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. 2000 Mar;154(3):1013–1023. doi: 10.1093/genetics/154.3.1013

Proteasome mutants, pre4-2 and ump1-2, suppress the essential function but not the mitochondrial RNase P function of the Saccharomyces cerevisiae gene RPM2.

M S Lutz 1, S R Ellis 1, N C Martin 1
PMCID: PMC1460975  PMID: 10757750

Abstract

The Saccharomyces cerevisiae nuclear gene RPM2 encodes a component of the mitochondrial tRNA-processing enzyme RNase P. Cells grown on fermentable carbon sources do not require mitochondrial tRNA processing activity, but still require RPM2, indicating an additional function for the Rpm2 protein. RPM2-null cells arrest after 25 generations on fermentable media. Spontaneous mutations that suppress arrest occur with a frequency of approximately 9 x 10(-6). The resultant mutants do not grow on nonfermentable carbon sources. We identified two loci responsible for this suppression, which encode proteins that influence proteasome function or assembly. PRE4 is an essential gene encoding the beta-7 subunit of the 20S proteasome core. A Val-to-Phe substitution within a highly conserved region of Pre4p that disrupts proteasome function suppresses the growth arrest of RPM2-null cells on fermentable media. The other locus, UMP1, encodes a chaperone involved in 20S proteasome assembly. A nonsense mutation in UMP1 also disrupts proteasome function and suppresses Deltarpm2 growth arrest. In an RPM2 wild-type background, pre4-2 and ump1-2 strains fail to grow at restrictive temperatures on nonfermentable carbon sources. These data link proteasome activity with Rpm2p and mitochondrial function.

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Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Arendt C. S., Hochstrasser M. Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation. Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7156–7161. doi: 10.1073/pnas.94.14.7156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Attardi G., Schatz G. Biogenesis of mitochondria. Annu Rev Cell Biol. 1988;4:289–333. doi: 10.1146/annurev.cb.04.110188.001445. [DOI] [PubMed] [Google Scholar]
  4. Campbell C. L., Tanaka N., White K. H., Thorsness P. E. Mitochondrial morphological and functional defects in yeast caused by yme1 are suppressed by mutation of a 26S protease subunit homologue. Mol Biol Cell. 1994 Aug;5(8):899–905. doi: 10.1091/mbc.5.8.899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen D. C., Yang B. C., Kuo T. T. One-step transformation of yeast in stationary phase. Curr Genet. 1992 Jan;21(1):83–84. doi: 10.1007/BF00318659. [DOI] [PubMed] [Google Scholar]
  6. Chen P., Hochstrasser M. Biogenesis, structure and function of the yeast 20S proteasome. EMBO J. 1995 Jun 1;14(11):2620–2630. doi: 10.1002/j.1460-2075.1995.tb07260.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chun K. T., Mathias N., Goebl M. G. Ubiquitin-dependent proteolysis and cell cycle control in yeast. Prog Cell Cycle Res. 1996;2:115–127. doi: 10.1007/978-1-4615-5873-6_12. [DOI] [PubMed] [Google Scholar]
  8. Fisk H. A., Yaffe M. P. A role for ubiquitination in mitochondrial inheritance in Saccharomyces cerevisiae. J Cell Biol. 1999 Jun 14;145(6):1199–1208. doi: 10.1083/jcb.145.6.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Glickman M. H., Rubin D. M., Fried V. A., Finley D. The regulatory particle of the Saccharomyces cerevisiae proteasome. Mol Cell Biol. 1998 Jun;18(6):3149–3162. doi: 10.1128/mcb.18.6.3149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Groom K. R., Heyman H. C., Steffen M. C., Hawkins L., Martin N. C. Kluyveromyces lactis SEF1 and its Saccharomyces cerevisiae homologue bypass the unknown essential function, but not the mitochondrial RNase P function, of the S. cerevisiae RPM2 gene. Yeast. 1998 Jan 15;14(1):77–87. doi: 10.1002/(SICI)1097-0061(19980115)14:1<77::AID-YEA201>3.0.CO;2-P. [DOI] [PubMed] [Google Scholar]
  11. Haas A. L., Bright P. M. The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates. J Biol Chem. 1985 Oct 15;260(23):12464–12473. [PubMed] [Google Scholar]
  12. Heinemeyer W., Fischer M., Krimmer T., Stachon U., Wolf D. H. The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing. J Biol Chem. 1997 Oct 3;272(40):25200–25209. doi: 10.1074/jbc.272.40.25200. [DOI] [PubMed] [Google Scholar]
  13. Hershko A. Roles of ubiquitin-mediated proteolysis in cell cycle control. Curr Opin Cell Biol. 1997 Dec;9(6):788–799. doi: 10.1016/s0955-0674(97)80079-8. [DOI] [PubMed] [Google Scholar]
  14. Hill K., Model K., Ryan M. T., Dietmeier K., Martin F., Wagner R., Pfanner N. Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins [see comment]. Nature. 1998 Oct 1;395(6701):516–521. doi: 10.1038/26780. [DOI] [PubMed] [Google Scholar]
  15. Hilt W., Enenkel C., Gruhler A., Singer T., Wolf D. H. The PRE4 gene codes for a subunit of the yeast proteasome necessary for peptidylglutamyl-peptide-hydrolyzing activity. Mutations link the proteasome to stress- and ubiquitin-dependent proteolysis. J Biol Chem. 1993 Feb 15;268(5):3479–3486. [PubMed] [Google Scholar]
  16. Hilt W., Wolf D. H. Proteasomes: destruction as a programme. Trends Biochem Sci. 1996 Mar;21(3):96–102. [PubMed] [Google Scholar]
  17. Kassenbrock C. K., Gao G. J., Groom K. R., Sulo P., Douglas M. G., Martin N. C. RPM2, independently of its mitochondrial RNase P function, suppresses an ISP42 mutant defective in mitochondrial import and is essential for normal growth. Mol Cell Biol. 1995 Sep;15(9):4763–4770. doi: 10.1128/mcb.15.9.4763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. King R. W., Deshaies R. J., Peters J. M., Kirschner M. W. How proteolysis drives the cell cycle. Science. 1996 Dec 6;274(5293):1652–1659. doi: 10.1126/science.274.5293.1652. [DOI] [PubMed] [Google Scholar]
  19. Kopito R. R. ER quality control: the cytoplasmic connection. Cell. 1997 Feb 21;88(4):427–430. doi: 10.1016/s0092-8674(00)81881-4. [DOI] [PubMed] [Google Scholar]
  20. Kopp F., Hendil K. B., Dahlmann B., Kristensen P., Sobek A., Uerkvitz W. Subunit arrangement in the human 20S proteasome. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2939–2944. doi: 10.1073/pnas.94.7.2939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leonhard K., Herrmann J. M., Stuart R. A., Mannhaupt G., Neupert W., Langer T. AAA proteases with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent degradation of inner membrane proteins in mitochondria. EMBO J. 1996 Aug 15;15(16):4218–4229. [PMC free article] [PubMed] [Google Scholar]
  22. Magnani M., Serafini G., Antonelli A., Malatesta M., Gazzanelli G. Evidence for a particulate location of ubiquitin conjugates and ubiquitin-conjugating enzymes in rabbit brain. J Biol Chem. 1991 Nov 5;266(31):21018–21024. [PubMed] [Google Scholar]
  23. Moczko M., Dietmeier K., Söllner T., Segui B., Steger H. F., Neupert W., Pfanner N. Identification of the mitochondrial receptor complex in Saccharomyces cerevisiae. FEBS Lett. 1992 Oct 5;310(3):265–268. doi: 10.1016/0014-5793(92)81345-m. [DOI] [PubMed] [Google Scholar]
  24. Morales M. J., Dang Y. L., Lou Y. C., Sulo P., Martin N. C. A 105-kDa protein is required for yeast mitochondrial RNase P activity. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9875–9879. doi: 10.1073/pnas.89.20.9875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Myers A. M., Pape L. K., Tzagoloff A. Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae. EMBO J. 1985 Aug;4(8):2087–2092. doi: 10.1002/j.1460-2075.1985.tb03896.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nandi D., Woodward E., Ginsburg D. B., Monaco J. J. Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor beta subunits. EMBO J. 1997 Sep 1;16(17):5363–5375. doi: 10.1093/emboj/16.17.5363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pearce D. A., Sherman F. Degradation of cytochrome oxidase subunits in mutants of yeast lacking cytochrome c and suppression of the degradation by mutation of yme1. J Biol Chem. 1995 Sep 8;270(36):20879–20882. doi: 10.1074/jbc.270.36.20879. [DOI] [PubMed] [Google Scholar]
  28. Plemper R. K., Wolf D. H. Retrograde protein translocation: ERADication of secretory proteins in health and disease. Trends Biochem Sci. 1999 Jul;24(7):266–270. doi: 10.1016/s0968-0004(99)01420-6. [DOI] [PubMed] [Google Scholar]
  29. Ramos P. C., Höckendorff J., Johnson E. S., Varshavsky A., Dohmen R. J. Ump1p is required for proper maturation of the 20S proteasome and becomes its substrate upon completion of the assembly. Cell. 1998 Feb 20;92(4):489–499. doi: 10.1016/s0092-8674(00)80942-3. [DOI] [PubMed] [Google Scholar]
  30. Rapaport D., Neupert W., Lill R. Mitochondrial protein import. Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence. J Biol Chem. 1997 Jul 25;272(30):18725–18731. doi: 10.1074/jbc.272.30.18725. [DOI] [PubMed] [Google Scholar]
  31. Rinaldi T., Bolotin-Fukuhara M., Frontali L. A Saccharomyces cerevisiae gene essential for viability has been conserved in evolution. Gene. 1995 Jul 4;160(1):135–136. doi: 10.1016/0378-1119(95)00212-o. [DOI] [PubMed] [Google Scholar]
  32. Rinaldi T., Francisci S., Zennaro E., Frontali L., Bolotin-Fukuhara M. Suppression of a mitochondrial point mutation in a tRNA gene can cast light on the mechanisms of 3' end-processing. Curr Genet. 1994 May;25(5):451–455. doi: 10.1007/BF00351785. [DOI] [PubMed] [Google Scholar]
  33. Rinaldi T., Ricci C., Porro D., Bolotin-Fukuhara M., Frontali L. A mutation in a novel yeast proteasomal gene, RPN11/MPR1, produces a cell cycle arrest, overreplication of nuclear and mitochondrial DNA, and an altered mitochondrial morphology. Mol Biol Cell. 1998 Oct;9(10):2917–2931. doi: 10.1091/mbc.9.10.2917. [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. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  36. Sherman F., Hicks J. Micromanipulation and dissection of asci. Methods Enzymol. 1991;194:21–37. doi: 10.1016/0076-6879(91)94005-w. [DOI] [PubMed] [Google Scholar]
  37. Sikorski R. S., Boeke J. D. In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol. 1991;194:302–318. doi: 10.1016/0076-6879(91)94023-6. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Smyth K. A., Belote J. M. The dominant temperature-sensitive lethal DTS7 of Drosophila melanogaster encodes an altered 20S proteasome beta-type subunit. Genetics. 1999 Jan;151(1):211–220. doi: 10.1093/genetics/151.1.211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stribinskis V., Gao G. J., Sulo P., Dang Y. L., Martin N. C. Yeast mitochondrial RNase P RNA synthesis is altered in an RNase P protein subunit mutant: insights into the biogenesis of a mitochondrial RNA-processing enzyme. Mol Cell Biol. 1996 Jul;16(7):3429–3436. doi: 10.1128/mcb.16.7.3429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Vestweber D., Brunner J., Baker A., Schatz G. A 42K outer-membrane protein is a component of the yeast mitochondrial protein import site. Nature. 1989 Sep 21;341(6239):205–209. doi: 10.1038/341205a0. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Wach A. PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae. Yeast. 1996 Mar 15;12(3):259–265. doi: 10.1002/(SICI)1097-0061(19960315)12:3%3C259::AID-YEA901%3E3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  45. Weber E. R., Hanekamp T., Thorsness P. E. Biochemical and functional analysis of the YME1 gene product, an ATP and zinc-dependent mitochondrial protease from S. cerevisiae. Mol Biol Cell. 1996 Feb;7(2):307–317. doi: 10.1091/mbc.7.2.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zennaro E., Francisci S., Ragnini A., Frontali L., Bolotin-Fukuhara M. A point mutation in a mitochondrial tRNA gene abolishes its 3' end processing. Nucleic Acids Res. 1989 Jul 25;17(14):5751–5764. doi: 10.1093/nar/17.14.5751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zhaung Z. P., McCauley R. Ubiquitin is involved in the in vitro insertion of monoamine oxidase B into mitochondrial outer membranes. J Biol Chem. 1989 Sep 5;264(25):14594–14596. [PubMed] [Google Scholar]
  48. Zieler H. A., Walberg M., Berg P. Suppression of mutations in two Saccharomyces cerevisiae genes by the adenovirus E1A protein. Mol Cell Biol. 1995 Jun;15(6):3227–3237. doi: 10.1128/mcb.15.6.3227. [DOI] [PMC free article] [PubMed] [Google Scholar]

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