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ESCRT ubiquitin-binding domains function cooperatively during MVB cargo sorting

¹ Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52240
² Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905

Correspondence to David J. Katzmann: katzmann.david{at}mayo.edu; or Robert Piper: Robert-piper{at}uiowa.edu

Ubiquitin (Ub) sorting receptors facilitate the targeting of ubiquitinated membrane proteins into multivesicular bodies (MVBs). Ub-binding domains (UBDs) have been described in several endosomal sorting complexes required for transport (ESCRT). Using available structural information, we have investigated the role of the multiple UBDs within ESCRTs during MVB cargo selection. We found a novel UBD within ESCRT-I and show that it contributes to MVB sorting in concert with the known UBDs within the ESCRT complexes. These experiments reveal an unexpected level of coordination among the ESCRT UBDs, suggesting that they collectively recognize a diverse set of cargo rather than act sequentially at discrete steps.

Abbreviations used in this paper: CPY, carboxypeptidase Y; ESCRT, endosomal sorting complexes required for transport; DIC, differential interference contrast; HSQC, heteronuclear single quantum coherence; MVB, multivesicular body; NMR, nuclear magnetic resonance; NZF, Npl4 zinc finger; TAP, tandem affinity purification; Ub, ubiquitin; UBD, Ub-binding domain; UEV, Ub E2 variant; UIM, Ub-interacting motif.

© 2009 Shields et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

	Introduction

Top Abstract Introduction Results and discussion Materials and methods References

 
Many integral membrane proteins targeted for lysosomal degradation are ubiquitinated and sorted into vesicles that bud from the limiting membrane into the lumen of endosomes during the biogenesis of multivesicular bodies (MVBs; Piper and Katzmann, 2007). Cargo selection during MVB sorting is dependent on several endosomal protein complexes known as endosomal sorting complexes required for transport (ESCRT). Several ESCRT components contain ubiquitin (Ub)-binding domains (UBDs) that may act as receptors for ubiquitinated membrane proteins. For instance, ESCRT-0, comprised of yeast Vps27 and Hse1 or mammalian Hrs and STAM (signal transducing adaptor molecule), contains Ub-interacting motifs (UIMs) required for MVB sorting of ubiquitinated cargo (Bilodeau et al., 2002; Shih et al., 2002; Piper and Katzmann, 2007). The Ub E2 variant (UEV) domain in Vps23/TSG101, an ESCRT-I component in yeast/mammalian cells, and the GLUE (GRAM-like Ub binding in EAP45) or Npl4 zinc finger (NZF) domains of Vps36, an ESCRT-II component, also contain UBDs that have been implicated in MVB cargo selection (Katzmann et al., 2001; Alam et al., 2004; Slagsvold et al., 2005). This had led to a model in which Ub cargo is passed sequentially from ESCRT-0 to ESCRT-I and to ESCRT-II for final deposit into the forming lumenal vesicles (Katzmann et al., 2002; Gruenberg and Stenmark, 2004; Hurley, 2008). Previous studies have indicated the importance of these UBDs during MVB sorting; however, recent high resolution structures that detail Ub interactions have enabled a more precise approach to dissect the contributions of these UBDs to MVB sorting (Alam et al., 2004, 2006; Sundquist et al., 2004; Teo et al., 2004; Hirano et al., 2006). Surprisingly, we found that mutant forms of Vps23 and Vps36 unable to bind Ub had no defects in MVB sorting, even in combination. This led us to search for additional UBDs within ESCRT-I and resulted in the discovery of a novel UBD within Mvb12. The UBD of Mvb12 works in concert with the UBDs of the ESCRTs to promote cargo recognition and MVB sorting.

	Results and discussion

Top Abstract Introduction Results and discussion Materials and methods References

 
Mutants of Vps23 and Vps36 defective for Ub binding
Cocrystal structures of Ub with the UEV domains of Vps23 and TSG101 were used to guide mutagenesis experiments rendering Vps23 unable to bind Ub (Sundquist et al., 2004; Teo et al. 2004). The structures show that the UEV domains bind to Ub via a β hairpin tongue and a lip (Fig. 1 A). The structure of the tongue interface is highly conserved and includes residues F₅₂-G₅₇ of Vps23 and Y₄₂-G₄₇ of TSG101. The lip of Vps23 contacts Ub with residues Q₁₁₇ and W₁₂₅, which is different from TSG101 (Pornillos et al., 2002a,b). Two triple-mutant alleles of VPS23 were generated: vps23^Ub1 (F₅₂A, Q₁₁₇A, and W₁₂₅A) and vps23^Ub2 (F₅₂A, M₁₀₇A, and W₁₂₅A). The corresponding UEV domains were expressed as V5 epitope–tagged proteins in bacteria and used for binding assays with GST-Ub. The wild-type UEV domain of Vps23 bound Ub, whereas no binding was observed for the mutant UEV domains (Fig. 1 B). However, all UEV domains bound to the C terminus of Vps27, which contains two PSDP motifs (P₄₄₈SDP and P₅₂₄SDP) that directly interact with Vps23 (Bilodeau et al., 2003). TSG101 has a similar interaction with PTAP motifs of viral gag proteins and Hrs (Pornillos et al., 2002a,b). Although the structural basis of the TSG101–PTAP interaction is known, it is not known for the Vps23–UEV–PSDP interaction, and the PTAP-binding pocket of TSG101 is not conserved in Vps23. Regardless, this demonstrated that the Vps23 UEV mutants are specifically defective for Ub binding and did not compromise expression of corresponding full-length Vps23 in vivo (Fig. 1 C).

View larger version (41K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Generation of Vps23 and Vps36 mutants incapable of binding Ub. (A) Model of the Vps23 UEV domain in complex with Ub (Protein Data Bank accession no. 1UZX). The residues mutated to create the vps23Ub alleles are shown in red. (bottom) A schematic of the position of the UEV domain and the core region of Vps23 involved in complex formation with other ESCRT-I subunits is shown. (B) Recombinant V5 epitope–tagged wild-type and mutant Vps23 UEV domains were used for binding experiments with GST, Ub-GST, or GST-Vps27 C terminus. Bound proteins were immunoblotted with 1 or 10% of the input lysates. (C) Cell lysates from strains (PLY335, PLY3529, and PLY3530) bearing the wild-type (WT) or mutant alleles of VPS23 were immunoblotted with -Vps23 and -PGK (3-phosphoglycerate kinase) antibodies. (D) Schematic of Vps36 showing the GLUE domain with an insertion of two NZF domains, the second of which binds Ub. The core region of Vps36 interacts with the rest of ESCRT-I. (E) Summary of HSQC NMR experiments with 15N-labeled Ub (25 µM) with the GLUE domains from wild-type Vps36 GLUE domain (40 µM) or Vps36Ub (240 µM). Chemical shift differences were quantified and plotted on the Ub sequence (right) or mapped on the Ub surface (left). Red indicates (0.2N2 + H2)1/2 0.03. Black residues were not observed. The models below show the NMR structure of Ub with the Npl4 NZF domain and a predicted structure of the Vps36 NZF domain. Yellow residues are the position of T187F188. (F) Portion of the HSQC spectra of 15N-labeled Ub in the presence (red) and absence (green) of GST fusions of Vps36 and Vp36Ub. ppm, parts per million. (G) V5 epitope–tagged Vps36 GLUE domains of wild-type or Ub were assayed for Ub binding with Ub-GST. Bound proteins were immunoblotted with 10% of the input lysates. (H) Cell lysates from wild-type strains expressing HA epitope–tagged alleles of wild-type VPS36 and vps36Ub from low copy plasmids were immunoblotted with anti-HA and -PGK.

Ub

Ub

Ub

Ub

Ub

vps36^Ub

vps36^Ub

Effect of inactivation of Vps23 and Vps36 Ub binding
To assess the role of Ub binding by Vps23 and Vps36, yeast strains were constructed in which the mutant alleles were integrated in place of the endogenous genes. We found that the inability of Vps23 or Vps36 to bind Ub had no effect on the sorting of GFP-Cps1, a model MVB cargo (Odorizzi et al., 1998), into the vacuolar lumen (Fig. 2 A). No difference was observed between the vps23^Ub1 or vps23^Ub2 alleles (unpublished data). Analysis of additional mutant vps23 alleles with mutations in other residues that line the Ub interface (S₅₆, G₅₇, F₁₀₅, and N₁₂₃) also did not display any sorting defects (Fig. S1). Likewise, a double mutant containing vps23^Ub1 and vps36^Ub showed normal sorting of GFP-Cps1 (Fig. 2 B). These results were confirmed using vps23 and vps36-null mutants transformed with wild-type and Ub alleles of VPS23 and VPS36 housed on low copy plasmids (unpublished data). Lastly, sorting of other MVB cargoes, including Ste3-GFP (Urbanowski and Piper, 2001), Fur4-GFP (Blondel et al., 2004), and Sna3-GFP (Reggiori and Pelham, 2001) was normal in the vps23^Ub1 and vps36^Ub alleles (unpublished data).

View larger version (92K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Loss of Mvb12 reveals sorting defect of vps23Ub and vps36Ub mutants. (A) GFP-Cps1 localization was assessed in vps23Ub1 (PLY3528) and vps36Ub (PLY3395) cells. GFP fluorescence images along with matching DIC images are shown. (B) Sorting of GFP-Cps1, Ste3-GFP–Ub, and Ste3-GFP in vps23Ub1 vps36Ub (PLY3522), mvb12-null (JPY21), vps23Ub1 vps36Ub mvb12 (PLY3623), and vps23Ub1 mvb12 (PLY3624) cells. (C) GFP-Cps1 from wild-type (WT) and vps23Ub1 vps36Ub mvb12 cells was immunoprecipitated with anti-GFP antibodies and immunoblotted with anti-Ub and anti-GFP antibodies. Black line indicates that intervening lanes have been spliced out. IP, immunoprecipitation. Bars, 5 µm.

vps23^Ub vps36^Ub

Mvb12 binds Ub
A potential candidate for a protein with a novel UBD was Mvb12, a recently identified subunit of ESCRT-I. Previous studies showed that deletion of MVB12 caused differential defects in MVB sorting; Ste3-GFP and Sna3-GFP display sorting defects, whereas GFP-Cps1, Ste2-GFP, and Ste3-GFP–Ub (in which Ub is fused in frame to the C terminus) sorting is largely unaffected (Chu et al., 2006; Curtiss et al., 2007; Oestreich et al., 2007). This indicates that Mvb12 plays a specific role in recognizing a subset of MVB cargoes. A role for Mvb12 cargo recognition was tested with strains that combined vps23^Ub1, vps36^ub, and mvb12 to probe for synthetic defects in MVB sorting. Combining these mutations caused dramatic sorting defects in GFP-Cps1 and Ste3-GFP (Fig. 2 B). The defect in sorting of GFP-Cps1 is not the result of a detectable loss of Cps1 ubiquitination (Fig. 2 C) but rather indicates a defect in recognition of Ub cargos. This was supported by examining the sorting of Ste3-GFP–Ub, which was normal in mvb12-null and vps23^Ub1 vps36^Ub double-mutant cells but defective in vps23^Ub1 vps36^Ub mvb12 triple-mutant cells (Fig. 2 B).

The genetic interactions between Mvb12 and ESCRT-I and -II UBDs prompted us to determine whether Mvb12 could bind Ub. Recombinant V5 epitope–tagged full-length Mvb12 from Saccharomyces cerevisiae and the related budding yeast Saccharomyces kluyveri were used in binding assays with Ub-GST. Both proteins bound specifically to Ub-GST (Fig. 3 A). Deletion analysis of Mvb12 mapped the Ub binding to the C terminus (W₅₀-S₁₀₁; unpublished data). This interaction was confirmed by NMR HSQC experiments of ¹⁵N-labeled Ub combined with the C terminus of Mvb12. Mvb12 induced chemical shift perturbations in the spectrum of ¹⁵N-labeled Ub that mapped to a surface of Ub that included residues R₄₂, V₇₀, and L₈ (Fig. 3 E). This overlaps with the surface engaged by the Vps23 UEV domain, the Vps36 NZF domain, and the UIM domains of Vps27 and Hse1, indicating that simultaneous binding of these components to a single Ub molecule is unlikely (Bilodeau et al., 2003; Alam et al., 2004; Teo et al., 2004).

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Binding of Mvb12 to Ub. (A) Recombinant V5 epitope–tagged Mvb12 for S. cerevisiae and S. kluyveri were assayed for Ub binding with Ub-GST. (B) V5 epitope–tagged fusions of the C terminus of either wild-type (WT) Mvb12 or the indicated mutants were expressed in bacteria and assayed for Ub binding with Ub-GST. (C) A sequence of Mvb12 with the Ub-binding region shown in red. Regions that make contact with Vps37 in the ESCRT-I crystal structure (Protein Data Bank accession no. 2P22) are underlined. The asterisk indicates the end of the protein sequence. (D) Cell lysates prepared from mvb12 cells transformed with low copy plasmids expressing Mvb12-HA (pPL3713), Mvb12Ub1-HA (pPL3709), or Mvb12Ub2-HA (pPL3711) were immunoblotted with anti-HA and anti-PGK antibodies. (E) Summary of NMR HSQC experiments with 15N-labeled Ub (50 µM) with the C-terminal fragment of Mvb12 (100 µM). (left) Part of the spectra of 15N-labeled Ub in the presence (red) and absence (green) of Mvb12 is shown. Chemical shift differences were quantified and plotted on the linear sequence of Ub (middle) or onto the surface of Ub (right). Red indicates (0.2N2 + H2)1/2 0.02. Black residues were not observed. ppm, parts per million.

Contribution of Mvb12 Ub binding to MVB sorting
We initially assessed the function of wild-type and the two Mvb12 Ub-binding mutants as HA epitope–tagged proteins expressed from low copy plasmids containing the native MVB12 promoter. All three proteins expressed at comparable levels (Fig. 3 D), and all coprecipitated with Vps23 and Vps28, demonstrating that they properly assembled with ESCRT-I (Fig. 4 A). Gel filtration showed that tandem affinity purification (TAP)–tagged Mvb12^Ub1 (Y₇₉-G₈₅>A₇₉AAAAA) supported assembly into a large complex with an apparent molecular mass of 350 kD, as has been previously observed for ESCRT-I (Fig. S2). Furthermore, GFP-tagged Mvb12^Ub1 localized to endosomal compartments similar to GFP-tagged wild-type Mvb12 (Fig. S2). These data indicate that these Mvb12 mutants were able to assemble into the ESCRT-I complex. This allowed us to ascribe phenotypes to a deficiency in Ub binding of Mvb12 rather than loss of ESCRT-I association.

View larger version (72K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Ub binding by Mvb12 contributes to GFP-Cps1 sorting. (A) Mvb12 mutants lacking Ub binding correctly assemble with ESCRT-I. HA epitope–tagged wild-type Mvb12 and two mutants, mvb12Ub1 and mvb12Ub2, were expressed in mvb12 cells (pPL23713, pPL3709, and pPL3711 in JPY21). Lysates were prepared and immunoprecipitated with anti-HA antibodies and immunoblotted with -Vps23, -Vps28, or -HA antibodies. IP, immunoprecipitation. (B) Sorting of Sna3-GFP and Ste3-GFP in mvb12 cells cotransformed with plasmids containing HA-tagged mvb12Ub alleles under the endogenous promoter. (C) Strains carrying the mvb12Ub alleles alone or in combination with vps23Ub and vps36Ub were assessed for MVB sorting using GFP-Cps1, Sna3-GFP, and Ste3-GFP. The MVB12 alleles were expressed as HA epitope–tagged proteins from low copy plasmids (pPL23713, pPL3709, and pPL3711). Wild-type (WT), mvb12-null, and vps23Ub2 vps36Ub mvb12–null cells were also analyzed. The GFP fluorescence images and corresponding DIC images are shown. Bars, 5 µm.

mvb12^Ub1

mvb12^Ub2

mvb12

mvb12^Ub

Together, these data indicate that the mutations used to ablate Ub binding did not indiscriminately compromise Mvb12 function. They also implied that the function of Ub binding by Mvb12 alone is not critical for MVB sorting. We next assessed the role of the Mvb12 UBD in cells lacking the UBDs of Vps23 and Vps36 (Fig. 4 C). The vps23^Ub1 vps36^Ub mvb12 triple mutant was cotransformed with low copy plasmids expressing wild-type MVB12, mvb12^Ub1, or mvb12^Ub2 and GFP-tagged MVB cargos. We found that wild-type MVB12 conferred proper sorting of all cargos, whereas both mvb12^Ub alleles showed defective sorting of GFP-Cps1 when combined with vps23^Ub and vps36^Ub. These data show that the UBDs of Mvb12, Vps23, and Vps36 work jointly to efficiently sort MVB cargo because sorting was defective only when mutations were combined (Fig. 2 B and Fig. 4 C).

In contrast to the sorting of GFP-Cps1, other MVB cargos were sorted normally in the vps23^Ub vps36^Ub mvb12^Ub triple mutants. These cargos included Sna3-GFP and Ste3-GFP (Fig. 4 C). This demonstrated that some degree of Ub-dependent MVB sorting was still intact and that there might be other ESCRT components that contribute to MVB cargo recognition; one candidate is Vps27–Hse1 (ESCRT-0). The Vps27–Hse1 complex contains three UIMs and binds the Vps23 UEV domain via its two PSDP motifs within the C terminus (P₄₄₈SDP and P₅₂₄SDP). Although deletion of VPS27 altogether blocks ESCRT-I localization to endosomes (Katzmann et al., 2003), little phenotype is observed when the protein–protein interface between ESCRT-0 and ESCRT-I is disrupted by mutation of the two PSDP motifs in Vps27 (Vps27^Vps23, AA₄₄₈ADP and P₅₂₄AAA; Bilodeau et al., 2003). Localization of Vps23-GFP to endosomes was unperturbed as was recruitment of mCherry-tagged ESCRT-III subunit Vps20 (Fig. S1). To investigate whether the interaction with ESCRT-0 helped maintain sorting of MVB cargos in the vps23^Ub1 vps36^Ub mvb12^Ub strain, these mutations were combined with the vps27^Vps23 mutation. The resulting quadruple mutant displayed very significant sorting defects (Fig. 5 A). Sorting of all MVB cargoes was defective in vps23^Ub1 vps36^Ub vps27^Vps23 mvb12^Ub mutant cells. This included Ste-GFP, Ste3-GFP–Ub, and Sna3-GFP, which otherwise sorted normally in vps23^Ub1 vps36^Ub mvb12^Ub mutant cells (Fig. 4 C) or vps23^Ub vps36^Ub vps27^Vps23 cells (Fig. 5 A). These experiments clearly showed a critical contribution of the Mvb12 UBD because dramatic sorting defects were observed when the mvb12^Ub vps23^Ub vps36^Ub and vps27^Vps23 alleles were combined. In spite of missorting MVB cargoes, the quadruple mutant cells were still capable of a residual level of sorting lipids into the interior lumenal membranes, indicated by sorting of the fluorescent lipid marker NBD-PC into the vacuole lumen. Unlike ESCRT-null mutants in which NBP-PC accumulates exclusively in endosomes (Bilodeau et al., 2002), NDB-PC accumulated within the vacuoles of vps23^Ub vps36^Ub vps27^Vps23 mvb12^Ub cells (Fig. 5 B). Disruption of the Vps27–Vps23 interaction combined with mutation of the UBDs within ESCRT-I and -II revealed a separation of cargo sorting and MVB biogenesis.

View larger version (51K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Synthetic defects revealed by uncoupling ESCRT-0 from ESCRT-I. (A) Strains (PLY3734, PLY3779, PLY3777, and PLY3781) of the indicated genotype were assessed for sorting of GFP-Cps1, Ste3-GFP, Ste3-GFP–Ub, and Sna3-GFP. All alleles were stably integrated into the genome as nonepitope alleles under the control of their endogenous promoter. The GFP fluorescence images and corresponding DIC images are shown. (B) The same strains as in A were analyzed for sorting of the lipid marker NBD-PC. (C) Model for function of UBDs in the ESCRT machinery. Model 1 depicts the UBDs of ESCRT-0, -I, and -II acting together to present multiple binding sites for cargo. This model predicts that other UBDs are present in the ESCRT complexes. Model 2 shows that although UBDs of ESCRT-0 bind Ub cargo, the UBDs of ESCRT-I and -II may interact with Ub in other proteins and may regulate the assembly or activity of the MVB sorting apparatus. (D) Schematic of the various UBDs within the ESCRTs and the connections that tie the ESCRTs together in a supercomplex. Bars, 5 µm.

vps27^Vps23

	Materials and methods

Top Abstract Introduction Results and discussion Materials and methods References

 
Materials, yeast strains, and plasmids
Chemicals, antibodies, and, growth methods were used as previously described (Bilodeau et al., 2003; Ren et al., 2007). S. cerevisiae strains used in this study are listed in Table I. Plasmids used in this study are listed in Table II. VPS36 was disrupted by inserting the HIS3 gene in place of codons 101–539 of VPS36. Wild-type or vps36^Ub (T₁₈₇F/G₁₈₈A) cloned into the integrating TRP1 plasmid pRS304 (pPL2646 and pPL2645, respectively) was linearized with ClaI and integrated at the vps36::HIS3 locus to make PLY3394 and PLY3395. The vps23::Kanr, mvb12::Kanr, and vps27::Kanr disruptions were made by amplifying the respective loci from the BY4742 deletion collection (Winzeler et al., 1999) and transforming the PCR product into the appropriate strain. Vps27^Vps23 was made by integrating linearized integration plasmid pLP2251 digested with BlgII into the URA3 loci of strains containing the vps27::LEU2 disruption. The vps23^Ub alleles were made by linearizing integrating pPL2940 into the VPS23 loci of PLY3405 and PLY3407 and looping out the insertion of the URA3 gene by selection of 5-FOA.

vps27::LEU2

HA epitope–tagged alleles of MVB12 were made by inserting a single HA tag at the C terminus of MVB12 followed by the PHO8 3' untranslated region. Expression was driven by the MVB12 promoter (500 bp region upstream of the MVB12 ORF). Untagged MVB12 alleles were made similarly but lacked the C-terminal tag. TAP- and GFP-tagged MVB12 alleles were made as previously described (Oestreich et al., 2007).

Glutathione agarose affinity chromatography
GST-fusion proteins were isolated from bacteria using glutathione-Sepharose beads as previously described (Smith and Johnson, 1988). For pull-down assays, 250 µg of each isolated GST fusion protein was bound to 50 µl glutathione-Sepharose in PBS by rotation for 30 min at 25°C. Bound GST or GST fusion proteins were pelleted and washed three times with PBS. Cell lysate was added to each protein/bead complex and incubated for 2 h at room temperature. Unbound proteins were removed from beads using three washes of cold PBS eluted with 50 µl 50 mM glutathione in PBS, pH 7.2, and the bound bead fraction was analyzed by SDS-PAGE and immunoblotting.

Fluorescence microscopy
Cells containing GFP-expressing plasmids were grown in synthetic dextrose minimal media to mid-log phase, resuspended in 0.2% NaN3 and 100 mM Tris, pH 8.0, and viewed using a 100x 1.4 NA aperture on a microscope (BX-60; Olympus) equipped with fluorescein isothiocyanate filters and Nomarski/differential interference contrast (DIC) optics at ambient temperature. Images were captured with a charge-coupled device camera (ORCA; Hamamatsu Photonics) with IPLABS software (BD) as previously described (Urbanowski and Piper, 1999). Image processing was performed in PhotoShop (CS; Adobe), where the original 12-bit images were converted to 8-bit images and the brightest pixel was set to 255. NBD-PC (1-myristoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocholine; Avanti Polar Lipids, Inc.) was used as previously described (Bilodeau et al., 2002).

NMR analysis
Recombinant proteins mixed with ¹⁵N-labeled Ub were prepared as previously described (Bilodeau et al., 2004) or purchased from Spectra Stable Isotopes. [¹⁵N]Ub HSQC spectra were collected on a 500-MHz spectrometer (Avance II US2; Bruker) and analyzed with SPARKY (T.D. Goddard and D.G. Kneller, SPARKY 3, University of California, San Francisco, San Francisco, CA) as previously described (Bilodeau et al., 2004). Chemical shift differences were calculated using the formula (0.2 N² + H²)^1/2. Binding experiments with Vps36 were performed in 50 mM NaPO₄, pH 7.2; binding experiments with Mvb12 were performed in 50 mM imidazole and 100 mM NaCl, pH 7.2. A spectrum of unbound ¹⁵N-labeled Ub in the same buffer was used as the reference.

Other methods
Metabolic [³⁵S]methionine labeling and immunoprecipitation of CPY were performed as described previously (Cooper and Stevens, 1996). Preparation of yeast lysates for gel filtration, immunoblotting, and CPY pulse-chase analysis were performed as described previously (Oestreich et al., 2007). Protein models were drawn with the PyMOL molecular graphics system (http://pymol.sourceforge.net). A model for the Vps36 NZF domain was computed using the Wurst protein server (Torda et al., 2004).

Online supplemental materials
Fig. S1 shows the localization of GFP-Cps1 in strains with a range of mutations in Vps23, the structure of Vps23 UEV domain in complex with Ub highlighting mutated residues, the localization of Vps23-GFP, and Vps20-mCherry in strains containing vps27^vps23 mutations. Fig. S2 shows the localization of Mvb12-GFP, gel filtration experiments performed on wild-type and mutant Mvb12, and the results of CPY pulse-chase analysis of all of the strains used in this study. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200811130/DC1.

Submitted: 24 November 2008

Accepted: 23 March 2009

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