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. 2003 Feb;163(2):495–506. doi: 10.1093/genetics/163.2.495

Saccharomyces cerevisiae Hsp70 mutations affect [PSI+] prion propagation and cell growth differently and implicate Hsp40 and tetratricopeptide repeat cochaperones in impairment of [PSI+].

Gary W Jones 1, Daniel C Masison 1
PMCID: PMC1462464  PMID: 12618389

Abstract

We previously described an Hsp70 mutant (Ssa1-21p), altered in a conserved residue (L483W), that dominantly impairs yeast [PSI(+)] prion propagation without affecting growth. We generated new SSA1 mutations that impaired [PSI(+)] propagation and second-site mutations in SSA1-21 that restored normal propagation. Effects of mutations on growth did not correlate with [PSI(+)] phenotype, revealing differences in Hsp70 function required for growth and [PSI(+)] propagation and suggesting that Hsp70 interacts differently with [PSI(+)] prion aggregates than with other cellular substrates. Complementary suppression of altered activity between forward and suppressing mutations suggests that mutations that impair [PSI(+)] affect a similar Hsp70 function and that suppressing mutations similarly overcome this effect. All new mutations that impaired [PSI(+)] propagation were located in the ATPase domain. Locations and homology of several suppressing substitutions suggest that they weaken Hsp70's substrate-trapping conformation, implying that impairment of [PSI(+)] by forward mutations is due to altered ability of the ATPase domain to regulate substrate binding. Other suppressing mutations are in residues important for interactions with Hsp40 or TPR-containing cochaperones, suggesting that such interactions are necessary for the impairment of [PSI(+)] propagation caused by mutant Ssa1p.

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Selected References

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  1. Becker J., Walter W., Yan W., Craig E. A. Functional interaction of cytosolic hsp70 and a DnaJ-related protein, Ydj1p, in protein translocation in vivo. Mol Cell Biol. 1996 Aug;16(8):4378–4386. doi: 10.1128/mcb.16.8.4378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  3. Boorstein W. R., Ziegelhoffer T., Craig E. A. Molecular evolution of the HSP70 multigene family. J Mol Evol. 1994 Jan;38(1):1–17. doi: 10.1007/BF00175490. [DOI] [PubMed] [Google Scholar]
  4. Brinker Achim, Scheufler Clemens, Von Der Mulbe Florian, Fleckenstein Burkhard, Herrmann Christian, Jung Gunther, Moarefi Ismail, Hartl F. Ulrich. Ligand discrimination by TPR domains. Relevance and selectivity of EEVD-recognition in Hsp70 x Hop x Hsp90 complexes. J Biol Chem. 2002 Mar 4;277(22):19265–19275. doi: 10.1074/jbc.M109002200. [DOI] [PubMed] [Google Scholar]
  5. Brown C. R., McCann J. A., Chiang H. L. The heat shock protein Ssa2p is required for import of fructose-1, 6-bisphosphatase into Vid vesicles. J Cell Biol. 2000 Jul 10;150(1):65–76. doi: 10.1083/jcb.150.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bukau B., Horwich A. L. The Hsp70 and Hsp60 chaperone machines. Cell. 1998 Feb 6;92(3):351–366. doi: 10.1016/s0092-8674(00)80928-9. [DOI] [PubMed] [Google Scholar]
  7. Chang H. C., Lindquist S. Conservation of Hsp90 macromolecular complexes in Saccharomyces cerevisiae. J Biol Chem. 1994 Oct 7;269(40):24983–24988. [PubMed] [Google Scholar]
  8. Chernoff Y. O., Lindquist S. L., Ono B., Inge-Vechtomov S. G., Liebman S. W. Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [psi+]. Science. 1995 May 12;268(5212):880–884. doi: 10.1126/science.7754373. [DOI] [PubMed] [Google Scholar]
  9. Cyr D. M., Lu X., Douglas M. G. Regulation of Hsp70 function by a eukaryotic DnaJ homolog. J Biol Chem. 1992 Oct 15;267(29):20927–20931. [PubMed] [Google Scholar]
  10. Derkatch I. L., Bradley M. E., Zhou P., Chernoff Y. O., Liebman S. W. Genetic and environmental factors affecting the de novo appearance of the [PSI+] prion in Saccharomyces cerevisiae. Genetics. 1997 Oct;147(2):507–519. doi: 10.1093/genetics/147.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eaglestone S. S., Ruddock L. W., Cox B. S., Tuite M. F. Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI(+)] of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):240–244. doi: 10.1073/pnas.97.1.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Edskes H. K., Gray V. T., Wickner R. B. The [URE3] prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments. Proc Natl Acad Sci U S A. 1999 Feb 16;96(4):1498–1503. doi: 10.1073/pnas.96.4.1498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  14. Glover J. R., Kowal A. S., Schirmer E. C., Patino M. M., Liu J. J., Lindquist S. Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae. Cell. 1997 May 30;89(5):811–819. doi: 10.1016/s0092-8674(00)80264-0. [DOI] [PubMed] [Google Scholar]
  15. Ha J. H., Hellman U., Johnson E. R., Li L., McKay D. B., Sousa M. C., Takeda S., Wernstedt C., Wilbanks S. M. Destabilization of peptide binding and interdomain communication by an E543K mutation in the bovine 70-kDa heat shock cognate protein, a molecular chaperone. J Biol Chem. 1997 Oct 31;272(44):27796–27803. doi: 10.1074/jbc.272.44.27796. [DOI] [PubMed] [Google Scholar]
  16. Hernández M. Patricia, Chadli Ahmed, Toft David O. HSP40 binding is the first step in the HSP90 chaperoning pathway for the progesterone receptor. J Biol Chem. 2002 Jan 23;277(14):11873–11881. doi: 10.1074/jbc.M111445200. [DOI] [PubMed] [Google Scholar]
  17. Jung G., Jones G., Wegrzyn R. D., Masison D. C. A role for cytosolic hsp70 in yeast [PSI(+)] prion propagation and [PSI(+)] as a cellular stress. Genetics. 2000 Oct;156(2):559–570. doi: 10.1093/genetics/156.2.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jung G., Masison D. C. Guanidine hydrochloride inhibits Hsp104 activity in vivo: a possible explanation for its effect in curing yeast prions. Curr Microbiol. 2001 Jul;43(1):7–10. doi: 10.1007/s002840010251. [DOI] [PubMed] [Google Scholar]
  19. Jung Giman, Jones Gary, Masison Daniel C. Amino acid residue 184 of yeast Hsp104 chaperone is critical for prion-curing by guanidine, prion propagation, and thermotolerance. Proc Natl Acad Sci U S A. 2002 Jul 8;99(15):9936–9941. doi: 10.1073/pnas.152333299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kryndushkin Dmitry S., Smirnov Vladimir N., Ter-Avanesyan Michael D., Kushnirov Vitaly V. Increased expression of Hsp40 chaperones, transcriptional factors, and ribosomal protein Rpp0 can cure yeast prions. J Biol Chem. 2002 Mar 28;277(26):23702–23708. doi: 10.1074/jbc.M111547200. [DOI] [PubMed] [Google Scholar]
  21. Kushnirov V. V., Kryndushkin D. S., Boguta M., Smirnov V. N., Ter-Avanesyan M. D. Chaperones that cure yeast artificial [PSI+] and their prion-specific effects. Curr Biol. 2000 Nov 16;10(22):1443–1446. doi: 10.1016/s0960-9822(00)00802-2. [DOI] [PubMed] [Google Scholar]
  22. Laufen T., Mayer M. P., Beisel C., Klostermeier D., Mogk A., Reinstein J., Bukau B. Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5452–5457. doi: 10.1073/pnas.96.10.5452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mayer M. P., Brehmer D., Gässler C. S., Bukau B. Hsp70 chaperone machines. Adv Protein Chem. 2001;59:1–44. doi: 10.1016/s0065-3233(01)59001-4. [DOI] [PubMed] [Google Scholar]
  24. Mayer M. P., Schröder H., Rüdiger S., Paal K., Laufen T., Bukau B. Multistep mechanism of substrate binding determines chaperone activity of Hsp70. Nat Struct Biol. 2000 Jul;7(7):586–593. doi: 10.1038/76819. [DOI] [PubMed] [Google Scholar]
  25. McCready S. J., Cox B. S., McLaughlin C. S. The extrachromosomal control of nonsense suppression in yeast: an analysis of the elimination of [psi+] in the presence of a nuclear gene PNM. Mol Gen Genet. 1977 Feb 15;150(3):265–270. doi: 10.1007/BF00268125. [DOI] [PubMed] [Google Scholar]
  26. Nelson R. J., Ziegelhoffer T., Nicolet C., Werner-Washburne M., Craig E. A. The translation machinery and 70 kd heat shock protein cooperate in protein synthesis. Cell. 1992 Oct 2;71(1):97–105. doi: 10.1016/0092-8674(92)90269-i. [DOI] [PubMed] [Google Scholar]
  27. Newnam G. P., Wegrzyn R. D., Lindquist S. L., Chernoff Y. O. Antagonistic interactions between yeast chaperones Hsp104 and Hsp70 in prion curing. Mol Cell Biol. 1999 Feb;19(2):1325–1333. doi: 10.1128/mcb.19.2.1325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Oka M., Nakai M., Endo T., Lim C. R., Kimata Y., Kohno K. Loss of Hsp70-Hsp40 chaperone activity causes abnormal nuclear distribution and aberrant microtubule formation in M-phase of Saccharomyces cerevisiae. J Biol Chem. 1998 Nov 6;273(45):29727–29737. doi: 10.1074/jbc.273.45.29727. [DOI] [PubMed] [Google Scholar]
  29. Patino M. M., Liu J. J., Glover J. R., Lindquist S. Support for the prion hypothesis for inheritance of a phenotypic trait in yeast. Science. 1996 Aug 2;273(5275):622–626. doi: 10.1126/science.273.5275.622. [DOI] [PubMed] [Google Scholar]
  30. Paushkin S. V., Kushnirov V. V., Smirnov V. N., Ter-Avanesyan M. D. Propagation of the yeast prion-like [psi+] determinant is mediated by oligomerization of the SUP35-encoded polypeptide chain release factor. EMBO J. 1996 Jun 17;15(12):3127–3134. [PMC free article] [PubMed] [Google Scholar]
  31. Pellecchia M., Montgomery D. L., Stevens S. Y., Vander Kooi C. W., Feng H. P., Gierasch L. M., Zuiderweg E. R. Structural insights into substrate binding by the molecular chaperone DnaK. Nat Struct Biol. 2000 Apr;7(4):298–303. doi: 10.1038/74062. [DOI] [PubMed] [Google Scholar]
  32. Pfund C., Huang P., Lopez-Hoyo N., Craig E. A. Divergent functional properties of the ribosome-associated molecular chaperone Ssb compared with other Hsp70s. Mol Biol Cell. 2001 Dec;12(12):3773–3782. doi: 10.1091/mbc.12.12.3773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pfund C., Lopez-Hoyo N., Ziegelhoffer T., Schilke B. A., Lopez-Buesa P., Walter W. A., Wiedmann M., Craig E. A. The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome-nascent chain complex. EMBO J. 1998 Jul 15;17(14):3981–3989. doi: 10.1093/emboj/17.14.3981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  35. Schatz P. J., Solomon F., Botstein D. Isolation and characterization of conditional-lethal mutations in the TUB1 alpha-tubulin gene of the yeast Saccharomyces cerevisiae. Genetics. 1988 Nov;120(3):681–695. doi: 10.1093/genetics/120.3.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Scheufler C., Brinker A., Bourenkov G., Pegoraro S., Moroder L., Bartunik H., Hartl F. U., Moarefi I. Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell. 2000 Apr 14;101(2):199–210. doi: 10.1016/S0092-8674(00)80830-2. [DOI] [PubMed] [Google Scholar]
  37. Schlossman D. M., Schmid S. L., Braell W. A., Rothman J. E. An enzyme that removes clathrin coats: purification of an uncoating ATPase. J Cell Biol. 1984 Aug;99(2):723–733. doi: 10.1083/jcb.99.2.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schwimmer Christine, Masison Daniel C. Antagonistic interactions between yeast [PSI(+)] and [URE3] prions and curing of [URE3] by Hsp70 protein chaperone Ssa1p but not by Ssa2p. Mol Cell Biol. 2002 Jun;22(11):3590–3598. doi: 10.1128/MCB.22.11.3590-3598.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stansfield I., Jones K. M., Kushnirov V. V., Dagkesamanskaya A. R., Poznyakovski A. I., Paushkin S. V., Nierras C. R., Cox B. S., Ter-Avanesyan M. D., Tuite M. F. The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO J. 1995 Sep 1;14(17):4365–4373. doi: 10.1002/j.1460-2075.1995.tb00111.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stone D. E., Craig E. A. Self-regulation of 70-kilodalton heat shock proteins in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Apr;10(4):1622–1632. doi: 10.1128/mcb.10.4.1622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Suh W. C., Burkholder W. F., Lu C. Z., Zhao X., Gottesman M. E., Gross C. A. Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ. Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15223–15228. doi: 10.1073/pnas.95.26.15223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Taylor K. L., Cheng N., Williams R. W., Steven A. C., Wickner R. B. Prion domain initiation of amyloid formation in vitro from native Ure2p. Science. 1999 Feb 26;283(5406):1339–1343. doi: 10.1126/science.283.5406.1339. [DOI] [PubMed] [Google Scholar]
  44. Van Der Spuy J., Kana B. D., Dirr H. W., Blatch G. L. Heat shock cognate protein 70 chaperone-binding site in the co-chaperone murine stress-inducible protein 1 maps to within three consecutive tetratricopeptide repeat motifs. Biochem J. 2000 Feb 1;345(Pt 3):645–651. doi: 10.1042/0264-6021:3450645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wach A., Brachat A., Pöhlmann R., Philippsen P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast. 1994 Dec;10(13):1793–1808. doi: 10.1002/yea.320101310. [DOI] [PubMed] [Google Scholar]
  46. Waldron C., Cox B. S., Wills N., Gesteland R. F., Piper P. W., Colby D., Guthrie C. Yeast ochre suppressor SUQ5-ol is an altered tRNA Ser UCA. Nucleic Acids Res. 1981 Jul 10;9(13):3077–3088. doi: 10.1093/nar/9.13.3077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Werner-Washburne M., Stone D. E., Craig E. A. Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jul;7(7):2568–2577. doi: 10.1128/mcb.7.7.2568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wickner R. B. [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. Science. 1994 Apr 22;264(5158):566–569. doi: 10.1126/science.7909170. [DOI] [PubMed] [Google Scholar]
  49. Zhouravleva G., Frolova L., Le Goff X., Le Guellec R., Inge-Vechtomov S., Kisselev L., Philippe M. Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. EMBO J. 1995 Aug 15;14(16):4065–4072. doi: 10.1002/j.1460-2075.1995.tb00078.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zhu X., Zhao X., Burkholder W. F., Gragerov A., Ogata C. M., Gottesman M. E., Hendrickson W. A. Structural analysis of substrate binding by the molecular chaperone DnaK. Science. 1996 Jun 14;272(5268):1606–1614. doi: 10.1126/science.272.5268.1606. [DOI] [PMC free article] [PubMed] [Google Scholar]

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