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Address correspondence to Robert C. Piper, Physiology 5-660 BSB, University of Iowa, Iowa City, IA 52242. Tel.: (319) 335-7842. Fax: (319) 335-7330. email: piperr{at}physiology.uiowa.edu
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Abstract |
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Key Words: endosome; ubiquitin; multivesicular body; endosome; lysosome
Abbreviations used in this paper: CPY, carboxy peptidase Y; MVB, multivesicular body; Ub, ubiquitin; UEV, Ub E2 variant; UIM, Ub interaction motif.
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Introduction |
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Two class E Vps protein complexes have been proposed as endosomal Ub-sorting receptors. Vps23p, and its mammalian counterpart Tsg101, is a component of the ESCRT-I complex (Babst et al., 2000; Garrus et al., 2001; Katzmann et al., 2001). The NH2 termini of Vps23 and Tsg101 contain a Ub E2 variant (UEV) domain, and the Tsg101 UEV domain binds directly to Ub (Pornillos et al., 2002). The Tsg101 UEV domain also binds to a PTAP motif within a region of viral gag proteins required for budding, providing a mechanism for recruiting other class E Vps components (Perez and Nolan, 2001). A complex of two other class E Vps proteins, Vps27-Hse1, as well as their mammalian equivalent, Hgs-STAM1/2, also binds Ub via Ub interaction motifs (UIMs) (Bilodeau et al., 2002; Raiborg et al., 2002; Shih et al., 2002). Both the yeast and mammalian complexes localize to early endosomes, and EM studies have pinpointed Hgs to clathrin-rich subdomains where MVB formation ensues (Komada and Kitamura, 2001; Sachse et al., 2002). Mutations within the UIM domains of both Vps27 and Hse1 prevent sorting of ubiquitinated proteins to the MVB interior while other functions provided by the Vps27-Hse1 complex remain intact (Bilodeau et al., 2002).
Although there are two potential Ub-sorting receptors on endosomes, it remains unclear whether each recognizes ubiquitinated cargo (Ub-cargo) in vivo or whether Ub binding serves another purpose. Vps23 can associate with Ub-cargo, but overexpression of other class E Vps proteins (ESCRT-II complex) obviates a requirement for Vps23 in MVB sorting (Babst et al., 2002). Also, while mutation of Vps23 in the UEV domain blocks MVB sorting and vacuolar protease sorting (Katzmann et al., 2001), the dual nature of the Tsg101 UEV domain (i.e., binding Ub and PTAP) may indicate another role of the Vps23-UEV domain. Likewise, while a specific function for sorting Ub-cargo can be assigned to the UIM domains of the Vps27-Hse1 complex (Bilodeau et al., 2002), UIM domains may also direct ubiquitination of the UIM-containing proteins themselves for a yet unknown function (Polo et al., 2002).
We examined the contribution of Vps27 and Vps23 in the recognition and sorting of Ub-cargo into the MVB interior by identifying two sets of Ub mutations: one that binds Vps27-Hse1 but not Vps23-UEV and vice versa. MVB sorting of Ub-conjugated reporter proteins is blocked by either set of mutations. We also find that Vps27 binds the Vps23 UEV domain by PTAP-related motifs, providing a mechanism for the cooperation of these two Ub-sorting complexes.
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Results and discussion |
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Mutations in either the putative Vps27 or Vps23 binding site on Ub were made to assess their contribution to the MVB sorting signal. We placed our mutations within a Ub that lacked the two COOH-terminal glycine residues and contained K29R, K48R and K63R mutations (R3GG) to ensure that polyubiquitination or formation of Ub adducts would not occur in vivo. Because Vps27 forms a complex with the UIM-containing protein Hse1, we performed binding analyses with yeast lysates containing both proteins to ensure that we assessed binding of the whole complex. This was done using yeast lacking VPS23 as previously described (Bilodeau et al., 2002). Analysis of Vps23 binding was performed using a recombinant Vps23 UEV domain. Despite key differences in the residues of TSG101 important for Ub binding and the aligned residues of Vps23 (Pornillos et al., 2002), the Vps23 UEV domain specifically bound GST-Ub and GST-Ub (R3
GG) (Fig. 2 C).
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We then assessed how well these mutant forms of Ub could direct MVB sorting by fusing them to the COOH terminus of an Fth1-GFP reporter protein, which otherwise localizes to the limiting membrane of the vacuole (Urbanowski and Piper, 2001). To prevent degradation of intravacuolar vesicles, we used a pep4 mutant strain (Fig. 2 A). Consistent with the Ub binding data, the L8A or R42A mutations had no effect on sorting Fth1-GFP-Ub to the vacuole lumen. I44A had a modest effect consistent with previous results (Urbanowski and Piper, 2001), while V70A also had a slight effect. Ub mutations that greatly inhibited Vps27 binding (I44A V70A and L8A V70A) were defective in sorting Fth1-GFP to the vacuole interior. Likewise, the Q62A E64A mutant was defective in MVB sorting, and the defect was accentuated by the addition of I44A. Thus, Ub mutations in either the Vps23- or Vps27-binding site blocked the ability of Ub to serve as an MVB sorting signal. These data indicate that both the Vps27-Hse1 complex and the Vps23-containing ESCRT-I complex recognize Ub-cargo proteins to effect their sorting into intralumenal membranes. One caveat is that there may yet be another endosomal Ub-binding protein that fulfills this function. No candidates have so far been identified, and given the correlation between loss of binding and the loss of MVB sorting in the various Ub mutants, these data support the idea that direct recognition of Ub-cargo by Vps27 and Vps23 is necessary for efficient MVB sorting.
We next used a series of reporter proteins with two tandem Ub molecules to show that the presence of a Vps27-bindingdefective Ub mutant could complement a Vps23-bindingdefective Ub mutant in a tandem array (Fig. 2 B). This was not simply due to the presence of multiple but defective Ub-sorting motifs, as an Fth1-GFP-Ub-Ub reporter containing either two I44A V70A mutant Ubs or I44A Q62A E64A mutant Ubs was still defective in sorting to the vacuole interior. The ability of these Ub alleles to complement indicated that the mutations specifically affected Vps27 or Vps23 binding.
The observation that Vps27-Hse1 and ESCRT-I recognize Ub-cargo but not necessarily the same Ub moiety suggests that Ub-cargo could interact sequentially with each Ub-receptor component for final incorporation into the MVB lumen. Tsg101 and UIM-containing proteins bind Ub with low affinity, which may allow for shuttling Ub conjugates between different sorting receptors (Pornillos et al., 2002). Indeed, the putative binding sites of Vps27 and TSG101 on Ub partly overlap, potentially prohibiting simultaneous binding. Such a model is attractive given the physical interaction of the Hgs-STAM complex with another Ub-binding protein, Eps15 (Komada and Kitamura, 2001; Bache et al., 2003). Eps15 is important for the clathrin-dependent internalization of Ub-cargo such as EGFR (Confalonieri et al., 2000). Perhaps Ub-cargo is passed from one Ub-sorting receptor to another to effect its final delivery into the MVB degradative pathway. One prediction from this model is that Vps27 may also directly associate with Vps23. As an initial test, we found that high levels of HA-tagged Vps23 expressed in yeast lysates could bind to various fragments of Vps27 expressed as GST fusion proteins (Fig. 3 A). The Vps27 COOH terminus also bound clathrin via a clathrin box motif (ELLIEL) located at the extreme COOH terminus. Deletion of the clathrin box did not affect binding of Vps23. Interestingly, a truncated version of Vps27 lacking the clathrin box motif was normal for vacuolar protease sorting and Ste3-GFP localization to the vacuole lumen and only showed a defect in the clearance of Ste3 from the cell surface (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200305007/DC1). The GST-Vps27 COOH-terminal fusion protein also bound high levels of recombinant Vps23-UEV (1161) and Vps23-UEV (1186) domains, demonstrating direct interaction (Fig. 3 B). Because the mammalian Vps23 homologue Tsg101 binds to PTAP motifs within viral gag proteins via its UEV domain, we suspected that a similar mechanism might explain the association of Vps23 with Vps27. A PTVP motif spans residues 581584 in the COOH terminus of Vps27. However, a GST-Vps27 truncation containing residues 1579 bound both Vps23-HA from yeast lysates as well as recombinant Vps23-UEV domain (Fig. 3, A and B). Truncation at residue 579 or beyond resulted in a class E phenotype (Fig. S1). To map the Vps23 binding site on Vps27, we performed coimmunoprecipitation studies using Vps27 truncation mutants. Approximately 10% of Vps23 could be immunoprecipitated with anti-Vps27 NH2-terminal antibodies when full-length Vps27 was present or when truncated at residues 617, 579, 524, or 485. Further truncation analysis showed that residues 430484 were required for Vps23 binding (Fig. 3 C). To determine what motifs serve as a binding interface between Vps23 and Vps27, we analyzed an alignment of Vps27 orthologues from several other budding yeast because the evolutionary distance of these related species might be close enough to conserve protein interaction sites but distant enough to allow irrelevant residues to vary. We identified a proline-containing region (PSDP 447450 [PSDP-1]) as a candidate interaction site for Vps23. Mutation of the PSDP-1 (447450) motif did not block Vps23 binding. However, additional truncation or alanine substitution of a second conserved PSDP motif (PSDP-2) resulted in loss of Vps23 coimmunoprecipitation (Fig. 3 E). Accordingly, the allele containing alanine substitutions in both PSDP motifs was renamed vps27-Vps23. This result was confirmed with recombinant protein in vitro (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200305007/DC1). The Vps27-
Vps23 protein could associate with Hse1, indicating that it did not undergo global misfolding (Fig. 4 B). A similar truncation and alanine scanning mutagenesis approach was undertaken to interrupt the Hse1 binding site on Vps27 (Fig. 3 C and Fig. 4 A). The resulting vps27-
Hse1 allele did not coimmunoprecipitate with Hse1 but did associate with Vps23 (Fig. 4 B).
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Materials and methods |
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NMR analysis
Recombinant Ub was expressed and purified as detailed previously (Sivaraman et al., 2001; Sundd et al., 2002). The 15N-labeling medium was from Spectra Stable Isotopes. 15N-Ub was mixed with 7 mg of GST-Vps27 fusion protein (Vps27 residues 1351 in pGEX-3x) or GST alone in 40 mM NaPO4, pH 7.2, containing 10% D2O at 25°C. Resonance assignments were used as previously described (Schneider et al., 1992).
Plasmids
Mutations in Ub used a derivative of p1717 that contained K29R, K48R, and K63R substitutions and deletion of G75G76 (Urbanowski and Piper, 2001). This cassette contained the 3' UT of PHO8 downstream of the Ub coding region. A BglII/NruI fragment encoding the various mutant Ub alleles was subcloned into the BglII/NruI site of Fth1-GFP-Ub plasmid p1717 to create GFP reporter proteins or into the BamHI/SmaI site of pGEX-3X to create GST-Ub proteins. The Fth1-GFP-Ub-Ub plasmids were similar to the Fth1-GFP-Ub constructs except that the last Ub moiety was preceded by a linker encoding SGSGTSGTR and an MluI site. The Vps23-UEV expression plasmids were made by subcloning a PCR fragment encoding 1161 or 1186 of Vps23 downstream of a GAL1 promoter and T7 promoter and upstream of a V5 epitope within pYes2.1 (Invitrogen). Expression of the Vps23-UEV was performed in BL21-DE3-RIL codon plus bacteria (Stratagene) with the addition of IPTG to induce production of T7 RNA polymerase. The vps27 alanine substitution mutants were made by creating a mutant PCR fragment encoding bp 11800 and subcloning relevant fragments into a CEN-based VPS27 plasmid (p1863). The Vps23-HA plasmid consisted of a centromeric plasmid expressing Vps23 flanked with a 3xHA tag expressed under the VPS23 promoter. GFP-CPS1 plasmid was used as previously described (Katzmann et al., 2001), except carried in a TRP1 low copy plasmid.
Binding studies
GST fusion protein (0.5 mg) bound to 100 µl of GSH-agarose was used to assess binding to Ub or the Vps27 COOH terminus. Yeast or bacterial lysates containing HA-tagged Vps27 and Hse1 (p1779 and p1808; Bilodeau et al., 2002) or the Vps23-UEV domain were added to the beads and incubated at 4°C for 2 h. Bacterial cells were lysed with a French Press, and yeast spheroplast lysates were produced with 600 mM sorbitol, 20 mM Hepes, 50 mM KCl, 100 mM potassium acetate, 1% TX-100 (lysis buffer). Beads were washed in lysis buffer and analyzed along with an equivalent of lysate by SDS-PAGE and immunoblotting with either monoclonal HA or V5 antibodies.
Immunoprecipitations
Cleared lysate from 100 OD of yeast spheroplasts resuspended in lysis buffer was divided and incubated with 2 µl of anti-Vps27, nonimmune, or anti-HA rabbit serum at 4°C for 2 h. Immune complexes were then isolated on fixed Staph-A beads, washed three times in lysis buffer, and analyzed by SDS-PAGE and immunoblotting.
Online supplemental material
The supplemental material (Figs. S1S3) is available a http://www.jcb.org/cgi/content/full/jcb.200305007/DC1. The effect of deleting the clathrin binding region of Vps27 is shown in Fig. S1. Confirmation of Vps23 binding to two PSDP motifs in Vps27 is shown in Fig. S2. Quantitation of sorting defects is shown in Fig. S3.
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Acknowledgments |
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Submitted: 2 May 2003
Accepted: 29 July 2003
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References |
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