Skip to main content
Genetics logoLink to Genetics
. 2003 Sep;165(1):35–45. doi: 10.1093/genetics/165.1.35

Suppression of a defect in mitochondrial protein import identifies cytosolic proteins required for viability of yeast cells lacking mitochondrial DNA.

Cory D Dunn 1, Robert E Jensen 1
PMCID: PMC1462761  PMID: 14504216

Abstract

The TIM22 complex, required for the insertion of imported polytopic proteins into the mitochondrial inner membrane, contains the nonessential Tim18p subunit. To learn more about the function of Tim18p, we screened for high-copy suppressors of the inability of tim18Delta mutants to live without mitochondrial DNA (mtDNA). We identified several genes encoding cytosolic proteins, including CCT6, SSB1, ICY1, TIP41, and PBP1, which, when overproduced, rescue the mtDNA dependence of tim18Delta cells. Furthermore, these same plasmids rescue the petite-negative phenotype of cells lacking other components of the mitochondrial protein import machinery. Strikingly, disruption of the genes identified by the different suppressors produces cells that are unable to grow without mtDNA. We speculate that loss of mtDNA leads to a lowered inner membrane potential, and subtle changes in import efficiency can no longer be tolerated. Our results suggest that increased amounts of Cct6p, Ssb1p, Icy1p, Tip41p, and Pbp1p help overcome the problems resulting from a defect in protein import.

Full Text

The Full Text of this article is available as a PDF (346.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beddoe Travis, Lithgow Trevor. Delivery of nascent polypeptides to the mitochondrial surface. Biochim Biophys Acta. 2002 Sep 2;1592(1):35–39. doi: 10.1016/s0167-4889(02)00262-8. [DOI] [PubMed] [Google Scholar]
  2. Belogrudov G. I., Lee P. T., Jonassen T., Hsu A. Y., Gin P., Clarke C. F. Yeast COQ4 encodes a mitochondrial protein required for coenzyme Q synthesis. Arch Biochem Biophys. 2001 Aug 1;392(1):48–58. doi: 10.1006/abbi.2001.2448. [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. Brachmann C. B., Davies A., Cost G. J., Caputo E., Li J., Hieter P., Boeke J. D. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast. 1998 Jan 30;14(2):115–132. doi: 10.1002/(SICI)1097-0061(19980130)14:2<115::AID-YEA204>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  5. Chen X. J., Clark-Walker G. D. Alpha and beta subunits of F1-ATPase are required for survival of petite mutants in Saccharomyces cerevisiae. Mol Gen Genet. 1999 Dec;262(4-5):898–908. doi: 10.1007/s004380051156. [DOI] [PubMed] [Google Scholar]
  6. Chen X. J., Clark-Walker G. D. The petite mutation in yeasts: 50 years on. Int Rev Cytol. 2000;194:197–238. doi: 10.1016/s0074-7696(08)62397-9. [DOI] [PubMed] [Google Scholar]
  7. Craig E. A., Jacobsen K. Mutations in cognate genes of Saccharomyces cerevisiae hsp70 result in reduced growth rates at low temperatures. Mol Cell Biol. 1985 Dec;5(12):3517–3524. doi: 10.1128/mcb.5.12.3517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crespo José L., Powers Ted, Fowler Brian, Hall Michael N. The TOR-controlled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine. Proc Natl Acad Sci U S A. 2002 May 7;99(10):6784–6789. doi: 10.1073/pnas.102687599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dupont C. H., Mazat J. P., Guerin B. The role of adenine nucleotide translocation in the energization of the inner membrane of mitochondria isolated from rho + and rho degree strains of Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1985 Nov 15;132(3):1116–1123. doi: 10.1016/0006-291x(85)91922-9. [DOI] [PubMed] [Google Scholar]
  10. Eilers M., Oppliger W., Schatz G. Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria. EMBO J. 1987 Apr;6(4):1073–1077. doi: 10.1002/j.1460-2075.1987.tb04860.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Epstein C. B., Waddle J. A., Hale W., 4th, Davé V., Thornton J., Macatee T. L., Garner H. R., Butow R. A. Genome-wide responses to mitochondrial dysfunction. Mol Biol Cell. 2001 Feb;12(2):297–308. doi: 10.1091/mbc.12.2.297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gakh Oleksandr, Cavadini Patrizia, Isaya Grazia. Mitochondrial processing peptidases. Biochim Biophys Acta. 2002 Sep 2;1592(1):63–77. doi: 10.1016/s0167-4889(02)00265-3. [DOI] [PubMed] [Google Scholar]
  13. Geissler Andreas, Chacinska Agnieszka, Truscott Kaye N., Wiedemann Nils, Brandner Katrin, Sickmann Albert, Meyer Helmut E., Meisinger Chris, Pfanner Nikolaus, Rehling Peter. The mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel. Cell. 2002 Nov 15;111(4):507–518. doi: 10.1016/s0092-8674(02)01073-5. [DOI] [PubMed] [Google Scholar]
  14. Giaever Guri, Chu Angela M., Ni Li, Connelly Carla, Riles Linda, Véronneau Steeve, Dow Sally, Lucau-Danila Ankuta, Anderson Keith, André Bruno. Functional profiling of the Saccharomyces cerevisiae genome. Nature. 2002 Jul 25;418(6896):387–391. doi: 10.1038/nature00935. [DOI] [PubMed] [Google Scholar]
  15. Giraud M. F., Velours J. The absence of the mitochondrial ATP synthase delta subunit promotes a slow growth phenotype of rho- yeast cells by a lack of assembly of the catalytic sector F1. Eur J Biochem. 1997 May 1;245(3):813–818. doi: 10.1111/j.1432-1033.1997.00813.x. [DOI] [PubMed] [Google Scholar]
  16. Goldring E. S., Grossman L. I., Krupnick D., Cryer D. R., Marmur J. The petite mutation in yeast. Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide. J Mol Biol. 1970 Sep 14;52(2):323–335. doi: 10.1016/0022-2836(70)90033-1. [DOI] [PubMed] [Google Scholar]
  17. Hallstrom T. C., Moye-Rowley W. S. Multiple signals from dysfunctional mitochondria activate the pleiotropic drug resistance pathway in Saccharomyces cerevisiae. J Biol Chem. 2000 Dec 1;275(48):37347–37356. doi: 10.1074/jbc.M007338200. [DOI] [PubMed] [Google Scholar]
  18. Hartl F. Ulrich, Hayer-Hartl Manajit. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science. 2002 Mar 8;295(5561):1852–1858. doi: 10.1126/science.1068408. [DOI] [PubMed] [Google Scholar]
  19. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  20. Hoogenraad Nicholas J., Ward Linda A., Ryan Michael T. Import and assembly of proteins into mitochondria of mammalian cells. Biochim Biophys Acta. 2002 Sep 2;1592(1):97–105. doi: 10.1016/s0167-4889(02)00268-9. [DOI] [PubMed] [Google Scholar]
  21. Hundley Heather, Eisenman Helene, Walter William, Evans Tara, Hotokezaka Yuka, Wiedmann Martin, Craig Elizabeth. The in vivo function of the ribosome-associated Hsp70, Ssz1, does not require its putative peptide-binding domain. Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4203–4208. doi: 10.1073/pnas.062048399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jacinto E., Guo B., Arndt K. T., Schmelzle T., Hall M. N. TIP41 interacts with TAP42 and negatively regulates the TOR signaling pathway. Mol Cell. 2001 Nov;8(5):1017–1026. doi: 10.1016/s1097-2765(01)00386-0. [DOI] [PubMed] [Google Scholar]
  23. Kerscher O., Holder J., Srinivasan M., Leung R. S., Jensen R. E. The Tim54p-Tim22p complex mediates insertion of proteins into the mitochondrial inner membrane. J Cell Biol. 1997 Dec 29;139(7):1663–1675. doi: 10.1083/jcb.139.7.1663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kerscher O., Sepuri N. B., Jensen R. E. Tim18p is a new component of the Tim54p-Tim22p translocon in the mitochondrial inner membrane. Mol Biol Cell. 2000 Jan;11(1):103–116. doi: 10.1091/mbc.11.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Koehler C. M., Merchant S., Schatz G. How membrane proteins travel across the mitochondrial intermembrane space. Trends Biochem Sci. 1999 Nov;24(11):428–432. doi: 10.1016/s0968-0004(99)01462-0. [DOI] [PubMed] [Google Scholar]
  26. Koehler C. M., Murphy M. P., Bally N. A., Leuenberger D., Oppliger W., Dolfini L., Junne T., Schatz G., Or E. Tim18p, a new subunit of the TIM22 complex that mediates insertion of imported proteins into the yeast mitochondrial inner membrane. Mol Cell Biol. 2000 Feb;20(4):1187–1193. doi: 10.1128/mcb.20.4.1187-1193.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kominsky D. J., Thorsness P. E. Expression of the Saccharomyces cerevisiae gene YME1 in the petite-negative yeast Schizosaccharomyces pombe converts it to petite-positive. Genetics. 2000 Jan;154(1):147–154. doi: 10.1093/genetics/154.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kominsky Douglas J., Brownson Mary P., Updike Dustin L., Thorsness Peter E. Genetic and biochemical basis for viability of yeast lacking mitochondrial genomes. Genetics. 2002 Dec;162(4):1595–1604. doi: 10.1093/genetics/162.4.1595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kovermann Peter, Truscott Kaye N., Guiard Bernard, Rehling Peter, Sepuri Naresh B., Müller Hanne, Jensen Robert E., Wagner Richard, Pfanner Nikolaus. Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel. Mol Cell. 2002 Feb;9(2):363–373. doi: 10.1016/s1097-2765(02)00446-x. [DOI] [PubMed] [Google Scholar]
  30. Kovácová V., Irmlerová J., Kovác L. Oxidative phosphorylatiion in yeast. IV. Combination of a nuclear mutation affecting oxidative phosphorylation with cytoplasmic mutation to respiratory deficiency. Biochim Biophys Acta. 1968 Aug 20;162(2):157–163. doi: 10.1016/0005-2728(68)90097-2. [DOI] [PubMed] [Google Scholar]
  31. Lee C. A., Beckwith J. Suppression of growth and protein secretion defects in Escherichia coli secA mutants by decreasing protein synthesis. J Bacteriol. 1986 Jun;166(3):878–883. doi: 10.1128/jb.166.3.878-883.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Li W. Z., Lin P., Frydman J., Boal T. R., Cardillo T. S., Richard L. M., Toth D., Lichtman M. A., Hartl F. U., Sherman F. Tcp20, a subunit of the eukaryotic TRiC chaperonin from humans and yeast. J Biol Chem. 1994 Jul 15;269(28):18616–18622. [PubMed] [Google Scholar]
  33. Lin P., Sherman F. The unique hetero-oligomeric nature of the subunits in the catalytic cooperativity of the yeast Cct chaperonin complex. Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10780–10785. doi: 10.1073/pnas.94.20.10780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Liu Z., Sekito T., Epstein C. B., Butow R. A. RTG-dependent mitochondria to nucleus signaling is negatively regulated by the seven WD-repeat protein Lst8p. EMBO J. 2001 Dec 17;20(24):7209–7219. doi: 10.1093/emboj/20.24.7209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mangus D. A., Amrani N., Jacobson A. Pbp1p, a factor interacting with Saccharomyces cerevisiae poly(A)-binding protein, regulates polyadenylation. Mol Cell Biol. 1998 Dec;18(12):7383–7396. doi: 10.1128/mcb.18.12.7383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Neupert Walter, Brunner Michael. The protein import motor of mitochondria. Nat Rev Mol Cell Biol. 2002 Aug;3(8):555–565. doi: 10.1038/nrm878. [DOI] [PubMed] [Google Scholar]
  38. Ogg S. C., Walter P. SRP samples nascent chains for the presence of signal sequences by interacting with ribosomes at a discrete step during translation elongation. Cell. 1995 Jun 30;81(7):1075–1084. doi: 10.1016/s0092-8674(05)80012-1. [DOI] [PubMed] [Google Scholar]
  39. Pause B., Diestelkötter P., Heid H., Just W. W. Cytosolic factors mediate protein insertion into the peroxisomal membrane. FEBS Lett. 1997 Sep 1;414(1):95–98. doi: 10.1016/s0014-5793(97)00975-7. [DOI] [PubMed] [Google Scholar]
  40. Plath K., Rapoport T. A. Spontaneous release of cytosolic proteins from posttranslational substrates before their transport into the endoplasmic reticulum. J Cell Biol. 2000 Oct 2;151(1):167–178. doi: 10.1083/jcb.151.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rine J. Gene overexpression in studies of Saccharomyces cerevisiae. Methods Enzymol. 1991;194:239–251. doi: 10.1016/0076-6879(91)94019-9. [DOI] [PubMed] [Google Scholar]
  42. Schleyer M., Schmidt B., Neupert W. Requirement of a membrane potential for the posttranslational transfer of proteins into mitochondria. Eur J Biochem. 1982 Jun 15;125(1):109–116. doi: 10.1111/j.1432-1033.1982.tb06657.x. [DOI] [PubMed] [Google Scholar]
  43. Schmelzle T., Hall M. N. TOR, a central controller of cell growth. Cell. 2000 Oct 13;103(2):253–262. doi: 10.1016/s0092-8674(00)00117-3. [DOI] [PubMed] [Google Scholar]
  44. Schneider J. C., Guarente L. Regulation of the yeast CYT1 gene encoding cytochrome c1 by HAP1 and HAP2/3/4. Mol Cell Biol. 1991 Oct;11(10):4934–4942. doi: 10.1128/mcb.11.10.4934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Sirrenberg C., Bauer M. F., Guiard B., Neupert W., Brunner M. Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22. Nature. 1996 Dec 12;384(6609):582–585. doi: 10.1038/384582a0. [DOI] [PubMed] [Google Scholar]
  47. Thorsness P. E., White K. H., Fox T. D. Inactivation of YME1, a member of the ftsH-SEC18-PAS1-CDC48 family of putative ATPase-encoding genes, causes increased escape of DNA from mitochondria in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Sep;13(9):5418–5426. doi: 10.1128/mcb.13.9.5418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Truscott K. N., Kovermann P., Geissler A., Merlin A., Meijer M., Driessen A. J., Rassow J., Pfanner N., Wagner R. A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23. Nat Struct Biol. 2001 Dec;8(12):1074–1082. doi: 10.1038/nsb726. [DOI] [PubMed] [Google Scholar]
  49. Voos Wolfgang, Röttgers Karin. Molecular chaperones as essential mediators of mitochondrial biogenesis. Biochim Biophys Acta. 2002 Sep 2;1592(1):51–62. doi: 10.1016/s0167-4889(02)00264-1. [DOI] [PubMed] [Google Scholar]
  50. Waldherr M., Ragnini A., Jank B., Teply R., Wiesenberger G., Schweyen R. J. A multitude of suppressors of group II intron-splicing defects in yeast. Curr Genet. 1993 Oct;24(4):301–306. doi: 10.1007/BF00336780. [DOI] [PubMed] [Google Scholar]
  51. Weber E. R., Rooks R. S., Shafer K. S., Chase J. W., Thorsness P. E. Mutations in the mitochondrial ATP synthase gamma subunit suppress a slow-growth phenotype of yme1 yeast lacking mitochondrial DNA. Genetics. 1995 Jun;140(2):435–442. doi: 10.1093/genetics/140.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wilkinson B. M., Tyson J. R., Stirling C. J. Ssh1p determines the translocation and dislocation capacities of the yeast endoplasmic reticulum. Dev Cell. 2001 Sep;1(3):401–409. doi: 10.1016/s1534-5807(01)00043-0. [DOI] [PubMed] [Google Scholar]
  53. Yamamoto Hayashi, Esaki Masatoshi, Kanamori Takashi, Tamura Yasushi, Nishikawa Shuh ichi, Endo Toshiya. Tim50 is a subunit of the TIM23 complex that links protein translocation across the outer and inner mitochondrial membranes. Cell. 2002 Nov 15;111(4):519–528. doi: 10.1016/s0092-8674(02)01053-x. [DOI] [PubMed] [Google Scholar]
  54. Young Jason C., Hoogenraad Nicholas J., Hartl F. Ulrich. Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70. Cell. 2003 Jan 10;112(1):41–50. doi: 10.1016/s0092-8674(02)01250-3. [DOI] [PubMed] [Google Scholar]
  55. Zwizinski C., Schleyer M., Neupert W. Transfer of proteins into mitochondria. Precursor to the ADP/ATP carrier binds to receptor sites on isolated mitochondria. J Biol Chem. 1983 Apr 10;258(7):4071–4074. [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES