A Global Census of Fission Yeast Deubiquitinating Enzyme Localization and Interaction Networks Reveals Distinct Compartmentalization Profiles and Overlapping Functions in Endocytosis and Polarity
Ubiquitination and deubiquitination are reciprocal processes that tune protein stability, function, and/or localization. The removal of ubiquitin and remodeling of ubiquitin chains is catalyzed by deubiquitinating enzymes (DUBs), which are cysteine proteases or metalloproteases. Although ubiquitination has been extensively studied for decades, the complexity of cellular roles for deubiquitinating enzymes has only recently been explored, and there are still several gaps in our understanding of when, where, and how these enzymes function to modulate the fate of polypeptides. To address these questions we performed a systematic analysis of the 20 Schizosaccharomyces pombe DUBs using confocal microscopy, proteomics, and enzymatic activity assays. Our results reveal that S. pombe DUBs are present in almost all cell compartments, and the majority are part of stable protein complexes essential for their function. Interestingly, DUB partners identified by our study include the homolog of a putative tumor suppressor gene not previously linked to the ubiquitin pathway, and two conserved tryptophan-aspartate (WD) repeat proteins that regulate Ubp9, a DUB that we show participates in endocytosis, actin dynamics, and cell polarity. In order to understand how DUB activity affects these processes we constructed multiple DUB mutants and find that a quintuple deletion of ubp4 ubp5 ubp9 ubp15 sst2/amsh displays severe growth, polarity, and endocytosis defects. This mutant allowed the identification of two common substrates for five cytoplasmic DUBs. Through these studies, a common regulatory theme emerged in which DUB localization and/or activity is modulated by interacting partners. Despite apparently distinct cytoplasmic localization patterns, several DUBs cooperate in regulating endocytosis and cell polarity. These studies provide a framework for dissecting DUB signaling pathways in S. pombe and may shed light on DUB functions in metazoans.
References
[1]
Ikeda F, Dikic I (2008) Atypical ubiquitin chains: new molecular signals. ‘Protein Modifications: Beyond the Usual Suspects’ review series. EMBO Rep 9: 536–542.
[2]
Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67: 425–479.
[3]
Reyes-Turcu F. E, Ventii K. H, Wilkinson K. D (2009) Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu Rev Biochem 78: 363–397.
[4]
Nijman S. M, Luna-Vargas M. P, Velds A, Brummelkamp T. R, Dirac A. M, et al. (2005) A genomic and functional inventory of deubiquitinating enzymes. Cell 123: 773–786.
[5]
Komander D, Clague M. J, Urbe S (2009) Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol 10: 550–563.
[6]
Verma R, Aravind L, Oania R, McDonald W. H, Yates J. R 3rd, et al. (2002) Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298: 611–615.
[7]
Yao T, Cohen R. E (2002) A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419: 403–407.
[8]
Lam Y. A, Xu W, DeMartino G. N, Cohen R. E (1997) Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature 385: 737–740.
[9]
Hanna J, Hathaway N. A, Tone Y, Crosas B, Elsasser S, et al. (2006) Deubiquitinating enzyme Ubp6 functions noncatalytically to delay proteasomal degradation. Cell 127: 99–111.
[10]
Borodovsky A, Kessler B. M, Casagrande R, Overkleeft H. S, Wilkinson K. D, et al. (2001) A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J 20: 5187–5196.
[11]
Henry K. W, Wyce A, Lo W. S, Duggan L. J, Emre N. C, et al. (2003) Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8. Genes Dev 17: 2648–2663.
[12]
Zhang X. Y, Varthi M, Sykes S. M, Phillips C, Warzecha C, et al. (2008) The putative cancer stem cell marker USP22 is a subunit of the human SAGA complex required for activated transcription and cell-cycle progression. Mol Cell 29: 102–111.
[13]
Trompouki E, Hatzivassiliou E, Tsichritzis T, Farmer H, Ashworth A, et al. (2003) CYLD is a deubiquitinating enzyme that negatively regulates NF-kappaB activation by TNFR family members. Nature 424: 793–796.
[14]
Nijman S. M, Huang T. T, Dirac A. M, Brummelkamp T. R, Kerkhoven R. M, et al. (2005) The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway. Mol Cell 17: 331–339.
[15]
Cohen M, Stutz F, Belgareh N, Haguenauer-Tsapis R, Dargemont C (2003) Ubp3 requires a cofactor, Bre5, to specifically de-ubiquitinate the COPII protein, Sec23. Nat Cell Biol 5: 661–667.
[16]
Cohen M, Stutz F, Dargemont C (2003) Deubiquitination, a new player in Golgi to endoplasmic reticulum retrograde transport. J Biol Chem 278: 51989–51992.
[17]
Ventii K. H, Wilkinson K. D (2008) Protein partners of deubiquitinating enzymes. Biochem J 414: 161–175.
[18]
Sowa M. E, Bennett E. J, Gygi S. P, Harper J. W (2009) Defining the human deubiquitinating enzyme interaction landscape. Cell 138: 389–403.
[19]
Tran H. J, Allen M. D, Lowe J, Bycroft M (2003) Structure of the Jab1/MPN domain and its implications for proteasome function. Biochemistry 42: 11460–11465.
[20]
Cope G. A, Suh G. S, Aravind L, Schwarz S. E, Zipursky S. L, et al. (2002) Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science 298: 608–611.
[21]
Zhou C, Wee S, Rhee E, Naumann M, Dubiel W, et al. (2003) Fission yeast COP9/signalosome suppresses cullin activity through recruitment of the deubiquitylating enzyme Ubp12p. Mol Cell 11: 927–938.
[22]
Iwaki T, Onishi M, Ikeuchi M, Kita A, Sugiura R, et al. (2007) Essential roles of class E Vps proteins for sorting into multivesicular bodies in Schizosaccharomyces pombe. Microbiology 153: 2753–2764.
[23]
Stone M, Hartmann-Petersen R, Seeger M, Bech-Otschir D, Wallace M, et al. (2004) Uch2/Uch37 is the major deubiquitinating enzyme associated with the 26S proteasome in fission yeast. J Mol Biol 344: 697–706.
[24]
Kim D. U, Hayles J, Kim D, Wood V, Park H. O, et al. (2010) Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe. Nat Biotechnol 28: 617–623.
[25]
Shimanuki M, Saka Y, Yanagida M, Toda T (1995) A novel essential fission yeast gene pad1+ positively regulates pap1(+)-dependent transcription and is implicated in the maintenance of chromosome structure. J Cell Sci 108(Part 2): 569–579.
[26]
Harada H, Nagai H, Tsuneizumi M, Mikami I, Sugano S, et al. (2001) Identification of DMC1, a novel gene in the TOC region on 17q25.1 that shows loss of expression in multiple human cancers. J Hum Genet 46: 90–95.
[27]
Li M, Chen D, Shiloh A, Luo J, Nikolaev A. Y, et al. (2002) Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature 416: 648–653.
[28]
Penney M, Wilkinson C, Wallace M, Javerzat J. P, Ferrell K, et al. (1998) The Pad1 gene encodes a subunit of the 26 S proteasome in fission yeast. J Biol Chem 273: 23938–23945.
[29]
Richert K, Schmidt H, Gross T, Kaufer F (2002) The deubiquitinating enzyme Ubp21p of fission yeast stabilizes a mutant form of protein kinase Prp4p. Mol Genet Genomics 267: 88–95.
[30]
Park J. H, Jensen B. C, Kifer C. T, Parsons M (2001) A novel nucleolar G-protein conserved in eukaryotes. J Cell Sci 114: 173–185.
[31]
Wilkinson C. R, Wallace M, Morphew M, Perry P, Allshire R, et al. (1998) Localization of the 26S proteasome during mitosis and meiosis in fission yeast. EMBO J 17: 6465–6476.
[32]
Li T, Naqvi N. I, Yang H, Teo T. S (2000) Identification of a 26S proteasome-associated UCH in fission yeast. Biochem Biophys Res Commun 272: 270–275.
[33]
Kaksonen M, Sun Y, Drubin D. G (2003) A pathway for association of receptors, adaptors, and actin during endocytic internalization. Cell 115: 475–487.
[34]
Kato M, Miyazawa K, Kitamura N (2000) A deubiquitinating enzyme UBPY interacts with the Src homology 3 domain of Hrs-binding protein via a novel binding motif PX(V/I)(D/N)RXXKP. J Biol Chem 275: 37481–37487.
[35]
Tanaka N, Kaneko K, Asao H, Kasai H, Endo Y, et al. (1999) Possible involvement of a novel STAM-associated molecule “AMSH” in intracellular signal transduction mediated by cytokines. J Biol Chem 274: 19129–19135.
[36]
McCullough J, Row P. E, Lorenzo O, Doherty M, Beynon R, et al. (2006) Activation of the endosome-associated ubiquitin isopeptidase AMSH by STAM, a component of the multivesicular body-sorting machinery. Curr Biol 16: 160–165.
[37]
Gachet Y, Hyams J. S (2005) Endocytosis in fission yeast is spatially associated with the actin cytoskeleton during polarised cell growth and cytokinesis. J Cell Sci 118: 4231–4242.
[38]
Losev E, Reinke C. A, Jellen J, Strongin D. E, Bevis B. J, et al. (2006) Golgi maturation visualized in living yeast. Nature 441: 1002–1006.
[39]
Vjestica A, Tang X. Z, Oliferenko S (2008) The actomyosin ring recruits early secretory compartments to the division site in fission yeast. Mol Biol Cell 19: 1125–1138.
[40]
Helmlinger D, Marguerat S, Villen J, Gygi S. P, Bahler J, et al. (2008) The S. pombe SAGA complex controls the switch from proliferation to sexual differentiation through the opposing roles of its subunits Gcn5 and Spt8. Genes Dev 22: 3184–3195.
[41]
Kohler A, Pascual-Garcia P, Llopis A, Zapater M, Posas F, et al. (2006) The mRNA export factor Sus1 is involved in Spt/Ada/Gcn5 acetyltransferase-mediated H2B deubiquitinylation through its interaction with Ubp8 and Sgf11. Mol Biol Cell 17: 4228–4236.
[42]
Kohler A, Schneider M, Cabal G. G, Nehrbass U, Hurt E (2008) Yeast Ataxin-7 links histone deubiquitination with gene gating and mRNA export. Nat Cell Biol 10: 707–715.
[43]
Leggett D. S, Hanna J, Borodovsky A, Crosas B, Schmidt M, et al. (2002) Multiple associated proteins regulate proteasome structure and function. Mol Cell 10: 495–507.
[44]
Hanna J, Meides A, Zhang D. P, Finley D (2007) A ubiquitin stress response induces altered proteasome composition. Cell 129: 747–759.
[45]
Yao T, Song L, Xu W, DeMartino G. N, Florens L, et al. (2006) Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1. Nat Cell Biol 8: 994–1002.
[46]
Qiu X. B, Ouyang S. Y, Li C. J, Miao S, Wang L, et al. (2006) hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37. EMBO J 25: 5742–5753.
[47]
Hamazaki J, Iemura S, Natsume T, Yashiroda H, Tanaka K, et al. (2006) A novel proteasome interacting protein recruits the deubiquitinating enzyme UCH37 to 26S proteasomes. EMBO J 25: 4524–4536.
[48]
Kee Y, Lyon N, Huibregtse J. M (2005) The Rsp5 ubiquitin ligase is coupled to and antagonized by the Ubp2 deubiquitinating enzyme. EMBO J 24: 2414–2424.
[49]
Lam M. H, Urban-Grimal D, Bugnicourt A, Greenblatt J. F, Haguenauer-Tsapis R, et al. (2009) Interaction of the deubiquitinating enzyme Ubp2 and the e3 ligase Rsp5 is required for transporter/receptor sorting in the multivesicular body pathway. PLoS ONE 4: e4259. doi:10.1371/journal.pone.0004259.
[50]
Kraft C, Deplazes A, Sohrmann M, Peter M (2008) Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease. Nat Cell Biol 10: 602–610.
[51]
Soncini C, Berdo I, Draetta G (2001) Ras-GAP SH3 domain binding protein (G3BP) is a modulator of USP10, a novel human ubiquitin specific protease. Oncogene 20: 3869–3879.
[52]
Dai R. M, Li C. C (2001) Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation. Nat Cell Biol 3: 740–744.
[53]
Vembar S. S, Brodsky J. L (2008) One step at a time: endoplasmic reticulum-associated degradation. Nat Rev Mol Cell Biol 9: 944–957.
[54]
Rumpf S, Jentsch S (2006) Functional division of substrate processing cofactors of the ubiquitin-selective Cdc48 chaperone. Mol Cell 21: 261–269.
[55]
Uchiyama K, Jokitalo E, Kano F, Murata M, Zhang X, et al. (2002) VCIP135, a novel essential factor for p97/p47-mediated membrane fusion, is required for Golgi and ER assembly in vivo. J Cell Biol 159: 855–866.
[56]
Ernst R, Mueller B, Ploegh H. L, Schlieker C (2009) The otubain YOD1 is a deubiquitinating enzyme that associates with p97 to facilitate protein dislocation from the ER. Mol Cell 36: 28–38.
[57]
Toya M, Iino Y, Yamamoto M (1999) Fission yeast Pob1p, which is homologous to budding yeast Boi proteins and exhibits subcellular localization close to actin patches, is essential for cell elongation and separation. Mol Biol Cell 10: 2745–2757.
[58]
Yamada K, Hirota K, Mizuno K, Shibata T, Ohta K (2008) Essential roles of Snf21, a Swi2/Snf2 family chromatin remodeler, in fission yeast mitosis. Genes Genet Syst 83: 361–372.
[59]
Monahan B. J, Villen J, Marguerat S, Bahler J, Gygi S. P, et al. (2008) Fission yeast SWI/SNF and RSC complexes show compositional and functional differences from budding yeast. Nat Struct Mol Biol 15: 873–880.
[60]
Miki F, Kurabayashi A, Tange Y, Okazaki K, Shimanuki M, et al. (2004) Two-hybrid search for proteins that interact with Sad1 and Kms1, two membrane-bound components of the spindle pole body in fission yeast. Mol Genet Genomics 270: 449–461.
[61]
Kaksonen M, Toret C. P, Drubin D. G (2006) Harnessing actin dynamics for clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 7: 404–414.
[62]
Castagnetti S, Behrens R, Nurse P (2005) End4/Sla2 is involved in establishment of a new growth zone in Schizosaccharomyces pombe. J Cell Sci 118: 1843–1850.
[63]
Sirotkin V, Beltzner C. C, Marchand J. B, Pollard T. D (2005) Interactions of WASp, myosin-I, and verprolin with Arp2/3 complex during actin patch assembly in fission yeast. J Cell Biol 170: 637–648.
[64]
Amerik A. Y, Li S. J, Hochstrasser M (2000) Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae. Biol Chem 381: 981–992.
[65]
Acconcia F, Sigismund S, Polo S (2009) Ubiquitin in trafficking: the network at work. Exp Cell Res 315: 1610–1618.
[66]
Matsuyama A, Arai R, Yashiroda Y, Shirai A, Kamata A, et al. (2006) ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nat Biotechnol 24: 841–847.
[67]
Nanao M. H, Tcherniuk S. O, Chroboczek J, Dideberg O, Dessen A, et al. (2004) Crystal structure of human otubain 2. EMBO Rep 5: 783–788.
[68]
Soboleva T. A, Jans D. A, Johnson-Saliba M, Baker R. T (2005) Nuclear-cytoplasmic shuttling of the oncogenic mouse UNP/USP4 deubiquitylating enzyme. J Biol Chem 280: 745–752.
[69]
Komander D, Lord C. J, Scheel H, Swift S, Hofmann K, et al. (2008) The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module. Mol Cell 29: 451–464.
[70]
Amerik A, Sindhi N, Hochstrasser M (2006) A conserved late endosome-targeting signal required for Doa4 deubiquitylating enzyme function. J Cell Biol 175: 825–835.
[71]
Richter C, West M, Odorizzi G (2007) Dual mechanisms specify Doa4-mediated deubiquitination at multivesicular bodies. EMBO J 26: 2454–2464.
[72]
Kimura Y, Yashiroda H, Kudo T, Koitabashi S, Murata S, et al. (2009) An inhibitor of a deubiquitinating enzyme regulates ubiquitin homeostasis. Cell 137: 549–559.
[73]
Row P. E, Liu H, Hayes S, Welchman R, Charalabous P, et al. (2007) The MIT domain of UBPY constitutes a CHMP binding and endosomal localization signal required for efficient epidermal growth factor receptor degradation. J Biol Chem 282: 30929–30937.
[74]
Li M, Brooks C. L, Kon N, Gu W (2004) A dynamic role of HAUSP in the p53-Mdm2 pathway. Mol Cell 13: 879–886.
[75]
Song M. S, Salmena L, Carracedo A, Egia A, Lo-Coco F, et al. (2008) The deubiquitinylation and localization of PTEN are regulated by a HAUSP-PML network. Nature 455: 813–817.
[76]
Ho Y, Gruhler A, Heilbut A, Bader G. D, Moore L, et al. (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415: 180–183.
[77]
Krogan N. J, Cagney G, Yu H, Zhong G, Guo X, et al. (2006) Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440: 637–643.
[78]
Cohn M. A, Kee Y, Haas W, Gygi S. P, D'Andrea A. D (2009) UAF1 is a subunit of multiple deubiquitinating enzyme complexes. J Biol Chem 284: 5343–5351.
[79]
Kee Y, Yang K, Cohn M. A, Haas W, Gygi S. P, et al. (2010) WDR20 regulates activity of the USP12 x UAF1 deubiquitinating enzyme complex. J Biol Chem 285: 11252–11257.
[80]
Prentice H. L (1992) High efficiency transformation of Schizosaccharomyces pombe by electroporation. Nucleic Acids Res 20: 621.
[81]
Moreno S, Klar A, Nurse P (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol 194: 795–823.
[82]
Bahler J, Wu J. Q, Longtine M. S, Shah N. G, McKenzie A 3rd, et al. (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14: 943–951.
[83]
Sandblad L, Busch K. E, Tittmann P, Gross H, Brunner D, et al. (2006) The Schizosaccharomyces pombe EB1 homolog Mal3p binds and stabilizes the microtubule lattice seam. Cell 127: 1415–1424.
[84]
Keeney J. B, Boeke J. D (1994) Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe. Genetics 136: 849–856.
[85]
Gould K. L, Moreno S, Owen D. J, Sazer S, Nurse P (1991) Phosphorylation at Thr167 is required for Schizosaccharomyces pombe p34cdc2 function. EMBO J 10: 3297–3309.
[86]
Wolfe B. A, McDonald W. H, Yates J. R 3rd, Gould K. L (2006) Phospho-regulation of the Cdc14/Clp1 phosphatase delays late mitotic events in S. pombe. Dev Cell 11: 423–430.
[87]
Tagwerker C, Flick K, Cui M, Guerrero C, Dou Y, et al. (2006) A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivocross-linking. Mol Cell Proteomics 5: 737–748.
[88]
Tasto J. J, Carnahan R. H, McDonald W. H, Gould K. L (2001) Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe. Yeast 18: 657–662.
[89]
McDonald W. H, Ohi R, Miyamoto D. T, Mitchison T. J, Yates J. R 3rd (2002) Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT. Int J Mass Spectrom 219: 245–251.
[90]
Yates J. R 3rd, Eng J. K, McCormack A. L, Schieltz D (1995) Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal Chem 67: 1426–1436.
[91]
McDonald W. H, Tabb D. L, Sadygov R. G, MacCoss M. J, Venable J, et al. (2004) MS1, MS2, and SQT-three unified, compact, and easily parsed file formats for the storage of shotgun proteomic spectra and identifications. Rapid Commun Mass Spectrom 18: 2162–2168.
[92]
Zhang B, Chambers M. C, Tabb D. L (2007) Proteomic parsimony through bipartite graph analysis improves accuracy and transparency. J Proteome Res 6: 3549–3557.
[93]
Ma Z. Q, Dasari S, Chambers M. C, Litton M. D, Sobecki S. M, et al. (2009) IDPicker 2.0: Improved protein assembly with high discrimination peptide identification filtering. J Proteome Res 8: 3872–3881.
[94]
Stark C, Breitkreutz B. J, Reguly T, Boucher L, Breitkreutz A, et al. (2006) BioGRID: a general repository for interaction datasets. Nucleic Acids Res 34: D535–D539.
[95]
Shannon P, Markiel A, Ozier O, Baliga N. S, Wang J. T, et al. (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13: 2498–2504.
[96]
Vida T. A, Emr S. D (1995) A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128: 779–792.
[97]
Jourdain I, Gachet Y, Hyams J. S (2009) The dynamin related protein Dnm1 fragments mitochondria in a microtubule-dependent manner during the fission yeast cell cycle. Cell Motil Cytoskeleton 66: 509–523.
[98]
Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16: 10881–10890.
