Vps9p CUE Domain Ubiquitin Binding Is Required for Efficient Endocytic Protein Traffic
2003; Elsevier BV; Volume: 278; Issue: 22 Linguagem: Inglês
10.1074/jbc.m301059200
ISSN1083-351X
AutoresBrian A. Davies, Justin D. Topp, Agnel Sfeir, David J. Katzmann, Darren S. Carney, Gregory G. Tall, A S Friedberg, Li Deng, Zhijian J. Chen, Bruce Horazdovsky,
Tópico(s)Pancreatic function and diabetes
ResumoRab5 GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway. The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p. Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole. In addition, Vps9p is itself monoubiquitylated. Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor endocytosis. These findings define a new ubiquitin binding domain and implicate ubiquitin as a modulator of Vps9p function in the endocytic pathway. Rab5 GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway. The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p. Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole. In addition, Vps9p is itself monoubiquitylated. Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor endocytosis. These findings define a new ubiquitin binding domain and implicate ubiquitin as a modulator of Vps9p function in the endocytic pathway. Rab proteins are critical regulators of the vesicle targeting and fusion events (reviewed in Refs. 1Pfeffer S.R. Nat. Cell Biol. 1999; 1: e17-e22Crossref PubMed Scopus (361) Google Scholar and 2Zerial M. McBride H.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-119Crossref PubMed Scopus (2709) Google Scholar). Discrete classes of these small GTPases mediate very specific transport events, showing little if any functional overlap. For example, the Rab5 family members appear to be involved exclusively in targeting events within the early stages of the endocytic pathway (3Gorvel J.P. Chavrier P. Zerial M. Gruenberg J. Cell. 1991; 64: 915-925Abstract Full Text PDF PubMed Scopus (860) Google Scholar, 4Bucci C. Parton R.G. Mather I.H. Stunnenberg H. Simons K. Hoflack B. Zerial M. Cell. 1992; 70: 715-728Abstract Full Text PDF PubMed Scopus (1121) Google Scholar). The activation of Rab5 GTPases like all Rab proteins is dependent on the state of bound nucleotide, GDP or GTP. Two classes of proteins that modulate the Rab nucleotide occupancy are the GTPase activating proteins (GAPs) 1The abbreviations used are: GAP, GTPase activating protein; GEF, guanine nucleotide exchange factor; Ni-NTA, nickel-nitrilotriacetic acid; HA, hemagglutinin; Ub, ubiquitin; MBP, maltose-binding protein; HRP, horseradish peroxidase; CPY, carboxypeptidase Y; CPS, carboxypeptidase S. and the guanine nucleotide exchange factors (GEFs). GAPs stimulate GTP hydrolysis, leaving the Rab in the GDP-bound, inactive state; conversely, GEFs initiate GDP release to permit GTP binding and thereby Rab activation. Multiple GAPs and GEFs for the Rab proteins have been identified, and an interesting distinction has been observed (reviewed in Ref. 5Segev N. Curr. Opin. Cell. Biol. 2001; 13: 500-511Crossref PubMed Scopus (247) Google Scholar). The Rab GAPs share a conserved sequence motif and exhibit substrate promiscuity among the Rab families (6Du L.L. Collins R.N. Novick P.J. J. Biol. Chem. 1998; 273: 3253-3256Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 7Albert S. Gallwitz D. J. Biol. Chem. 1999; 274: 33186-33189Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) (reviewed in Ref. 8Scheffzek K. Ahmadian M.R. Wittinghofer A. Trends Biochem. Sci. 1998; 23: 257-262Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). In contrast, the GEF proteins for different Rab families are dissimilar at the sequence level and show great specificity for their cognate Rab proteins. Consequently, the GEFs appear to be the primary mechanism to control specific Rab activity. A number of exchange factors for Vps21p/Rab5 family members have been identified in mammalian and yeast systems. In yeast, Vps9p is the exchange factor for the Rab5 ortholog Vps21p (9Hama H. Tall G.G. Horazdovsky B.F. J. Biol. Chem. 1999; 274: 15284-15291Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). VPS9 was initially identified in genetic screens for mutants defective in vacuolar protein sorting (10Burd C.G. Mustol P.A. Schu P.V. Emr S.D. Mol. Cell. Biol. 1996; 16: 2369-2377Crossref PubMed Scopus (72) Google Scholar). In vitro reconstitution of Vps9p-stimulated GDP release and GTP loading onto Vps21p demonstrated that Vps9p is the GEF for Vps21p (9Hama H. Tall G.G. Horazdovsky B.F. J. Biol. Chem. 1999; 274: 15284-15291Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Concurrently, Rabex5 was identified as a Rab5-binding protein and demonstrated to exhibit in vitro GEF activity (11Horiuchi H. Lippe R. McBride H.M. Rubino M. Woodman P. Stenmark H. Rybin V. Wilm M. Ashman K. Mann M. Zerial M. Cell. 1997; 90: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). In addition to conserved functions in activating Rab5 proteins, yeast Vps9p (451 amino acids) and human Rabex5 (491 amino acids) share 27% overall sequence identity (11Horiuchi H. Lippe R. McBride H.M. Rubino M. Woodman P. Stenmark H. Rybin V. Wilm M. Ashman K. Mann M. Zerial M. Cell. 1997; 90: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). A peptide comprised of residues 158–347 of Vps9p was identified as the domain necessary and sufficient for GEF catalytic activity. 2G. G. Tall, D. S. Carney, and B. F. Horazdovsky, manuscript in preparation. Although Vps9p is the only known GEF for Vps21p in yeast, six human proteins have been identified with the Vps9 domain in addition to human Rabex5 (SMART data base) (12Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (3019) Google Scholar, 13Letunic I. Goodstadt L. Dickens N.J. Doerks T. Schultz J. Mott R. Ciccarelli F. Copley R.R. Ponting C.P. Bork P. Nucleic Acids Res. 2002; 30: 242-244Crossref PubMed Scopus (567) Google Scholar). 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The coincidence of these signaling motifs with the Vps9 domain suggests that these proteins serve as key integrators of signal transduction pathways and the receptor endocytosis machinery. This concept has been demonstrated for the Rab5 GEF, Rin1 (15Tall G.G. Barbieri M.A. Stahl P.D. Horazdovsky B.F. Dev. Cell. 2001; 1: 73-82Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). In contrast to Rin1, the mechanisms regulating Vps9p and Rabex5 are unclear. Here we provide evidence that ubiquitin binding and monoubiquitylation regulate yeast Vps9p. The CUE domain of Vps9p is shown to mediate an interaction with ubiquitin, and we show that this interaction is required to potentiate Vps9p function in endocytic traffic to the vacuole. We demonstrate that Vps9p is monoubiquitylated and that this modification is dependent on CUE-dependent ubiquitin binding and the E3 ubiquitin ligase Rsp5p. Together, these findings identify a novel role for ubiquitin in regulating endocytosis by the Vps21p/Rab5 exchange factor Vps9p. Strains and Reagents—Bacterial strains used in this study were DH5α (Invitrogen) and HMS174 DE3 (Novagen). The Saccharomyces cerevisiae strains used in this study were SEY6210 (MATa trp1 lys2 leu2 his3 ura3 suc2Δ9) (22Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (736) Google Scholar), CBY1 (SEY6210; vps9Δ1::HIS3) (10Burd C.G. Mustol P.A. Schu P.V. Emr S.D. Mol. Cell. Biol. 1996; 16: 2369-2377Crossref PubMed Scopus (72) Google Scholar), PSY83 (SEY6210; vps8Δ1::HIS3) (23Horazdovsky B.F. Cowles C.R. Mustol P. Holmes M. Emr S.D. J. Biol. Chem. 1996; 271: 33607-33615Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), MYY290 (MATa his3 leu2 ura3) (24Smith B.J. Yaffe M.P. Mol. Cell. Biol. 1991; 11: 2647-2655Crossref PubMed Scopus (73) Google Scholar), MYY808 (MYY290; smm1/rsp5ts) (25Fisk H.A. Yaffe M.P. J. Cell Biol. 1999; 145: 1199-1208Crossref PubMed Scopus (160) Google Scholar), and L40 (MATa trp1 leu2 his3 LYS2::lexAop)4-HIS3 URA3::(lexAop)4-lacZ) (26Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar). The bacterial strains were grown in LB medium supplemented with ampicillin (100 μg ml–1) and kanamycin (50 μg ml–1) when necessary (27Miller J. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar). Yeast strains were grown in YPD medium (containing 2% peptone, 1% yeast extract, and 2% glucose) or synthetic medium supplemented with appropriate amino acids as required (28Sherman F. Fink G.R. Lawrence L.W. Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1979Google Scholar). Thermostable DNA polymerases, restriction endonucleases, and DNA modifying enzymes were purchased from Invitrogen, Roche Molecular Biochemicals, or New England Biolabs (Beverly, MA). EasyTag Expre35S35S protein labeling mix was purchased from PerkinElmer Life Sciences. Protein A-Sepharose was purchased from Amersham Biosciences. Zymolase 100T was purchased from Seikagaku (Tokyo, Japan). Glass beads (0.5 mm) were purchased from Biospec Products, Inc. (Bartlesville, OK). Ni-NTA-agarose and penta-His antibody were purchased from Qiagen, Inc. (Valencia, CA). Bioscale Q2 was purchased from Bio-Rad. HA.11 monoclonal antibody (raw ascites fluid) was purchased from Covance Inc. (Princeton, NJ). Antiserum for Vps9p was obtained from Scott Emr (University of California at San Diego). SuperSignal West Femto maximum sensitivity substrate was purchased from Pierce. FM4–64 was purchased from Molecular Probes, Inc. (Eugene, OR). Plasmid Construction—VPS9 was amplified with Vent DNA polymerase and oligos Vps9–20 (5′-TCCTCTCGAGAATAGTACCGCAATAGGAGA-3′) with Vps9–21 (5′-CCGCGGCTAGCGGCCGCCTTCTGACAGAGAAAGTAGAGC-3′) and Vps9–22 (5′-GGCGGCCGCTAGCCGCGGTGATCTCATGCACATATTTC-3′) with Vps9–23 (5′-TATAGAGCTCTGGCAGGCCCGTTTACGTAGGC-3′), and the products were used as template in overlapping PCR with Vps9–20 and Vps9–23. The resultant 2.9-kb fragment was subcloned into pRS416 (29Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene. 1992; 110: 119-122Crossref PubMed Scopus (1434) Google Scholar) via the XhoI and SstI sites present in the oligos to generate pRS416 Vps9. Vps9ΔCUE was amplified in two portions, via Vps9–20 with Vps9–24 (5′GCTAGCGGCCGCCGTTCTCTTCAATTTTCTTAATTAACG-3′) and Vps9–22 with Vps9–23. The resultant fragments were then used as a template for overlapping PCR with Vps9–20 and Vps9–23 and subcloned into pRS416 via the XhoI, SstI sites to yield pRS416 Vps9ΔCUE. Vps9 M419A was amplified in two portions via Vps9–20 with Vps9 M419A Noncoding (5′-TCCATATCTGGAAACGCGTTCTGTAATGTGTTC-3′) and Vps9 M419A Coding (5′-GAACACATTACAGAACGCGTTTCCAGATATGGA-3′) with Vps9–23. The resultant fragments were digested with XhoI, MluI, and MluI, SstI, respectively, and cloned into the XhoI, SstI sites of pRS315 (29Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene. 1992; 110: 119-122Crossref PubMed Scopus (1434) Google Scholar) to generate pRS315 Vps9 M419A. VPS9 M419A open reading frame was then amplified with Vps9–3 (5′-AATCGGATCCCATGACTGATGATGAAAAGAGG-3′) and Vps9–4(5′-TGTGCATGGTCGACTTATTCTGACAGAGAAAGTAG-3′) and subcloned into the BamHI, SalI sites of pMBP parallel 1 (30Sheffield P. Garrard S. Derewenda Z. Protein Expr. Purif. 1999; 15: 34-39Crossref PubMed Scopus (530) Google Scholar) to generate pMBP Vps9 M419A. The BamHI, SalI fragment from pGT9–1 (9Hama H. Tall G.G. Horazdovsky B.F. J. Biol. Chem. 1999; 274: 15284-15291Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) was subcloned into pMBP parallel 1 (pMBP Vps9) and pET28b (pET28 Vps9; Novagen), as well. The CUE domains from VPS9 wild-type and M419A were amplified with Vps9–26 (5′-TTGAGGATCCCGAACGAAAGGACACGTTGAACAC-3′) and Vps9–4 and subcloned into pMBP parallel 1 (pMBP Vps9 CUE wild-type and M419A). To generate His6-Vps9p carboxyl-terminal (ΔC) and amino-terminal (ΔN) truncations, Vps9–3 with Vps9–13 (5′-GGGTTTCAGTAAAGTGTCGACTGGCTGTAACTAATCCGG-3′) and Vps9–8 (5′-TCTTTAGGATCCTATGCAGAAACCATTAGACGATGAGCAT-3′) with Vps9–4 were used in PCR amplifications with template pGT9–1, and the resultant fragments were cloned into the BamHI, SalI sites of pET28b (pET28 Vps9 ΔC and ΔN). The Vps9–8/4 PCR product was also subcloned into the BamHI, SalI sites of pVJL11 (31Jullien-Flores V. Dorseuil O. Romero F. Letourneur F. Saragosti S. Berger R. Tavitian A. Gacon G. Camonis J.H. J. Biol. Chem. 1995; 270: 22473-22477Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) (Vps9 carboxyl-terminal bait). pET28 Vps9 and the truncation constructs (pET28 Vps9 ΔC and ΔN) were used as templates in PCR reactions with pET28–1 (5′-CTTTAAGAAGGAGATCTACCATGGGCAGCA-3′) and Vps9–15 (FL, ΔN; 5′-TGTGCATGAGATCTTTATTCTGACAGAGAAAGTAG-3′) or Vps9–17 (ΔC; 5′-GGGTTTCAGTAAAGTAGATCTTGGCTGTAACTAATCCGG-3′); the resultant fragments were digested with BglII and cloned into the BglII site of pPGK415 to generate pHis9–1 (1–451), pHis9–2 (1–347), and pHis9–3 (158–451). pPGK415 was generated by subcloning the HindIII fragment from pEMBLye30/2 (32Banroques J. Delahodde A. Jacq C. Cell. 1986; 46: 837-844Abstract Full Text PDF PubMed Scopus (98) Google Scholar) into the HindIII site of pRS415. Oligos HA 5′ (5′-GGATCCAATGTACCCATACGATGTTCCTGAC-3′) and ubiquitin 3′ (5′-GAATTCTCAACCACCTCTTAGCCTTAAGAC-3′) were used with pEF-HA-Ub (from L. Deng and Z. Chen, University of Texas Southwestern Medical Center) to amplify HA-ubiquitin, and the product was cloned into the BamHI and EcoRI sites of pGPD416 (33Mumberg D. Muller R. Funk M. Gene. 1995; 156: 119-122Crossref PubMed Scopus (1594) Google Scholar) and pET28b. Ste3–1 (5′-TGATCTCGAGGCGAATCGCACATTGCGCAAC-3′) and Ste3–2 (5′-GTGTTAGCGGCCGCCAGGGCCTGCAGTATTTTC-3′) were used to amplify the STE3 promoter and open reading frame from genomic DNA; the product was digested with XhoI, NotI and subcloned into the XhoI, NotI sites of the pRS414 vector (29Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene. 1992; 110: 119-122Crossref PubMed Scopus (1434) Google Scholar) containing the HA3 coding and VPS26 terminator sequences from the NotI to KspI sites (pRS414 Ste3HA). UBC5 was amplified from genomic DNA with Ubc5 5′ (5′-GGATTCAATGTCTTCCTCCAAGCGTATTG-3′) and Ubc5 3′ (5′-CAGCTGTCAAACAGCATATTTTTTAG-3′); the PCR product was cloned into the SmaI site of pBluescript (Stratagene), and the BamHI, SalI fragment was then subcloned into the BamHI, SalI sites of pMBP parallel 1 (pMBP Ubc5). Rsp5–1 (5′-AAAGAGATCTAATGCCTTCATCCATATCCGTC-3′) and Rsp5–2 (5′-TGCGCTCGAGTCATTCTTGACCAAACCCTATGG-3′) were used with genomic DNA to amplify the RSP5 open reading frame; the PCR product was digested with BglII, XhoI and subcloned into the pMBP parallel 1 BamHI, SalI sites (pMBP Rsp5). Protein Expression and Purification—pMBP Vps9, pMBP Vps9 M419A, pMBP Vps9 CUE, pMBP Vps9 CUE M419A, pMBP Ubc5, pMBP Rsp5, pQE31 Vps9, pET28 Vps21, and pET28 HA-ubiquitin were transformed into HMS174 DE3. For expression of His6HA-Ub and His6Vps21, Escherichia coli were cultured at 37 °C, induced with 500 μm isopropyl-β-d-thiogalactoside, and harvested after 4 h of protein production at 37 °C. For the remaining protein fusions, cultures were shifted from 37 °C to room temperature prior to induction with 500 μm isopropyl-β-d-thiogalactoside and harvested after 10–15 h of protein production at room temperature. MBP-protein fusions were affinity purified with amylose resin following the manufacturer's instructions (New England Biolabs, Beverly, MA). His6-tagged proteins were affinity purified with Ni-NTA-agarose following the manufacturer's instructions (Qiagen, Inc, Valencia, CA), and His6Vps9p was further purified with the Bioscale Q2 column following the manufacturer's recommended conditions (Bio-Rad). Protein fusions were concentrated, and buffer was switched to 50 mm Tris, pH 7.5, 150 mm NaCl and stored at –80 °C for later use. Ubiquitin Binding Assay—Putative ubiquitin-binding protein (MBP-Vps9p, MBP-Vps9p M419A; 4 μg ml–1) was incubated with either His6HA-ubiquitin (13 μg ml–1) or bovine serum albumin (5 μg ml–1) in 1 ml of binding buffer (50 mm Tris, pH 7.5, 300 mm KOAc with protease inhibitors (N-tosyl-l-phenlalanine-chloromethyl ketone, Nα-p-tosyl-l-lysine-chloromethyl ketone, phenylmethylsulfonyl fluoride, leupeptin, and trypsin inhibitor)) with Ni-NTA-agarose (40 μl). Binding reactions were incubated for >1 h at 4 °C in a Rotator. The Ni-NTA-agarose was then washed six times with 1 ml of binding buffer. 50 μl of elution buffer (binding buffer with 200 mm imidazole) was added, and the samples were incubated for 10 min on ice. The supernatant was transferred, 5× Laemmli sample buffer (0.312 m Tris, pH 6.8, 10% SDS, 25% β-mercaptoethanol, 0.05% bromphenol blue) was added, and the sample was incubated at 37 °C for 10 min. The eluate material was resolved by SDS-PAGE, and Western analyses were performed with Vps9p (1: 2,000) or Rabex5 (1:1,000) antisera or HA.11 monoclonal antibody (1: 10,000), appropriate HRP-conjugated antibody (1:2,000), and SuperSignal West Femto maximum sensitivity substrate (1:4 in 50 mm Tris, pH 7.5, 150 mm NaCl). Analysis of Vps9 CUE domain ubiquitin binding was conducted similarly except that His6Vps21p (equimolar to His6HA-Ub) was used as the negative control, binding buffer was 50 mm NaPO4, pH 7.5, 300 mm KOAc, and Western analysis utilized MBP antiserum (1:5,000; New England Biolabs, Beverly, MA). Whole Cell Western Analysis—Yeast strains were grown in YPD or yeast nitrogen base-glucose with appropriate amino acids, and 1 A600 equivalent was harvested during log-phase growth (0.5–0.8 A600 ml–1). Samples were resuspended in 100 μl of 5× Laemmli sample buffer, and ∼150 μl of glass beads (0.5 mm) were added. Samples were vortexed in mass for 10 min and heated at 95 °C for 4 min. 0.1 A600 equivalent was resolved by SDS-PAGE, and Western analysis was performed with Vps9p antiserum (1:2,000), HRP-conjugated anti-rabbit antibody (1: 2,000), and SuperSignal West Femto maximum sensitivity substrate (1:4 in 50 mm Tris, pH 7.5, 150 mm NaCl). CPY Immunoprecipitation Assay—CPY immunoprecipitation experiments were performed as described previously (23Horazdovsky B.F. Cowles C.R. Mustol P. Holmes M. Emr S.D. J. Biol. Chem. 1996; 271: 33607-33615Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Yeast strains were grown in YNB-glucose and labeled at 30 °C for 10 min with EasyTag Expre35S35S protein labeling mix (30 μCi A600–1) before the addition of excess methionine and cysteine. Following the 30-min chase, samples were precipitated with trichloroacetic acid (final concentration 10%) and resuspended in boiling buffer (50 mm Tris, pH 7.5, 1 mm EDTA, 1% SDS). Glass beads were added, samples were vortexed, and CPY was isolated as described previously. Immunoprecipitated material was resolved by SDS-PAGE and visualized by fluorography. Ste3p Degradation Assay—Yeast strains harboring the pRS414 Ste3HA plasmid were grown in YNB-glucose, 0.2% yeast extract with appropriate amino acids past log growth phase. Cultures were diluted to 0.2 A600 ml–1 and cultured for 4 h. Three A600 equivalents were harvested and resuspended at 1 A600 ml–1 in YNB-glucose, and cyclohexamide (1 mg ml–1 in ethanol) was added to 1.3 μg ml–1. 0.5 A600 equivalents were removed at 0, 20, 40, and 60 min after cyclohexamide addition, and NaN3 and NaF were added to 10 mm. Samples were resuspended in 100 μl of 2× urea sample buffer (6 m urea, 125 mm Tris, pH 6.8, 6% SDS, 10% β-mercaptoethanol, 0.01% bromphenol blue), and ∼150 μl of glass beads (0.5 mm) were added. Samples were vortexed in mass for 10 min and heated at 65 °C for 4 min. An additional 100 μl of 2× urea sample buffer was added, and samples were subjected to centrifugation for 5 min at 14,000 rpm. 0.05 A600 equivalent was resolved by SDS-PAGE, and Western analysis was performed with HA.11 monoclonal antibody (1:5,000), HRP-conjugated anti-mouse antibody (1:2,000), and SuperSignal West Femto maximum sensitivity substrate (1:5 in 50 mm Tris, pH 7.5, 150 mm NaCl). The ABI computing densitometer 300A was used with ImageQuant V1.2 for quantitation. Similar degradation patterns were observed when analyses were performed using Ste3p antiserum (from G. Payne, UCLA) to detect endogenous receptors, indicating that the Ste3HA reporter recapitulates Ste3p trafficking (data not shown). In Vivo Ubiquitylation Assay—Yeast strains harboring the pGPD416 HA-ubiquitin plasmid were grown in YNB-glucose with appropriate amino acids. 10 A600 equivalents were harvested in late log growth phase (∼1 A600 ml–1) and resuspended in 100 mm Tris, pH 9.4, 10 mm dithiothreitol. Following a 10-min room temperature incubation, samples were resuspended in spheroplasting buffer (25 mm Tris, pH 7.5, 1 m sorbitol, 1× YNB, 4% glucose, 1× amino acids, 100 μg ml–1 zymolase 100T) and incubated 10 min at 30 °C. Samples were osmotically lysed in 10 mm NaPO4, pH 8.0, with Roche Molecular Biochemicals EDTA-free protease inhibitor mixture. The lysate was then cleared by a 10-min, 16,000 × g spin at 4 °C. The cleared lysate was then adjusted to ∼25 mm NaPO4, pH 8.0, 300 mm NaCl, and 2% glycerol, and 50 μl of Ni-NTA-agarose was added. Samples were incubated for >1 h at 4 °C and washed four times with wash buffer (50 mm NaPO4, pH 8.0, 300 mm NaCl, 5% glycerol). 100 μl of elution buffer (wash buffer with 200 mm imidazole) was added, and the samples were incubated for 10 min on ice. The supernatant was transferred, 5× Laemmli sample buffer was added, and the sample was incubated at 37 °C for 10 min. The eluate material was resolved by SDS-PAGE, and Western analyses were performed with penta-His (1:2,000) or HA.11 antibody (1:10,000), HRP-conjugated anti-mouse antibody (1:2,000), and SuperSignal West Femto maximum sensitivity substrate (1:1 in 50 mm Tris, pH 7.5, 150 mm NaCl). In Vitro Ubiquitylation Assay—The in vitro ubiquitylation assay was modified from Huibregtse et al. (34Huibregtse J.M. Yang J.C. Beaudenon S.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3656-3661Crossref PubMed Scopus (182) Google Scholar). MYY290 (RSP5) and MYY808 (rsp5ts) cultures were grown in YPD at 25 °C to ∼5 A600 ml–1. Pelleted yeast were resuspended in 25 mm Tris, pH 7.5, 150 mm NaCl with protease inhibitors (N-tosyl-l-phenlalanine-chloromethyl ketone, Nα-p-tosyl-l-lysine-chloromethyl ketone, phenylmethylsulfonyl fluoride, leupeptin, and trypsin inhibitor). The samples were frozen at –80 °C, thawed, and lysed by vortexing with glass beads. The lysate was then cleared by a 5-min, 16,000 × g spin. The 20-μl ubiquitylation reaction was set up on ice with 25 mm Tris, pH 8.0, 125 mm NaCl, 2 mm MgCl2, 2.5 mm ATP, MYY290 or MYY808 cleared lysate (0.8 mg ml–1), His6Vps9p (0.34 mg ml–1), His6hE1 (3.25 μg ml–1; generously provided by J. Chen laboratory, UTSW), MBP-Ubc5p (0.8 mg ml–1), with or without MBP-Rsp5p (27 μg ml–1), added. Reactions were incubated at 25 °C for 1 h and terminated with the addition of 5× Laemmli sample buffer and incubation at 95 °C for 4 min. One-third of the reaction was then resolved by SDS-PAGE, and Western analysis was performed with Vps9p antiserum (1:2,000) or HA.11 monoclonal antibody (1:2,000), appropriate HRP-conjugated secondary antibodies (1:2,000), and SuperSignal West Femto maximum sensitivity substrate (1:4 in 50 mm Tris, pH 7.5, 150 mm NaCl). Vps9p CUE Domain Binds Ubiquitin—To identify potential regulators of Vps9p function, a yeast two-hybrid screen was conducted using the carboxyl-terminal portion of Vps9p (amino acids 158–451). From this screen, ubiquitin was repeatedly isolated as a potent Vps9p interaction partner. The specificity of this interaction in the yeast two-hybrid system was verified using a variety of unrelated protein expression constructs (data not shown). This region of Vps9p contains a sequence motif called CUE (Fig. 1A). This domain (amino acids 408–450) (Fig. 1B) was originally identified by reiterative sequence homology searches initiated with the yeast protein Cue1p (35Ponting C.P. Biochem. J. 2000; 351: 527-535Crossref PubMed Scopus (120) Google Scholar) and can also be found in the SMART and PFAM databases (12Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (3019) Google Scholar, 13Letunic I. Goodstadt L. Dickens N.J. Doerks T. Schultz J. Mott R. Ciccarelli F. Copley R.R. Ponting C.P. Bork P. Nucleic Acids Res. 2002; 30: 242-244Crossref PubMed Scopus (567) Google Scholar, 36Bateman A. Birney E. Cerruti L. Durbin R. Etwiller L. Eddy S.R. Griffiths-Jones S. Howe K.L. Marshall M. Sonnhammer E.L. Nucleic Acids Res. 2002; 30: 276-280Crossref PubMed Scopus (2014) Google Scholar). The CUE domain is found in organisms from yeast to humans (35Ponting C.P. Biochem. J. 2000; 351: 527-535Crossref PubMed Scopus (120) Google Scholar) (Fig. 1B), but its functional role was not defined. Of the Vps21p/Rab5 GEF proteins, the canonical CUE domain has been identified in only S. cerevisiae Vps9p (Fig. 1A); however, human Rabex5 may also harbor a highly divergent CUE domain (37Donaldson K.M. Yin H. Gekakis N. Supek F. Joazeiro C.A.P. Curr. Biol. 2003; 13: 258-262Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) (Fig. 1B). To confirm the Vps9p-ubiquitin interaction and to examine the role of the CUE domain in this interaction, an in vitro ubiquitin binding assay was utilized. A gene fusion between the MBP and VPS9 coding sequences was constructed, and the recombinant protein (MBP-Vps9p) was expressed in E. coli and affinity purified (Fig. 2A). A His6- and HA-tagged version of a human ubiquitin coding sequence was also constructed (His6-HA-Ub), expressed in E. coli, and affinity purified. MBP-Vps9p was then incubated with His6-HA-Ub (or bovine serum albumin as a control), and His6-HA-Ub, together with the potential His6-HA-Ub·MBP-Vps9p complexes, were isolated using Ni-NTA-agarose. Following extensive washing, His6-HA-Ub was eluted from the Ni-NTA-agarose with imidazole, and the presence of Vps9p in the eluate was determined by Western analysis. As shown in Fig. 2B, MBP-Vps9p copurified with His6-HA-Ub (lane 5). When bovine serum albumin was substituted for His6-HA-Ub, only a very small amount of MBP-Vps9p was detected representing the level of nonspecific association with the Ni-NTA resin (Fig. 2B, lane 4). These results confirm the Vps9p-ubiquitin interaction uncovered in the yeast two-hybrid screen. To explore the possibility that the CUE domain mediates the interaction between Vps9p and ubiquitin, an allele was generated in which the first residue of the highly conserved signature sequence MFP (35Ponting C.P. Biochem. J. 2000; 351: 527-535Crossref PubMed Scopus (120) Google Scholar) (methionine at position 419) was mutated to alanine (M419A) (Fig. 1B). The methionine was chosen for mutagenesis to minimize potential structural perturbations of this domain. MBP-Vps9p M419A was expressed in E. coli and affinity purified. Soluble protein yields from the M419A allele were equivalent to that of wild-type, suggesting that global protein folding and stability were largely unaffected (Fig. 2A) (see below). When the mutant protein was tested for its ability to bind ubiquitin, the M419A mutation precluded the ability of this protein to bind ubiquitin in vitro (Fig. 2B, lane 6). This finding indicates that the CUE domain is necessary for the Vps9p-ubiquitin association and suggests that th
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