Skip to main content
Log in

Novel interaction of the Hsp90 chaperone machine with Ssl2, an essential DNA helicase in Saccharomyces cerevisiae

  • Research Article
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

Hsp90 is an essential molecular chaperone that is critical for the activity of diverse cellular proteins. Hsp90 functions with a number of co-chaperone proteins, including Sti1/Hop. We conducted a genetic screen in Saccharomyces cerevisiae to isolate mutations that exhibit enhanced growth defects in the absence of STI1. We obtained mutations in genes encoding components of the Hsp90 chaperone machine, HSC82, CPR7 and YDJ1, and two essential genes, SSL2 and UTP21, not previously linked to Hsp90. Ssl2, the yeast homologue of XPB, is an ATP-dependent DNA helicase that is a component of the TFIIH multiprotein complex and has dual functions in transcription and DNA repair. In order to determine whether Ssl2 function is dependent on Hsp90, we further examined the interaction between Ssl2 and Hsp90. Multiple mutant alleles of SSL2 exhibited a pronounced growth defect when co-expressed with a mutant allele of Hsp90. In addition, isolation of Ssl2 protein resulted in the co-purification of Hsp90 and Sti1, suggesting that Ssl2 and Hsp90 are in the same protein complexes in vivo. These results suggest a novel role for Hsp90 in the essential cellular functions of transcription and DNA repair.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Abbas-Terki T, Briand PA, Donze O, Picard D (2002) The Hsp90 co-chaperones Cdc37 and Sti1 interact physically and genetically. Biol Chem 383:1335–1342

    CAS  PubMed  Google Scholar 

  • Atencio DP, Yaffe MP (1992) MAS5, a yeast homolog of DnaJ involved in mitochondrial protein import. Mol Cell Biol 12:283–291

    CAS  PubMed  Google Scholar 

  • Bali M, Zhang B, Morano KA, Michels CA (2003) The Hsp90 molecular chaperone complex regulates maltose induction and stability of the Saccharomyces MAL gene transcription activator Mal63p. J Biol Chem 278:47441–47448

    CAS  PubMed  Google Scholar 

  • Bansal PK, Abdulle R, Kitagawa K (2004) Sgt1 associates with Hsp90: an initial step of assembly of the core kinetochore complex. Mol Cell Biol 24:8069–8079

    CAS  PubMed  Google Scholar 

  • Bender A, Pringle JR (1991) Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae. Mol Cell Biol 11:1295–1305

    CAS  PubMed  Google Scholar 

  • Bisht KS, et al (2003) Geldanamycin and 17-allylamino-17-demethoxygeldanamycin potentiate the in vitro and in vivo radiation response of cervical tumor cells via the heat shock protein 90-mediated intracellular signaling and cytotoxicity. Cancer Res 63:8984–8995

    CAS  PubMed  Google Scholar 

  • Bohen SP, Yamamoto KR (1993) Isolation of Hsp90 mutants by screening for decreased steroid receptor function. Proc Natl Acad Sci USA 90:11424–11428

    CAS  PubMed  Google Scholar 

  • Borkovich KA, Farrelly FW, Finkelstein DB, Taulien J, Lindquist S (1989) hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures. Mol Cell Biol 9:3919–3930

    CAS  PubMed  Google Scholar 

  • Brugge J, Yonemoto W, Darrow D (1983) Interaction between the Rous sarcoma virus transforming protein and two cellular phosphoproteins: analysis of the turnover and distribution of this complex. Mol Cell Biol 3:9–19

    CAS  PubMed  Google Scholar 

  • Camphausen K, Tofilon PJ (2004) Combining radiation and molecular targeting in cancer therapy. Cancer Biol Ther 3:247–250

    CAS  PubMed  Google Scholar 

  • Caplan AJ, Douglas MG (1991) Characterization of YDJ1: a yeast homologue of the bacterial dnaJ protein. J Cell Biol 114:609–621

    CAS  PubMed  Google Scholar 

  • Carr-Schmid A, Pfund C, Craig EA, Kinzy TG (2002) Novel G-protein complex whose requirement is linked to the translational status of the cell. Mol Cell Biol 22:2564–2574

    CAS  PubMed  Google Scholar 

  • Chang HC, Nathan DF, Lindquist S (1997) In vivo analysis of the Hsp90 cochaperone Sti1 (p60). Mol Cell Biol 17:318–325

    CAS  PubMed  Google Scholar 

  • Chen S, Smith DF (1998) Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery. J Biol Chem 273:35194–35200

    CAS  PubMed  Google Scholar 

  • Chen S, Prapapanich V, Rimerman RA, Honore B, Smith DF (1996) Interactions of p60, a mediator of progesterone receptor assembly, with heat shock proteins hsp90 and hsp70. Mol Endocrinol 10:682–693

    CAS  PubMed  Google Scholar 

  • Dey B, Caplan AJ, Boschelli F (1996) The Ydj1 molecular chaperone facilitates formation of active p60v-src in yeast. Mol Biol Cell 7:91–100

    CAS  PubMed  Google Scholar 

  • Donze O, Picard D (1999) Hsp90 binds and regulates Gcn2, the ligand-inducible kinase of the alpha subunit of eukaryotic translation initiation factor 2 [corrected]. Mol Cell Biol 19:8422–8432

    CAS  PubMed  Google Scholar 

  • Dosil M, Bustelo XR (2004) Functional characterization of Pwp2, a WD family protein essential for the assembly of the 90 S pre-ribosomal particle. J Biol Chem 279:37385–37397

    CAS  PubMed  Google Scholar 

  • Dragon F, et al (2002) A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature 417:967–970

    CAS  PubMed  Google Scholar 

  • Duina AA, Marsh JA, Gaber RF (1996) Identification of two CyP-40-like cyclophilins in Saccharomyces cerevisiae, one of which is required for normal growth. Yeast 12:943–952

    CAS  PubMed  Google Scholar 

  • Duina AA, Kalton HM, Gaber RF (1998) Requirement for Hsp90 and a CyP-40-type cyclophilin in negative regulation of the heat shock response. J Biol Chem 273:18974–18978

    CAS  PubMed  Google Scholar 

  • Fang Y, Fliss AE, Rao J, Caplan AJ (1998) SBA1 encodes a yeast hsp90 cochaperone that is homologous to vertebrate p23 proteins. Mol Cell Biol 18:3727–3734

    CAS  PubMed  Google Scholar 

  • Feaver WJ et al (1993) Dual roles of a multiprotein complex from S. cerevisiae in transcription and DNA repair. Cell 75:1379–1387

    CAS  PubMed  Google Scholar 

  • Freeman BC, Yamamoto KR (2002) Disassembly of transcriptional regulatory complexes by molecular chaperones. Science 296:2232–2235

    CAS  PubMed  Google Scholar 

  • Ghaemmaghami S, et al (2003) Global analysis of protein expression in yeast. Nature 425:737–741

    CAS  PubMed  Google Scholar 

  • Giglia-Mari G, et al (2004) A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A. Nat Genet 36:714–719

    CAS  PubMed  Google Scholar 

  • Gulyas KD, Donahue TF (1992) SSL2, a suppressor of a stem-loop mutation in the HIS4 leader encodes the yeast homolog of human ERCC-3. Cell 69:1031–1042

    CAS  PubMed  Google Scholar 

  • Guzder SN, Sung P, Bailly V, Prakash L, Prakash S (1994) RAD25 is a DNA helicase required for DNA repair and RNA polymerase II transcription. Nature 369:578–581

    CAS  PubMed  Google Scholar 

  • Hernandez MP, Chadli A, Toft DO (2002) HSP40 binding is the first step in the HSP90 chaperoning pathway for the progesterone receptor. J Biol Chem 277:11873–11881

    CAS  PubMed  Google Scholar 

  • Ho Y, et al (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415:180–183

    CAS  PubMed  Google Scholar 

  • Imai J, Yahara I (2000) Role of HSP90 in salt stress tolerance via stabilization and regulation of calcineurin. Mol Cell Biol 20:9262–9270

    CAS  PubMed  Google Scholar 

  • Imai J, Maruya M, Yashiroda H, Yahara I, Tanaka K (2003) The molecular chaperone Hsp90 plays a role in the assembly and maintenance of the 26S proteasome. EMBO J 22:3557–3567

    CAS  PubMed  Google Scholar 

  • Isaacs JS, Xu W, Neckers L (2003) Heat shock protein 90 as a molecular target for cancer therapeutics. Cancer Cell 3:213–217

    CAS  PubMed  Google Scholar 

  • Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168

    CAS  PubMed  Google Scholar 

  • Itoh H, Ogura M, Komatsuda A, Wakui H, Miura AB, Tashima Y (1999) A novel chaperone-activity-reducing mechanism of the 90-kDa molecular chaperone HSP90. Biochem J 343:697–703

    CAS  PubMed  Google Scholar 

  • Johnson JL, Craig EA (2000) A role for the Hsp40 Ydj1 in repression of basal steroid receptor activity in yeast. Mol Cell Biol 20:3027–3036

    CAS  PubMed  Google Scholar 

  • Johnson JL, Craig EA (2002) Identifying functional interactions with molecular chaperones. Methods Enzymol 351:442–453

    CAS  PubMed  Google Scholar 

  • Kimura Y, Matsumoto S, Yahara I (1994) Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae. Mol Gen Genet 242:517–527

    CAS  PubMed  Google Scholar 

  • Kimura Y, Yahara I, Lindquist S (1995) Role of the protein chaperone YDJ1 in establishing Hsp90-mediated signal transduction pathways. Science 268:1362–1365

    CAS  PubMed  Google Scholar 

  • Lee P, Shabbir A, Cardozo C, Caplan AJ (2004) Sti1 and Cdc37 can stabilize Hsp90 in chaperone complexes with a protein kinase. Mol Biol Cell 15:1785–1792

    CAS  PubMed  Google Scholar 

  • Liu J, DeFranco DB (1999) Chromatin recycling of glucocorticoid receptors: implications for multiple roles of heat shock protein 90. Mol Endocrinol 13:355–365

    CAS  PubMed  Google Scholar 

  • Liu XD, Morano KA, Thiele DJ (1999) The yeast Hsp110 family member, Sse1, is an Hsp90 cochaperone. J Biol Chem 274:26654–26660

    CAS  PubMed  Google Scholar 

  • Louvion JF, Abbas-Terki T, Picard D (1998) Hsp90 is required for pheromone signaling in yeast. Mol Biol Cell 9:3071–3083

    CAS  PubMed  Google Scholar 

  • Machida H, Matsumoto Y, Shirai M, Kubota N (2003) Geldanamycin, an inhibitor of Hsp90, sensitizes human tumour cells to radiation. Int J Radiat Biol 79:973–980

    CAS  PubMed  Google Scholar 

  • Mayer MP, Bukau B (1999) Molecular chaperones: the busy life of Hsp90. Curr Biol 9:R322–325

    CAS  PubMed  Google Scholar 

  • Morishima Y, Murphy PJ, Li DP, Sanchez ER, Pratt WB (2000) Stepwise assembly of a glucocorticoid receptor. hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket. J Biol Chem 275:18054–18060

    CAS  PubMed  Google Scholar 

  • Mumberg D, Muller R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119–122

    CAS  PubMed  Google Scholar 

  • Nathan DF, Lindquist S (1995) Mutational analysis of Hsp90 function: interactions with a steroid receptor and a protein kinase. Mol Cell Biol 15:3917–3925

    CAS  PubMed  Google Scholar 

  • Nathan DF, Vos MH, Lindquist S (1997) In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone. Proc Natl Acad Sci USA 94:12949–12956

    CAS  PubMed  Google Scholar 

  • Neckers L (2002) Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends Mol Med 8:S55–S61

    CAS  PubMed  Google Scholar 

  • Nicolet CM, Craig EA (1989) Isolation and characterization of STI1, a stress-inducible gene from Saccharomyces cerevisiae. Mol Cell Biol 9:3638–3646

    CAS  PubMed  Google Scholar 

  • Park E et al (1992) RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability. Proc Natl Acad Sci USA 89:11416–11420

    CAS  PubMed  Google Scholar 

  • Pearl LH, Prodromou C (2001) Structure, function, and mechanism of the Hsp90 molecular chaperone. Adv Protein Chem 59:157–186

    CAS  PubMed  Google Scholar 

  • Pratt WB, Toft DO (2003) Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp Biol Med 228:111–133

    CAS  Google Scholar 

  • Prodromou C, et al (1999) Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones. EMBO J 18:754–762

    CAS  PubMed  Google Scholar 

  • Prodromou C, Pearl LH (2003) Structure and functional relationships of Hsp90. Curr Cancer Drug Targets 3:301–323

    CAS  PubMed  Google Scholar 

  • Qiu H, Park E, Prakash L, Prakash S (1993) The Saccharomyces cerevisiae DNA repair gene RAD25 is required for transcription by RNA polymerase II. Genes Dev 7:2161–2171

    CAS  PubMed  Google Scholar 

  • Ranish JA, et al (2004) Identification of TFB5, a new component of general transcription and DNA repair factor IIH. Nat Genet 36:707–713

    CAS  PubMed  Google Scholar 

  • Richter K, Muschler P, Hainzl O, Reinstein J, Buchner J (2003) Sti1 Is a Non-competitive Inhibitor of the Hsp90 ATPase. Binding prevents the N-terminal dimerization reaction during the atpase cycle. J Biol Chem 278:10328–10333

    CAS  PubMed  Google Scholar 

  • Rose MD, Novick P, Thomas JH, Botstein D, Fink GR (1987) A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60:237–243

    CAS  PubMed  Google Scholar 

  • Rosenhagen MC et al (2003) The heat shock protein 90-targeting drug cisplatin selectively inhibits steroid receptor activation. Mol Endocrinol 17:1991–2001

    CAS  PubMed  Google Scholar 

  • Rothstein R (1991) Targeting, disruption, replacement and allele rescue: intergrative DNA transformation in yeast. In: Guthrie C, Fink GR (eds) Methods in enzymology: guide to yeast genetics and molecular biology. Cold Harbor Laboratory, Cold Harbor, N.Y., pp 281–301

  • Russell JS, Burgan W, Oswald KA, Camphausen K, Tofilon PJ (2003) Enhanced cell killing induced by the combination of radiation and the heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin: a multitarget approach to radiosensitization. Clin Cancer Res 9:3749–3755

    CAS  PubMed  Google Scholar 

  • Schafer T, Strauss D, Petfalski E, Tollervey D, Hurt E (2003) The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes. EMBO J 22:1370–1380

    PubMed  Google Scholar 

  • Scheufler C et al (2000) Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell 101:199–210

    CAS  PubMed  Google Scholar 

  • Schiestl RH, Gietz RD (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 16:339–346

    CAS  PubMed  Google Scholar 

  • Schultz P, Fribourg S, Poterszman A, Mallouh V, Moras D, Egly JM (2000) Molecular structure of human TFIIH. Cell 102:599–607

    CAS  PubMed  Google Scholar 

  • Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Harbor Laboratory, Cold Harbor, N.Y.

  • Sikorski RS, Boeke JD (1991) In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. In: Guthrie C, Fink GR (eds) Metholds in enzymology: guide to yeast genetics and molecular biology. Cold Harbor Laboratory, Cold Harbor, N.Y., pp 302–318

  • Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27

    CAS  PubMed  Google Scholar 

  • Smith DF (1993) Dynamics of heat shock protein 90-progesterone receptor binding and the disactivation loop model for steroid receptor complexes. Mol Endocrinol 7:1418–1429

    CAS  PubMed  Google Scholar 

  • Smith DF, et al (1993) Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70. Mol Cell Biol 13:869–876

    CAS  PubMed  Google Scholar 

  • Soti C, Racz A, Csermely P (2002) A Nucleotide-dependent molecular switch controls ATP binding at the C-terminal domain of Hsp90. N-terminal nucleotide binding unmasks a C-terminal binding pocket. J Biol Chem 277:7066–7075

    CAS  PubMed  Google Scholar 

  • Sweder KS, Hanawalt PC (1994) The COOH terminus of suppressor of stem loop (SSL2/RAD25) in yeast is essential for overall genomic excision repair and transcription-coupled repair. J Biol Chem 269:1852–1857

    CAS  PubMed  Google Scholar 

  • Thomas BJ, Rothstein R (1989) Elevated recombination rates in transcriptionally active DNA. Cell 56:619–630

    CAS  PubMed  Google Scholar 

  • Toledano MB, Delaunay A, Biteau B, Spector D, Azevedo D (2003) Oxidative stress responses in yeast. In: Hohmann S, Mager WH (eds) Yeast stress responses. Springer, Berlin Heidelberg New York, pp 241–303

    Google Scholar 

  • Valay JG, Simon M, Dubois MF, Bensaude O, Facca C, Faye G (1995) The KIN28 gene is required both for RNA polymerase II mediated transcription and phosphorylation of the Rpb1p CTD. J Mol Biol 249:535–544

    CAS  PubMed  Google Scholar 

  • van Brabant AJ, Stan R, Ellis NA (2000) DNA helicases, genomic instability, and human genetic disease. Annu Rev Genomics Hum Genet 1:409–459

    PubMed  Google Scholar 

  • Wegele H, Haslbeck M, Reinstein J, Buchner J (2003) Sti1 is a novel activator of the Ssa proteins. J Biol Chem 278:25970–25976

    Article  CAS  PubMed  Google Scholar 

  • Workman P (2003) Overview: translating Hsp90 biology into Hsp90 drugs. Curr Cancer Drug Targets 3:297–300

    CAS  PubMed  Google Scholar 

  • Yan W, Craig EA (1999) The glycine-phenylalanine-rich region determines the specificity of the yeast Hsp40 Sis1. Mol Cell Biol 19:7751–7758

    CAS  PubMed  Google Scholar 

  • Young JC, Hartl FU (2000) Polypeptide release by Hsp90 involves ATP hydrolysis and is enhanced by the co-chaperone p23. EMBO J 19:5930–5940

    CAS  PubMed  Google Scholar 

  • Young JC, Moarefi I, Hartl FU (2001) Hsp90: a specialized but essential protein-folding tool. J Cell Biol 154:267–273

    CAS  PubMed  Google Scholar 

  • Zhang L, Hach A, Wang C (1998) Molecular mechanism governing heme signaling in yeast: a higher-order complex mediates heme regulation of the transcriptional activator HAP1. Mol Cell Biol 18:3819–3828

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Chris Pfund, Brenda Schilke, Willy Walter, Kevin Morano, Brian Freeman, Avrom Caplan, Sue Lindquist, Lee Fortunato, David Smith and David Toft for reagents. We also thank Gary Daughdrill, Doug Cole and Elizabeth Craig for helpful advice and Lee Fortunato for comments on this manuscript. This work was supported by NIH-COBRE P20 RR15587 and NIH Grant P20 RR16454 from the BRIN/INBRE Program of the National Center for Research Resources.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jill L. Johnson.

Additional information

Communicated by S. Hohmann

Rights and permissions

Reprints and permissions

About this article

Cite this article

Flom, G., Weekes, J. & Johnson, J.L. Novel interaction of the Hsp90 chaperone machine with Ssl2, an essential DNA helicase in Saccharomyces cerevisiae. Curr Genet 47, 368–380 (2005). https://doi.org/10.1007/s00294-005-0580-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-005-0580-6

Keywords

Navigation