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. 1993 Oct 1;123(1):119–126. doi: 10.1083/jcb.123.1.119

Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix

PMCID: PMC2119817  PMID: 8408192

Abstract

To test the hypothesis that 70-kD mitochondrial heat shock protein (mt- hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome b2 (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the amino-terminal 167 residues of the precursor of cyt. b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein. In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. b2 portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function.

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Selected References

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  1. Baker K. P., Schaniel A., Vestweber D., Schatz G. A yeast mitochondrial outer membrane protein essential for protein import and cell viability. Nature. 1990 Dec 13;348(6302):605–609. doi: 10.1038/348605a0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Craig E. A., Kramer J., Kosic-Smithers J. SSC1, a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae, is essential for growth. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4156–4160. doi: 10.1073/pnas.84.12.4156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Craig E. A., Kramer J., Shilling J., Werner-Washburne M., Holmes S., Kosic-Smithers J., Nicolet C. M. SSC1, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial protein. Mol Cell Biol. 1989 Jul;9(7):3000–3008. doi: 10.1128/mcb.9.7.3000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Daum G., Gasser S. M., Schatz G. Import of proteins into mitochondria. Energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria. J Biol Chem. 1982 Nov 10;257(21):13075–13080. [PubMed] [Google Scholar]
  6. Eilers M., Schatz G. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature. 1986 Jul 17;322(6076):228–232. doi: 10.1038/322228a0. [DOI] [PubMed] [Google Scholar]
  7. Ernst V., Levin D. H., Ranu R. S., London I. M. Control of protein synthesis in reticulocyte lysates: effects of 3':5'-cyclic AMP, ATP, and GTP on inhibitions induced by hemedeficiency, double-stranded RNA, and a reticulocyte translationa inhibitor. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1112–1116. doi: 10.1073/pnas.73.4.1112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gambill B. D., Voos W., Kang P. J., Miao B., Langer T., Craig E. A., Pfanner N. A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins. J Cell Biol. 1993 Oct;123(1):109–117. doi: 10.1083/jcb.123.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Glick B. S., Beasley E. M., Schatz G. Protein sorting in mitochondria. Trends Biochem Sci. 1992 Nov;17(11):453–459. doi: 10.1016/0968-0004(92)90487-t. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Guiard B. Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L(+)-lactate cytochrome c oxidoreductase (cytochrome b2). EMBO J. 1985 Dec 1;4(12):3265–3272. doi: 10.1002/j.1460-2075.1985.tb04076.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hartl F. U., Ostermann J., Guiard B., Neupert W. Successive translocation into and out of the mitochondrial matrix: targeting of proteins to the intermembrane space by a bipartite signal peptide. Cell. 1987 Dec 24;51(6):1027–1037. doi: 10.1016/0092-8674(87)90589-7. [DOI] [PubMed] [Google Scholar]
  13. Hawlitschek G., Schneider H., Schmidt B., Tropschug M., Hartl F. U., Neupert W. Mitochondrial protein import: identification of processing peptidase and of PEP, a processing enhancing protein. Cell. 1988 Jun 3;53(5):795–806. doi: 10.1016/0092-8674(88)90096-7. [DOI] [PubMed] [Google Scholar]
  14. Horwich A. Protein import into mitochondria and peroxisomes. Curr Opin Cell Biol. 1990 Aug;2(4):625–633. doi: 10.1016/0955-0674(90)90103-l. [DOI] [PubMed] [Google Scholar]
  15. Kang P. J., Ostermann J., Shilling J., Neupert W., Craig E. A., Pfanner N. Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature. 1990 Nov 8;348(6297):137–143. doi: 10.1038/348137a0. [DOI] [PubMed] [Google Scholar]
  16. Kiebler M., Pfaller R., Söllner T., Griffiths G., Horstmann H., Pfanner N., Neupert W. Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins. Nature. 1990 Dec 13;348(6302):610–616. doi: 10.1038/348610a0. [DOI] [PubMed] [Google Scholar]
  17. Koll H., Guiard B., Rassow J., Ostermann J., Horwich A. L., Neupert W., Hartl F. U. Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space. Cell. 1992 Mar 20;68(6):1163–1175. doi: 10.1016/0092-8674(92)90086-r. [DOI] [PubMed] [Google Scholar]
  18. Lathrop J. T., Timko M. P. Regulation by heme of mitochondrial protein transport through a conserved amino acid motif. Science. 1993 Jan 22;259(5094):522–525. doi: 10.1126/science.8424176. [DOI] [PubMed] [Google Scholar]
  19. Maarse A. C., Blom J., Grivell L. A., Meijer M. MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria. EMBO J. 1992 Oct;11(10):3619–3628. doi: 10.1002/j.1460-2075.1992.tb05446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Martin J., Mahlke K., Pfanner N. Role of an energized inner membrane in mitochondrial protein import. Delta psi drives the movement of presequences. J Biol Chem. 1991 Sep 25;266(27):18051–18057. [PubMed] [Google Scholar]
  22. Neupert W., Hartl F. U., Craig E. A., Pfanner N. How do polypeptides cross the mitochondrial membranes? Cell. 1990 Nov 2;63(3):447–450. doi: 10.1016/0092-8674(90)90437-j. [DOI] [PubMed] [Google Scholar]
  23. Nicholson D. W., Köhler H., Neupert W. Import of cytochrome c into mitochondria. Cytochrome c heme lyase. Eur J Biochem. 1987 Apr 1;164(1):147–157. doi: 10.1111/j.1432-1033.1987.tb11006.x. [DOI] [PubMed] [Google Scholar]
  24. Ostermann J., Horwich A. L., Neupert W., Hartl F. U. Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature. 1989 Sep 14;341(6238):125–130. doi: 10.1038/341125a0. [DOI] [PubMed] [Google Scholar]
  25. Ostermann J., Voos W., Kang P. J., Craig E. A., Neupert W., Pfanner N. Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix. FEBS Lett. 1990 Dec 17;277(1-2):281–284. doi: 10.1016/0014-5793(90)80865-g. [DOI] [PubMed] [Google Scholar]
  26. Pace C. N. Conformational stability of globular proteins. Trends Biochem Sci. 1990 Jan;15(1):14–17. doi: 10.1016/0968-0004(90)90124-t. [DOI] [PubMed] [Google Scholar]
  27. Pfanner N., Neupert W. Distinct steps in the import of ADP/ATP carrier into mitochondria. J Biol Chem. 1987 Jun 5;262(16):7528–7536. [PubMed] [Google Scholar]
  28. Pfanner N., Rassow J., Guiard B., Söllner T., Hartl F. U., Neupert W. Energy requirements for unfolding and membrane translocation of precursor proteins during import into mitochondria. J Biol Chem. 1990 Sep 25;265(27):16324–16329. [PubMed] [Google Scholar]
  29. Pollock R. A., Hartl F. U., Cheng M. Y., Ostermann J., Horwich A., Neupert W. The processing peptidase of yeast mitochondria: the two co-operating components MPP and PEP are structurally related. EMBO J. 1988 Nov;7(11):3493–3500. doi: 10.1002/j.1460-2075.1988.tb03225.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rassow J., Guiard B., Wienhues U., Herzog V., Hartl F. U., Neupert W. Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites. J Cell Biol. 1989 Oct;109(4 Pt 1):1421–1428. doi: 10.1083/jcb.109.4.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rassow J., Hartl F. U., Guiard B., Pfanner N., Neupert W. Polypeptides traverse the mitochondrial envelope in an extended state. FEBS Lett. 1990 Nov 26;275(1-2):190–194. doi: 10.1016/0014-5793(90)81469-5. [DOI] [PubMed] [Google Scholar]
  32. Scherer P. E., Krieg U. C., Hwang S. T., Vestweber D., Schatz G. A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein. EMBO J. 1990 Dec;9(13):4315–4322. doi: 10.1002/j.1460-2075.1990.tb07880.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schneider A., Behrens M., Scherer P., Pratje E., Michaelis G., Schatz G. Inner membrane protease I, an enzyme mediating intramitochondrial protein sorting in yeast. EMBO J. 1991 Feb;10(2):247–254. doi: 10.1002/j.1460-2075.1991.tb07944.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Söllner T., Rassow J., Pfanner N. Analysis of mitochondrial protein import using translocation intermediates and specific antibodies. Methods Cell Biol. 1991;34:345–358. doi: 10.1016/s0091-679x(08)61689-1. [DOI] [PubMed] [Google Scholar]
  35. Wickner W., Driessen A. J., Hartl F. U. The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu Rev Biochem. 1991;60:101–124. doi: 10.1146/annurev.bi.60.070191.000533. [DOI] [PubMed] [Google Scholar]
  36. Witte C., Jensen R. E., Yaffe M. P., Schatz G. MAS1, a gene essential for yeast mitochondrial assembly, encodes a subunit of the mitochondrial processing protease. EMBO J. 1988 May;7(5):1439–1447. doi: 10.1002/j.1460-2075.1988.tb02961.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Xia Z. X., Mathews F. S. Molecular structure of flavocytochrome b2 at 2.4 A resolution. J Mol Biol. 1990 Apr 20;212(4):837–863. doi: 10.1016/0022-2836(90)90240-M. [DOI] [PubMed] [Google Scholar]
  38. Yang M., Jensen R. E., Yaffe M. P., Oppliger W., Schatz G. Import of proteins into yeast mitochondria: the purified matrix processing protease contains two subunits which are encoded by the nuclear MAS1 and MAS2 genes. EMBO J. 1988 Dec 1;7(12):3857–3862. doi: 10.1002/j.1460-2075.1988.tb03271.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. van Loon A. P., Brändli A. W., Schatz G. The presequences of two imported mitochondrial proteins contain information for intracellular and intramitochondrial sorting. Cell. 1986 Mar 14;44(5):801–812. doi: 10.1016/0092-8674(86)90846-9. [DOI] [PubMed] [Google Scholar]

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