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. 1997 Mar;145(3):595–603. doi: 10.1093/genetics/145.3.595

Mdp1, a Saccharomyces Cerevisiae Gene Involved in Mitochondrial/Cytoplasmic Protein Distribution, Is Identical to the Ubiquitin-Protein Ligase Gene Rsp5

T Zoladek 1, A Tobiasz 1, G Vaduva 1, M Boguta 1, N C Martin 1, A K Hopper 1
PMCID: PMC1207845  PMID: 9055070

Abstract

Alteration of the subcellular distribution of Mod5p-I, a tRNA modification enzyme, member of the sorting isozyme family, affects tRNA-mediated nonsense suppression. Altered suppression efficiency was used to identify MDP genes, which, when mutant, change the mitochondrial/cytosolic distribution of Mod5p-I,KR6. MDP2 is the previously identified VRP1, which encodes verprolin, required for proper organization of the actin cytoskeleton. MDP3 is identical to PAN1, which encodes a protein involved in initiation of translation and actin cytoskeleton organization. We report here the cloning and characterization of wild-type and mutant MDP1 alleles and the isolation and characterization of a multicopy suppressor of mdp1 mutations. MDP1 is identical to RSP5, which encodes ubiquitin-protein ligase, and mdp1 mutations are suppressed by high copy expression of ubiquitin. All four characterized mdp1 mutations cause missense changes located in the hect domain of Rsp5p that is highly conserved among ubiquitin-protein ligases. In addition to its well-known function in protein turnover, ubiquitination has been proposed to play roles in subcellular sorting of proteins via endocytosis and in delivery of proteins to peroxisomes, the endoplasmic reticulum and mitochondria. mdp1, as well as mdp2/vrp1 and mdp3/pan1 mutations, affect endocytosis. Further, mdp1 mutations show synthetic interactions with mdp2/vrp1 and mdp3/pan1. Identification of MDP1 as RSP5, along with our previous identification of MDP2/VRP1 and MDP3/PAN1, implicate interactions of the ubiquitin system, the actin cytoskeleton and protein synthesis in the subcellular distribution of proteins.

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Selected References

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  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. André B., Springael J. Y. WWP, a new amino acid motif present in single or multiple copies in various proteins including dystrophin and the SH3-binding Yes-associated protein YAP65. Biochem Biophys Res Commun. 1994 Dec 15;205(2):1201–1205. doi: 10.1006/bbrc.1994.2793. [DOI] [PubMed] [Google Scholar]
  3. Azzi A., Boscoboinik D., Hensey C. The protein kinase C family. Eur J Biochem. 1992 Sep 15;208(3):547–557. doi: 10.1111/j.1432-1033.1992.tb17219.x. [DOI] [PubMed] [Google Scholar]
  4. Bork P., Sudol M. The WW domain: a signalling site in dystrophin? Trends Biochem Sci. 1994 Dec;19(12):531–533. doi: 10.1016/0968-0004(94)90053-1. [DOI] [PubMed] [Google Scholar]
  5. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Chen Z. J., Parent L., Maniatis T. Site-specific phosphorylation of IkappaBalpha by a novel ubiquitination-dependent protein kinase activity. Cell. 1996 Mar 22;84(6):853–862. doi: 10.1016/s0092-8674(00)81064-8. [DOI] [PubMed] [Google Scholar]
  8. Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell. 1994 Oct 7;79(1):13–21. doi: 10.1016/0092-8674(94)90396-4. [DOI] [PubMed] [Google Scholar]
  9. Clark J. D., Lin L. L., Kriz R. W., Ramesha C. S., Sultzman L. A., Lin A. Y., Milona N., Knopf J. L. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell. 1991 Jun 14;65(6):1043–1051. doi: 10.1016/0092-8674(91)90556-e. [DOI] [PubMed] [Google Scholar]
  10. Crane D. I., Kalish J. E., Gould S. J. The Pichia pastoris PAS4 gene encodes a ubiquitin-conjugating enzyme required for peroxisome assembly. J Biol Chem. 1994 Aug 26;269(34):21835–21844. [PubMed] [Google Scholar]
  11. DiDonato J., Mercurio F., Rosette C., Wu-Li J., Suyang H., Ghosh S., Karin M. Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation. Mol Cell Biol. 1996 Apr;16(4):1295–1304. doi: 10.1128/mcb.16.4.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Donnelly S. F., Pocklington M. J., Pallotta D., Orr E. A proline-rich protein, verprolin, involved in cytoskeletal organization and cellular growth in the yeast Saccharomyces cerevisiae. Mol Microbiol. 1993 Nov;10(3):585–596. doi: 10.1111/j.1365-2958.1993.tb00930.x. [DOI] [PubMed] [Google Scholar]
  13. Dulic V., Egerton M., Elguindi I., Raths S., Singer B., Riezman H. Yeast endocytosis assays. Methods Enzymol. 1991;194:697–710. doi: 10.1016/0076-6879(91)94051-d. [DOI] [PubMed] [Google Scholar]
  14. Ecker D. J., Khan M. I., Marsh J., Butt T. R., Crooke S. T. Chemical synthesis and expression of a cassette adapted ubiquitin gene. J Biol Chem. 1987 Mar 15;262(8):3524–3527. [PubMed] [Google Scholar]
  15. Egner R., Kuchler K. The yeast multidrug transporter Pdr5 of the plasma membrane is ubiquitinated prior to endocytosis and degradation in the vacuole. FEBS Lett. 1996 Jan 8;378(2):177–181. doi: 10.1016/0014-5793(95)01450-0. [DOI] [PubMed] [Google Scholar]
  16. Eisenmann D. M., Arndt K. M., Ricupero S. L., Rooney J. W., Winston F. SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae. Genes Dev. 1992 Jul;6(7):1319–1331. doi: 10.1101/gad.6.7.1319. [DOI] [PubMed] [Google Scholar]
  17. Galan J. M., Moreau V., Andre B., Volland C., Haguenauer-Tsapis R. Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease. J Biol Chem. 1996 May 3;271(18):10946–10952. doi: 10.1074/jbc.271.18.10946. [DOI] [PubMed] [Google Scholar]
  18. Galan J. M., Volland C., Urban-Grimal D., Haguenauer-Tsapis R. The yeast plasma membrane uracil permease is stabilized against stress induced degradation by a point mutation in a cyclin-like "destruction box". Biochem Biophys Res Commun. 1994 Jun 15;201(2):769–775. doi: 10.1006/bbrc.1994.1767. [DOI] [PubMed] [Google Scholar]
  19. Hein C., Springael J. Y., Volland C., Haguenauer-Tsapis R., André B. NPl1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase. Mol Microbiol. 1995 Oct;18(1):77–87. doi: 10.1111/j.1365-2958.1995.mmi_18010077.x. [DOI] [PubMed] [Google Scholar]
  20. Hicke L., Riezman H. Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell. 1996 Jan 26;84(2):277–287. doi: 10.1016/s0092-8674(00)80982-4. [DOI] [PubMed] [Google Scholar]
  21. Hochstrasser M. Protein degradation or regulation: Ub the judge. Cell. 1996 Mar 22;84(6):813–815. doi: 10.1016/s0092-8674(00)81058-2. [DOI] [PubMed] [Google Scholar]
  22. Ireland L. S., Johnston G. C., Drebot M. A., Dhillon N., DeMaggio A. J., Hoekstra M. F., Singer R. A. A member of a novel family of yeast 'zn-finger' proteins mediates the transition from stationary phase to cell proliferation. EMBO J. 1994 Aug 15;13(16):3812–3821. doi: 10.1002/j.1460-2075.1994.tb06692.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jentsch S. The ubiquitin-conjugation system. Annu Rev Genet. 1992;26:179–207. doi: 10.1146/annurev.ge.26.120192.001143. [DOI] [PubMed] [Google Scholar]
  24. Kumar S., Tomooka Y., Noda M. Identification of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem Biophys Res Commun. 1992 Jun 30;185(3):1155–1161. doi: 10.1016/0006-291x(92)91747-e. [DOI] [PubMed] [Google Scholar]
  25. Kölling R., Hollenberg C. P. The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants. EMBO J. 1994 Jul 15;13(14):3261–3271. doi: 10.1002/j.1460-2075.1994.tb06627.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Marchuk D., Drumm M., Saulino A., Collins F. S. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 1991 Mar 11;19(5):1154–1154. doi: 10.1093/nar/19.5.1154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mihara K., Omura T. Cytoplasmic chaperones in precursor targeting to mitochondria: the role of MSF and hsp 70. Trends Cell Biol. 1996 Mar;6(3):104–108. doi: 10.1016/0962-8924(96)81000-2. [DOI] [PubMed] [Google Scholar]
  28. Munn A. L., Stevenson B. J., Geli M. I., Riezman H. end5, end6, and end7: mutations that cause actin delocalization and block the internalization step of endocytosis in Saccharomyces cerevisiae. Mol Biol Cell. 1995 Dec;6(12):1721–1742. doi: 10.1091/mbc.6.12.1721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nefsky B., Beach D. Pub1 acts as an E6-AP-like protein ubiquitiin ligase in the degradation of cdc25. EMBO J. 1996 Mar 15;15(6):1301–1312. [PMC free article] [PubMed] [Google Scholar]
  30. Pfanner N., Douglas M. G., Endo T., Hoogenraad N. J., Jensen R. E., Meijer M., Neupert W., Schatz G., Schmitz U. K., Shore G. C. Uniform nomenclature for the protein transport machinery of the mitochondrial membranes. Trends Biochem Sci. 1996 Feb;21(2):51–52. [PubMed] [Google Scholar]
  31. Riles L., Dutchik J. E., Baktha A., McCauley B. K., Thayer E. C., Leckie M. P., Braden V. V., Depke J. E., Olson M. V. Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs. Genetics. 1993 May;134(1):81–150. doi: 10.1093/genetics/134.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Sommer T., Jentsch S. A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature. 1993 Sep 9;365(6442):176–179. doi: 10.1038/365176a0. [DOI] [PubMed] [Google Scholar]
  34. Tang H. Y., Cai M. The EH-domain-containing protein Pan1 is required for normal organization of the actin cytoskeleton in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Sep;16(9):4897–4914. doi: 10.1128/mcb.16.9.4897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Taussig R., Carlson M. Nucleotide sequence of the yeast SUC2 gene for invertase. Nucleic Acids Res. 1983 Mar 25;11(6):1943–1954. doi: 10.1093/nar/11.6.1943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Verner K., Schatz G. Protein translocation across membranes. Science. 1988 Sep 9;241(4871):1307–1313. doi: 10.1126/science.2842866. [DOI] [PubMed] [Google Scholar]
  37. Wiebel F. F., Kunau W. H. The Pas2 protein essential for peroxisome biogenesis is related to ubiquitin-conjugating enzymes. Nature. 1992 Sep 3;359(6390):73–76. doi: 10.1038/359073a0. [DOI] [PubMed] [Google Scholar]
  38. von Heijne G. Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 1986 Jun;5(6):1335–1342. doi: 10.1002/j.1460-2075.1986.tb04364.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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