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Mutational Uncoupling of the Role of Sus1 in Nuclear Pore Complex Targeting of an mRNA Export Complex and Histone H2B Deubiquitination

2009; Elsevier BV; Volume: 284; Issue: 18 Linguagem: Inglês

10.1074/jbc.m900502200

ISSN

1083-351X

Autores

Christoph Klöckner, Maren Schneider, Sheila Lutz, Divyang Jani, Dieter Kressler, Murray Stewart, Ed Hurt, Alwin Köhler,

Tópico(s)

Genomics and Chromatin Dynamics

Resumo

Sus1 is an evolutionary conserved protein that functions both in transcription and mRNA export and has been proposed to contribute to coupling these processes in yeast. Sus1 mediates its different roles as a component of both the histone H2B deubiquitinating module (Sus1-Sgf11-Ubp8-Sgf73) of the SAGA (Spt-Ada-Gcn5 acetyltransferase) transcriptional co-activator and the mRNA export complex, TREX-2 (Sus1-Sac3-Thp1-Cdc31). We have dissected the different functions of Sus1 with respect to its partitioning in transcription and export complexes using a mutational approach. Here we show that the sus1–10 (E18A, S19A, and G20A) and sus1–12 (V73A and D75A) alleles of Sus1 can be dissociated from TREX-2 while leaving its interaction with SAGA largely intact. Conversely, the binding to both TREX-2 and SAGA was impaired in the sus1–11 allele (G37A and W38A), in which two highly conserved residues were mutated. In vitro experiments demonstrated that dissociation of mutant Sus1 from its partners is caused by a reduced affinity toward the TREX-2 subunit, Sac3, and the SAGA factor, Sgf11, respectively. Consistent with the biochemical data, these sus1 mutant alleles showed differential genetic relationships with SAGA and mRNA export mutants. In vivo, all three sus1 mutants were impaired in targeting TREX-2 (i.e. Sac3) to the nuclear pore complexes and exhibited nuclear mRNA export defects. This study has implications for how Sus1, in combination with distinct interaction partners, can regulate diverse aspects of gene expression. Sus1 is an evolutionary conserved protein that functions both in transcription and mRNA export and has been proposed to contribute to coupling these processes in yeast. Sus1 mediates its different roles as a component of both the histone H2B deubiquitinating module (Sus1-Sgf11-Ubp8-Sgf73) of the SAGA (Spt-Ada-Gcn5 acetyltransferase) transcriptional co-activator and the mRNA export complex, TREX-2 (Sus1-Sac3-Thp1-Cdc31). We have dissected the different functions of Sus1 with respect to its partitioning in transcription and export complexes using a mutational approach. Here we show that the sus1–10 (E18A, S19A, and G20A) and sus1–12 (V73A and D75A) alleles of Sus1 can be dissociated from TREX-2 while leaving its interaction with SAGA largely intact. Conversely, the binding to both TREX-2 and SAGA was impaired in the sus1–11 allele (G37A and W38A), in which two highly conserved residues were mutated. In vitro experiments demonstrated that dissociation of mutant Sus1 from its partners is caused by a reduced affinity toward the TREX-2 subunit, Sac3, and the SAGA factor, Sgf11, respectively. Consistent with the biochemical data, these sus1 mutant alleles showed differential genetic relationships with SAGA and mRNA export mutants. In vivo, all three sus1 mutants were impaired in targeting TREX-2 (i.e. Sac3) to the nuclear pore complexes and exhibited nuclear mRNA export defects. This study has implications for how Sus1, in combination with distinct interaction partners, can regulate diverse aspects of gene expression. Gene expression machineries are functionally and physically coupled to ensure that transcription, RNA processing, RNA quality control, and nuclear mRNA export take place with high fidelity and efficiency (1Maniatis T. Reed R. Nature. 2002; 416: 499-506Crossref PubMed Scopus (920) Google Scholar, 2Köhler A. Hurt E. Nat. Rev. Mol. Cell Biol. 2007; 8: 761-773Crossref PubMed Scopus (553) Google Scholar, 3Luna R. Gaillard H. Gonzalez-Aguilera C. Aguilera A. Chromosoma. 2008; 117: 319-331Crossref PubMed Scopus (88) Google Scholar, 4Iglesias N. Stutz F. 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Studies in Saccharomyces cerevisiae have shown that nuclear pore complexes (NPCs) 2The abbreviations used are: NPC, nuclear pore complex; SAGA, Spt-Ada-Gcn5 acetyltransferase; DUB, deubiquitinating; GST, glutathione S-transferase; MES, 4-morpholineethanesulfonic acid; 5-FOA, 5-fluoroorotic acid; MOPS, 4-morpholinepropanesulfonic acid; TAP, tandem affinity purification; CID, Cdc31 interaction domain. 2The abbreviations used are: NPC, nuclear pore complex; SAGA, Spt-Ada-Gcn5 acetyltransferase; DUB, deubiquitinating; GST, glutathione S-transferase; MES, 4-morpholineethanesulfonic acid; 5-FOA, 5-fluoroorotic acid; MOPS, 4-morpholinepropanesulfonic acid; TAP, tandem affinity purification; CID, Cdc31 interaction domain. mediate tethering of activated genes to the nuclear periphery, thereby providing a platform for the integration of transcription and mRNA export (7Cabal G.G. Genovesio A. Rodriguez-Navarro S. Zimmer C. Gadal O. Lesne A. Buc H. Feuerbach-Fournier F. Olivo-Marin J.C. Hurt E.C. 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Sus1 was proposed to play a role in transcription-coupled mRNA export because of its presence in the SAGA transcriptional co-activator and the NPC-based TREX-2 mRNA exporter (14Rodriguez-Navarro S. Fischer T. Luo M.J. Antunez O. Brettschneider S. Lechner J. Perez-Ortin J.E. Reed R. Hurt E. Cell. 2004; 116: 75-86Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Moreover, both complexes are involved in the repositioning of the activated GAL1 gene (and possibly other genes) to the NPCs (7Cabal G.G. Genovesio A. Rodriguez-Navarro S. Zimmer C. Gadal O. Lesne A. Buc H. Feuerbach-Fournier F. Olivo-Marin J.C. Hurt E.C. Nehrbass U. Nature. 2006; 441: 770-773Crossref PubMed Scopus (366) Google Scholar, 8Chekanova J.A. Abruzzi K.C. Rosbash M. Belostotsky D.A. RNA (N. Y.). 2008; 14: 66-77Crossref PubMed Scopus (48) Google Scholar, 15Köhler A. Schneider M. Cabal G.G. Nehrbass U. Hurt E. Nat. Cell Biol. 2008; 10: 707-715Crossref PubMed Scopus (160) Google Scholar).SAGA (Spt-Ada-Gcn5 acetyltransferase) functions include histone acetylation and deubiquitination, nucleosome remodeling activity, and interactions with gene-specific activators and general transcription factors (16Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (875) Google Scholar, 17Larschan E. Winston F. Genes Dev. 2001; 15: 1946-1956Crossref PubMed Scopus (243) Google Scholar, 18Daniel J.A. Grant P.A. Mutat. Res. 2007; 618: 135-148Crossref PubMed Scopus (80) Google Scholar). Within SAGA, the 96-residue protein Sus1 has been shown to be an integral part of a tetrameric histone H2B-deubiquitinating (DUB) module (15Köhler A. Schneider M. Cabal G.G. Nehrbass U. Hurt E. Nat. Cell Biol. 2008; 10: 707-715Crossref PubMed Scopus (160) Google Scholar, 19Shukla A. Stanojevic N. 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The DUB module contains the protease Ubp8, Sus1, the small zinc finger protein Sgf11, and Sgf73. Sgf73 is the adaptor protein that anchors Sus1-Sgf11-Ubp8 at its N terminus and connects it to SAGA. Deletion of either SGF11 or SUS1 results in the dissociation of Ubp8 from Sgf73, implicating these proteins in the structural integrity of the DUB module (20Ingvarsdottir K. Krogan N.J. Emre N.C. Wyce A. Thompson N.J. Emili A. Hughes T.R. Greenblatt J.F. Berger S.L. Mol. Cell. Biol. 2005; 25: 1162-1172Crossref PubMed Scopus (116) Google Scholar, 21Lee K.K. Florens L. Swanson S.K. Washburn M.P. Workman J.L. Mol. Cell. Biol. 2005; 25: 1173-1182Crossref PubMed Scopus (131) Google Scholar, 25Köhler A. Pascual-Garcia P. Llopis A. Zapater M. Posas F. Hurt E. Rodriguez-Navarro S. Mol. Biol. Cell. 2006; 17: 4228-4236Crossref PubMed Scopus (102) Google Scholar). Importantly, Sgf73 together with Sus1 and Sgf11 are required for activation of Ubp8, which by itself is enzymatically inactive. The precise architecture of the DUB module has not been determined so far; however, Sus1 was shown to interact directly with the 99-residue protein Sgf11 both in vivo and in vitro (15Köhler A. Schneider M. Cabal G.G. Nehrbass U. Hurt E. Nat. Cell Biol. 2008; 10: 707-715Crossref PubMed Scopus (160) Google Scholar). Overall, a regulated cycle of histone H2B ubiquitin addition (catalyzed by Rad6/Bre1) and removal (Ubp8) at the promoter and coding region of a gene triggers multiple steps of gene activation and influences both transcription initiation and elongation (26Weake V.M. Workman J.L. Mol. Cell. 2008; 29: 653-663Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 27Wyce A. Xiao T. Whelan K.A. Kosman C. Walter W. Eick D. Hughes T.R. Krogan N.J. Strahl B.D. Berger S.L. Mol. Cell. 2007; 27: 275-288Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 28Fleming A.B. Kao C.F. Hillyer C. Pikaart M. Osley M.A. Mol. Cell. 2008; 31: 57-66Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 29Pavri R. Zhu B. Li G. Trojer P. Mandal S. Shilatifard A. Reinberg D. Cell. 2006; 125: 703-717Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 30Pascual-Garcia P. Govind C.K. Queralt E. Cuenca-Bono B. Llopis A. Chavez S. Hinnebusch A.G. Rodriguez-Navarro S. Genes Dev. 2008; 22: 2811-2822Crossref PubMed Scopus (83) Google Scholar). Moreover, recent work has shown that SAGA-associated Sgf73 creates a link to TREX-2 by regulating TREX-2 assembly or stability, possibly through a chaperone activity (15Köhler A. Schneider M. Cabal G.G. Nehrbass U. Hurt E. Nat. Cell Biol. 2008; 10: 707-715Crossref PubMed Scopus (160) Google Scholar). Specifically, Sgf73 is required for recruiting the TREX-2 factors Sac3 and Thp1 to SAGA and promotes the association of Sus1 with a distinct domain of Sac3. This assembly event is critical for TREX-2 function (see below).The other Sus1-containing complex, TREX-2, functions in mRNA export as well as promoting transcription elongation and preventing DNA:RNA hybrid formation and genome instability (31Fischer T. Strässer K. Racz A. Rodriguez-Navarro S. Oppizzi M. Ihrig P. Lechner J. Hurt E. EMBO J. 2002; 21: 5843-5852Crossref PubMed Scopus (222) Google Scholar, 32Fischer T. Rodriguez-Navarro S. Pereira G. Racz A. Schiebel E. Hurt E. Nat. Cell Biol. 2004; 6: 840-848Crossref PubMed Scopus (141) Google Scholar, 33Jones A.L. Quimby B.B. Hood J.K. Ferrigno P. Keshava P.H. Silver P.A. Corbett A.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3224-3229Crossref PubMed Scopus (36) Google Scholar, 34Lei P. Stern C.A. Fahrenkrog B. Krebber H. Moy T.I. Aebi U. Silver P.A. Mol. Biol. Cell. 2003; 14: 836-847Crossref PubMed Scopus (61) Google Scholar, 35Gonzalez-Aguilera C. Tous C. Gomez-Gonzalez B. Huertas P. Luna R. Aguilera A. Mol. Biol. Cell. 2008; 19: 4310-4318Crossref PubMed Scopus (117) Google Scholar, 36Bauer A. Kölling R. J. Cell Sci. 1996; 109: 1575-1583PubMed Google Scholar). TREX-2 is composed of Sac3, Thp1, Cdc31, and Sus1. Recently, Sem1 was described as an additional TREX-2 subunit, but it is still unclear whether Sem1 is a stoichiometric component (37Wilmes G.M. Bergkessel M. Bandyopadhyay S. Shales M. Braberg H. Cagney G. Collins S.R. Whitworth G.B. Kress T.L. Weissman J.S. Ideker T. Guthrie C. Krogan N.J. Mol. Cell. 2008; 32: 735-746Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Within TREX-2, Sus1 directly interacts with the Sac3 CID motif that also harbors a binding site for the calmodulin-like centrin Cdc31 (32Fischer T. Rodriguez-Navarro S. Pereira G. Racz A. Schiebel E. Hurt E. Nat. Cell Biol. 2004; 6: 840-848Crossref PubMed Scopus (141) Google Scholar). TREX-2 is mainly localized at the nuclear periphery and interacts physically and functionally with the general mRNA export receptor Mex67/Mtr2 (31Fischer T. Strässer K. Racz A. Rodriguez-Navarro S. Oppizzi M. Ihrig P. Lechner J. Hurt E. EMBO J. 2002; 21: 5843-5852Crossref PubMed Scopus (222) Google Scholar). NPC tethering of TREX-2 depends on the nuclear basket protein, Nup1, and possibly other nucleoporins (31Fischer T. Strässer K. Racz A. Rodriguez-Navarro S. Oppizzi M. Ihrig P. Lechner J. Hurt E. EMBO J. 2002; 21: 5843-5852Crossref PubMed Scopus (222) Google Scholar). Removal of the Sac3 CID strongly impairs TREX-2 targeting to the NPCs in vivo and triggers an mRNA export defect. Notably, the small Sus1 protein is important for NPC targeting of TREX-2, because SUS1 deletion causes TREX-2 dissociation from the NPCs (15Köhler A. Schneider M. Cabal G.G. Nehrbass U. Hurt E. Nat. Cell Biol. 2008; 10: 707-715Crossref PubMed Scopus (160) Google Scholar).In evolutionary terms, SAGA is well conserved in subunit composition and structural appearance and plays broad and important regulatory roles in transcription from yeast to flies and humans (18Daniel J.A. Grant P.A. Mutat. Res. 2007; 618: 135-148Crossref PubMed Scopus (80) Google Scholar). Specifically, the Sus1-containing histone H2B DUB module of SAGA has human orthologues, which include ENY2 (Sus1), the protease USP22 (Ubp8), ATXN7L3 (Sgf11), and ATXN7 (Sgf73) (38Zhao Y. Lang G. Ito S. Bonnet J. Metzger E. Sawatsubashi S. Suzuki E. Le Guezennec X. Stunnenberg H.G. Krasnov A. Georgieva S.G. Schule R. Takeyama K. Kato S. Tora L. Devys D. Mol. Cell. 2008; 29: 92-101Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 39Zhang X.Y. Varthi M. Sykes S.M. Phillips C. Warzecha C. Zhu W. Wyce A. Thorne A.W. Berger S.L. McMahon S.B. Mol. Cell. 2008; 29: 102-111Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 40Weake V.M. Lee K.K. Guelman S. Lin C.H. Seidel C. Abmayr S.M. Workman J.L. EMBO J. 2008; 27: 394-405Crossref PubMed Scopus (95) Google Scholar). In analogy to yeast TREX-2, the Drosophila orthologue of Sus1, E(y)2, forms a complex with the Sac3 counterpart X-linked male sterile 2 (Xmas-2) and functions in mRNA export and gene-NPC anchorage (41Kurshakova M.M. Krasnov A.N. Kopytova D.V. Shidlovskii Y.V. Nikolenko J.V. Nabirochkina E.N. Spehner D. Schultz P. Tora L. Georgieva S.G. EMBO J. 2007; 26: 4956-4965Crossref PubMed Scopus (142) Google Scholar). The Sac3 orthologue GANP was reported to suppress DNA recombination in mammalian cells, but whether GANP operates in mRNA export and gene positioning remains to be explored (42Yoshida M. Kuwahara K. Shimasaki T. Nakagata N. Matsuoka M. Sakaguchi N. Genes Cells. 2007; 12: 1205-1213Crossref PubMed Scopus (18) Google Scholar, 43Kuwahara K. Tomiyasu S. Fujimura S. Nomura K. Xing Y. Nishiyama N. Ogawa M. Imajoh-Ohmi S. Izuta S. Sakaguchi N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10279-10283Crossref PubMed Scopus (37) Google Scholar, 44Takei Y. Swietlik M. Tanoue A. Tsujimoto G. Kouzarides T. Laskey R. EMBO Rep. 2001; 2: 119-123Crossref PubMed Scopus (73) Google Scholar). Notably, a potential human orthologue of Cdc31, centrin 2, is associated with the NPCs and plays a role in the export of mRNA (45Resendes K.K. Rasala B.A. Forbes D.J. Mol. Cell. Biol. 2008; 28: 1755-1769Crossref PubMed Scopus (63) Google Scholar). The functional diversification of Sus1 as a component of both the SAGA histone DUB module and the TREX-2 mRNA export complex represents an intriguing example of molecular innovation during evolution. The small Sus1 protein can support both a sophisticated enzymatic mechanism and confer positional information for an NPC targeting event. Understanding Sus1 function therefore requires dissection of its separate SAGA- and TREX-2-related roles, integrated with an analysis of how SAGA and TREX-2 interact functionally.In this study we report a comprehensive mutational analysis of Sus1 aimed at defining the molecular requirements for its association with either SAGA or TREX-2. Our data show that mutational uncoupling of Sus1-ligand interactions results in selective functional impairments in transcription-coupled mRNA export.EXPERIMENTAL PROCEDURESYeast Strains, Plasmids, and Microbiological Techniques—The S. cerevisiae strains used in this study are listed in supplemental Table 1. Deletion disruption and C-terminal TAP tagging at the genomic locus were performed as described previously (46Longtine M.S. McKenzie A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 10: 953-961Crossref Scopus (4106) Google Scholar, 47Janke C. Magiera M.M. Rathfelder N. Taxis C. Reber S. Maekawa H. Moreno-Borchart A. Doenges G. Schwob E. Schiebel E. Knop M. Yeast. 2004; 21: 947-962Crossref PubMed Scopus (1346) Google Scholar, 48Kressler D. Roser D. Pertschy B. Hurt E. J. Cell Biol. 2008; 181: 935-944Crossref PubMed Scopus (62) Google Scholar). A two-step allele replacement method was devised to generate strains expressing nontagged and C-terminally TAP- or FLAG-tagged Sus1 wild-type or mutant variants (see below).Plasmids used in this study are listed in supplemental Table 2. The site-directed sus1 mutants were generated by fusion PCR, and the correctness of the cloned DNA fragments was verified by sequencing. All recombinant DNA techniques were done according to standard procedures using Escherichia coli DH5α for cloning and plasmid propagation.Preparation of media, yeast transformation, and genetic manipulations were performed according to established methods. For selection of yeast transformants on nourseothricin (clonNAT)-containing plates, YPD plates were supplemented with 100 μg/ml nourseothricin (Werner BioAgents). Tetrad dissection was performed using a Singer MSM micromanipulator.Genomic SUS1 Gene Replacement—To replace genomic wild-type SUS1 with nontagged and TAP- or FLAG-tagged sus1 alleles, we devised a novel two-step allele replacement strategy. In a first step, haploid sus1::klURA3 (URA3 gene from Kluyveromyces lactis) deletion disruption strains were obtained by tetrad dissection of a heterozygous diploid SUS1/sus1::klURA3 strain that had been created by transformation of a klURA3 PCR cassette, bearing short flanking homology regions of the SUS1 promoter and terminator sequence into the diploid W303 strain background. The haploid sus1::klURA3 null mutant strains were then co-transformed with DNA fragments (1 μg) containing wild-type and mutant SUS1, SUS1-TAP, or SUS1-FLAG alleles, excised by XhoI/BamHI digestion from pRS315-SUS1/sus1, pRS315-SUS1/sus1-TAP, or pRS316-SUS1/sus1-FLAG, and empty pRS315 vector (100 ng) for selection of transformants on SDC-Leu plates. To select for clones that had lost the klURA3 marker because of a site-specific recombination event, transformants were replica-plated onto 5-FOA-containing plates. To verify the correctness of the allele replacement, clones that grew on 5-FOA-containing medium were analyzed by colony PCR and sequencing.Affinity Purifications—TAP-tagged proteins were affinity-purified according to published methods (49Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Séraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2275) Google Scholar). Proteins were detected by SDS-PAGE on NuPAGE 4–12% polyacrylamide gels (Invitrogen) with subsequent colloidal Coomassie Brilliant Blue G (Sigma) staining or by Western blot analysis. Mass spectrometric identification of the proteins contained in Coomassie-stained bands was performed as described (50Nissan T.A. Bassler J. Petfalski E. Tollervey D. Hurt E.C. EMBO J. 2002; 21: 5539-5547Crossref PubMed Scopus (289) Google Scholar). The following primary antibodies were used for Western analysis: anti-Arc1 (Hurt laboratory), anti-CBP (BioCat), anti-Cdc31 (from E. Schiebel, ZMBH, Heidelberg University, Germany), anti-FLAG (Sigma), and anti-Sac3 (from R. Kölling, Hohenheim University, Germany).In Vitro Binding Assays—Recombinant proteins were expressed in LB medium in E. coli BL21 codon plus RIL cells (Stratagene). Expression was induced by addition of 0.5 mm isopropyl 1-thio-β-d-galactopyranoside at 23 °C for 3 h. Hexahistidine-tagged Sus1 was purified by metal affinity chromatography and imidazole elution. The GST-Sac3-(573–805)-Cdc31 heterodimer was created by co-expression of GST-Sac3-(573–805) and Cdc31. GST-tagged proteins were purified on GSH beads and eluted with GSH. Recombinant Sus1 proteins were then mixed with GST-Sgf11 or with GST-Sac3-(573–805)-Cdc31 at a 2:1 molar ratio, respectively, in a buffer containing 100 mm NaCl, 0.5 mm dithiothreitol, 50 mm HEPES, pH 7.5. Proteins were incubated with GSH beads for 30 min at 16 °C, washed in the same buffer (4 °C), and eluted with GSH. After trichloroacetic acid precipitation, the samples were separated by SDS-PAGE (12% gel, MES buffer) and visualized by Coomassie staining.Live Cell Imaging and Fluorescence Microscopy—Prior to live imaging, cells were grown to mid-log phase in YPD (integration strains) or SDC-Leu (plasmid-based) liquid medium. Fluorescence microscopy was performed using an Imager Z1 (Carl Zeiss) microscope equipped with a 100×/63× NA 1.4 Plan-Apo-Chromat oil immersion lens (Carl Zeiss) and using DICIII, HE-EGFP, or 4,6-diamidino-2-phenylindole filters. Images were acquired with an AxioCam MRm camera and AxioVision 4.3 software (Carl Zeiss).In situ hybridization of poly(A)+ RNA was performed according to Ref. 51Segref A. Sharma K. Doye V. Hellwig A. Huber J. Lührmann R. Hurt E.C. EMBO J. 1997; 16: 3256-3271Crossref PubMed Scopus (433) Google Scholar. Prior to fixation, cells were grown to an A600 of 0.3 at 30 °C and then shifted to 37 °C for 2 h.Measurement of Global H2B Ubiquitin Levels—Immunoprecipitation of FLAG-tagged histone H2B was performed as described previously (52Kao C.F. Osley M.A. Methods (San Diego). 2003; 31: 59-66Crossref PubMed Scopus (29) Google Scholar).RESULTSSus1 is an evolutionary conserved protein in eukaryotes (Fig. 1a) with no homologues in viral, archaeal, and eubacterial genomes. Several Sus1 core residues are strongly conserved, whereas the N and C termini of the protein exhibit variable lengths and little sequence conservation. Secondary structure predictions indicate a primarily α-helical topology for Sus1 with five putative α-helices connected by short loop regions (Fig. 1b). We sought to generate a battery of sus1 mutants that could establish whether Sus1 employs distinct or overlapping interaction surfaces to bind to its two known ligands as follows: the CID (Cdc31 interaction domain) within the C terminus of Sac3 (32Fischer T. Rodriguez-Navarro S. Pereira G. Racz A. Schiebel E. Hurt E. Nat. Cell Biol. 2004; 6: 840-848Crossref PubMed Scopus (141) Google Scholar) or Sgf11, a subunit of the SAGA histone H2B DUB module (15Köhler A. Schneider M. Cabal G.G. Nehrbass U. Hurt E. Nat. Cell Biol. 2008; 10: 707-715Crossref PubMed Scopus (160) Google Scholar, 25Köhler A. Pascual-Garcia P. Llopis A. Zapater M. Posas F. Hurt E. Rodriguez-Navarro S. Mol. Biol. Cell. 2006; 17: 4228-4236Crossref PubMed Scopus (102) Google Scholar). A "clustered charged-to-alanine mutagenesis" strategy (53Wertman K.F. Drubin D.G. Botstein D. Genetics. 1992; 132: 337-350Crossref PubMed Google Scholar) was initially used to probe for potential interaction surfaces on Sus1 (Fig. 1b). This approach exploits the fact that charged residues are generally exposed at the protein surface rather than being buried in the hydrophobic core of the molecule. Sus1-TAP purifications were then performed to biochemically check whether the interaction with either SAGA or TREX-2 or both was perturbed. Normally, Sus1 affinity-purified by the TAP method efficiently co-enriches both SAGA (including the SAGA-like SLIK(SALSA) complex) and TREX-2 (14Rodriguez-Navarro S. Fischer T. Luo M.J. Antunez O. Brettschneider S. Lechner J. Perez-Ortin J.E. Reed R. Hurt E. Cell. 2004; 116: 75-86Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Surprisingly, none of the nine mutants (sus1-1 to 9) significantly altered Sus1 binding to SAGA or TREX-2 (supplemental Fig. S1A). We found that deleting the less conserved N- and C-terminal parts of the protein (sus1ΔN1–10 and sus1ΔC91–96; see Fig. 1b) also did not impair the interaction of Sus1 with either SAGA or TREX-2 (data not shown). The remarkable tolerance of Sus1 toward this systematic replacement of charged residues may suggest that the interaction of Sus1 with its partners is based on other types of interaction (e.g. hydrophobic) or requires more extensive mutations to be disrupted.Next, we engineered point-specific mutations in putative loop regions in Sus1 that lie between the α-helices predicted by sequence analysis (Fig. 1b; termed sus1–10, sus1–11, and sus1–12). Each of these amino acid changes involved residues, such as Gly and Asp, that are characteristically present in flexible surface loops in proteins. Additionally, some of the selected residues (e.g. the Gly37Trp38 pair) are highly conserved in evolution (Fig. 1a). Notably, affinity purification of TAP-tagged Sus1–10 and Sus1–12 mutant proteins from yeast showed a pronounced loss of TREX-2 factors (i.e. Sac3, Thp1, and Cdc31), but typical SAGA factors (e.g. Tra1 and Spt7), including the DUB module components Sgf73, Sgf11, and Ubp8, were still co-enriched (Fig. 1c). Western blot analysis revealed that the loss of TREX-2 factors was more severe in sus1–10 than sus1–12 (Fig. 1c). These data indicate that whereas Sus1–10 and Sus1–12 are impaired in their interaction with TREX-2, the structural integrity of the SAGA DUB module (Sus1-Sgf11-Ubp8-Sgf73) remains largely unaffected in these sus1 mutants. In contrast, the purification of TAP-tagged Sus1–11 displayed a striking loss of both TREX-2 and SAGA subunits (Fig. 1c). Because Sgf11 was also absent from this purification, it is conceivable that a reduced affinity between Sus1 and its direct interaction partner Sgf11 may have dissociated Sus1 from the DUB module and hence from the entire SAGA complex (see below). The decrease or lack of a biochemical interaction observed for the different Sus1 mutant proteins is not because of major alterations in Sus1 protein levels. Wild-type and mutant Sus1 proteins exhibited similar expression levels in yeast, although we noticed a slight reduction in the total amount of Sus1–11 (Fig. 1c).To characterize the effects of the different sus1 alleles on TREX-2 subunit composition, we purified TREX-2 via TAP-tagged Thp1 and used Western blotting to determine the amount of bound mutant Sus1 proteins. Consistent with the results of the Sus1-TAP purifications, FLAG-tagged Sus1–10, Sus1–11, and Sus1–12 were specifically absent from the TREX-2 complex, whereas the other TREX-2 subunits Sac3 and Cdc31 were efficiently co-enriched with Thp1 (Fig. 1d). Taken together, the biochemical data indicate that sus1–10 and sus1–12 uncouple Sus1 from TREX-2, whereas the sus1–11 mutation effectively impairs the Sus1 interaction with both TREX-2 and SAGA.To confirm that the dissociation of Sus1 from TREX-2 or SAGA is caused primarily by a reduced affinity between Sus1-Sac3 and Sus1-Sgf11, we reconstituted these Sus1-ligand interactions in vitro (Fig. 2). Recombinant wild-type and mutant Sus1 proteins were first assayed for their ability to bind to a Sac3-(573–805)-Cdc31 heterodimer. This C-terminal fragment of Sac3 harbors the binding sites for Sus1 and Cdc31. Wild-type Sus1 bound to Sac3-(573–805)-Cdc31 very efficiently, whereas the binding was reduced with Sus1–10 and Sus1–12 and largely abolished for the Sus1–11 mutant. On the other hand, binding of Sus1–10 and Sus1–12 to Sgf11 was largely unaffected, whereas Sus1–11 failed to interact with Sgf11 (Fig. 2). We consider it unlikely that the perturbed Sus1-ligand interactions were caused by severe misfolding of the Sus1 mutants. CD spectra for the mutant Sus1 proteins were determined and found to be virtually indistinguishable from those of the wild-type protein (all showed prominent negative ellipticities at 220 nm consistent with the presence of an α-helical conformation; data not shown). In summary, the in vitro experiments largely recapitulate the Sus1-TAP affinity purifications, with Sus1–11 showing a global binding defect, whereas Sus1–10/-12 display a selective Sac3 interaction defect. We observed that the extent of Sus1–10/-12 dissociation from Sac3

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