Vps9p CUE Domain Ubiquitin Binding Is Required for Efficient Endocytic Protein Traffic*

Brian A. Davies {ddagger}, Justin D. Topp {ddagger}, Agnel J. Sfeir §, David J. Katzmann {ddagger}, Darren S. Carney {ddagger}, Gregory G. Tall ¶, Andrew S. Friedberg ||, Li Deng **, Zhijian Chen ** and Bruce F. Horazdovsky {ddagger} {ddagger}{ddagger}

From the {ddagger}Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, §Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, ||Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, **Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received for publication, January 31, 2003 , and in revised form, March 17, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Rab proteins are critical regulators of the vesicle targeting and fusion events (reviewed in Refs. 1 and 2). 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 (3, 4). 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)1 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. 5). The Rab GAPs share a conserved sequence motif and exhibit substrate promiscuity among the Rab families (6, 7) (reviewed in Ref. 8). 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 (9). VPS9 was initially identified in genetic screens for mutants defective in vacuolar protein sorting (10). In vitro reconstitution of Vps9p-stimulated GDP release and GTP loading onto Vps21p demonstrated that Vps9p is the GEF for Vps21p (9). Concurrently, Rabex5 was identified as a Rab5-binding protein and demonstrated to exhibit in vitro GEF activity (11). 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 (11). A peptide comprised of residues 158–347 of Vps9p was identified as the domain necessary and sufficient for GEF catalytic activity.2 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) (12, 13). These human Rab5 GEF proteins contain well defined signaling, protein-protein interaction, and structural domains, including Src homology 2 and Ras association motifs (14, 15, 16), Rho GEF and seven-bladed {beta} propeller domains (17, 18, 19), a Ras GAP motif (20), and Ankyrin repeats (21). 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 (15).

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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Reagents—Bacterial strains used in this study were DH5{alpha} (Invitrogen) and HMS174 DE3 (Novagen). The Saccharomyces cerevisiae strains used in this study were SEY6210 (MATa trp1 lys2 leu2 his3 ura3 suc2{Delta}9) (22), CBY1 (SEY6210; vps9{Delta}1::HIS3) (10), PSY83 (SEY6210; vps8{Delta}1::HIS3) (23), MYY290 (MATa his3 leu2 ura3) (24), MYY808 (MYY290; smm1/rsp5ts) (25), and L40 (MATa trp1 leu2 his3 LYS2::lexAop)4-HIS3 URA3::(lexAop)4-lacZ) (26). The bacterial strains were grown in LB medium supplemented with ampicillin (100 µg ml1) and kanamycin (50 µg ml1) when necessary (27). 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 (28). 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 (29) via the XhoI and SstI sites present in the oligos to generate pRS416 Vps9. Vps9{Delta}CUE 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 Vps9{Delta}CUE. 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 (29) 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 (30) to generate pMBP Vps9 M419A. The BamHI, SalI fragment from pGT9–1 (9) 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 ({Delta}C) and amino-terminal ({Delta}N) 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 {Delta}C and {Delta}N). The Vps9–8/4 PCR product was also subcloned into the BamHI, SalI sites of pVJL11 (31) (Vps9 carboxyl-terminal bait). pET28 Vps9 and the truncation constructs (pET28 Vps9 {Delta}C and {Delta}N) were used as templates in PCR reactions with pET28–1 (5'-CTTTAAGAAGGAGATCTACCATGGGCAGCA-3') and Vps9–15 (FL, {Delta}N; 5'-TGTGCATGAGATCTTTATTCTGACAGAGAAAGTAG-3') or Vps9–17 ({Delta}C; 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 (32) 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 (33) 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 (29) 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-{beta}-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-{beta}-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 ml1) was incubated with either His6HA-ubiquitin (13 µg ml1) or bovine serum albumin (5 µg ml1) in 1 ml of binding buffer (50 mM Tris, pH 7.5, 300 mM KOAc with protease inhibitors (N-tosyl-L-phenlalanine-chloromethyl ketone, N{alpha}-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, 5x Laemmli sample buffer (0.312 M Tris, pH 6.8, 10% SDS, 25% {beta}-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 ml1). Samples were resuspended in 100 µl of 5x 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 (23). Yeast strains were grown in YNB-glucose and labeled at 30 °C for 10 min with EasyTag Expre35S35S protein labeling mix (30 µCi A6001) 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 ml1 and cultured for 4 h. Three A600 equivalents were harvested and resuspended at 1 A600 ml1 in YNB-glucose, and cyclohexamide (1 mg ml1 in ethanol) was added to 1.3 µg ml1. 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 2x urea sample buffer (6 M urea, 125 mM Tris, pH 6.8, 6% SDS, 10% {beta}-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 2x 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 ml1) 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, 1x YNB, 4% glucose, 1x amino acids, 100 µg ml1 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 x 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, 5x 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. (34). MYY290 (RSP5) and MYY808 (rsp5ts) cultures were grown in YPD at 25 °C to ~5 A600 ml1. Pelleted yeast were resuspended in 25 mM Tris, pH 7.5, 150 mM NaCl with protease inhibitors (N-tosyl-L-phenlalanine-chloromethyl ketone, N{alpha}-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 x 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 ml1), His6Vps9p (0.34 mg ml1), His6hE1 (3.25 µg ml1; generously provided by J. Chen laboratory, UTSW), MBP-Ubc5p (0.8 mg ml1), with or without MBP-Rsp5p (27 µg ml1), added. Reactions were incubated at 25 °C for 1 h and terminated with the addition of 5x 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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (35) and can also be found in the SMART and PFAM databases (12, 13, 36). The CUE domain is found in organisms from yeast to humans (35) (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 (37) (Fig. 1B).



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FIG. 1.
Analysis of the CUE domain. A, depiction of the Vps9p and Rabex5 proteins in S. cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, mouse, and human with domains identified in the SMART data base. The Vps9 domain (black) is associated with catalytic exchange on Vps21p/Rab5 family proteins. The CUE domain is indicated in gray. B, ClustalW alignment of a subset of CUE domains from yeast, flies, and mammals described by Ponting (35). The carboxyl terminus of human Rabex5 (residues 449–491) alignment is also shown. Position of the methionine mutated to alanine (M419A) is denoted by an asterisk.

 

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.



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FIG. 2.
Vps9p binds ubiquitin. A, MBP-Vps9p (lane 1) and MBP-Vps9p M419A (lane 2) were expressed in E. coli and affinity purified with amylose resin as described under "Experimental Procedures." Approximately 1 µg of the purified proteins were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. The sizes of relevant proteins markers are indicated. B, Vps9p proteins were combined with His6HA-ubiquitin (lanes 2, 3, 5, and 6) or bovine serum albumin (lanes 1 and 4) and affinity purified with Ni-NTA-agarose as described under "Experimental Procedures." Western analysis of the eluate and starting material was performed with Vps9 antiserum (upper panels) or HA.11 monoclonal antibody (lower panels). C, the CUE domains (residues 408–451) from Vps9p wild-type (WT) and M419A were expressed in E. coli as MBP fusion proteins. Purified proteins were combined with His6HA-ubiquitin (lanes 1 and 3) or His6Vps21p (lanes 2 and 4) and affinity purified with Ni-NTA-agarose as described under "Experimental Procedures." Western analysis of the eluate was performed with MBP antiserum.

 

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 (35) (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 the CUE domain itself is a ubiquitin binding motif.

To confirm that the Vps9p CUE domain mediates the ubiquitin interaction, the isolated domain was examined for ubiquitin binding. Residues 408–451 were expressed in E. coli as a MBP fusion (MBP-Vps9p CUE). Purified protein was incubated with His6-HA-Ub (or His6Vps21p as a control), and His6-HA-Ub, together with the potential His6-HA-Ub·MBP-Vps9p CUE complexes, was isolated using Ni-NTA-agarose. As shown in Fig. 2C, MBP-Vps9p CUE was highly enriched in eluates upon inclusion of His6-HA-Ub (lane 1) as compared with His6-Vps21p (lane 2) indicating a specific association with ubiquitin. Mutation of methionine 419 to alanine (MBP-Vps9p CUE M419A) eliminated the ubiquitin-dependent enrichment (lane 3), consistent with results observed with the full-length protein. Starting material analysis confirmed that equal amounts of Vps9p CUE domain were used in each experiment (data not shown). These results demonstrate that the Vps9p CUE domain is both necessary and sufficient for ubiquitin binding.

CUE Domain Involvement in the Endocytic Pathway—Because an intact CUE domain is required for Vps9p ubiquitin binding in vitro, the role this domain plays in the in vivo function of Vps9p was examined. Wild-type VPS9, the M419A mutant allele, and a truncated allele that eliminated the CUE domain ({Delta}CUE; 1–407) were expressed in a yeast strain that lacked the chromosomal copy of VPS9 ({Delta}9). Western analysis of whole cell lysates from yeast expressing these forms of Vps9p indicated that all proteins were expressed at or near wild-type levels (Fig. 3A). It was demonstrated previously (10) that Vps9p is required for vacuolar delivery of soluble proteases such as CPY. In the absence of Vps9p, cells secrete CPY in its Golgi-modified p2 precursor form. To examine the role of the CUE domain in this biosynthetic pathway, maturation of CPY was assessed in strains expressing wild-type Vps9p, {Delta}CUE, or M419A (Fig. 3B, lanes 1, 3, and 4). CPY immunoprecipitated from 35S pulse-labeled wild-type or CUE mutant strains was found in its mature form indicative of vacuolar delivery (Fig. 3B). Similar analysis of carboxypeptidase S (CPS) processing indicated that CPS maturation is also unaffected in the CUE mutant strains (data not shown). In addition, a time course analysis of CPY processing in strains expressing wild-type Vps9p or the M419A allele was performed, and the patterns of CPY maturation are indistinguishable (data not shown). These results demonstrate that the Vps9p CUE domain, and thus ubiquitin binding, is not required for biosynthetic transport of CPY or CPS to the vacuole.



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FIG. 3.
Vps9p CUE domain is not required for CPY sorting. A, lysates were generated from wild-type (WT; lane 1), {Delta}vps9 (lane 2), and {Delta}vps9 yeast strains expressing Vps9p {Delta}Cue (lane 3), wild-type (lane 4), or M419A (lane 5) encoded on a plasmid. Western analysis with Vps9p antiserum detected the predominant unmodified form of Vps9p, as well as a covalently modified form (Vps9p*). B, yeast strains expressing Vps9p wild-type (lane 1), {Delta}Cue (lane 3), or M419A (lane 4) proteins were labeled with EasyTag Expre35S35S protein labeling mix for 10 min and chased with excess methionine and cysteine for 30 min. Proteins were precipitated with trichloroacetic acid, and CPY was immunoprecipitated from the resolublized fraction as described under "Experimental Procedures." Samples were resolved by SDS-PAGE and detected by fluorography. The positions of mature vacuolar CPY (mCPY) and Golgi-modified precursor CPY (p2CPY) are indicated.

 

Ubiquitylation is a critical signal for the endocytosis and vacuolar degradation of plasma membrane proteins (reviewed in Ref. 38), and Vps21p has been demonstrated to participate in endocytosis (39). To address a potential role for Vps9p and the Vps9p CUE domain in the endocytic process, trafficking of the a-factor receptor, Ste3p, was analyzed. In the absence of pheromone, Ste3p is constitutively ubiquitylated, internalized, and transported to the vacuole where it is degraded (40, 41). The extent and rate of Ste3p degradation is therefore a good indicator of traffic through the yeast endocytic pathway. To quantitatively examine this trafficking process, degradation of Ste3p was examined. Extracts from wild-type and vps9 mutant strains expressing an HA-tagged Ste3p (Ste3HAp) were generated 0, 20, 40, and 60 min after the addition of cyclohexamide (to block further production of Ste3HAp), and the amount or Ste3HAp remaining at each time point was determined by Western analysis (Fig. 4A and B). In the wild-type strain (VPS9), the majority of Ste3HAp had been degraded at 60 min, with only 17% remaining. By contrast, complete elimination of Vps9p ({Delta}vps9) resulted in Ste3HAp stabilization; however, this effect can be attributed to defects in vacuole proteolytic capacity in addition to trafficking defects in the endocytic pathway. Mutation of the CUE domain (vps9 M419A) resulted in decreased Ste3p turnover, with 42% Ste3HAp remaining at 60 min. A similar defect was observed with the CUE domain deletion allele (data not shown). Because the vacuole is proteolytically active in the vps9 CUE mutant strains (Fig. 3B), these findings indicate that the Vps9p CUE domain is required for efficient delivery of Ste3p to the vacuole for degradation.



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FIG. 4.
Vps9p CUE domain functions in endocytic trafficking. A, 0, 20, 40, and 60 min after the addition of cyclohexamide, lysates were generated from wild-type (panel 1), {Delta}vps9 (panel 2), and {Delta}vps9 yeast strains expressing Vps9p M419A (panel 3) encoded on a plasmid as described under "Experimental Procedures." Western analysis with HA.11 monoclonal antibody was performed to detect Ste3HAp. B, densitometry was performed to quantitative the Ste3HAp degradation at 60 min as normalized to the amount at 0 min.

 

Identification of Ubiquityl-Vps9p—Western analysis of the various Vps9p alleles (Fig. 3A) revealed the presence of an immunoreactive band ~8 kDa larger than full-length Vps9p (denoted by asterisk; Vps9p*). The presence of this band was dependent on Vps9p expression (lane 2) and was also apparent in Western analysis of HA-tagged Vps9p (data not shown). These results, in conjunction with serial dilutions (data not shown), indicate that ~10% of Vps9p is covalently modified. This modification is dependent on ubiquitin binding as deletion or mutation of the CUE domain eliminated (lane 3) or diminished (lane 5) the amount of Vps9p*. These findings suggested that Vps9p might be a target for monoubiquitylation.

To demonstrate that Vps9p* is the monoubiquitylated form of Vps9p, His6-tagged Vps9p was coexpressed with HA-tagged ubiquitin in yeast. Vps9p was affinity purified from the lysates with Ni-NTA-agarose, and the presence of ubiquitylated Vps9p was detected by Western analysis using the HA.11 monoclonal antibody (Fig. 5). When full-length His6Vps9p was expressed and purified, an HA cross-reactive band ~8 kDa larger than His6Vps9p copurified with His6Vps9p (lanes 1 and 5); this band is also apparent in penta-His Westerns with longer exposure (data not shown). This result indicates that Vps9p* is ubiquityl-Vps9p. A slightly faster migrating HA-tagged band was also detected and is believed to result from partial proteolysis of ubiquityl-Vps9p. Expression of a mutant form of Vps9p that lacked residues 347–451 (Vps9p {Delta}C), including the CUE domain, was robust (Fig. 5, lane 2), but no corresponding HA-reactive band was detected (lane 6). These results indicate that Vps9p lacking residues 347–451, including the CUE domain, is not ubiquitylated, as observed in the Vps9p {Delta}CUE whole cell Western analysis shown in Fig. 3A. However, ubiquitylation of Vps9p was unaffected by removal of the amino terminus (Vps9p {Delta}N, 158–451; lanes 3 and 7). These observations demonstrate that Vps9p is subject to monoubiquitylation and that this modification is dependent on the CUE domain.



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FIG. 5.
Vps9p ubiquitylation in vivo. A, depiction of the His6-tagged Vps9p proteins expressed in yeast. B, lysates were generated from wild-type (WT) yeast strains expressing HA-ubiquitin and His6Vps9p full-length (FL; lanes 1 and 5), {Delta}C (1–347; lanes 2 and 6), or {Delta}N (158–451; lanes 3 and 7) and from {Delta}vps8 yeast strain expressing HA-ubiquitin and His6Vps9p (lanes 4 and 8). Nickel affinity purification was performed as described under "Experimental Procedures," and eluates were analyzed by Western analyses with penta-His (lanes 1–4) and HA.11 (lanes 5–8) monoclonal antibodies.

 

Vps9p Ubiquitylation Is Rsp5p-dependent—Ubiquitylation of proteins involves a multiprotein complex comprised of E1, E2, and E3 components (reviewed in Ref. 42). The E3 of this system is involved in substrate recognition and therefore imparts specificity. Several E3 ubiquitin ligases were examined to uncover their possible roles in Vps9p ubiquitylation. Vps8p was the first candidate investigated. Vps8p is a 134-kDa protein required for CPY sorting to the vacuole (23, 43). Like many E3 ubiquitin ligases, Vps8p contains a carboxyl-terminal H2 RING finger and has been demonstrated to function as an E3 ubiquitin-protein ligase in vitro.3 Because both Vps9p and Vps8p are thought to act at similar sites in the endocytic/vacuolar protein sorting pathway, Vps8p was examined as the putative Vps9p E3 ligase. However, wild-type levels of ubiquitylated Vps9p were still found in extracts generated from cells that lacked Vps8p ({Delta}vps8) (Fig. 5B, lanes 4 and 8). This result indicates that Vps8p is not the E3 ligase that modifies Vps9p.

Rsp5p is a yeast HECT domain E3 ubiquitin-protein ligase of the Nedd4 family (reviewed in Ref. 44). Rsp5p participates in the endocytosis of plasma membrane proteins both through ubiquitylation of the substrate proteins themselves and as components of the endocytic machinery (45, 46, 47). To determine whether Rsp5p is the E3 ubiquitin-protein ligase that modifies Vps9p, we examined ubiquitylation of Vps9p in a yeast strain that carried a temperature conditional allele of RSP5 (rsp5ts). Western analysis of cell extracts generated from the rsp5ts, wild-type, and {Delta}vps9 strains at permissive temperature (25 °C) revealed the presence of ubiquityl-Vps9p in both wild-type and the rsp5ts strains, though the level of ubiquitylated material was slightly reduced in the rsp5ts strain (Fig. 6A, lanes 2 and 3). When cells extracts generated from these stains were examined after a 30-min shift to nonpermissive temperature (37 °C), no ubiquitylated Vps9p was observed in the rsp5ts strain (lane 6). The level of ubiquityl-Vps9p in wild-type cells was unaffected by this temperature shift (lane 5). This finding demonstrates that Rsp5p is required for Vps9p ubiquitylation in vivo.



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FIG. 6.
Vps9p ubiquitylation is dependent on Rsp5p. A, lysates were generated from {Delta}vps9 (lanes 1 and 4), wild-type (WT; lanes 2 and 4), and rsp5ts yeast strains (ts; lanes 3 and 6) grown at permissive temperature (25 °C; lanes 1–3) or restrictive temperature (37 °C) for 30 min (lanes 4–6). Western analysis with Vps9p antiserum was performed. Positions of unmodified Vps9p and ubiquityl-Vps9p are indicated. B, in vitro Vps9p ubiquitylation reactions were performed with His6Vps9p, His6HA-ubiquitin, and lysates generated from wild-type (lanes 1 and 5)or rsp5ts yeast strains (lanes 2 and 6) or with MBP-Rsp5p added to the wild-type (lanes 5 and 7) or rsp5ts lysates (lanes 4 and 8), as described under "Experimental Procedures." Products were resolved by SDS-PAGE, and Western analyses with Vps9p antiserum (lanes 1–4) or HA.11 monoclonal antibody (lanes 5–8) were performed.

 

To further examine the Rsp5p dependence of ubiquityl-Vps9p, an in vitro ubiquitylation assay was utilized. Cleared lysates from wild-type and rsp5ts strains were generated and incubated with recombinant His6Vps9p, His6HA-ubiquitin, and ATP. Following a 1-h incubation period, Western analyses with Vps9p and HA antibodies were performed to detect ubiquityl-Vps9p. Addition of wild-type lysate could support the covalent modification of Vps9p with ubiquitin (Fig. 6B, lanes 1 and 5), with monoubiquitylation being the predominant form. A small amount of polyubiquitylated Vps9p was generated (Fig. 6B, lane 1) in these in vitro reactions. The faster migrating form of Vps9p seen in both the Vps9p and HA Western is likely a ubiquityl-Vps9p degradation product described earlier. In contrast, ubiquitylation of Vps9p was not supported by the rsp5ts extract (lanes 2 and 6); however, reintroduction of recombinant Rsp5p, expressed in E. coli as an MBP fusion, was able to restore Vps9p ubiquitylation to the rsp5ts extract (lanes 4 and 8). Additionally, HA Western analysis indicated increased incorporation of HA-ubiquitin in higher molecular weight species with the addition of Rsp5p (Fig. 6B, lanes 7 and 8); however, Vps9p Western analysis demonstrated that the vast majority of these species are not Vps9p (compare lanes 3 and 7). These results indicate that Vps9p ubiquitylation is dependent on Rsp5p both in vitro and in vivo and support Rsp5p as the Vps9p E3 ubiquitin-protein ligase.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vps9p and Rabex5 are guanine nucleotide exchange factors for Vps21p/Rab5 protein family members, but how these proteins are regulated has yet to be completely defined. Here, we have demonstrated that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. The interaction with ubiquitin is required to potentiate Vps9p function in the endocytic pathway; however, a requirement for the CUE domain in the CPY and CPS biosynthetic pathways is not apparent. Vps9p also is shown to be monoubiquitylated, and this modification is dependent on both the CUE domain and the E3 ubiquitin-protein ligase Rsp5p. These findings implicate ubiquitin as a regulator of Vps9p and identify Vps9p as an intermediary in ubiquitin-regulation of endocytosis.

The CUE domain is found in a large number of proteins that have been implicated in a variety of cellular processes (35), but the role this domain plays in protein function was unclear. We have demonstrated the CUE domain binds monoubiquitin. In addition, recent reports from Donaldson et al. (37) and Shih et al. (48) have demonstrated that the CUE domain binds polyubiquitin, as well as monoubiquitin. In what manner might ubiquitin binding or ubiquitylation modulate Vps9p? Two mechanisms can be proposed drawn from the functions of the Ras association and Src homology 2 domains in Rin1, a specialized mammalian Rab5 GEF. Binding of GTP·Ras to the Ras association domain has been shown to stimulate the in vitro GEF activity of Rin1, indicating an allosteric regulation that potentiates catalytic activity (15). Ubiquitylation or ubiquitin binding may serve a similar role to potentiate or depress the GEF activity of Vps9p by altering the accessibility to or the conformation of the exchange domain. Alternatively, ubiquitin binding by the CUE domain of Vps9p may serve to localize the exchange factor. The Rin1 Src homology 2 domain has been shown to bind to activated EGF receptors, thereby concentrating the GEF at sites of stimulated endocytosis.4 Vps9p binding to free ubiquitin has been demonstrated here; however, CUE domain binding may be preferential toward protein-conjugated ubiquityl moieties and thereby target Vps9p to ubiquitylated membrane proteins during endocytosis. A similar function has been suggested for the ubiquitin binding motifs found in the yeast proteins Ede1p, Vps27p, Ent1p, and Ent2p (49). A third possibility is that ubiquitylation or ubiquitin binding regulates Vps9p interaction with other unidentified components of the endocytosis machinery. Potential targets include components of the ubiquitylation machinery itself, by analogy to Cue1p interaction with E2 ubiquitin conjugating enzymes (50), or modulators of the actin cytoskeleton. A fourth possible function for ubiquitylation and ubiquitin binding is an intramolecular interaction whereby the CUE domain binds the ubiquityl moiety on Vps9p. The potential physiological effects of such an interaction are unclear, but this binding could explain the apparent stability of Vps9p monoubiquitylation in lysates. Of these possibilities, we favor the CUE domain serving to localize the exchange factor and Vps9p ubiquitylation serving to modulate its GEF catalytic activity by analogy to Rin1 regulation.

Ubiquitylation of yeast plasma membrane proteins is an essential step in the endocytosis of pheromone receptors (Ste2p, Ste3p) and small molecule transporters, including the general amino acid permease (Gap1p) and uracil permease (Fur4p) (reviewed in Refs. 38 and 44). This modification is required for the initial internalization step and is dependent on the E3 ubiquitin-protein ligase Rsp5p. However, ubiquitylation of the endocytic machinery by Rsp5p is also required for transport to the vacuole, as indicated by Dunn and Hicke (47). Because Vps9p is required for the efficient endocytosis of Ste3p, and Vps9p ubiquitylation is dependent on Rsp5p, Vps9p appears to be one piece of the endocytic machinery positively regulated by Rsp5p ubiquitylation. In addition, ubiquitin binding by the CUE domain positively impacts Vps9p function in the endocytic pathway. We suggest that the CUE domain may target Vps9p to transport intermediates harboring ubiquitylated receptors and subsequently stimulates their fusion with endosomal structures by the activation of the Rab protein Vps21p. In contrast, the CUE domain and ubiquitylation appear to be dispensable for Vps9p function in the biosynthetic pathway by which CPY and CPS are transported to the yeast vacuole. These findings do not address a role for Vps9p ubiquitin binding in the sorting of other ubiquitin-dependent biosynthetic pathway cargos, including Gap1p; these additional proteins will be examined in the near future.

Although further experimentation is required to define the precise role of ubiquitin in Vps9p function, similar regulation may occur in mammalian systems. Although a canonical CUE domain is not apparent in mammalian Rabex5 proteins, a highly divergent form may be present in the carboxyl terminus (37) (Fig. 1B). Recombinant human Rabex5 binds ubiquitin weakly in pull down experiments, and we have detected monoubiquitylation of green fluorescent protein-hRabex5 transiently transfected in Chinese hamster ovary cells.5 In addition, the Rsp5p orthologous Nedd4 E3 ubiquitin-protein ligase family has been shown to regulate mammalian receptor trafficking (reviewed in Ref. 44). These preliminary observations suggest that the same ubiquitin-dependent mechanisms regulating Vps9p may modulate mammalian Rabex5 proteins and will be examined further.


    FOOTNOTES
 
* This work was supported in part by grants from the National Institutes of Health (GM-055301) and by predoctoral fellowships from the Howard Hughes Medical Institute (to B. A. D.) and the National Science Foundation (to J. D. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger}{ddagger} To whom correspondence should be addressed. Tel.: 507-284-0308; Fax: 507-284-2053; E-mail: Horazdovsky.Bruce{at}mayo.edu.

1 The 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. Back

2 G. G. Tall, D. S. Carney, and B. F. Horazdovsky, manuscript in preparation. Back

3 A. S. Friedberg, P. Marshall, and B. F. Horazdovsky, manuscript in preparation. Back

4 M. A. Barbieri, C. Kong, B. F. Horazdovsky, and P. D. Stahl, submitted for publication. Back

5 J. D. Topp, B. A. Davies, and B. F. Horazdovsky, unpublished results. Back


    ACKNOWLEDGMENTS
 
We thank Claudio Joazeiro of the Genomics Institute of the Novartis Research Foundation for helpful scientific conversations. We also thank S. Emr for Vps9p antiserum, G. Payne for Ste3p antiserum, and members of the Horazdovsky and Roth laboratories for helpful discussions. We also acknowledge the early contributions of S. Straud.



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