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• W2037179957 abstract "Induction of the Saccharomyces MAL structural genes encoding maltose permease and maltase requires the MAL activator, a DNA-binding transcription activator. Genetic analysis of MAL activator mutations suggested that protein folding and stability play an important role in MAL activator regulation and led us to explore the role of the Hsp90 molecular chaperone complex in the regulation of the MAL activator. Strains carrying mutations in genes encoding components of the Hsp90 chaperone complex, hsc82Δ hsp82-T101I and hsc82Δ cpr7Δ, are defective for maltase induction and exhibit significantly reduced growth rates on media containing a limiting concentration of maltose (0.05%). This growth defect is suppressed by providing maltose in excess. Using epitope-tagged alleles of the MAL63 MAL activator, we showed that Mal63p levels are drastically reduced following depletion of cellular Hsp90. Overexpression (∼3-fold) of Mal63p in the hsc82Δ hsp82-T101I and hsc82Δ cpr7Δ strains suppresses their Mal– growth phenotype, suggesting that Mal63p levels are limiting for maltose utilization in strains with abrogated Hsp90 activity. Consistent with this, the half-life of Mal63p is significantly shorter in the hsc82Δ cpr7Δ strain (reduced about 6-fold) and modestly affected in the Hsp90-ts strain (reduced about 2-fold). Most importantly, triple hemagglutinin-tagged Mal63p protein is found in association with Hsp90 as demonstrated by co-immunoprecipitation. Taken together, these results identify the inducible MAL activator as a client protein of the Hsp90 molecular chaperone complex and point to a critical role for chaperone function in alternate carbon source utilization in Saccharomyces cerevisiae. Induction of the Saccharomyces MAL structural genes encoding maltose permease and maltase requires the MAL activator, a DNA-binding transcription activator. Genetic analysis of MAL activator mutations suggested that protein folding and stability play an important role in MAL activator regulation and led us to explore the role of the Hsp90 molecular chaperone complex in the regulation of the MAL activator. Strains carrying mutations in genes encoding components of the Hsp90 chaperone complex, hsc82Δ hsp82-T101I and hsc82Δ cpr7Δ, are defective for maltase induction and exhibit significantly reduced growth rates on media containing a limiting concentration of maltose (0.05%). This growth defect is suppressed by providing maltose in excess. Using epitope-tagged alleles of the MAL63 MAL activator, we showed that Mal63p levels are drastically reduced following depletion of cellular Hsp90. Overexpression (∼3-fold) of Mal63p in the hsc82Δ hsp82-T101I and hsc82Δ cpr7Δ strains suppresses their Mal– growth phenotype, suggesting that Mal63p levels are limiting for maltose utilization in strains with abrogated Hsp90 activity. Consistent with this, the half-life of Mal63p is significantly shorter in the hsc82Δ cpr7Δ strain (reduced about 6-fold) and modestly affected in the Hsp90-ts strain (reduced about 2-fold). Most importantly, triple hemagglutinin-tagged Mal63p protein is found in association with Hsp90 as demonstrated by co-immunoprecipitation. Taken together, these results identify the inducible MAL activator as a client protein of the Hsp90 molecular chaperone complex and point to a critical role for chaperone function in alternate carbon source utilization in Saccharomyces cerevisiae. In Saccharomyces cerevisiae, maltose fermentation requires maltose permease, a proton symporter that transports maltose across the plasma membrane; maltase, an α-glucosidase that hydrolyzes maltose to produce glucose; and the MAL activator, a DNA-binding transcription activator (reviewed in Refs. 1Needleman R.B. Mol. Microbiol. 1991; 5: 2079-2084Crossref PubMed Scopus (87) Google Scholar and 2Hu Z. Gibson A.W. Kim J.H. Wojciechowicz L.A. Zhang B. Michels C.A. Curr. Genet. 1999; 36: 1-12Crossref PubMed Scopus (31) Google Scholar). Maltose-induced expression of maltase and maltose permease requires the MAL activator and maltose permease, and strains lacking either gene are noninducible (3Needleman R.B. Kaback D.B. Dubin R.A. Perkins E.L. Rosenberg N.G. Sutherland K.A. Forrest D.B. Michels C.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2811-2815Crossref PubMed Scopus (81) Google Scholar). Wang et al. (4Wang X. Bali M. Medintz I. Michels C.A. Eukaryotic Cell. 2002; 1: 696-703Crossref PubMed Scopus (27) Google Scholar) showed that intracellular maltose is sufficient to stimulate induction, thereby demonstrating that the role of the permease in induction is simply to provide sufficient intracellular inducer to activate the maltose sensor. The maltose sensor has not been identified, but the MAL activator is a candidate. The genes encoding maltose permease, maltase, and the MAL activator are clustered in a complex MAL locus. S. cerevisiae yeast strains can carry anywhere from one to five unlinked copies of a MAL locus, named MAL1–4 and MAL6, which map to sites near the telomere of different chromosomes (5Charron M.J. Read E Haut S.R. Michels C.A. Genetics. 1989; 122: 307-316Crossref PubMed Google Scholar). The presence of any one of these five loci is sufficient for maltose fermentation. All of the five MAL loci are highly homologous both structurally and functionally. The genes encoding maltose permease and maltase share a bi-directional promoter that contains the DNA-binding site of the MAL activator, thereby providing for the coordinate expression of these maltose utilization enzymes (2Hu Z. Gibson A.W. Kim J.H. Wojciechowicz L.A. Zhang B. Michels C.A. Curr. Genet. 1999; 36: 1-12Crossref PubMed Scopus (31) Google Scholar, 6Levine J. Tanouye L. Michels C.A. Curr. Genet. 1992; 22: 181-189Crossref PubMed Scopus (50) Google Scholar). MAL63 encodes an inducible allele of the MAL activator at MAL6 (3Needleman R.B. Kaback D.B. Dubin R.A. Perkins E.L. Rosenberg N.G. Sutherland K.A. Forrest D.B. Michels C.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2811-2815Crossref PubMed Scopus (81) Google Scholar, 5Charron M.J. Read E Haut S.R. Michels C.A. Genetics. 1989; 122: 307-316Crossref PubMed Google Scholar). Mal63p is 470 residues in length and contains a six-cysteine zinc finger DNA-binding domain in the N-terminal ∼60–100 residues, a single transcription activation domain in approximately residues 60–250, and a C-terminal regulatory domain in approximately residues 250–470 (2Hu Z. Gibson A.W. Kim J.H. Wojciechowicz L.A. Zhang B. Michels C.A. Curr. Genet. 1999; 36: 1-12Crossref PubMed Scopus (31) Google Scholar, 7Kim J.H. Michels C.A. Curr. Genet. 1988; 14: 319-323Crossref PubMed Scopus (47) Google Scholar). Mal23p, the inducible MAL activator allele encoded by MAL2, is 95% identical to Mal63p (7Kim J.H. Michels C.A. Curr. Genet. 1988; 14: 319-323Crossref PubMed Scopus (47) Google Scholar, 8Gibson A.W. Wojciechowicz L.A. Danzi S.E. Zhang B. Kim J.H. Hu Z. Michels C.A. Genetics. 1997; 146: 1287-1298Crossref PubMed Google Scholar). Our genetic studies of the MAL activator suggest that protein folding and stability play important roles in MAL activator regulation (2Hu Z. Gibson A.W. Kim J.H. Wojciechowicz L.A. Zhang B. Michels C.A. Curr. Genet. 1999; 36: 1-12Crossref PubMed Scopus (31) Google Scholar, 8Gibson A.W. Wojciechowicz L.A. Danzi S.E. Zhang B. Kim J.H. Hu Z. Michels C.A. Genetics. 1997; 146: 1287-1298Crossref PubMed Google Scholar, 9Danzi S.E. Zhang B. Michels C.A. Curr. Genet. 2000; 38: 233-240Crossref PubMed Scopus (14) Google Scholar, 10Danzi S.E. Bali M. Michels C.A. Curr. Genet. 2003; (in press)PubMed Google Scholar). Characterization of constitutive alleles of the MAL activator localized the maltose-responsive regulatory domain to the C-terminal ∼200 residues and demonstrated that this region is a negative regulator of MAL activator function. mal64 is a nonfunctional homologue of MAL63 but can be activated to a constitutive MAL activator by mutation (11Dubin R.A. Charron M.J. Haut S.R. Needleman R.B. Michels C.A. Mol. Cell. Biol. 1988; 8: 1027-1035Crossref PubMed Scopus (24) Google Scholar). These MAL64-C mutations are nonsense mutations at codons 282 and 307 (8Gibson A.W. Wojciechowicz L.A. Danzi S.E. Zhang B. Kim J.H. Hu Z. Michels C.A. Genetics. 1997; 146: 1287-1298Crossref PubMed Google Scholar). The sequence of the MAL activator constitutive mutants MAL23-C and MAL43-C of the MAL2 and MAL4 loci, respectively, reported in Gibson et al. (8Gibson A.W. Wojciechowicz L.A. Danzi S.E. Zhang B. Kim J.H. Hu Z. Michels C.A. Genetics. 1997; 146: 1287-1298Crossref PubMed Google Scholar) contain multiple sequence alterations located largely in the C-terminal regulatory domain. Danzi et al. (9Danzi S.E. Zhang B. Michels C.A. Curr. Genet. 2000; 38: 233-240Crossref PubMed Scopus (14) Google Scholar) used in vitro mutagenesis to localize those residues in the MAL activator that are important for negative regulation. They identified clustered alterations in three regions (residues 250–307, 343–357, and 419–461), and the introduction of multiple alterations in any one of these regions fully relieves negative regulation by this C-terminal domain. They also found that other sites throughout the protein could modulate the constitutive phenotype of mutations in these regions. But the C-terminal region also plays a positive role in MAL activator induction. Charged cluster to alanine scanning mutagenesis of this C-terminal negative regulatory domain of Mal63p produced a series of noninducible alleles that alter residues in regions adjacent to or overlapping two of the three negative regulatory regions defined by Danzi et al. (9Danzi S.E. Zhang B. Michels C.A. Curr. Genet. 2000; 38: 233-240Crossref PubMed Scopus (14) Google Scholar, 10Danzi S.E. Bali M. Michels C.A. Curr. Genet. 2003; (in press)PubMed Google Scholar). Taken together, this genetic analysis suggests that conformational changes involving complex protein-protein interactions regulate MAL activator activity. All efforts to identify interactions between different domains of Mal63p using two-hybrid analysis were unsuccessful, and Hu et al. (2Hu Z. Gibson A.W. Kim J.H. Wojciechowicz L.A. Zhang B. Michels C.A. Curr. Genet. 1999; 36: 1-12Crossref PubMed Scopus (31) Google Scholar) proposed that the interactions were likely to be intermolecular. The well documented role for the Hsp90 molecular chaperone complex in the inducer binding and regulation of other transcription activators such as the steroid hormone receptors raised the possibility that the Hsp90 chaperone complex could be a candidate for this MAL activator-interacting protein(s) and thus may be involved in the maltose stimulation of the MAL activator. The results reported here explore this possibility. Hsp90 is a highly conserved, abundantly expressed, essential protein in eukaryotes that is localized to the cytoplasm and nucleus (specific references may be found in Refs. 12Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1542) Google Scholar, 13Johnson J.L. Craig E.A. Cell. 1997; 90: 201-214Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 14Caplan A.J. Cell Biol. 1999; 9: 262-268Scopus (164) Google Scholar, 15Mayer M.P. Bukau B. Curr. Genet. 1999; 9: R322-R325Scopus (139) Google Scholar, 16Frydman J. Annu. Rev. Biochem. 2001; 70: 603-647Crossref PubMed Scopus (944) Google Scholar, 17Pearl L.H. Prodromou C. Adv. Protein Chem. 2001; 59: 157-186Crossref PubMed Scopus (178) Google Scholar, 18Richter K. Buchner J. J. Cell. Physiol. 2001; 188: 281-290Crossref PubMed Scopus (509) Google Scholar, 19Picard D. Cell. Mol. Life Sci. 2002; 59: 1640-1648Crossref PubMed Scopus (666) Google Scholar, 20Pratt W.B. Toft D.O. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1276) Google Scholar). Hsp90 is the key component of a large complex of proteins, many of them molecular chaperones, that function together assisting certain proteins to achieve an activated conformation in response to external or physiological signals. Despite its abundance, Hsp90 is unlikely to be required for de novo folding of the bulk of newly synthesized proteins. Instead it appears to play a role in the maturation of a specific set of newly synthesized proteins, so-called client or substrate proteins, and in the refolding and assembly of misfolded proteins that accumulate in cells following exposure to mild heat shock or other stresses. Therefore, Hsp90 has both stress-related and housekeeping functions and, as such, plays an essential role in processes controlling cell growth and differentiation in unstressed cells. Many of the components of the Hsp90 chaperone complex are conserved both in structure and function from S. cerevisiae to mammals. In Saccharomyces this includes the following genes: HSC82 and HSP82 encode Hsp90; the SSA and SSB family of genes encode Hsp70 isoforms (21Chang H.-C.J. Lindquist S. J. Biol. Chem. 1994; 269: 24983-24988Abstract Full Text PDF PubMed Google Scholar, 22Bohen S.P. Mol. Cell. Biol. 1998; 18: 3330-3339Crossref PubMed Scopus (97) Google Scholar); YDJ1 encodes Hsp40 (23Kimura Y. Yahara I. Lindquist S. Science. 1995; 268: 1362-1365Crossref PubMed Scopus (219) Google Scholar); STI1 encodes Hop/p60 protein (24Chang H.-C.J. Nathan D.F. Lindquist S. Mol. Cell. Biol. 1997; 17: 318-325Crossref PubMed Scopus (193) Google Scholar); SBA1 encodes p23 (22Bohen S.P. Mol. Cell. Biol. 1998; 18: 3330-3339Crossref PubMed Scopus (97) Google Scholar, 25Fang Y. Fliss A.E. Rao J. Caplan A.J. Mol. Cell. Biol. 1998; 18: 3727-3734Crossref PubMed Scopus (133) Google Scholar); CPR6 and CPR7 encode cyclophilins (26Duina A.A. Chang H.-C.J. Marsh J.A. Lindquist S. Gaber R.F. Science. 1996; 274: 1713-1715Crossref PubMed Scopus (186) Google Scholar); CDC37 encodes p50Cdc37 (27Hunter T. Poon R.Y.C. Trends Cell Biol. 1997; 7: 157-161Abstract Full Text PDF PubMed Scopus (101) Google Scholar); AHA1 and HCH1 encode hAha1 (28Panaretou B. Siligardi G. Meyer P. Maloney A. Sulivan J.K. Singh S. Millison S.H. Clarke P.A. Naaby-Hansen S. Stein R. Cramer R. Mollapour M. Workman P. Piper P.W. Pearl L.H. Prodromou C. Mol. Cell. 2002; 10: 1307-1318Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar); and SSE1 encodes a yeast Hsp110 family member (29Liu X.-D. Morano K.A. Thiele D.J. J. Biol. Chem. 1999; 274: 26654-26660Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Saccharomyces does not encode a homologue of Hip/p48. HSC82 and CPR7 are constitutively expressed, whereas their homologues, HSP82 and CPR6, respectively, are stress-induced. Thus, although the different isoforms are functionally overlapping, they are differentially expressed and probably play distinct cellular roles. Hsp90 is an ATPase, and its ATPase activity is essential for its chaperone function (reviewed in Ref. 20Pratt W.B. Toft D.O. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1276) Google Scholar). Several of the components listed above, including Hsp70, Hsp40, Hip/p48, and Hop/p60, are involved in client protein selection and the assembly of the chaperone complex (reviewed in Ref. 20Pratt W.B. Toft D.O. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1276) Google Scholar). Others such as p23, p50Cdc37, and Aha1 modulate the ATPase activity of Hsp90. The immunophilins, which include cyclophilins and FBPK proteins, have peptidyl proline isomerase activity and tetratricopeptide repeat domains and work to modulate client protein maturation and activation. Saccharomyces has been used for the study of mammalian steroid hormone receptor activation (particularly glucocorticoid and androgen receptors) and Src protein kinase maturation. Neither of these proteins is a natural substrate of the yeast Hsp90 chaperone complex, and although much important information has been obtained, it would be valuable to identify and characterize endogenous yeast substrates. To date the only Saccharomyces proteins identified as substrates of the Hsp90 chaperone complex are Ste11 protein kinase, Gcn2 kinase, and the heme-regulated transcription activator HapI (30Louvion J.F. Abbas-Terki T. Picard D. Yeast. 1998; 9: 3071-3983Google Scholar, 31Donze O. Picard D. Mol. Cell. Biol. 1999; 19: 8422-8432Crossref PubMed Scopus (57) Google Scholar, 32Lee H.C. Hon T. Zhang L. J. Biol. Chem. 2002; 277: 7430-7437Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Here we investigate the role of Hsp90 chaperone complex in MAL gene regulation. We find that in strains carrying mutations in components of the Hsp90 complex maltose-dependent MAL gene induction is defective. Depletion of Hsp90 causes the rapid loss of Mal63p MAL activator protein, and in Hsp90 chaperone mutant strains, Mal63p half-life is reduced up to 5-fold. Most significantly, Mal63 MAL activator immunoprecipitates with His-tagged Hsp90 from native cell extracts. Thus, the Saccharomyces MAL activator is shown to be a novel signal transducing protein client of the Hsp90 chaperone complex, further demonstrating the integration of chaperone function into nonstress cellular metabolism. Yeast Strains and Plasmids—The Saccharomyces strains used in this study are listed in Table I. Strain W303 carries naturally occurring defective copies of the MAL1 and MAL3 loci (33Han E.-K. Cotty F. Sottas C. Jiang H. Michels C.A. Mol. Microbiol. 1995; 17: 1093-1107Crossref PubMed Scopus (129) Google Scholar). Both loci contain functional maltose permease and maltase genes, referred to as MAL11 (also known as AGT1) and MAL12, respectively, at MAL1, and MAL31 and MAL33, respectively, at MAL3. Sequences homologous to the MAL63 MAL activator gene are found at both MAL1 and MAL3, referred to as mal13 and mal33, respectively, but these genes are nonfunctional. Thus, strain W303 does not ferment maltose. Plasmid-borne copies of MAL63 complement the defective chromosomal copies of the MAL activator genes. Plasmid pMAL63 was constructed by subcloning the BamHI-SalI fragment carrying MAL63 on its native promoter into the Escherichia coli yeast/shuttle vector YCp50 as described by Gibson et al. (8Gibson A.W. Wojciechowicz L.A. Danzi S.E. Zhang B. Kim J.H. Hu Z. Michels C.A. Genetics. 1997; 146: 1287-1298Crossref PubMed Google Scholar).Table IList of strainsStrainGenotypeSourceW303MATa leu2-3, 112 ura3-1 trp1-1 his3-11, 15 can 1-100 GAL SUC2S. Lindquisthsc82ΔIsogenic to W303 except hsc82Δ::LEU2Ref. 26Duina A.A. Chang H.-C.J. Marsh J.A. Lindquist S. Gaber R.F. Science. 1996; 274: 1713-1715Crossref PubMed Scopus (186) Google ScholarS153MATa leu2-3, 112 ura3-1 trp1-1 ade2-1 can1-100 GAL SUC hsc82Δ::LEU2 hsp82Δ::LEU2 pGPD-hsp82-T1011Ref. 36Nathan D.F. Lindquist S. Mol. Cell. Biol. 1995; 15: 3917-3925Crossref PubMed Scopus (370) Google Scholarcpr7ΔIsogenic to W303 except cpr7Δ::HIS3Ref. 26Duina A.A. Chang H.-C.J. Marsh J.A. Lindquist S. Gaber R.F. Science. 1996; 274: 1713-1715Crossref PubMed Scopus (186) Google Scholarhsc82Δ cpr7ΔIsogenic to W303 except hsc82Δ::LEU2 cpr7Δ::TRP1Ref. 26Duina A.A. Chang H.-C.J. Marsh J.A. Lindquist S. Gaber R.F. Science. 1996; 274: 1713-1715Crossref PubMed Scopus (186) Google Scholar5CG2MATα ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 hsc82::URA3 hsp82::GAL1-HSP82::LEU2S. Lindquist Open table in a new tab The following series of plasmids were constructed using vectors described by Mumberg et al. (34Mumberg D. Muller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1603) Google Scholar). Plasmid p414GPD of this series was used to construct p414GPD-MAL63/FLAG. It is a CEN vector containing the TRP1 selection marker and the promoter of TDH1, encoding glyceraldehyde-3-phosphate dehydrogenase, which is a highly expressed constitutive promoter. The MAL63 coding region was amplified by PCR using an upstream primer that inserts a BamHI site and the sequence encoding the FLAG epitope at the 5′ end of the MAL63 open reading frame (primer B11; Table II) and a downstream primer that inserts a SalI site immediately following the MAL63 termination codon (primer B5; Table II). The amplified BamHI-SalI fragment was inserted into the BamHI and SalI sites in the multiple cloning sequence of p414GPD creating a GPD promoter-MAL63/FLAG fusion gene and plasmid p414GPD-MAL63/FLAG.Table IIList of primersPrimerPrimer sequenceB115′-GGGGGATCCATGGATTATAAGGATGACGATGACAAGGGTATTGCGAAACAGTCTTGC-3′B55′-GGGGGTCGACAACGGCGTGAACAATAAA-3′KM-N5′-GGGGATCCAAAATGGGCGGCCGCATTGCGAAACAGTCTTGC-3′MB-45′-CCAAAATGGGCGGCCGCATCTTTTACC-3′MB-55′-CGCAATACCGCGGCCGCACTGAGCAG-3′ Open table in a new tab The FLAG tag sequence in plasmid p414GPD-MAL63/FLAG was replaced with a triple HA 1The abbreviations used are: HAhemagglutininPGKphosphoglycerol kinase. tag sequence to create plasmid p414GPD-MAL63/HA3 as follows. The fragment encoding the 5′ half of the MAL63 open reading frame was amplified using a 5′ primer (KM-N; Table II) that inserts a NotI site between codons 2 and 3 of MAL63 and a 3′ primer complementary to a sequence just downstream of the EcoRI site at codons 215/216 of MAL63. This amplified product was digested with BamHI and EcoRI and used to replace the BamHI-EcoRI fragment containing the 5′ end of the tagged MAL63 gene in p414GPD-MAL63/FLAG, thereby removing the FLAG sequence. A 115-base pair NotI fragment containing three copies of the sequences encoding the HA epitope was inserted into the NotI site in the proper orientation, creating a GPD promoter-MAL63/HA3 fusion gene and plasmid p414GPD-MAL63/HA3. hemagglutinin phosphoglycerol kinase. Plasmid p416GPD from the Mumberg et al. (34Mumberg D. Muller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1603) Google Scholar) series contains URA3 as the selectable marker but is otherwise the same as p414. The SacI-KpnI fragment containing the entire GPD promoter-MAL63/HA3 tagged fusion gene was released from p414GPD-MAL63/HA3 by digestion with SacI and KpnI and inserted into SacI-KpnI-digested plasmid p416 to create plasmid p416GPD-MAL63/HA3. Vector plasmid p416TEF is another from the Mumberg et al. (34Mumberg D. Muller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1603) Google Scholar) series. It is a CEN plasmid and contains URA3 as the selectable marker gene and the promoter of the TEF1 gene, a lower level constitutive promoter. The full MAL63 open reading frame was amplified by PCR using primer KM-N (Table II) as the 5′ primer, which inserts a NotI site between codons 2 and 3 of MAL63, and a 3′ primer complementary to the sequence just downstream of the natural MAL63 termination codon (B5; Table II). The amplified product was digested with BamHI and SalI and inserted into BamHI- and SalI-digested plasmid p416TEF to create a TEF1 promoter-MAL63 fusion gene. A 115-base pair NotI fragment containing three copies of the sequences encoding the HA epitope was amplified using primers MB-4 and MB-5 (Table II) from p414GPD-MAL63/HA3 and inserted into the NotI site of the TEF1 promoter-MAL63 fusion gene in the proper orientation producing a TEF1 promoter-MAL63/HA3 fusion gene and plasmid pTEF-MAL63/HA3. Preparation of Cell Extracts and Immunoblot Analysis—The strains were grown in the appropriate selective minimal medium to mid-log phase (A600 of 0.2–0.5). An aliquot of the culture, volume in milliliters approximately equal to 15 divided by A600, was harvested by filtration, washed with 50 mm KPO4 buffer, pH 7.4, plus 2% sodium azide, and frozen while still on the filter paper at –80 °C for at least 20 min. The frozen cells were defrosted and resuspended in 1 ml of 50 mm HEPES buffer, pH 7.5, containing a mixture of protease inhibitors (Roche Applied Science; complete, mini, EDTA-free protease Inhibitor tablets (catalogue number 1836170) plus Sigma yeast protease inhibitor mixture (catalogue number P8215)), pelleted by centrifugation, and resuspended in 300 μl of SB buffer. SB buffer is prepared by dissolving one tablet of Roche Applied Science protease inhibitor, 8 μl of Sigma yeast protease inhibitor mixture, 0.1 g of SDS, and a toothpick tip-full of sodium bisulfite in 2 ml of H2O. An equal volume of glass beads was added to the cell suspension, and the samples were vortexed at a medium speed at 4 °C for 20 min. The samples were placed in a 37 °C water bath for 20 min, after which an additional 50 μl of SB buffer was added to each sample, and the samples were vortexed again for 2 min at 4 °C. The glass beads were separated from the extract by centrifugation at 4 °C, and the supernatant was removed. The samples were boiled for 4 min and stored at –80 °C. The protein concentration of the cell extract was determined by the Lowry assay method. Western blot analysis was carried out using standard methods, and the proteins were detected using an Amersham Vistra ECF kit in which the secondary antibody is conjugated to a fluorescent dye. The signal was visualized using a Molecular Dynamics Storm 860 and quantitated using software provided by the manufacturer. This method allows relatively accurate quantitation of the signal that is linear over ∼5 logs. M2 anti-FLAG antibody was obtained from Sigma. The anti-Hsp90 antibody was a gift from Susan Lindquist. Phosphoglycerol kinase (PGK) was detected by anti-PGK antibody from Molecular Probes. PGK levels are relatively constant at different growth conditions, and thus PGK levels were used as a control to adjust for loading variations in those experiments in which accurate quantitation was needed. Co-immunoprecipitation—The tagged proteins were co-expressed in wild-type cells and grown in selective media. The cells were harvested, resuspended in a nondenaturing buffer containing 50 mm sodium molybdate and extensive protease inhibitors, and flash frozen in liquid nitrogen. The protein extracts were made via glass bead lysis at 4 °C and quantitated by Lowry assay. 50 μg of extract was mixed with 2× sample loading buffer and boiled for 3 min. Approximately 1 mg of total cell extract was then combined with 100 μl of a 50% slurry of Ni2+-nitrilotriacetic acid-agarose (Qiagen) and incubated on ice for 15 min. The resin was washed twice with lysis buffer containing 5 mm imidazole and twice again with lysis buffer containing 10 mm imidazole. The remaining protein was eluted by boiling in sample buffer for 3 min. All of the protein samples were electrophoresed using 10% SDS-PAGE and electroblotted to nitrocellulose, and specific proteins were detected using the indicated antibodies and enhanced chemiluminescence. Maltase Assay—The cells were grown to mid-log, harvested by centrifugation, and resuspended in 0.5 ml of potassium phosphate buffer, and an equal volume of glass beads was added. The extracts were prepared by votexing the cell suspension three times for 1 min each, keeping the mixture cooled on ice. Maltase activity was measured in whole cell extracts as described in Dubin et al. (35Dubin R.A. Needleman R.B. Gossett D. Michels C.A. J. Bacteriol. 1985; 164: 605-610Crossref PubMed Google Scholar). Activity is expressed as nmol of p-nitrophenol-α-glucopyranoside hydrolyzed per min/mg of protein. Protein concentration of the cell extracts was measured using the Bio-Rad protein assay dye reagent. The assay values are the averages of results from three independent transformants assayed in duplicate. Variation is ∼20%. Maltase Induction and Maltose Utilization Are Defective in Strains Carrying Mutations in Components of the Hsp90 Chaperone Complex—We investigated the effects of Hsp90 chaperone mutations using an isogenic strain series derived from strain W303 carrying mutations in the genes encoding the two differentially expressed Hsp90 isoforms, HSC82 and HSP82, or in the gene encoding the constitutively expressed cyclophilin isoform, CPR7. Strain hsc82Δ (hsc82Δ HSP82) lacks the gene encoding the constitutive Hsp90 isoform, HSC82. Strain S153 (hsc82Δ hsp82-T101I) contains a null mutation in both HSC82 and HSP82 but carries a plasmid-borne copy of the temperature-sensitive allele hsp82-T101I expressed from the high level constitutive glyceraldehyde-3-phosphate dehydrogenase gene promoter. The hsp82-T101I product exhibits reduced activity even at the permissive temperature (24 °C) but is inactivated further at higher temperatures (36Nathan D.F. Lindquist S. Mol. Cell. Biol. 1995; 15: 3917-3925Crossref PubMed Scopus (370) Google Scholar). In strains expressing only hsp82-T101I, no growth is observed on media containing glucose as the sole carbon source at temperatures above ∼35 °C, indicating full loss of Hsp90 activity. Strains carrying a null mutation in CPR7 and null mutations in both CPR7 and HSC82 were also studied. Duina et al. (26Duina A.A. Chang H.-C.J. Marsh J.A. Lindquist S. Gaber R.F. Science. 1996; 274: 1713-1715Crossref PubMed Scopus (186) Google Scholar) found that growth on glucose-containing medium at 30 °C was slowed in the cpr7Δ strain, unaffected in the hsc82Δ strain, but reduced in the cpr7Δ hsc82Δ double null strain. Strain W303 carries two copies of the genes encoding maltose permease (MAL11, also called AGT1, and MAL31) and maltase (MAL12 and MAL32) but does not ferment maltose because it lacks a functional MAL activator gene. To study maltose utilization in this strain series MAL63, the MAL activator gene from the MAL6 locus was introduced into the Hsp90 mutant strains by transformation with the CEN plasmid pMAL63. The ability of these strains to induce maltase expression was determined. Transformants were grown in selective medium under uninduced conditions at 24 °C to mid-log phase and induced with maltose at either 24 or 35 °C. Maltase activity was assayed at time 0 and at 4 h after the addition of maltose. The results are shown in Fig. 1. The wild-type strain is able to induce to similar levels at both 24 and 35 °C. Loss of HSC82 alone causes a modest decrease in the rate of induction at 24 °C," @default.
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• W2037179957 date "2003-11-01" @default.
• W2037179957 modified "2023-10-18" @default.
• W2037179957 title "The Hsp90 Molecular Chaperone Complex Regulates Maltose Induction and Stability of the Saccharomyces MAL Gene Transcription Activator Mal63p" @default.
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