ESCRT-III Family Members Stimulate Vps4 ATPase Activity Directly or via Vta1

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1 Article ESCRT-III Family Members Stimulate Vps4 ATPase Activity Directly or via Vta1 Ishara F. Azmi, 1,4 Brian A. Davies, 1,4 Junyu Xiao, 2 Markus Babst, 3 Zhaohui Xu, 2 and David J. Katzmann 1, * 1 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA 2 Life Sciences Institute and Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor, MI 48109, USA 3 Department of Biology, University of Utah, Salt Lake City, UT 84112, USA 4 These authors contributed equally to this work. *Correspondence: katzmann.david@mayo.edu DOI /j.devcel SUMMARY The AAA-ATPase Vps4 is critical for function of the MVB sorting pathway, which in turn impacts cellular phenomena ranging from receptor downregulation to viral budding to cytokinesis. Vps4 dissociates ESCRTs from endosomal membranes during MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. Vta1 potentiates Vps4 activity and interacts with ESCRT-III family members. We have investigated the impact of Vta1 and ESCRT-III family members on Vps4 ATPase activity. Two distinct mechanisms of Vps4 stimulation are described: Vps2 can directly stimulate Vps4 via its MIT domain, whereas Vps60 stimulates via Vta1. Moreover, Did2 can stimulate Vps4 by both mechanisms in distinct contexts. Recent structural determination of the ESCRT-III-binding region of Vta1 unexpectedly revealed a MIT-like region. These data support a model wherein a network of MIT and MITlike domain interactions with ESCRT-III subunits contributes to the regulation of Vps4 activity during MVB sorting. INTRODUCTION Multivesicular bodies (MVBs) are endocytic intermediates formed when the limiting membrane of the endosome invaginates and buds into its lumen, actively selecting transmembrane protein cargoes in the process. Fusion of an MVB with the lysosome represents a mechanism by which eukaryotic cells degrade endocytosed transmembrane proteins (Gruenberg and Stenmark, 2004). MVB function is critical for maintaining cellular homeostasis, highlighted by its role in modulation of growth factor receptor signaling. Exvagination of the endosomal membrane from the cytosol is topologically similar to the budding of retroviral particles and cytokinesis (Carlton and Martin-Serrano, 2007; Morita and Sundquist, 2004), wherein membranes bud away from the cytoplasm, and the machinery responsible for MVB sorting has been implicated in these phenomena (recently reviewed in Hurley and Emr, 2006; Piper and Katzmann, 2007). The machinery responsible for executing MVB sorting is highly conserved in all eukaryotes. Originally identified in yeast, the class E Vps (vacuolar protein sorting) gene products have been implicated in MVB sorting in plants, fungi, and animals (recently reviewed in Piper and Katzmann, 2007; Winter and Hauser, 2006). Biochemical and genetic studies have demonstrated that the majority of class E Vps proteins assemble into distinct multimeric complexes, the endosomal sorting complexes required for transport (ESCRT)-I, -II, and -III (Hurley and Emr, 2006; Piper and Katzmann, 2007). ESCRT-I and -II are transiently recruited to the endosomal membrane, where they are thought to play a role in cargo selection as well as the proper recruitment and assembly of ESCRT-III (Babst, 2005; Piper and Katzmann, 2007). ESCRT-III is responsible for coordination of activities found in the deubiquitinating enzyme Doa4 (together with Bro1) (Amerik et al., 2000; Luhtala and Odorizzi, 2004) and the AAA-ATPase Vps4/SKD1 (Babst et al., 2002a). ESCRT-III is therefore thought to play a later role during the sorting process. Vps4-mediated disassembly and membrane release of ESCRTs are critical for MVB sorting, but the precise mechanism coordinating ATP hydrolysis, ESCRT release, and intralumenal vesicle formation is unclear. Vta1/SBP/Lip5 has been characterized as a positive regulator of Vps4 ATPase activity that also interacts with the accessory ESCRT-III subunits Vps60/Mos10/CHMP5 and Did2/Fti1/CHMP1 (Azmi et al., 2006; Haas et al., 2007; Lottridge et al., 2006; Shiflett et al., 2004; Vajjhala et al., 2006). Additional interactions between ESCRT-III family members and Vps4 have also been described that confer proper membrane targeting of Vps4, but are presumably also relevant for membrane dissociation of these factors. ESCRT-III family members can be broken into core elements, defined by the four subunits Snf7/CHMP4, Vps20/CHMP6, Vps2/CHMP2, and Vps24/CHMP3, and the accessory elements, Vps60 and Did2 (Babst et al., 2002a; von Schwedler et al., 2003). All are relatively small (20 25 kda), highly charged, coiled-coil domain-containing proteins. The structure of CHMP3 suggests that all ESCRT-III family members share a conserved core structure (Muziol et al., 2006) and a C-terminal portion implicated in binding to Vps4 (Scott et al., 2005a), Doa4, and Bro1 (Kim et al., 2005). Data in yeast support a model wherein the recruitment of Vps20-Snf7 to the endosomal membrane precedes Vps2- Vps24, with appropriate release requiring association of all four core elements and Vps4 activity (Babst et al., 2002a). Vps2- Vps24 is also required for the recruitment of the accessory ESCRT-III subunit Did2, and loss of Did2 function stabilizes core ESCRT-III components on endosomes (Nickerson et al., 2006). These observations support the idea that Did2 modulates 50 Developmental Cell 14, 50 61, January 2008 ª2008 Elsevier Inc.

2 ESCRT-III endosomal association in concert with Vps4. Similarly, loss of Vps60 displays a less severe phenotype than loss of core ESCRT-III components, suggesting a regulatory role during MVB sorting (Babst et al., 2002a). Vps4 is critical for the function of the MVB sorting pathway, and its dysfunction results in the accumulation of ESCRTs on endosomal membranes (most notably ESCRT-III) (Babst et al., 1998, 2002a, 2002b; Katzmann et al., 2001). In addition to an AAA-ATPase domain, Vps4 contains a microtubule interacting and trafficking (MIT) domain that is required for binding to a subset of ESCRT-III family members (Obita et al., 2007; Scott et al., 2005a, 2005b; Stuchell-Brereton et al., 2007; Tsang et al., 2006). (Refer below for schematic representation of pertinent domains and interactions.) The b domain within the AAA-ATPase domain of Vps4 interacts with Vta1 via the C-terminal VSL region of Vta1, and this is required for Vta1-dependent potentiation of Vps4 ATPase activity (Azmi et al., 2006; Scott et al., 2005a). Additionally, Vta1 contains an N-terminal region that is critical for binding to Vps60 (Azmi et al., 2006) and Did2 (Xiao et al., 2008 [this issue of Developmental Cell]), and structural determination of this domain has identified that it is related to the MIT domain of Vps4 (Xiao et al., 2008). This MIT-like domain is dispensable for Vta1-dependent stimulation of Vps4 in vitro yet is critical for in vivo function, suggesting yet another level of Vps4 ATPase regulation through the accessory ESCRT-III factors (Azmi et al., 2006). To better understand the interactions that contribute to regulation of Vps4 ATPase activity and ESCRT-III membrane release, we have performed analyses of the relationships between ESCRT-III family members, Vta1, and Vps4 as they impact Vps4 function in vitro and in vivo. Two mechanisms of Vps4 activation are described: Vps2 can stimulate Vps4 directly through its MIT domain, whereas Vps60 potentiates Vps4 activity in a Vta1- dependent manner that does not require the MIT domain of Vps4. Additionally, Did2 can stimulate Vps4 by both mechanisms in distinct contexts. In addition, we demonstrate that Vta1 endosomal association is dependent on Did2 and Vps4, whereas Vps60 endosomal association is dependent upon Vta1. We conclude that a previously unappreciated level of interactions between Vta1 and the accessory ESCRT-III members contribute to coordination of Vps4 ATPase activity and ESCRT-III function. RESULTS Vta1 Localization Is Impacted by ESCRT-III Family Members Previous reports have indicated that Vta1 interacts with both the core ESCRT-III subunits Snf7 and Vps20 (Bowers et al., 2004; Lottridge et al., 2006; Yeo et al., 2003) and the accessory ESCRT-III subunits Did2 and Vps60, although direct interaction has only been demonstrated with Did2 (Azmi et al., 2006; Lottridge et al., 2006; Shiflett et al., 2004). To further understand the contributions of the ESCRT-III subunits to Vta1 function, a complete analysis of Vta1-ESCRT-III binding was performed with purified proteins to assess direct interactions. Bacterially expressed Snf7, Vps2, Vps20, Vps24, Vps60, and Did2 GST fusion proteins were immobilized and incubated with purified Vta1, and bound material was visualized by both western blotting and Coomassie staining (Figure 1A). Under these conditions, both GST- Vps60 and GST-Did2 were observed to bind Vta1 (Figure 1A, Figure 1. Vta1 Interactions with Core ESCRT-III and Accessory ESCRT-III Proteins (A) In vitro binding analysis of Vta1 with core ESCRT-III and accessory ESCRT- III subunits. Purified Vta1 was incubated with affinity-purified GST or GSTtagged Vps60, Did2, Snf7, Vps2, Vps20, and Vps24. Bound materials were visualized by western blotting with anti-vta1 and Coomassie blue staining. (B) Determinants impacting Vta1 localization to endosomal compartments. Fluorescence microscopy was utilized on wild-type and indicated deletion strains coexpressing Vta1-GFP and DsRed-FYVE. Developmental Cell 14, 50 61, January 2008 ª2008 Elsevier Inc. 51

3 lanes 2 and 3), whereas GST alone, Snf7, Vps2, Vps20, and Vps24 GST fusions did not (Figure 1A, lanes 1 and 4 7). These findings contrast to previously published results using yeast lysates or two-hybrid analyses that reported interactions between Vta1 and Snf7 or Vps20 (Bowers et al., 2004; Lottridge et al., 2006; Yeo et al., 2003). These results suggest that Vta1 directly binds the accessory ESCRT-III subunits Vps60 and Did2, whereas the interactions with the core ESCRT-III subunits Snf7 and Vps20 appear to be indirect or impacted by regulation in vivo. The site of Vta1/Vps4 function would appear to be the endosomal membrane, and therefore the impact of ESCRT-III family members on Vta1-GFP subcellular localization was analyzed. Vta1-GFP displays both a soluble signal and colocalization with PI(3)P-positive endsomes (decorated with DsRed-FYVE) in wild-type cells (Azmi et al., 2006; Figure 1B). Loss of Vps4 confers increased solubility of Vta1-GFP combined with decreased colocalization with DsRed-FYVE positive structures (Azmi et al., 2006; Figure 1B); however, residual association with endocytic compartments remains (based on staining with the endocytic dye FM4-64) (see Figure S1 in the Supplemental Data available with this article online). Loss of the accessory ESCRT-III subunits Did2 and Vps60 has disparate effects on Vta1-GFP localization. Loss of Vps60 resulted in increased accumulation of Vta1-GFP on endosomal structures (Figure 1B). By contrast, loss of Did2 displayed a relatively minor impact on Vta1-GFP distribution, with only a subtle increase in the apparent soluble signal. Double and triple mutants were used to examine further the contributions of Vps60, Vps4, and Did2 to Vta1-GFP endosomal localization. Loss of both Vps4 and Did2 (vps4d did2d and vps4d did2d vps60d) eliminated the residual Vta1-GFP endosomal association apparent in strains that contained either Vps4 or Did2 (vps60d did2d or vps60d vps4d)(figure S1). These results suggest that Vps4 and Did2 are the primary determinants of Vta1 endosomal localization, whereas Vps60 antagonizes Vta1 endosomal association. However, these analyses do not eliminate additional contributions from unknown cellular factors. Although no direct interaction was detected between Vta1 and the core ESCRT-III subunits Snf7, Vps20, and Vps24 (Figure 1A), yeast lacking these factors were also analyzed for defects in Vta1-GFP localization as these factors impact Did2 and Vps4 localization (Babst et al., 2002a; Nickerson et al., 2006). Consistent with previously published results (Lottridge et al., 2006), loss of Snf7 resulted in a diffuse Vta1-GFP pattern similar to our observations with did2d vps4d and did2d vps4d vps60d (Figure 1B). Similar phenotypes were observed for vps24d and vps20d mutants (Figure S2). Loss of Snf7 perturbs membrane association and assembly of the other core ESCRT-III subunits and Vps4 (Babst et al., 2002a), and Vps2 and Vps24 have been implicated in Did2 endosomal association (Nickerson et al., 2006). It would therefore appear that the defect in Vta1-GFP localization observed with the loss of core ESCRT-III subunits (Snf7, Vps20, and Vps24) is a secondary consequence of Did2 and Vps4 mislocalization. These results are consistent with direct Vta1 interactions with Did2, Vps4, and Vps60 observed in vitro regulating Vta1 endosomal association in vivo. Vta1 Interaction Determinants within Vps60 and Did2 The ESCRT-III proteins share a conserved core structure, elucidated by determination of the CHMP3 structure and sequence alignments (Muziol et al., 2006). Despite the similarity exhibited throughout the ESCRT-III family, Vta1 specifically binds the accessory ESCRT-III subunits Vps60 and Did2 (Figure 1A). To define the portion of Vps60 conferring this specificity, a series of GST-Vps60 truncations was generated to identify the Vta1 interaction surface. Design of the Vps60 truncations was guided by the CHMP3 structural studies containing the five conserved core a helices of ESCRT-III family members (Muziol et al., 2006; Figure 2A). These Vps60 truncations were expressed in bacteria as GST fusions and used to perform in vitro binding studies. These results indicate that the a4-5 region (amino acids ) of Vps60 is necessary and sufficient for binding to Vta1 at levels comparable to full-length GST-Vps60 (Figures 2A and 2B). Further examination of this region revealed that the Vps60 a4 helix alone (GST-Vps ) is capable of binding Vta1 (Figure S3A), although the association appears to be much less stable than the interaction between full-length Vps60 or a4-5 Vps60 and Vta1. To address whether the a4-5 region might be masked in other ESCRT-III subunits that did not bind to Vta1, the a4-5 regions of the core ESCRT-III subunits Snf7, Vps2, and Vps20 were also expressed as GST fusions and examined for Vta1 binding in vitro. Vta1 was not isolated by these GST fusions or by GST alone at levels detectable in this assay, consistent with observations using full-length GST fusion proteins (Figure S3B). These results suggest that the ability of the Vps60 a4-5 region to bind Vta1 is not shared by core ESCRT-III subunits. Alanine scanning mutagenesis of Vps60 (residues ) was utilized to further map the Vta1 interaction surface. These mutant forms of Vps60 were expressed in bacteria as GST fusions and examined for Vta1 binding in vitro (Figure S3C; Figure 2B, lane 10). Mutation of residues , , and partially compromise Vta1 binding (Figure S3C, lanes 4, 7, and 9), whereas mutation of residues resulted in a form of Vps60 that was incapable of binding Vta1 to levels detectable in this assay (Figure 2B, lane 10; Figure S3B, lane 5). These results suggest that residues within Vps60 a4 are critical for Vta1 association, whereas additional residues within the a4-5 region contribute to this interaction to a lesser extent. Similar analyses were utilized to examine whether the analogous region of Did2 was responsible for interacting with Vta1. A GST fusion containing a1-3 or a4-5 was unable to bind Vta1, whereas a GST fusion containing more distal C-terminal amino acids ( ) conferred Vta1 binding, although to a lesser extent than the full-length protein (Figure 2D, lane 2 compared to lane 6). Did2 has been reported to bind Vps4 (Nickerson et al., 2006); these fragments were therefore analyzed for this ability. Although we did not detect interaction between Vps4 E233Q and full-length GST-Did2 under these conditions, binding was observed with GST-Did (Figure 3A). This result indicated that the C-terminal portion of Did2 (amino acids ) was also sufficient for interaction with both Vps4 and Vta1. As full-length Did2 binds Vta1 but not Vps4 under these conditions, these observations suggest a conformational change is required for binding Vps4 but not Vta1 and suggest an inverse relationship between Did2 binding to Vps4 or Vta1. Furthermore, distinct regions of Did2 and Vps60 are required for binding to Vta1. 52 Developmental Cell 14, 50 61, January 2008 ª2008 Elsevier Inc.

4 Figure 2. Mapping Vta1 Binding Regions in Vps60 and Did2 (A) Schematic of predicted Vps60 a helices and truncations tested for binding to Vta1. Residues are highlighted with an asterisk. (B) In vitro binding analysis of Vps60 truncations and Vta1. GST pull-down assay was performed with GST, full-length Vps60, and corresponding Vps60 truncations and the point mutant. GST fusions were incubated with recombinant Vta1, and bound materials were visualized by western blotting with anti-vta1 and Coomassie blue staining. (C) Schematic of predicted Did2 a helices and truncations tested for binding to Vta1. (D) In vitro binding analysis of Did2 truncations with Vta1. Bound materials were visualized by western blotting with anti-vta1 and Coomassie blue staining. Vps60 and Did2 Stimulate Vps4 ATPase Activity in a Vta1-Dependent Manner Vta1 has been demonstrated to stimulate Vps4 ATPase activity (Azmi et al., 2006; Lottridge et al., 2006; Haas et al., 2007). To evaluate the impact of Vps60 and Did2 binding to Vta1 on its ability to stimulate Vps4 activity, ATPase assays were performed. Equivalent amounts of GST, GST-Vps60, or GST-Did2 (2 mg) together with Vps4 had no effect on the ATPase activity of Vps4 (ADP/Vps4/min) nor exhibited any activity alone (Figure 3B and data not shown). In contrast, 2 mm Vta1 was able to stimulate 500 nm Vps4 activity from 15 ADP/Vps4/min to 45 ADP/Vps4/ min (Figure 3B). Interestingly, both GST-Vps60 and GST-Did2 augmented the Vta1-dependent stimulation of Vps4 by an additional 3-fold (132 and 130 ADP/Vps4/min, respectively) (Figure 3B). This result indicated that the accessory ESCRT-III family members Did2 and Vps60 are capable of stimulating Vps4 ATPase activity in a Vta1-dependent manner. Interactions between ESCRT-III components and Vps4 have been reported, and Vps20 was found to stimulate Vps4 ATPase activity (Bowers et al., 2004; Vajjhala et al., 2006). We therefore sought to determine whether the core ESCRT-III subunits could stimulate Vps4 ATPase activity or augment Vta1-dependent stimulation of Vps4. Vps4 ATPase assays were performed with purified ESCRT-III GST fusions in the presence or absence of Vta1. The core ESCRT-III GST fusions did not exhibit intrinsic ATPase activity, and Snf7 and Vps24 did not enhance Vps4 ATPase activity alone or in combination with Vta1 (Figure 3B). Although Vps20 has been reported to stimulate Vps4 (Vajjhala et al., 2006), we observed GST-Vps20 binding to Vps4 E233Q but no additional stimulation of Vps4 ATPase activity with or without Vta1 (Figures 3A and 3B). In contrast, GST-Vps2 bound Vps4 E233Q and stimulated Vps4 ATPase activity by 3-fold to 50 ADP/Vps4/min (Figures 3A and 3B). These results suggest that Vps4 binding by ESCRT-III subunits can result in distinct outcomes, as only Vps2 stimulates Vps4 in this assay. GST-Vps2 in combination with Vta1 further stimulated Vps4 activity to 145 ADP/Vps4/min. This activity is similar to the levels observed for Vps4 and Vta1 with Vps60 or Did2 (Figure 3B). However, no interaction between Vta1 and Vps2 was detected (Figure 1A) and Vps2 can stimulate Vps4 ATPase activity directly (Figure 3B), raising the question as to whether Vps2 was modulating Vta1-dependent Vps4 activation or was acting independently. Vps4 interacts with Vps2 and Did2 through the aminoterminal MIT domain (Bowers et al., 2004; Nickerson et al., 2006; Vajjhala et al., 2007). However, the MIT domain is not required for intrinsic Vps4 ATPase activity (Babst et al., 1998) or Developmental Cell 14, 50 61, January 2008 ª2008 Elsevier Inc. 53

5 Figure 3. Vps60 and Did2 Augment Vps4 ATPase Activity through Vta1 (A) Analysis of ESCRT-III subunits binding Vps4 E233Q in the presence of ATP. GST or the indicated GST fusion proteins were incubated with purified Vps4 E233Q, and bound material was visualized by Coomassie staining and western blotting with anti-vps4 antibody. (B) ATPase activity of Vps4 (0.5 mm) was measured with GST, GST-Vps60, GST- Did2, GST-Snf7, GST-Vps2, GST-Vps20, and GST-Vps24 in the presence or absence of Vta1 (2 mm). Activity is expressed as ADP/Vps4/min. (C) ATPase activities of the wild-type and Vps4 lacking a MIT domain (Vps4 DN ) were measured with GST, GST-Vps60, GST-Did2, and GST-Vps2 in the presence or absence of Vta1. Vta1-Vps4 interaction (Azmi et al., 2006). ATPase assays were performed with Vps4 or Vps4 lacking the MIT domain (Vps4 DN ) to address the role of the Vps4 MIT domain in these activities. Both GST-Vps60 and GST-Did2 were capable of stimulating Vta1-dependent activation of Vps4 and Vps4 DN (Figure 3C). Although GST-Vps2 enhanced Vps4 activity to 50 ADP/Vps4/ min (Figures 3B and 3C), GST-Vps2 failed to stimulate Vps4 DN (Figure 3C). Similarly, the activity observed with GST-Vps2, Vta1, and Vps4 DN was indistinguishable from the activity of Vta1 and Vps4 DN alone (Figure 3C). These results are consistent with the interpretation that Vps2 stimulates Vps4 ATPase activity via the Vps4 MIT domain independently of Vta1. These results also indicate that Vps60 and Did2 stimulate Vps4 via Vta1 and independently of the Vps4 MIT domain under these conditions. These two mechanisms of activation are therefore distinct. The Vps60 a4-5 region is necessary and sufficient for binding to Vta1 at levels comparable to full-length Vps60. We next addressed whether this region is sufficient for Vta1-dependent stimulation of Vps4. GST-Vps (a4-5 peptide) was tested for enhancement of Vps4 ATPase activity in combination with Vta1. This fusion did not exhibit intrinsic ATPase activity nor did it stimulate Vps4 ATPase activity by itself (Figure 4A and data not shown). However, GST-Vps in conjunction with Vta1 enhanced Vps4 activity to approximately 200 ADP/ Vps4/min (Figure 4A). The single a4 and a5 helix GST fusions were also tested for ATPase stimulation, as residual Vta1 binding was observed for the a4 construct (GST-Vps ). Whereas GST-Vps was capable of conferring partial stimulation of ATPase activity, GST-Vps (the a5 construct) failed to enhance Vps4 activity in combination with Vta1 (Figure 4A). These results indicated that the Vps60 a4-5 region, and to a lesser extent a4 alone, are sufficient to potentiate Vta1 activation of Vps4. Mutational analysis of the a4-5 region identified five residues ( ) within a4 required for the interaction with Vta1 (Figure 2B). To examine the requirement for Vps60 residues in this process, Vps4 ATPase enhancement by Vps60 ( )Ala was examined. GST-Vps60 ( )Ala did not exhibit intrinsic ATPase activity nor stimulate Vps4 by itself, and addition of GST-Vps60 ( )Ala to Vta1 and Vps4 yielded activity similar to GST alone with Vta1 and Vps4 (Figure 4B). These results indicate that Vps60 residues within a4 are necessary for both Vta1 binding and Vta1-dependent enhancement of Vps4 ATPase activity and are consistent with the finding that the Vps60 a4 helix is capable of these activities. The C terminus of Did2 is sufficient for binding to both Vta1 and Vps4, and was therefore analyzed for its ability to stimulate Vps4 ATPase activity. Did was tested for enhancement of Vps4 and Vps4 DN ATPase activity in combination with Vta1. GST-Did did not display intrinsic ATPase activity (data not shown), but was capable of stimulating Vps4 ATPase activity 3-fold while no Vps4 DN stimulation was observed (Figure 4C). This result indicated that the C terminus of Did2 is capable of stimulating Vps4 directly through its MIT domain. This result contrasts with our observations with full-length Did2 and may reflect a loss of N-terminal inhibition, a previously suggested regulatory mechanism (Shim et al., 2007). In combination with Vta1, GST- Did stimulated Vps4 ATPase activity to 153 ADP/Vps4/ min and Vps4 DN to 66 ADP/Vps4 DN /min (Figure 4C). This reduced stimulation of Vps4 DN as compared to activity with full-length GST-Did2 indicates that Did is a less potent enhancer of Vta1-dependent Vps4 activity, consistent with the reduction observed for Vta1 binding by GST-Did (Figure 2D). However, 54 Developmental Cell 14, 50 61, January 2008 ª2008 Elsevier Inc.

6 Figure 4. Domain Analyses of Vps60 and Did2 Regions Critical for Stimulation of Vps4 ATPase Activity (A) ATPase activities were measured with either GST or GST fusions corresponding to the a4-5 ( ), a4 ( ), and a5 ( ) helices of Vps60 in the presence or absence of Vta1. (B) ATPase activities of Vps4 were analyzed with GST, GST-Vps60, or GST- Vps60 ( )Ala mutant in the presence or absence of Vta1. (C) ATPase activities of wild-type Vps4 and Vps4 DN were measured with GST, GST-Did2, and GST-Did (C-terminal Did2 peptide) in the presence or absence of Vta1. the activity observed with GST-Did , Vta1, and full-length Vps4 is comparable to enhancement by full-length GST-Did2, indicating that the reduced Vta1-dependent Vps4 activation of Did is offset by an increase in direct activation of Vps4 by Did These observations are consistent with an inverse relationship between Vta1 and Vps4 binding by Did2 (Figures 2D and 3A). These results indicate that the C terminus of Did2 can stimulate Vps4 ATPase activity through both Vta1 and the MIT domain of Vps4. The Vta1 amino terminus is necessary and sufficient for Did2 and Vps60 binding, and the structure of this region has recently been solved (Xiao et al., 2008). The Vta1 amino terminus (residues 1 163) is comprised of a seven-helix arrangement similar to tandem MIT domains (MIT1 and MIT2), and two point mutations (Vta1 W122A and Vta1 K152A ) have been identified in MIT2 that perturb binding to Vps60 and Did2 (Xiao et al., 2008). Purified Vta1, Vta1 W122A, Vta1 K152A, and Vta (amino-terminal truncation) were incubated with Vps4 and GST alone, GST- Vps60, or GST-Did2. All three mutants retained Vps4 stimulation at levels comparable to wild-type (Figure 5A). However, whereas combining Vta1 with Vps60 or Did2 enhanced Vps4 activity to 156 or 154 ADP/Vps4/min, respectively, the Vta1 mutants exhibited reduced or no enhancement in combination with Vps60 or Did2. Vta1 W122A in conjunction with Vps60 or Did2 yielded 108 and 104 ADP/Vps4/min, respectively, whereas Vta1 K152A yielded 71 and 66 ADP/Vps4/min, respectively (Figure 5A). By contrast, removal of the Vta1 MIT-like domain (Vta ) eliminated Vps60- and Did2-enhanced Vps4 activity (Figure 5A). These results suggest that Vta1 W122A and Vta1 K152A retain some binding to Vps60 and Did2 not apparent in in vitro binding assays (Xiao et al., 2008) and that Vps60 and Did2 binding to the Vta1 MIT-like domain is important for enhanced but not basal Vta1 stimulation of Vps4 ATPase activity. To further examine the role of the Vta1 N terminus in mediating Vps60- and Did2-enhanced Vta1 stimulation of Vps4, a mutant altered in MIT1 (Vta1 E57A ) was also examined. In contrast to tryptophan 122 and lysine 152, mutation of glutamate 57 does not compromise Vta1 binding to GST-Vps60 or GST-Did2 (Figure S4; Xiao et al., 2008). Examination of Vta1 E57A stimulation of Vps4 revealed comparable activity to wild-type (Figure 5B). However, Vta1 E57A in combination with Vps4 and GST-Vps60 exhibited less ATPase activity than wild-type Vta1 (p < 0.05) (Figure 5B), and similar results were obtained in combination with GST-Did2 (data not shown). The observation that Vta1 E57A binds Vps60 and Did2, but exhibits a reduced ability to enhance Vps4 ATPase activity in concert with Vps60 or Did2, suggests that MIT1 is also involved in this enhancement. Together, these results indicate that the accessory ESCRT-III subunits Vps60 and Did2 enhance Vps4 ATPase activity through Vta1 MIT-like domains whereas the core ESCRT-III subunit Vps2 enhances Vps4 ATPase activity by binding the Vps4 MIT domain. However, the C-terminal portion of Did2 was additionally able to stimulate Vps4 directly through its MIT domain, raising the possibility that in vivo regulation of ESCRT-III conformation impacts Vps4 activation as well. In Vivo Analyses of Vta1 Interactions with Vps60 and Did2 Whereas Did2 contributes to appropriate association of Vta1 with the endosomal membrane, Vps60 negatively regulates Vta1 membrane association (Figure 1B). This raised the possibility that Vps60 functions downstream of Vta1. To investigate this model, localization of a functional form of Cherry-Vps60 was Developmental Cell 14, 50 61, January 2008 ª2008 Elsevier Inc. 55

7 Dvta1 cells harboring an empty plasmid or plasmids encoding Vta1, Vta1 W122A,Vta1 K152A, or Vta LossofVta1(Dvta1 + vector) resulted in only a partial defect in MVB sorting with 43% CPS maturation at 60 min compared to 70% CPS maturation with Vta1 (Figure 6C). Expression of the Vta1 mutants defective for Vps60 and Did2 binding exhibits partial complementation, as would be expected due to the retention of basal Vps4 stimulation. Despite expression equivalent to wild-type, all three exhibit a reduction in CPS maturation that is significantly reduced from Vta1 (p < 0.005) (Figure 6C and data not shown). Normalizing maturation at 60 min to access percent Vta1 function reveals that Vta1 W122A confers 70% function, Vta1 K152A confers 66% function, and Vta confers 63% function (Figure 6D). This trend correlates with Vps60- and Did2-enhanced Vta1 stimulation of Vps4 observed in vitro (Figure 5A). These results indicate that Vta1 MIT-like domain interactions significantly potentiate Vta1 function in yeast. To more directly assess Vps4 function in vivo, the membrane association of the ESCRT-III subunit Snf7 was examined in yeast expressing Vta1 mutants. As previously demonstrated (Azmi et al., 2006), loss of Vta1 increased Snf7 membrane association from 28% in wild-type cells to 53% in vta1d cells (Figure 6E). Expression of either Vta1 mutant (W122A or K152A) only partially restored ESCRT-III recycling, resulting in 37% and 39% Snf7 membrane association (Figure 6E). These results are consistent with a deficit in Vps4 function as a consequence of compromised interactions between Vta1 and the accessory ESCRT-III subunits Vps60 and Did2. DISCUSSION Figure 5. Vta1 MIT Regions Contribute to Vta1-Dependent Accessory ESCRT-III Stimulation of Vps4 ATPase Activity (A) ATPase activities of wild-type Vps4 were measured with GST, GST-Vps60, and GST-Did2 in the presence of wild-type Vta1, Vta1 MIT2 mutants (Vta1 W122A and Vta1 K152A ), or Vta1 lacking both MIT1 and MIT2 (Vta ). (B) ATPase activities of wild-type Vps4 were measured with GST, GST-Vps60 in the presence of wild-type Vta1, or a Vta1 MIT1 mutant (Vta1 E57A ). investigated in wild-type, vps4d, and vta1d strains. In wild-type cells, Vps60 displays a largely soluble distribution with interspersed puncta (Figure 6A). These puncta were not apparent or were greatly reduced in the vta1d and vps4d strains. Similarly, analysis of Cherry-Vps60 ( )Ala, the mutant defective for Vta1 binding, also exhibited reduced association with puncta in wild-type cells (Figure 6A). The punctate structures visible with Cherry-Vps60 are also positive for Vta1-GFP in wild-type cells, indicating that these proteins colocalize on endosomes as expected (Figure 6B). However, this colocalization was reduced in the context of Cherry-Vps60 ( )Ala. These results suggest that Vps60 endosomal association is dependent upon Vta1 binding. To ascertain the impact of loss of interactions between Vta1 and accessory ESCRT-III subunits on MVB sorting, Vta1 mutants defective for binding to Vps60 and Did2 were analyzed for suppression of vta1d MVB sorting defects. Pulse-chase immunoprecipitation was used to examine maturation kinetics of the biosynthetic MVB cargo carboxypeptidase S (CPS) in The ESCRT machinery was initially characterized with respect to its ability to sort lysosomal/vacuolar lumenal cargoes into the intralumenal vesicles of the multivesicular body (MVB). More recently, the role of this machinery in diverse processes ranging from retroviral budding to exosome formation to cytokinesis has begun to be appreciated. Common to these processes is exvagination from the cytosol, either into the lumen of the endosome or extracellular milieu. Currently, it appears that the last stages of this common ESCRT-mediated process involve assembly of ESCRT-III on the appropriate membrane followed by release of this complex through the hydrolysis of ATP by Vps4. However, the mechanism wherein assembly of ESCRT-III is coupled with the activation of Vps4 to disassemble ESCRT-III has been elusive. In the present study, we have documented two distinct mechanisms by which Vps4 activity can be modulated by ESCRT-III family members: Vps2 can activate directly via the MIT domain of Vps4, whereas Vps60 and Did2 stimulate Vps4 activity through Vta1 in a manner that is not dependent upon the MIT of Vps4 under our assay conditions. The Did2 C terminus (Did ) can stimulate by both mechanisms to a lesser extent, resulting in activity with Vta1 and Vps4 at levels comparable to full-length Did2 stimulation. Vta1 has been shown to interact with the b domain in Vps4, further highlighting the notion that Vps4 activation can occur via its MIT domain or b domain. Interestingly, Vps60 and Did2 execute Vta1-dependent Vps4 activation through an N-terminal MIT-like region within Vta1. This indicates that both mechanisms of Vps4 activation via the ESCRT-III family members involve MIT domains. Additionally, we found that 56 Developmental Cell 14, 50 61, January 2008 ª2008 Elsevier Inc.

8 Figure 6. In Vivo Analysis of Vta1 and Vps60 Mutants (A) Cherry-Vps60 and Cherry-Vps60 ( )Ala subcellular localizations were analyzed in the indicated genetic backgrounds. (B) Cherry-Vps60 and Cherry-Vps60 ( )Ala subcellular localizations were analyzed in vps60d vta1d cells expressing Vta1-GFP. (C) CPS pulse-chase immunoprecipitation was performed to analyze the effects of Vta1 N-terminal mutations on CPS maturation. Percent CPS maturation was determined by comparing the amount of mature CPS to total CPS for each time point. (D) Percent CPS maturation at 60 min was normalized to determine percent Vta1 function relative to wild-type. (E) Subcellular fractionation was performed on lysates generated from vta1d cells or vta1d cells expressing wild-type Vta1, Vta1 W122A, or Vta1 K152A. Endogenous Snf7 was visualized by a-snf7 antibody. a-pgk antibody was utilized as a marker for soluble fractions. Vta1 function is required for Vps60 endosomal localization, and that Vta1 localization requires both Vps4 and Did2. Together, these results depict a complicated orchestration of ESCRT-III subunit recruitment along with activation of factors mediating disassembly of the complex in order to maintain the maximum fidelity of the MVB sorting process (Figures 7A and 7B). These studies raise the question as to how Vps60 and Did2 binding enhance Vta1 stimulation of Vps4 ATPase activity. One model is that accessory ESCRT-III subunit binding to the MIT2 domain allosterically regulates Vta1 and potentiates its ability to activate Vps4. Generally, allosteric regulation can be divided into two forms: relief of an autoinhibitory mechanism, or inducement of further activating interactions. Removal of the Vta1 tandem MIT domains (Vta ) does not enhance the basal activation of Vps4 by Vta1 (Figure 5A). This observation suggests that the Vta1 MIT domains are not autoinhibiting Vps4 activation and that Did2 and Vps60 binding to the Vta1 MIT2 domain may be inducing additional Vta1-Vps4 interactions. Another possibility is that interaction of Vta1 and Did2 or Vps60 increases the local concentration of Vta1-Vps4 in vitro to enhance Vta1-Vps4 binding and thereby stimulate Vps4 activity. However, the Vta1 MIT1 mutant (Vta1 E57A ) was capable of binding to Vps60 and Did2 but did not fully potentiate increased Vps4 ATPase activity (Figure 5B), suggesting that concentration cannot account for all aspects of activation. As these mechanisms are compatible, it seems reasonable to suggest that both allosteric and concentration mechanisms may contribute to Did2 and Vps60 enhancement of Vta1-Vps4 function in vivo. We also demonstrated that Vps2 binding to Vps4 enhances ATPase activity. ESCRT-III subunits have been suggested to bind Vps4 and to enhance Vps4 ATPase activity, but this property has been previously demonstrated only with Vps20 (Vajjhala et al., 2006). We observed that both Vps2 and Vps20 bound Vps4 E233Q to similar extents (Figure 3A); however, stimulation of Vps4 ATPase activity was observed with Vps2 but not with Vps20 under these assay conditions (Figure 3B). Although both Vps2 and Vps20 binding to Vps4 would be expected to concentrate Vps4 in a similar manner in these experiments, concomitant Vps4 ATPase stimulation was not observed with Vps20. Similar to Vps2, the Did2 C terminus (Did ) was able to directly stimulate ATPase activity via the Vps4 MIT domain (Figure 4C). Structural analyses have recently revealed that conserved residues Developmental Cell 14, 50 61, January 2008 ª2008 Elsevier Inc. 57

9 Figure 7. Model of ESCRT-III Recruitment and Dissociation by Vps4-Vta1 (A) Schematic representation of Vps2, Vps4, Vps60, Did2, and Vta1, highlighting regions critical for interactions that contribute to increased Vps4 ATPase activity. The MIM (MIT interacting motif) in Vps2 and Did2 has recently been characterized as responsible for interacting with the MIT domain of Vps4 and is included for clarity (Obita et al., 2007; Stuchell-Brereton et al., 2007). (B) Stepwise recruitment of Vps20-Snf7 1 and Vps2-Vps24 2 provides a docking site for Vps4-Vta1. This association allows for stimulation of Vps4 ATPase activity by Vps2 and dissociation of the core ESCRT-III subunits A and may represent the major mode of Vps4 activation (based on the relatively minor phenotypes observed upon loss of Vps60 or Did2). Dissociation step A may not require Vta1 function, as Vps2 can stimulate Vps4 directly. Did2 recruitment by Vps2-Vps24 3 represents a second point at which Vps4 ATPase activity can be stimulated through Vta1, leading to dissociation step B. Last, recruitment of Vps60 by Vta1 can lead to dissociation step C. At present, the precise function of dissociation steps B and C are not entirely clear, but they appear to be required for maximal function of the MVB pathway. Several possible scenarios can be invoked as explanations for these three modes of dissociation, including a specific response to environmental or growth conditions, a proofreading activity by Vps4 to prevent inappropriate assembly of ESCRT-III, or distinct sites of action within the cell. (MIT interacting motif, or MIM) within a helix in C-terminal tails of both Vps2 and Did2/CHMP1B mediate interaction with the Vps4 MIT domain (Obita et al., 2007; Stuchell-Brereton et al., 2007). Vps20 lacks this C-terminal MIM, and the region binding to the Vps4 MIT domain does not map to the Vps20 C terminus (Stuchell-Brereton et al., 2007). These distinctions suggest that specific aspects of Vps2 and Did2 MIM binding to Vps4 allow stimulation of ATPase activity and that these effects do not occur upon Vps20 binding to Vps4 under these same conditions. In addition to enhancing Vta1 stimulation of Vps4 activity, interactions with the accessory ESCRT-III subunits also serve to regulate Vta1 endosomal association, as does interaction with Vps4. The ESCRT-III subunits are thought to be recruited to the site of MVB sorting in a sequential manner, with Snf7 58 Developmental Cell 14, 50 61, January 2008 ª2008 Elsevier Inc.

10 and the myristoylated Vps20 recruited by ESCRT-II and thereby allowing association of Vps2 and Vps24. Recruitment of Did2 to endosomes is dependent on Vps2 and Vps24, and Vps60 would appear to be the final ESCRT-III subunit recruited (Figure 7). Loss of core ESCRT-III subunits disrupted Vta1 endosomal localization as an apparent secondary effect of mislocalizing Did2 and Vps4. By comparison, loss of both Did2 and Vps4 reduced Vta1 endosomal localization to undetectable levels (Figure 1B). Although loss of Did2 reduces Vps4 membrane association (Lottridge et al., 2006; Nickerson et al., 2006), direct Vps4 interactions with the core ESCRT-III subunits have been observed and suggest that lingering endosomal Vps4 could confer residual Vta1 association in did2d cells. By contrast, loss of Vps60 resulted in exaggerated Vta1 accumulation on endosomal structures in vps60d (Figure 1B; Figure S1). In striking contrast to core ESCRT-III subunits and Did2, Vps60 endosomal localization is perturbed in both vps4d and vta1d mutants (Figure 6A). Together, this suggests that Vps60 recruitment to endosomes occurs through binding Vta1. Although these analyses are complicated by the inherent difficulties of analyzing a terminal phenotype in a living cell, they suggest the following model regarding the membrane recruitment of these factors: Did2 localizes to endosomes via interactions with Vps2-Vps24, Vps4 is recruited via interactions with Did2 (and ESCRT-III), Vta1 is recruited concomitantly or subsequently to Vps4, and finally Vps60 is recruited by Vta1 (Figure 7). Inherent in this model are several levels of potential modulation of Vps4 activity that may have distinct consequences resulting from the factors assembled. These observations concerning endosomal recruitment and Vps4 stimulation enhancement raise an apparent paradox: Vta1 interactions serve to promote both assembly (Vps60 recruitment) and disassembly of ESCRT-III. This apparent contradiction suggests that assembly and disassembly of ESCRT-III on the endosome is a dynamic process during MVB sorting. Furthermore, the ability to stimulate Vps4 activity via distinct modes suggests that Vps4 ATPase activity may impact discrete events during the MVB sorting reaction. At least two obvious consequences of ATP hydrolysis can be envisioned pertaining to ESCRT-III family members. First, Vps4 may prevent misassembly/overassembly of ESCRT-III. Second, Vps4 likely participates in the disassembly and dissociation of ESCRT subunits concomitant with or subsequent to the completion of ESCRT-III function during the MVB sorting reaction. We suggest that if Vps4 detects spurious, nonproductive accumulations of ESCRT-III subunits, Vps4 ATP hydrolysis would stimulate dissolution of these aberrant structures. One possibility is that Vps2 stimulation of Vps4 might be involved in this proofreading activity. By contrast, if ESCRT-III properly assembles with Did2 and Vps60, the Vta1 interactions with these two accessory ESCRT-III subunits drive a rapid disassembly of ESCRT-III via activation of Vps4 subsequent to completion of MVB sorting. Whether this Vps4 activation or ESCRT-III release may drive membrane invagination is yet to be determined, but this appears to represent the last step in the MVB sorting process identified to date and may represent yet another step during which ATP is hydrolyzed. Although both Did2 and Vps60 enhance Vta1 stimulation of Vps4 in vitro, Did2 and Vps60 serve distinct functions in the later stages of MVB sorting that require ESCRT-III function. Did2 is recruited to the site of MVB sorting subsequent to Vps2-Vps24 recruitment. Did2 in turn recruits Vta1 and Vps4, although the order of recruitment is unclear. Furthermore, additional interactions between Vps4 and the core ESCRT-III subunits, in particular Vps2, may further contribute to both recruitment and stimulation of Vps4. We observed that full-length Did2 binding to Vps4 was less robust than Did , leading us to speculate the presentation of the Vps4 binding site within the Did2 C terminus may be hidden in the context explored in these experiments. Moreover, comparison of Did2 and Did stimulation of Vta1-dependent and -independent Vps4 activity suggests that Did2 direct binding to Vps4 or Vta1 represents alternative interactions. Binding to the endosomal membrane, to other ESCRT-III subunits or to other unknown factors, may alter Did2 confirmation to facilitate the preference for Vps4 or Vta1 binding. Having been recruited to the site of MVB sorting, Vta1 in turn recruits Vps60. In such a model, Vps60 might function as a capping ESCRT-III subunit, preventing the further spreading of the ESCRT-III complex either directly or through stimulating Vta1- Vps4 activity. As a final aspect of Did2 and Vps60 function, we suggest that the binding of Did2 and Vps60 to the Vta1 N terminus stimulates Vps4 activity to recycle ESCRT-III subunits from the endosome. In the absence of Did2 or Vps60, we suggest that ESCRT-III recycling can still occur via interaction between Vps2 and the Vta1-Vps4 complex or Vps4 alone; however, the efficiency of this process is reduced and leads to the formation of aberrant intralumenal vesicles within the MVB (Nickerson et al., 2006). Similarly, in the absence of Vta1, MVB sorting is less efficient but can still occur, presumably due to stimulation of Vps4 by Vps2 (and possibly Did2). Loss of Vta1 interaction with Did2 and Vps60 conferred a significant reduction in Vta1 function. Although this reduced function is not dramatic under normal experimental conditions, the observation that the N termini of Vta1/SBP1/LIP5 are conserved from yeast to human (Xiao et al., 2008) suggests this coordination provides a fitness advantage to the organisms. Further studies of the dynamics of ESCRT-III assembly and disassembly will be required to better understand the precise function of Vta1 in this process. However, we have now established that interactions between the accessory ESCRT-III subunits Did2 and Vps60 enhance Vta1 stimulation of Vps4 in a previously unanticipated manner. EXPERIMENTAL PROCEDURES Plasmid Construction and Yeast Strains VPS60, DID2, VPS2, VPS20, VPS24, and SNF7 full-length and a4-5 coding sequences were amplified from yeast genomic DNA, cloned into pcr2.1-topo (Invitrogen), and subcloned into the BamHI and SalI sites of pgst parallel l (pgst Vps60, pgst Did2, etc.) (Sheffield et al., 1999). GST-Did (pdn106) and Did (pdn104) were kindly provided by D. Nickerson and G. Odorizzi (Nickerson et al., 2006). Vps60 truncations and additional Did2 truncations were generated by amplifying the relevant regions and subcloning into the BamHI and SalI sites of pgst parallel 1. Vps60 and Did2 alanine point mutants were constructed using the TOPO-Vps60 and TOPO-Did2 plasmids with the Gene tailor site-directed mutagenesis system (Invitrogen) and then subcloned into the pgst parallel 1 BamHI and SalI sites. Vta1-GFP, including the VTA1 promoter, was amplified from the genomic DNA of JPY42 (VTA1-GFP::HIS3; Azmi et al., 2006) and subcloned into prs415 and prs416 (Sikorski and Hieter, 1989). To construct pet28 Vta , the relevant coding sequence was amplified from pet28 Vta1 (Azmi et al., 2006) and cloned into pet28b (Novagen). Vta1 W122A, Vta1 K152A, and Vta1 E57A mutants were generated using pbs Vta1 with the Gene tailor Developmental Cell 14, 50 61, January 2008 ª2008 Elsevier Inc. 59

11 site-directed mutagenesis system and subcloned into pet28b and pntap415 (Oestreich et al., 2007). The mcherry-vps60 plasmid was constructed by cloning the VPS21 promoter upstream of the mcherry N-terminal tag coding sequence in a prs416 backbone vector and subcloning the Vps60 BamHI, SalI fragment into BglII, XhoI sites. All PCR and mutagenesis clones were sequenced to ensure the absence of unexpected mutations. Standard yeast genetics techniques were employed to generate strains (Table S1) utilized in these studies. Protein Expression and Purification GST fusion proteins were expressed in BL21 DE3 bacteria for 3 hr at 30 C with 0.5 mm IPTG, lysed in PBS, cleared by a 30 min 50,000 3 g spin, and purified with glutathione Sepharose 4B (GE Healthcare Bio-Sciences). Only GST- Vps24 a4-5 failed to yield sufficient soluble protein for examination in these studies. Vta1, Vta , Vta1 E57A, Vta1 W122A, and Vta1 K152A proteins were expressed in BL21 DE3 bacteria with 0.5 mm IPTG at 25 C for hr and purified as previously described (Azmi et al., 2006)byNi 2+ -affinity chromatography, thrombin cleavage, ATP treatment, and ion-exchange chromatography. Biochemical Analyses GST pull-down experiments were performed as previously described (Katzmann et al., 2001) with slight modifications [PBS with 0.05% Tween 20 (PBST)]. PBST was used with 2 mg/ml Vta1. Bound materials were visualized by Coomassie staining or western blotting with anti-vta1 (see anti-vta1 polyclonal antibody production below). CPS pulse chase was performed as previously described (Babst et al., 2002a). ATPase Assay ATPase assays for GST fusion proteins were performed as previously described (Azmi et al., 2006). GST fusion proteins (approximately 2 mg, yielding an apparent concentration of approximately 2 mm in20 ml reaction) were bound to glutathione beads (in PBST) and washed with PBST (43) and ATPase reaction buffer % Tween 20 (43). Residual buffer was removed by aspiration with a 30 gauge needle and 500 nm Vps4 or 2 mm Vta1 or both were added in a total of 18 ml of ATPase reaction buffer. Subsequently, 2 ml of a 10 mm ATP mixture containing [a- 32 P]ATP was added and time points were taken at 1 min 20 s, 2 min 40 s, and 4 min, resolved by thin-layer chromatography, and processed as described previously (Azmi et al., 2006). Experiments were performed at least three times and results were analyzed using Excel and Prism. Error bars indicating standard error from the mean are presented in all graphs. Subcellular Localization Subcellular fractionation was performed as previously described (Babst et al., 1997) with slight modifications. The trichloroacetic acid pellets were washed twice with acetone and dried. The samples were then resuspended in 100 ml of urea cracking buffer before adding 25 mlof 53 Laemmli sample buffer. Samples were incubated for 5 min at 50 C, resolved by SDS-PAGE, and transferred to nitrocellulose for western blotting. Snf7 protein was detected using Snf7 polyclonal antiserum (Babst et al., 1998). Anti-PGK monoclonal antibody (Molecular Probes) was used as a soluble marker. Microscopy was carried out on living yeast cells using a fluorescence microscope (Nikon) with GFP and DsRed filters and a digital camera (Coolsnap HQ; Photometrics) as previously described (Azmi et al., 2006). Vta1-GFP images (except in combination with Cherry-Vps60) were deconvolved using DeltaVision software (Applied Precision). Labeling with FM4-64 was performed as previously described (Oestreich et al., 2007). Anti-Vta1 Polyclonal Antibody Production Full-length Vta1 protein was purified for antiserum production against Vta1 as previously described (Azmi et al., 2006). A New Zealand white rabbit was immunized with 1 mg/ml Vta1 and bleeds were collected every 2 weeks for a period of 2 months (Covance). Bleeds were tested for detection of Vta1 in SEY6210 (Figure S5). Supplemental Data Supplemental Data include five figures and one table and are available with this article online at 50/DC1/. ACKNOWLEDGMENTS We thank members of the Katzmann and Horazdovsky labs, as well as Greg Odorizzi and Daniel Nickerson for helpful discussions and plasmids pdn104 and pdn106, Bradley Bellin, Bob Sikkink, and Johanna Payne for technical assistance, and Bruce Horazdovsky for critical evaluation of the manuscript. This work was supported by grants RO1 GM (D.J.K.), RO1 DK65980 (Z.X.), and RO1 GM (M.B.) from the National Institutes of Health. I.F.A. is supported by a predoctoral fellowship from the American Heart Association (AHA ). Received: September 7, 2007 Revised: October 29, 2007 Accepted: October 30, 2007 Published: January 14, 2008 REFERENCES Amerik, A.Y., Nowak, J., Swaminathan, S., and Hochstrasser, M. (2000). The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar proteinsorting and endocytic pathways. Mol. Biol. Cell 11, Azmi, I., Davies, B., Dimaano, C., Payne, J., Eckert, D., Babst, M., and Katzmann, D.J. (2006). Recycling of ESCRTs by the AAA-ATPase Vps4 is regulated by a conserved VSL region in Vta1. J. Cell Biol. 172, Babst, M. (2005). A protein s final ESCRT. Traffic 6, 2 9. Babst, M., Sato, T.K., Banta, L.M., and Emr, S.D. (1997). Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p. EMBO J. 16, Babst, M., Wendland, B., Estepa, E.J., and Emr, S.D. (1998). 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