A genome-wide synthetic dosage lethality screen reveals multiple pathways that require the functioning of ubiquitin-binding proteins Rad23 and Dsk2
Tóm tắt
Ubiquitin regulates a myriad of important cellular processes through covalent attachment to its substrates. A classic role for ubiquitin is to flag proteins for destruction by the proteasome. Recent studies indicate that ubiquitin-binding proteins (e.g. Rad23, Dsk2, Rpn10) play a pivotal role in transferring ubiquitylated proteins to the proteasome. However, the specific role of these ubiquitin receptors remains poorly defined. A key to unraveling the functions of these ubiquitin receptors is to identify their cellular substrates and biological circuits they are involved in. Although many strategies have been developed for substrate isolation, the identification of physiological targets of proteolytic pathways has proven to be quite challenging. Using a genome-wide functional screen, we have identified 11 yeast genes that cause slower growth upon their overexpression in cells lacking two ubiquitin-binding proteins Rad23 and Dsk2. Our results suggest that proper functioning of Rad23 and Dsk2 is required for efficient pheromone response, transcription, amino acid metabolism, and DNA damage response. Two proteins identified by the screen are shown to be proteolytic substrates of Dsk2, validating the large scale synthetic dosage lethality screen as a new strategy for identifying substrates of a specific degradation pathway. In conclusion, as proof-of-concept, we show that a synthetic dosage lethality screen, which is based on the toxicity induced by gene overexpression, offers an effective, complementary method to elucidating biological functions of proteolytic pathways.
Tài liệu tham khảo
DeMartino GN, Slaughter CA: The proteasome, a novel protease regulated by multiple mechanisms. J Biol Chem. 1999, 274: 22123-6. 10.1074/jbc.274.32.22123.
Finley D: Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem. 2009, 78: 477-513. 10.1146/annurev.biochem.78.081507.101607.
Kerscher O, Felberbaum R, Hochstrasser M: Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu Rev Cell Dev Biol. 2006, 22: 159-80. 10.1146/annurev.cellbio.22.010605.093503.
Madura K: Rad23 and Rpn10: perennial wallflowers join the melee. Trends Biochem Sci. 2004, 29: 637-40. 10.1016/j.tibs.2004.10.008.
Elsasser S, Finley D: Delivery of ubiquitinated substrates to protein-unfolding machines. Nat Cell Biol. 2005, 7: 742-9. 10.1038/ncb0805-742.
Kim I, Rao H: What's Ub chain linkage got to do with it?. Sci STKE. 2006, 18.
Verma R, Oania R, Graumann J, Deshaies R: Multiubiquitin Chain Receptors Define a Layer of Substrate Selectivity in the Ubiquitin-Proteasome System. Cell. 2004, 118: 99-110. 10.1016/j.cell.2004.06.014.
Richly H, Rape M, Braun S, Rumpf S, Hoege C, Jentsch S: A series of ubiquitin binding factors connects CDC48/p97 to substrate multiubiquitylation and proteasomal targeting. Cell. 2005, 120: 73-84. 10.1016/j.cell.2004.11.013.
Ravid T, Kreft SG, Hochstrasser M: Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways. Embo J. 2006, 25: 533-43. 10.1038/sj.emboj.7600946.
Bertolaet BL, Clarke DJ, Wolff M, Watson MH, Henze M, Divita G, Reed SI: UBA domains of DNA damage-inducible proteins interact with ubiquitin. Nat Struct Biol. 2001, 8: 417-422. 10.1038/87575.
Wilkinson CRM, Seeger M, Hartmann-Petersen R, Stone M, Wallace M, Semple C, Gordon C: Proteins containing the UBA domain are able to bind to multi- ubiquitin chains. Nat Cell Biol. 2001, 3: 939-943. 10.1038/ncb1001-939.
Rao H, Sastry A: Recognition of specific ubiquitin conjugates is important for the proteolytic functions of the ubiquitin-associated domain proteins Dsk2 and Rad23. J Biol Chem. 2002, 277: 11691-5. 10.1074/jbc.M200245200.
Schauber C, Chen L, Tongaonkar P, Vega I, Lambertson D, Potts W, Madura K: Rad23 links DNA repair to the ubiquitin/proteasome pathway. Nature. 1998, 391: 715-718. 10.1038/35661.
Elsasser S, Gali RR, Schwickart M, Larsen CN, Leggett DS, Muller B, Feng MT, Tubing F, Dittmar GA, Finley D: Proteasome subunit Rpn1 binds ubiquitin-like protein domains. Nat Cell Biol. 2002, 4: 725-30. 10.1038/ncb845.
Lambertson D, Chen L, Madura K: Pleiotropic defects caused by loss of the proteasome- interacting factors Rad23 and Rpn10 of Saccharomyces cerevisiae. Genetics. 1999, 153: 69-79.
Medicherla B, Kostova Z, Schaefer A, Wolf DH: A genomic screen identifies Dsk2p and Rad23p as essential components of ER-associated degradation. EMBO Rep. 2004, 5: 692-7. 10.1038/sj.embor.7400164.
Boutet SC, Disatnik MH, Chan LS, Iori K, Rando TA: Regulation of Pax3 by proteasomal degradation of monoubiquitinated protein in skeletal muscle progenitors. Cell. 2007, 130: 349-62. 10.1016/j.cell.2007.05.044.
Glockzin S, Ogi FX, Hengstermann A, Scheffner M, Blattner C: Involvement of the DNA repair protein hHR23 in p53 degradation. Mol Cell Biol. 2003, 23: 8960-9. 10.1128/MCB.23.24.8960-8969.2003.
Biggins S, Ivanovska I, Rose MD: Yeast ubiquitin-like genes are involved in duplication of the microtubule organizing center. J Cell Biol. 1996, 133: 1331-46. 10.1083/jcb.133.6.1331.
Kim I, Mi K, Rao H: Multiple interactions of rad23 suggest a mechanism for ubiquitylated substrate delivery important in proteolysis. Mol Biol Cell. 2004, 15: 3357-65. 10.1091/mbc.E03-11-0835.
Schreiner P, Chen X, Husnjak K, Randles L, Zhang N, Elsasser S, Finley D, Dikic I, Walters KJ, Groll M: Ubiquitin docking at the proteasome through a novel pleckstrin-homology domain interaction. Nature. 2008, 453: 548-52. 10.1038/nature06924.
Mayor T, Lipford JR, Graumann J, Smith GT, Deshaies RJ: Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets. Mol Cell Proteomics. 2005, 4: 741-51. 10.1074/mcp.M400220-MCP200.
Mayor T, Graumann J, Bryan J, Maccoss MJ, Deshaies RJ: Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway. Mol Cell Proteomics. 2007, 6 (11): 1885-95. 10.1074/mcp.M700264-MCP200.
Rao H, Uhlmann F, Nasmyth K, Varshavsky A: Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability. Nature. 2001, 410: 955-9. 10.1038/35073627.
Johnson ES, Ma PCM, Ota IM, Varshavsky A: A Proteolytic Pathway That Recognizes Ubiquitin as a Degradation Signal. J Biol Chem. 1995, 270: 17442-17456. 10.1074/jbc.270.29.17442.
Kim I, Ahn J, Liu C, Tanabe K, Apodaca J, Suzuki T, Rao H: The Png1-Rad23 complex regulates glycoprotein turnover. J Cell Biol. 2006, 172: 211-9. 10.1083/jcb.200507149.
Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A, Bertone P, Lan N, Jansen R, Bidlingmaier S, Houfek T, et al: Global analysis of protein activities using proteome chips. Science. 2001, 293: 2101-5. 10.1126/science.1062191.
Sopko R, Huang D, Preston N, Chua G, Papp B, Kafadar K, Snyder M, Oliver SG, Cyert M, Hughes TR, et al: Mapping pathways and phenotypes by systematic gene overexpression. Mol Cell. 2006, 21: 319-30. 10.1016/j.molcel.2005.12.011.
Tong AH, Lesage G, Bader GD, Ding H, Xu H, Xin X, Young J, Berriz GF, Brost RL, Chang M, et al: Global mapping of the yeast genetic interaction network. Science. 2004, 303: 808-13. 10.1126/science.1091317.
Ausubel FM, Brent R, Kingston RE, Moore DD, Smith JA, Seidman JG, Struhl K, eds: Current Protocols in Molecular Biology. 1996, Wiley-Interscience, New York
Liu C, Apodaca J, Davis LE, Rao H: Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells. Biotechniques. 2007, 42: 158-10.2144/000112389. 160, 162
Kroll ES, Hyland KM, Hieter P, Li JJ: Establishing genetic interactions by a synthetic dosage lethality phenotype. Genetics. 1996, 143: 95-102.
Boone C, Bussey H, Andrews BJ: Exploring genetic interactions and networks with yeast. Nat Rev Genet. 2007, 8: 437-49. 10.1038/nrg2085.
Henchoz S, Chi Y, Catarin B, Herskowitz I, Deshaies RJ, Peter M: Phosphorylation- and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Far1p in budding yeast. Genes Dev. 1997, 11: 3046-60. 10.1101/gad.11.22.3046.
Irniger S, Piatti S, Michaelis C, Nasmyth K: Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast. Cell. 1995, 81: 269-78. 10.1016/0092-8674(95)90337-2.
Bartel B, Wunning I, Varshavsky A: The recognition component of the N-end rule pathway. Embo J. 1990, 9: 3179-89.
Bachmair A, Finley D, Varshavsky A: In vivo half-life of a protein is a function of its amino-terminal residue. Science. 1986, 234: 179-186. 10.1126/science.3018930.
Wilson WA, Wang Z, Roach PJ: Systematic identification of the genes affecting glycogen storage in the yeast Saccharomyces cerevisiae: implication of the vacuole as a determinant of glycogen level. Mol Cell Proteomics. 2002, 1: 232-42. 10.1074/mcp.M100024-MCP200.
Dimmer KS, Fritz S, Fuchs F, Messerschmitt M, Weinbach N, Neupert W, Westermann B: Genetic basis of mitochondrial function and morphology in Saccharomyces cerevisiae. Mol Biol Cell. 2002, 13: 847-53. 10.1091/mbc.01-12-0588.
Chang M, Bellaoui M, Boone C, Brown GW: A genome-wide screen for methyl methanesulfonate-sensitive mutants reveals genes required for S phase progression in the presence of DNA damage. Proc Natl Acad Sci USA. 2002, 99: 16934-9. 10.1073/pnas.262669299.
Geissler S, Pereira G, Spang A, Knop M, Soues S, Kilmartin J, Schiebel E: The spindle pole body component Spc98p interacts with the gamma-tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment. Embo J. 1996, 15: 3899-911.
Matiuhin Y, Kirkpatrick DS, Ziv I, Kim W, Dakshinamurthy A, Kleifeld O, Gygi SP, Reis N, Glickman MH: Extraproteasomal Rpn10 restricts access of the polyubiquitin-binding protein Dsk2 to proteasome. Mol Cell. 2008, 32: 415-25. 10.1016/j.molcel.2008.10.011.
Varshavsky A: Regulated protein degradation. Trends Biochem Sci. 2005, 30: 283-6. 10.1016/j.tibs.2005.04.005.
Uhlmann F, Lottspeich F, Nasmyth K: Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature. 1999, 400: 37-42. 10.1038/21831.