by glucose, indicating that Bmh1 and Bmh2 are functionally redundant in their ability to repress ADH2 expression.2 This is consistent with the strong two-hybrid interaction between each Bmh protein and Reg1 observed by us and others (24).2 Restoration of Bmh function by transformation of {Delta}bmh1{Delta}bmh2 cells with a BMH2 expression plasmid completely suppressed constitutive ADH2 expression confirming that the lack of functional Bmh protein in these cells was responsible for the defect in glucose repression.2



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  		FIG. 1.
Constitutive ADH2 expression in {Delta}bmh1{Delta}bmh2 cells. ADH2 expression in {Delta}bmh1{Delta}bmh2 cells was measured as ADHII enzyme activity with an in-gel ADH activity assay of 50 µg of native protein extract prepared from each of the following yeast strains: 10560-4d (WT), RRY1216 ({Delta}bmh1{Delta}bmh2), KDY116 ({Delta}adr1), KDY118 ({Delta}bmh1{Delta}bmh2{Delta}adr1), KDY119 ({Delta}bcy1), and KDY120 ({Delta}bmh1- {Delta}bmh2{Delta}bcy1). All of these strains have the genetic background of {sum}1278b and are congenic with 10560-4d. Cells were grown in YEPD broth to late exponential phase. After checking that the level of glucose in the medium was greater than 1% (w/v), one-half of each culture was harvested as repressed (R) cells. The other half was washed once with cold YEP broth and then was suspended in YEPE broth. After incubating at 30 °C for 24 h, these cultures were harvested as derepressed (DR) cells.

 
Constitutive ADH2 Expression in {Delta}bmh1{Delta}bmh2 Cells Has the Same Genetic Dependences as in {Delta}reg1 Cells—If Reg1 and Bmh proteins form a multisubunit complex having a role in glucose repression and each subunit contributes to the activity of that complex, then constitutive ADH2 expression in {Delta}bmh1{Delta}bmh2 cells should exhibit the same genetic dependences as constitutive ADH2 expression in {Delta}reg1 cells. Constitutive ADH2 expression in {Delta}reg1 cells is dependent on SNF1 and ADR1, encoding the major transcriptional activator of the ADH2 gene. Adr1 binds to UAS1 in the ADH2 promoter to activate expression in a glucose-regulated, SNF1-dependent manner (45). Its binding is inhibited by Reg1 during growth on glucose. Constitutive ADH2 expression in {Delta}reg1 cells also requires a functional BCY1 gene, which encodes the regulatory subunit of cAPK in S. cerevisiae. It is suppressed by the cAPK activity that is released from cAMP control when the BCY1 gene is deleted (13). We were unable to test whether SNF1 was required for constitutive expression in {Delta}bmh1{Delta}bmh2 cells, because attempts to construct a {Delta}bmh1{Delta}bmh2{Delta}snf1 strain were unsuccessful. Neither directly deleting SNF1 in {Delta}bmh1{Delta}bmh2 cells nor first deleting SNF1 in the wild type {sum}1278b strain and then crossing it into the {Delta}bmh1{Delta}bmh2 strain yielded viable cells of the desired genotype. It is possible that the combination of the {Delta}snf1 and {Delta}bmh1{Delta}bmh2 alleles is synthetically lethal. However, this possibility was not tested. Constitutive ADH2 expression in {Delta}bmh1{Delta}bmh2 cells did exhibit the same genetic dependences for ADR1 and BCY1 as constitutive ADH2 expression in {Delta}reg1 cells. Deletion of either ADR1 or BCY1 eliminated constitutive ADH2 expression in repressed {Delta}bmh1{Delta}bmh2 cells (Fig. 1). This supports the hypothesis that Reg1 and Bmh proteins form a complex that plays a role in glucose repression.

Deletion of BCY1 or ADR1 also severely impaired derepression of ADH2 expression in wild type {sum}1278b cells (Fig. 1). Therefore, ADH2 expression in the {sum} strain genetic background used in this study has the same genetic dependences as it does in the S288C strain genetic background used in previous studies (28). Deletion of BCY1 also blocked ADH2 expression in derepressed {Delta}bmh1{Delta}bmh2 cells, as was shown previously for {Delta}reg1 cells (13). However, the derepressed level of ADH2 expression was only modestly lower in {Delta}adr1{Delta}bmh1{Delta}bmh2 cells than in wild type cells (Fig. 1). A much larger decrease in expression was observed previously for {Delta}adr1{Delta}reg1 cells (22). This indicates a partial bypass of the ADR1 requirement for ADH2 derepression and suggests that Bmh proteins can act through an ADR1-independent process to inhibit derepression of ADH2 expression. The ADR1-independent process, however, must be sensitive to unregulated cAPK activity because derepression was eliminated in {Delta}bcy1{Delta}bmh1{Delta}bmh2 cells.

The Level of Adr1 Protein Is Not Significantly Elevated in {Delta}bmh1{Delta}bmh2 Cells—The ADR1 dependence of constitutive ADH2 expression in repressed {Delta}bmh1bmh2 cells and the ability of artificially high levels of ADR1 expression to relieve glucose repression (46) suggested that this constitutive ADH2 expression might be caused by abnormally high levels of Adr1 protein. Western blot analysis showed that the level of Adr1 protein in repressed {Delta}bmh1bmh2 cells was only slightly higher than in wild type cells and less than 5% of the level in {Delta}reg1 cells (Fig. 2). This level of Adr1 protein is not normally sufficient to allow ADH2 to escape repression, because ADH2 expression in repressed cells having a derepressed level of Adr1 protein is still completely repressed (47). Therefore, relief of glucose repression is not caused by abnormally high levels of Adr1 protein.



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  		FIG. 2.
Effect of {Delta}bmh1{Delta}bmh2 on the level of Adr1 protein. The amount of Adr1 protein in {Delta}bmh1bmh2 cells was measured by quantitative Western blot analysis of denatured protein extract prepared from repressed 10560-4d (WT), RRY1216 ({Delta}bmh1{Delta}bmh2), KDY126 ({Delta}reg1), and KDY116 ({Delta}adr1) cells. The proteins in 33.3 and 100 µg of each extract were separated on a NuPAGE 3-8% acrylamide Tris acetate denaturing, reducing gel (Invitrogen) and then electrophoretically transferred to a nitrocellulose membrane. The membrane was probed with anti-Adr1-pTY67 rabbit polyclonal antibody (28) using the conditions recommended by Licor (Lincoln, NE) for detection with an Odyssey fluorescence scanner. After scanning the probed blot, Adr1 bands were quantified using Odyssey Image Analysis Software version 1.2 (Licor). Because no Adr1 was detectable in the wild type extract, all values were normalized to the amount of Adr1 in the {Delta}bmh1{Delta}bmh2 extract. The number below each pair of lanes is the average value determined for both amounts of extract loaded.

 
Bmh Proteins Also Play a Reg1-independent Role in Repression of ADH2 Expression by Glucose—To determine whether the Bmh proteins act exclusively with Reg1, the REG1 gene was deleted in the wild type {sum} strain and then crossed into {Delta}bmh1{Delta}bmh2 cells. ADH2 expression was examined in {Delta}bmh1{Delta}bmh2{Delta}reg1 random segregants obtained from the cross. If Bmh protein acts exclusively in a complex with Reg1, then the level of constitutive ADH2 expression in {Delta}bmh1{Delta}bmh2 cells should be unaffected by the {Delta}reg1 deletion. To the contrary, however, ADH2 expression was synergistically activated in all of the random segregants examined. ADHII activity and {beta}-galactosidase activity expressed from an ADH2-lacZ reporter in repressed {Delta}bmh1{Delta}bmh2{Delta}reg1 cells were greater than the sum of the individual activities in {Delta}reg1 and {Delta}bmh1{Delta}bmh2 cells (Fig. 3). ADH2 expression in {Delta}bmh1{Delta}bmh2{Delta}reg1 cells was 65-fold higher than in wild type cells, whereas expression in {Delta}reg1 cells and {Delta}bmh1{Delta}bmh2 cells was only about 10-fold higher than in wild type cells. This suggests that the Bmh proteins act independently of Reg1. The level of activity in the {Delta}bmh1{Delta}bmh2{Delta}reg1 transformants was only 38% of the fully derepressed level in wild type cells, indicating that factors in addition to Bmh1, Bmh2, and Reg1 also may contribute to the repression of ADH2 expression.



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  		FIG. 3.
ADH2 expression in {Delta}bmh1{Delta}bmh2{Delta}reg1 cells. ADH2 expression in yeast strains 10560-4d (WT), RRY1216 ({Delta}bmh1{Delta}bmh2), KDY126 ({Delta}reg1), and KDY151 ({Delta}bmh1{Delta}bmh2{Delta}reg1) was first assessed by an in-gel ADH activity assay as described in the legend to Fig. 1. The protein band migrating between the ADHI and ADHII tetramers is the heterotetramer of ADHI and ADHII homodimers. This activity gel shows one of five {Delta}bmh1{Delta}bmh2{Delta}reg1 random segregants tested. Each of the other four segregants also had a similar level of ADHII activity.2 A quantitative assessment of ADH2 expression was obtained by measuring {beta}-galactosidase activity in native cell extracts prepared from these strains after transformation with the ADH2-lacZ reporter plasmid pBGM18. Each value is the average for three independent transformants and has an S.D. value of less than 30%. n.d., not done.

 
Amino Acids 86-247, Containing Highly Conserved Regions of Reg1, Are Sufficient for the Interaction with Bmh Proteins—The Bmh interaction site on Reg1 was mapped by a two-hybrid strategy using LexA-Reg1 fusion proteins with ~200-amino acid overlapping deletions covering the entire length of the REG1 open reading frame. Of the deletions tested, only the two covering the N terminus of the protein, Reg1({Delta}2-198) and Reg1({Delta}166-369), did not produce a two-hybrid signal (Fig. 4). This suggests that amino acids in the region missing in both deletions might be required for the interaction with Bmh proteins. A LexA fusion with amino acids 86-247 of Reg1, which includes the region missing from both overlapping deletions, produced a two-hybrid signal as strong as that with the full-length Reg1 protein. This shows that amino acids 86-247 of Reg1 are sufficient to mediate the interaction with Bmh protein. This portion of Reg1 lies within the N-terminal 313 amino acids previously reported to be sufficient for the Bmh interaction (24).



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  		FIG. 4.
Two-hybrid mapping of Reg1 sequences required for the Bmh interaction. Yeast strain KDY107 was transformed with either pKD134 (+Bmh1) or pGAD-C1 (-Bmh1) and a lexA-REG1 two-hybrid expression plasmid containing the sequence of the REG1 open reading frame to be tested for interaction with Bmh1. The three LexA-Reg1 expression plasmids having deletions covering the C-terminal portion of REG1, pKD131 (Reg1({Delta}521-724)), pKD129 (Reg1({Delta}694-904)), and pKD132 (Reg1({Delta}869-1014)), exhibited two-hybrid interactions with GAD-Bmh1 similar to that of the full-length LexA-Reg1 fusion.2 At least three independent transformants for each combination of plasmids were tested and three independent clones of plasmids pKD142 (Reg1(86-247)) and pKD143 (Reg1({Delta}187-232)) were tested. Transformants were grown in SD broth lacking leucine and uracil until late exponential phase. {beta}-Galactosidase activity was measured in permeabilized whole cells and is reported in Miller units. Each value is listed with its associated S.D. value.

 
If this region of Reg1 were important for function, then it should be evolutionarily conserved. To delineate conserved sequences, the translated REG1 open reading frame of S. cerevisiae was compared with those from representatives of three other classes of Ascomycota and one representative from a class of Basidiomycota (Fig. 5). This analysis identified three blocks of sequence that were highly conserved in all of these fungal species. Blocks 1 and 2 are located in the region of Reg1 that is sufficient for the interaction with Bmh proteins. Block 3 contains the Glc7-binding motif (12, 13). The order of these blocks in the linear amino acid sequence is conserved, although their spacing is not. This signature of sequence blocks suggests that these proteins belong to the same functional class. Identities with the S. cerevisiae sequence blocks ranged from 37 to 70%. Identities outside of the three blocks of sequence homology were very low, less than 10% for the 50 amino acids immediately following Block 2. This confirms the conserved nature of the three identified sequence blocks and lends support to the idea that they are of functional importance for the Reg1 protein.



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  		FIG. 5.
Alignment of blocks of amino acid sequence homology in Reg1 homologs. The translated REG1 open reading frame of S. cerevisiae, an Ascomycete of the class Saccharomycotina, was compared with the translated open reading frames from representatives of three other classes of Ascomycota, A. nidulans of the class Eurotiomycetes, N. crassa of the class Sordariomycetes, and S. pombe of the class Taphrinomycotina, and one class of Basidiomycota, U. maydis of the class Ustilaginomyctese (67). A graphic representation of the amino acid alignment of Reg1 homologs created by the MACAW program (A) shows the three most highly conserved blocks of amino acid sequence. A textual alignment of each block (B) is shown below the graphic representation. The numbers marking the beginning and end of each block indicate the location in the S. cerevisiae sequence. A majority rule consensus with X denoting the divergent residues is shown below each alignment. Identity is shown as follows: lowercase letter, conserved; uppercase letter, not conserved.

 
The Reg1-Bmh Interaction Plays a Minor Role in the Repression of ADH2 Expression—Since the multiple alignments of Block 1 and Block 2 sequences showed that Block 2 was more highly conserved (Fig. 5B), the requirement of Block 2 for the interaction with Bmh1 was tested. LexA-Reg1({Delta}187-232), which is missing all but the first four amino acids of Block 2, was unable to interact with GAD-Bmh1 in a two-hybrid assay, illustrating that this sequence is required for the Bmh interaction (Fig. 4). It was expected that a Reg1 protein with this deletion would have only a minor effect on ADH2 expression, because deletions {Delta}2-198 and {Delta}166-369 had only minor effects (13). This is what was observed (Fig. 6A). Cells with an integrated ADH2-lacZ reporter and expressing the Reg1({Delta}187-232) protein showed a significant but slight 40% increase in expression of the reporter over wild type cells. Cells expressing no Reg1 protein showed a much larger 20-fold increase in activity. The amount of Reg1({Delta}187-232) protein was equal to that of the wild type protein (Fig. 6B). Therefore, differences in the abundance of the Reg1({Delta}187-232) protein and the wild type protein cannot account for these observations. Although the presence of a secondary Bmh binding site in the Reg1({Delta}187-232) protein cannot be ruled out, this seems unlikely, since LexA-Reg1({Delta}187-232) failed to show an interaction with GADBmh1 (Fig. 4). These results support the idea of the Reg1-Bmh interaction playing a minor role in mediating ADH2 repression and indicate a greater role for Bmh protein in Reg1-independent processes.



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  		FIG. 6.
Effect of the {Delta}187-232 deletion on glucose repression. A, effect of the {Delta}187-232 deletion on ADH2 and SUC2 expression. ADH2 expression was measured as {beta}-galactosidase activity expressed from an integrated ADH2-lacZ reporter and SUC2 expression was measured as invertase activity. Yeast strain KDY82 was transformed with plasmids pKD89 (REG1), three independent clones of pKD143 ({Delta}187-232), and pRS316 ({Delta}reg1) and grown in SD broth lacking leucine and uracil until late exponential phase. These data are the means for at least three independent transformants and have S.D. values of 10% or less. B, Western analysis of Reg1 protein levels. A Western blot of denatured proteins from each transformant was prepared, probed with HA probe mouse monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and quantitatively analyzed as described in the legend to Fig. 2. n.a., not analyzed.

 
Bmh Proteins Have Broad Reg1-dependent and Reg1-independent Roles in Glucose Repression—It has been reported that {Delta}bmh1{Delta}bmh2 cells hyperaccumulate glycogen and that this hyperaccumulation is suppressible by abnormally high levels of cAPK activity (27). This phenotype has also been observed for {Delta}reg1 cells (48), suggesting that Bmh protein may play a broad role with Reg1 in maintaining glucose repression. In an attempt to extend this observation, the expression of SUC2, another gene whose repression by glucose is completely dependent on REG1 (49), was examined in {Delta}bmh1{Delta}bmh2 cells. Invertase activity as a measure of SUC2 expression was undetectable in {Delta}bmh1bmh2 cells growing on glucose.2 This confirmed a similar observation made by Mayordomo et al. (24). However, we were also unable to detect invertase activity in either the mutant or wild type {sum} strains growing in derepressing medium. Therefore, no conclusion can be drawn from this experiment regarding a broad role for Bmh protein in glucose repression. The reg1({Delta}187-232) deletion allele, however, provided a tool for examining this issue in other genetic backgrounds. This allele was expressed in an S288C strain, a popular strain for investigations of SUC2 gene regulation, and SUC2 expression was measured as secreted invertase activity. Invertase activity was elevated 5-fold in repressed cells expressing the Reg1({Delta}187-232) protein (Fig. 6A). This suggests that binding of Bmh protein to Reg1 is required for full repression of SUC2 expression and supports a more general role for Bmh protein in glucose repression. Repression of SUC2 expression, however, was not completely dependent on the Block 2 sequence. The level of SUC2 expression in cells expressing the Reg1({Delta}187-232) protein was only 25% of that observed in {Delta}reg1 cells. This suggests that the Reg1({Delta}187-232) protein is partially functional in glucose repression.

Purification of TAP-tagged Reg1 Confirms the in Vivo Association with Bmh Proteins and Identifies Ssb Proteins as Other Members of the Complex—To confirm the existence of a Reg1-Bmh complex in vivo and to possibly identify other components of the complex, HA3-TAP-tagged Reg1 protein was purified from repressed cells, and the proteins associated with it were identified. Western blot analysis of TAP-purified proteins showed that Bmh1 and Bmh2 copurify with Reg1 (Fig. 7A). Four major constituents of the TAP-purified protein preparation were identified on a silver-stained gel (Fig. 7B). Based on their mobility in a denaturing polyacrylamide gel, the slowest migrating protein band was identified as Reg1-HA3-TAP, and the two fastest migrating bands were identified as Bmh1 and Bmh2. A band of unknown protein migrated slightly slower than the 64-kDa standard. To identify this protein and to confirm the identities of the others, HPLC-coupled mass spectrometry of trypsin-digested TAP-purified proteins was performed. This analysis identified the unknown band as a mixture of Ssb1 and Ssb2 proteins.



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  		FIG. 7.
Identification of proteins that copurified with HA3-TAP-tagged Reg1. A, Western blot analysis of Bmh1 and Bmh2 proteins. Cell extracts prepared from repressed (R) and derepressed (DR) KDY80 (-) and KDY121 (+TAP) were subjected to the TAP purification protocol as described under "Experimental Procedures." 2% of the total TAP purified protein was run on a Novex bis-tris 4-12% acrylamide gel (Invitrogen), and the proteins were transferred from the gel to polyvinylidene difluoride membrane. The membrane was first probed with polyclonal rabbit anti-Bmh2 antibodies ({alpha}-Bmh2), which strongly cross-react with the Bmh1 protein (35). The blot was then stripped and reprobed with monoclonal mouse anti-HA antibodies ({alpha}-HA) (Roche Applied Science) to visualize the HA3-TAP-tagged Reg1 protein. B, silver-stained gel. 20% of the total TAP-purified protein was trichloroacetic acid-precipitated and run on an acrylamide gel as described for the Western blot in A. The gel was then silver-stained using the nonkit-based method of the Hahn laboratory (available on the World Wide Web at www.fhcrc.org/labs/hahn/methods/biochem_meth/silver_stain.html). The identity of each protein band was assigned based on its mobility in the gel relative to that of the molecular weight markers, whose masses in kDa are shown on the left of the gel, and the molecular weight of proteins in the TAP-purified preparation identified by mass spectrometry.

 
To determine whether the composition of the Reg1 complex was glucose-regulated, HA3-TAP-tagged Reg1 protein was also purified from derepressed cells. All of the major proteins found associated with Reg1 from repressed cells were also found associated with Reg1 from derepressed cells (Fig. 7B). Thus, the composition of the Reg1 protein complex does not appear to be glucose-regulated. The same conclusion for the Bmh-Reg1 interaction was also reached by Mayordomo et al. (24).

Deletion of SSB1 and SSB2 Modestly Activates ADH2 Expression during Growth on Glucose—Ssb proteins are nonessential, highly abundant, nonconventional heat shock proteins found in all fungi (29). They are encoded by the SSB1 and SSB2 genes and have nearly identical amino acid sequences. One of their functions appears to be in protein synthesis as ribosome-associated molecular chaperones that facilitate proper folding of nascent polypeptide chains exiting translating ribosomes (50). To determine whether these proteins also play a role in glucose repression, ADH2 expression was examined in yeast strains of two different genetic backgrounds having both of the SSB genes deleted. Strains of different genetic backgrounds were tested, because after the introduction of the SSB1 and SSB2 deletions into BY4741, we discovered that growth of these cells was not inhibited by the aminoglycoside paromomycin,2 a phenotype previously reported for cells of the DS10 genetic background having these deletions (34). In both genetic backgrounds, expression of an ADH2-lacZ reporter gene was activated to a low but significant level (Fig. 8). This level of activation was ~20% of the level observed in BY4741 cells having REG1 deleted.2 Thus, it appears that the Ssb proteins play a small role in glucose repression of ADH2 expression.



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  		FIG. 8.
ADH2 expression in {Delta}ssb1ssb2 cells growing on glucose. This bar graph shows the level of ADH2 expression in wild type (WT) and {Delta}ssb1{Delta}ssb2 cells as measured by {beta}-galactosidase activity expressed from an ADH2-lacZ reporter gene. Yeast strains of the DS10 genetic background, JH27A (WT) and NL164 ({Delta}ssb1{Delta}ssb2), and of the BY4741 genetic background, BY4741 (WT) and KDY157 ({Delta}ssb1{Delta}ssb2), were transformed with the ADH2-lacZ reporter plasmid pBGM18. Transformants were grown in SD medium lacking uracil and harvested while greater than 2% (w/v) glucose was still present in the medium. {beta}-Galactosidase activity was measured in permeabilized whole cells and is reported in Miller units. Each bar is the average for three independent transformants and has an S.D. value indicated by its associated error bar.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this paper, we show that the yeast homologs of mammalian 14-3-3 proteins, Bmh1 and Bmh2, are required for the repression of ADH2 expression by glucose. We also provide evidence suggesting that this requirement is not limited to ADH2 expression. Repression of SUC2 expression was partially relieved when the sequence required for their binding to Reg1 was deleted. In addition to constitutive ADH2 expression, {Delta}bmh1{Delta}bmh2 and {Delta}reg1 cells also share a number of other phenotypes: glycogen hyperaccumulation (27, 48), slow growth (13, 27), constitutive haploid invasive growth (27, 48),2 and loss of catabolite inactivation of the maltose permease (24). Common phenotypes would be expected if Bmh and Reg1 proteins act together in a complex. We also provide evidence supporting a small role for Ssb proteins in glucose repression. Deletion of the SSB1 and SSB2 genes modestly relieved repression of ADH2 expression. Deleting the Bmh binding site in Reg1 did not relieve repression to the same extent as deleting the REG1 gene or deleting the Glc7-binding motif in Reg1 (12, 13). These observations suggest that the interactions with the Ssb and Bmh proteins are not as essential for the function of Reg1 as is binding to Glc7.

In addition to their role as a component of the Reg1-Glc7 phosphatase complex, our data also suggest that Bmh proteins function independently of Reg1 in maintaining glucose repression. This is not surprising, considering that there are 80-fold more molecules of Bmh protein per cell than Reg1 (51). One candidate Reg1-independent role for Bmh proteins is in cAMP signal transduction. High levels of unregulated cAPK activity block ADH2 expression (28), and Bmh proteins appear to be needed for cAMP signaling mediated by the cAPK catalytic subunits (35). Low cAPK activity in {Delta}bmh1{Delta}bmh2 cells in combination with the partial loss of repression by Reg1-Glc7 could be responsible for the constitutive ADH2 expression observed in glucose-repressed cells. This is consistent with the suppression of constitutive ADH2 expression observed previously in {Delta}reg1 cells (13) and currently in {Delta}bmh1{Delta}bmh2 cells when the BCY1 gene was deleted. Repression of ADH2 expression is less dependent on Reg1 and its Bmh binding site than is repression of SUC2 expression. This may reflect the additional control of ADH2 expression by cAPK that is absent from the regulation of SUC2 expression (52).

Two-hybrid binding analyses and alignment of Reg1 homologs showed that amino acids 187-232 are required for the interaction with Bmh1 and suggested that the presence of other Bmh binding sites in Reg1 is unlikely. The sequence requirements of many 14-3-3 binding sites have been studied in detail, and phosphoserine motifs that mediate binding have been described (25, 53). Ligand selection from a degenerate phosphoserine peptide library identified the motif RK(S/F/H/A/Y)(H/R/K)pS(L/A/Y/M/W/F)P (where pS represents phosphoserine) as an optimal Bmh1 and Bmh2 binding site (53). However, this motif is not present in the yeast proteome.2 Little is known about the amino acid sequences recognized by Bmh proteins in yeast cells (54). No phosphorylated or nonphosphorylated motifs recognized by mammalian 14-3-3 proteins are present in this region of the Reg1 protein (25, 55). Therefore, the precise mode of interaction between Reg1 and Bmh proteins remains to be determined. It is interesting that amino acids 187-232 are within the region of Reg1 reported to undergo glucose-regulated phosphorylation, because many ligands have a much higher affinity for 14-3-3 proteins when they are phosphorylated than when they are dephosphorylated (53, 56).

Our results demonstrate that Reg1 is associated with Bmh and Ssb proteins in vivo. Reg1-HA3-TAP protein expressed at a physiologically relevant level behaved identically to the untagged protein. Unexpectedly, Glc7 was not identified as a copurifying protein, consistent with the results of a previous study in which Reg1 did not copurify with Protein A-tagged Glc7 (10). Analyses of affinity-purified FLAG-tagged proteins did identify Reg1, Bmh1, Bmh2, and Ssb1 as proteins that copurify with FLAG-tagged Glc7 (57). However, except for Reg1, these identifications did not meet background-filtering criteria established in the study and suffer from the complication that Glc7 was expressed from a strong promoter. Perhaps the inability to purify Glc7 with Reg1 reflects competition with other regulatory subunits for access to the hydrophobic pocket on Glc7 that binds to their RVXF motif (58, 59). Alternatively, the interaction of Reg1 with Glc7 may require a regulatory signal that is eliminated during the purification of the complex.

Snf1 was also not identified in the mixture of proteins associated with TAP-purified Reg1. It was expected that Snf1 would be found in the associated proteins from derepressed cells because Reg1 interacts with the kinase domain of Snf1 in a two-hybrid assay in derepressed cells (18). However, it was absent in the mixture of proteins purified from either repressed or derepressed cells. It has been reported that Reg1 can be copurified with TAP-tagged Snf1 from exponentially growing repressed cells (60). The reason for these different observations is unclear. One possible explanation is that the HA3-TAP tag on the C terminus of Reg1 blocks or weakens the binding to Snf1. However, this seems unlikely because the tag does not interfere with the function of Reg1.

The Bmh and Ssb interactions suggest several mechanisms by which the activity of the Reg1 complex might be regulated. One possible function of Bmh protein might be as an adaptor molecule that helps to target the Reg1-Glc7 phosphatase to relevant phosphorylated substrates. Bmh proteins might also play a role as energy status sensors that regulate this targeting. Interaction of AMP with the Arabidopsis 14-3-3 protein GF14{omega} partially inhibits binding to nitrate reductase (61), and Bmh1 and Bmh2 are highly homologous to GF14{omega} (62). This suggests the possibility that the accumulation of AMP that occurs when cells are starved for glucose dissociates Bmh protein from Reg1, making it less able to target the Glc7 phosphatase complex to specific phospholigand substrates (63). Although our Reg1-HA3-TAP purification data suggest that the Reg1-Bmh interaction is not glucose-regulated, this is still a viable model, because small molecule regulation of the complex composition was probably compromised by the dialysis step of the purification protocol. Likewise, the Ssb proteins could also serve as metabolic sensors to modulate the activity of the Reg1-Glc7 complex. As with other members of the Hsp70 family of proteins, the Ssb proteins have an N-terminal ATPase domain that can bind and hydrolyze ATP. It has been reported that ATP can cause dissociation of Ssb proteins from their binding partners and can also alter intracomplex interactions as reported for the interaction of nascent chains exiting from translating ribosomes with Ssb proteins (29, 64). The high ATP/AMP ratio present in cells growing exponentially on glucose might alter the interaction with Reg1 and, thus, influence its activity (63).

Another possible function of the Bmh and Ssb proteins could be in maintaining the cytosolic localization of Reg1. The amino acid sequence of Reg1 contains several regions that resemble nuclear targeting signals, and a protein composed of the first 316 amino acids of Reg1 fused to LacZ can be detected in a nuclear fraction at significant levels (19). Therefore, Reg1 has sequences that can target it to the nucleus. We have shown previously that a fully functional Reg1-green fluorescent protein fusion protein is constitutively localized to the cytoplasm (13). This raises the question of how Reg1 is sequestered in the cytoplasm and prevented from accumulating in the nucleus. Bmh proteins appear to act as cytoplasmic anchors for the stress-regulated transcription factors Msn2 and Msn4 (65). Perhaps they are doing the same for Reg1. The Ssb proteins have been shown to contain a functional nuclear export sequence (66). This suggests the possibility that the Ssb proteins could prevent the accumulation of Reg1 protein in the nucleus by promoting the rapid export of any protein that might have escaped the cytoplasmic anchoring of the Bmh proteins.

Our results implicate the Bmh proteins in glucose repression and suggest that they partially exert their influence over this pathway through an interaction with Reg1. A minor role for the Ssb proteins is also suggested by their copurification with Reg1 and their modest contribution to the repression of ADH2 expression. Glc7 and Bmh proteins are likely to form a ternary complex with Reg1 because they have nonoverlapping binding sites. Whether this is also true for the Ssb proteins is unknown, because their binding sites on Reg1 have yet to be identified. The molecular basis of these interactions, the possible mechanisms for the regulation of Reg1-Glc7 function suggested by them, and the Reg1-independent role of Bmh in glucose repression remain to be elucidated.


   FOOTNOTES
 
* This work was supported by NIGMS Research Grant GM26079 from the National Institutes of Health (to E. T. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195-7350. Tel.: 206-543-6035; Fax: 206-685-1792; E-mail: kmd{at}u.washington.edu.

1 The abbreviations used are: PP1c, type 1 protein phosphatase catalytic subunit; HA3-TAP, a C-terminal epitope tag having three repeats of the influenza hemagglutinin epitope followed by one TAP tag consisting of a calmodulin binding peptide and Protein A; GAD, Gal4 activation domain; cAPK, cAMP-dependent protein kinase; ADHI, alcohol dehydrogenase isozyme I; ADHII, glucose-repressible alcohol dehydrogenase isozyme II; PP1c, type 1 protein phosphatase catalytic subunit; HPLC, high pressure liquid chromatography; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Back

2 K. M. Dombek, data not shown. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Gerald Fink for yeast strains derived from {sum}1278b, Dr. Elizabeth Craig for yeast strains of the DS10 genetic background, Dan Gelperin for providing Bmh2 polyclonal antiserum and BMH2 expression plasmids, and Dr. David Shore for providing the TAP-tagging plasmid pYM12-HA3-TAP. Thanks also go to Thahn-Nga Nguyen who worked on this project during undergraduate studies. Finally, we acknowledge the support of other members of the Young laboratory for materials, logistics, and useful discussions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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