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. 1998 Oct 15;17(20):5868–5876. doi: 10.1093/emboj/17.20.5868

Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non-identical requirement for hsp60 and hsp10.

Y Dubaquié 1, R Looser 1, U Fünfschilling 1, P Jenö 1, S Rospert 1
PMCID: PMC1170914  PMID: 9774331

Abstract

The mechanism of chaperonin-assisted protein folding has been mostly analyzed in vitro using non-homologous substrate proteins. In order to understand the relative importance of hsp60 and hsp10 in the living cell, homologous substrate proteins need to be identified and analyzed. We have devised a novel screen to test the folding of a large variety of homologous substrates in the mitochondrial matrix in the absence or presence of functional hsp60 or hsp10. The identified substrates have an Mr of 15-90 kDa and fall into three groups: (i) proteins that require both hsp60 and hsp10 for correct folding; (ii) proteins that completely fail to fold after inactivation of hsp60 but are unaffected by the inactivation of hsp10; and (iii) newly imported hsp60 itself, which is more severely affected by inactivation of hsp10 than by inactivation of pre-existing hsp60. The majority of the identified substrates are group I proteins. For these, the lack of hsp60 function has a more pronounced effect than inactivation of hsp10. We suggest that homologous substrate proteins have differential chaperonin requirements, indicating that hsp60 and hsp10 do not always act as a single functional unit in vivo.

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Selected References

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  1. 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]
  2. Braig K., Otwinowski Z., Hegde R., Boisvert D. C., Joachimiak A., Horwich A. L., Sigler P. B. The crystal structure of the bacterial chaperonin GroEL at 2.8 A. Nature. 1994 Oct 13;371(6498):578–586. doi: 10.1038/371578a0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Cheng M. Y., Hartl F. U., Horwich A. L. The mitochondrial chaperonin hsp60 is required for its own assembly. Nature. 1990 Nov 29;348(6300):455–458. doi: 10.1038/348455a0. [DOI] [PubMed] [Google Scholar]
  5. Cheng M. Y., Hartl F. U., Martin J., Pollock R. A., Kalousek F., Neupert W., Hallberg E. M., Hallberg R. L., Horwich A. L. Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature. 1989 Feb 16;337(6208):620–625. doi: 10.1038/337620a0. [DOI] [PubMed] [Google Scholar]
  6. Clark A. C., Frieden C. GroEL-mediated folding of structurally homologous dihydrofolate reductases. J Mol Biol. 1997 May 2;268(2):512–525. doi: 10.1006/jmbi.1997.0969. [DOI] [PubMed] [Google Scholar]
  7. Davis M. T., Stahl D. C., Hefta S. A., Lee T. D. A microscale electrospray interface for on-line, capillary liquid chromatography/tandem mass spectrometry of complex peptide mixtures. Anal Chem. 1995 Dec 15;67(24):4549–4556. doi: 10.1021/ac00120a019. [DOI] [PubMed] [Google Scholar]
  8. Dubaquié Y., Looser R., Rospert S. Significance of chaperonin 10-mediated inhibition of ATP hydrolysis by chaperonin 60. Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):9011–9016. doi: 10.1073/pnas.94.17.9011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ellis R. J., Hartl F. U. Protein folding in the cell: competing models of chaperonin function. FASEB J. 1996 Jan;10(1):20–26. doi: 10.1096/fasebj.10.1.8566542. [DOI] [PubMed] [Google Scholar]
  10. Ewalt K. L., Hendrick J. P., Houry W. A., Hartl F. U. In vivo observation of polypeptide flux through the bacterial chaperonin system. Cell. 1997 Aug 8;90(3):491–500. doi: 10.1016/s0092-8674(00)80509-7. [DOI] [PubMed] [Google Scholar]
  11. Fenton W. A., Horwich A. L. GroEL-mediated protein folding. Protein Sci. 1997 Apr;6(4):743–760. doi: 10.1002/pro.5560060401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fisher M. T. GroE chaperonin-assisted folding and assembly of dodecameric glutamine synthetase. Biochemistry (Mosc) 1998 Apr;63(4):382–398. [PubMed] [Google Scholar]
  13. Garcia P. D., Hansen W., Walter P. In vitro protein translocation across microsomal membranes of Saccharomyces cerevisiae. Methods Enzymol. 1991;194:675–682. doi: 10.1016/0076-6879(91)94049-i. [DOI] [PubMed] [Google Scholar]
  14. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  15. Glick B. S., Brandt A., Cunningham K., Müller S., Hallberg R. L., Schatz G. Cytochromes c1 and b2 are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism. Cell. 1992 May 29;69(5):809–822. doi: 10.1016/0092-8674(92)90292-k. [DOI] [PubMed] [Google Scholar]
  16. Glick B. S., Pon L. A. Isolation of highly purified mitochondria from Saccharomyces cerevisiae. Methods Enzymol. 1995;260:213–223. doi: 10.1016/0076-6879(95)60139-2. [DOI] [PubMed] [Google Scholar]
  17. Goloubinoff P., Christeller J. T., Gatenby A. A., Lorimer G. H. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfoleded state depends on two chaperonin proteins and Mg-ATP. Nature. 1989 Dec 21;342(6252):884–889. doi: 10.1038/342884a0. [DOI] [PubMed] [Google Scholar]
  18. Gray T. E., Eder J., Bycroft M., Day A. G., Fersht A. R. Refolding of barnase mutants and pro-barnase in the presence and absence of GroEL. EMBO J. 1993 Nov;12(11):4145–4150. doi: 10.1002/j.1460-2075.1993.tb06098.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hallberg E. M., Shu Y., Hallberg R. L. Loss of mitochondrial hsp60 function: nonequivalent effects on matrix-targeted and intermembrane-targeted proteins. Mol Cell Biol. 1993 May;13(5):3050–3057. doi: 10.1128/mcb.13.5.3050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Heitman J., Movva N. R., Hiestand P. C., Hall M. N. FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1948–1952. doi: 10.1073/pnas.88.5.1948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heyrovská N., Frydman J., Höhfeld J., Hartl F. U. Directionality of polypeptide transfer in the mitochondrial pathway of chaperone-mediated protein folding. Biol Chem. 1998 Mar;379(3):301–309. doi: 10.1515/bchm.1998.379.3.301. [DOI] [PubMed] [Google Scholar]
  22. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  23. Horovitz A. Structural aspects of GroEL function. Curr Opin Struct Biol. 1998 Feb;8(1):93–100. doi: 10.1016/s0959-440x(98)80015-8. [DOI] [PubMed] [Google Scholar]
  24. Horwich A. L., Low K. B., Fenton W. A., Hirshfield I. N., Furtak K. Folding in vivo of bacterial cytoplasmic proteins: role of GroEL. Cell. 1993 Sep 10;74(5):909–917. doi: 10.1016/0092-8674(93)90470-b. [DOI] [PubMed] [Google Scholar]
  25. Hunt J. F., Weaver A. J., Landry S. J., Gierasch L., Deisenhofer J. The crystal structure of the GroES co-chaperonin at 2.8 A resolution. Nature. 1996 Jan 4;379(6560):37–45. doi: 10.1038/379037a0. [DOI] [PubMed] [Google Scholar]
  26. Hunt J. F., van der Vies S. M., Henry L., Deisenhofer J. Structural adaptations in the specialized bacteriophage T4 co-chaperonin Gp31 expand the size of the Anfinsen cage. Cell. 1997 Jul 25;90(2):361–371. doi: 10.1016/s0092-8674(00)80343-8. [DOI] [PubMed] [Google Scholar]
  27. Hutchinson J. P., el-Thaher T. S., Miller A. D. Refolding and recognition of mitochondrial malate dehydrogenase by Escherichia coli chaperonins cpn 60 (groEL) and cpn10 (groES). Biochem J. 1994 Sep 1;302(Pt 2):405–410. doi: 10.1042/bj3020405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Höhfeld J., Hartl F. U. Role of the chaperonin cofactor Hsp10 in protein folding and sorting in yeast mitochondria. J Cell Biol. 1994 Jul;126(2):305–315. doi: 10.1083/jcb.126.2.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lorimer G. H. A quantitative assessment of the role of the chaperonin proteins in protein folding in vivo. FASEB J. 1996 Jan;10(1):5–9. doi: 10.1096/fasebj.10.1.8566548. [DOI] [PubMed] [Google Scholar]
  30. Lorimer G. H., Todd M. J., Viitanen P. V. Chaperonins and protein folding: unity and disunity of mechanisms. Philos Trans R Soc Lond B Biol Sci. 1993 Mar 29;339(1289):297–304. doi: 10.1098/rstb.1993.0028. [DOI] [PubMed] [Google Scholar]
  31. Manning-Krieg U. C., Scherer P. E., Schatz G. Sequential action of mitochondrial chaperones in protein import into the matrix. EMBO J. 1991 Nov;10(11):3273–3280. doi: 10.1002/j.1460-2075.1991.tb04891.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Martin J., Langer T., Boteva R., Schramel A., Horwich A. L., Hartl F. U. Chaperonin-mediated protein folding at the surface of groEL through a 'molten globule'-like intermediate. Nature. 1991 Jul 4;352(6330):36–42. doi: 10.1038/352036a0. [DOI] [PubMed] [Google Scholar]
  33. Mattingly J. R., Jr, Iriarte A., Martinez-Carrion M. Homologous proteins with different affinities for groEL. The refolding of the aspartate aminotransferase isozymes at varying temperatures. J Biol Chem. 1995 Jan 20;270(3):1138–1148. doi: 10.1074/jbc.270.3.1138. [DOI] [PubMed] [Google Scholar]
  34. Mayhew M., da Silva A. C., Martin J., Erdjument-Bromage H., Tempst P., Hartl F. U. Protein folding in the central cavity of the GroEL-GroES chaperonin complex. Nature. 1996 Feb 1;379(6564):420–426. doi: 10.1038/379420a0. [DOI] [PubMed] [Google Scholar]
  35. McLennan N., Masters M. GroE is vital for cell-wall synthesis. Nature. 1998 Mar 12;392(6672):139–139. doi: 10.1038/32317. [DOI] [PubMed] [Google Scholar]
  36. Mendoza J. A., Rogers E., Lorimer G. H., Horowitz P. M. Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese. J Biol Chem. 1991 Jul 15;266(20):13044–13049. [PubMed] [Google Scholar]
  37. Neupert W. Protein import into mitochondria. Annu Rev Biochem. 1997;66:863–917. doi: 10.1146/annurev.biochem.66.1.863. [DOI] [PubMed] [Google Scholar]
  38. Robbins A. H., Stout C. D. The structure of aconitase. Proteins. 1989;5(4):289–312. doi: 10.1002/prot.340050406. [DOI] [PubMed] [Google Scholar]
  39. Roseman A. M., Chen S., White H., Braig K., Saibil H. R. The chaperonin ATPase cycle: mechanism of allosteric switching and movements of substrate-binding domains in GroEL. Cell. 1996 Oct 18;87(2):241–251. doi: 10.1016/s0092-8674(00)81342-2. [DOI] [PubMed] [Google Scholar]
  40. Rospert S., Junne T., Glick B. S., Schatz G. Cloning and disruption of the gene encoding yeast mitochondrial chaperonin 10, the homolog of E. coli groES. FEBS Lett. 1993 Dec 13;335(3):358–360. doi: 10.1016/0014-5793(93)80419-u. [DOI] [PubMed] [Google Scholar]
  41. Rospert S., Looser R., Dubaquie Y., Matouschek A., Glick B. S., Schatz G. Hsp60-independent protein folding in the matrix of yeast mitochondria. EMBO J. 1996 Feb 15;15(4):764–774. [PMC free article] [PubMed] [Google Scholar]
  42. Rospert S., Müller S., Schatz G., Glick B. S. Fusion proteins containing the cytochrome b2 presequence are sorted to the mitochondrial intermembrane space independently of hsp60. J Biol Chem. 1994 Jun 24;269(25):17279–17288. [PubMed] [Google Scholar]
  43. Saijo T., Welch W. J., Tanaka K. Intramitochondrial folding and assembly of medium-chain acyl-CoA dehydrogenase (MCAD). Demonstration of impaired transfer of K304E-variant MCAD from its complex with hsp60 to the native tetramer. J Biol Chem. 1994 Feb 11;269(6):4401–4408. [PubMed] [Google Scholar]
  44. Schmidt M., Bücheler U., Kaluza B., Buchner J. Correlation between the stability of the GroEL-protein ligand complex and the release mechanism. J Biol Chem. 1994 Nov 11;269(45):27964–27972. [PubMed] [Google Scholar]
  45. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  46. Viitanen P. V., Donaldson G. K., Lorimer G. H., Lubben T. H., Gatenby A. A. Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase. Biochemistry. 1991 Oct 8;30(40):9716–9723. doi: 10.1021/bi00104a021. [DOI] [PubMed] [Google Scholar]
  47. Weissman J. S., Hohl C. M., Kovalenko O., Kashi Y., Chen S., Braig K., Saibil H. R., Fenton W. A., Horwich A. L. Mechanism of GroEL action: productive release of polypeptide from a sequestered position under GroES. Cell. 1995 Nov 17;83(4):577–587. doi: 10.1016/0092-8674(95)90098-5. [DOI] [PubMed] [Google Scholar]
  48. Widmann M., Christen P. Differential effects of molecular chaperones on refolding of homologous proteins. FEBS Lett. 1995 Dec 27;377(3):481–484. doi: 10.1016/0014-5793(95)01406-3. [DOI] [PubMed] [Google Scholar]
  49. Xu Z., Horwich A. L., Sigler P. B. The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature. 1997 Aug 21;388(6644):741–750. doi: 10.1038/41944. [DOI] [PubMed] [Google Scholar]
  50. Yates J. R., 3rd, Eng J. K., McCormack A. L., Schieltz D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal Chem. 1995 Apr 15;67(8):1426–1436. doi: 10.1021/ac00104a020. [DOI] [PubMed] [Google Scholar]
  51. Zahn R., Lindner P., Axmann S. E., Plückthun A. Effect of single point mutations in citrate synthase on binding to GroEL. FEBS Lett. 1996 Feb 12;380(1-2):152–156. doi: 10.1016/0014-5793(96)00013-0. [DOI] [PubMed] [Google Scholar]
  52. van der Vies S. M., Gatenby A. A., Georgopoulos C. Bacteriophage T4 encodes a co-chaperonin that can substitute for Escherichia coli GroES in protein folding. Nature. 1994 Apr 14;368(6472):654–656. doi: 10.1038/368654a0. [DOI] [PubMed] [Google Scholar]

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