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Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast

Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast JCB: Article Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast 1 2 1 2 2 1 Megan Wemmer, Ishara Azmi, Matthew West, Brian Davies, David Katzmann, and Greg Odorizzi Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905 ndosomal sorting complexes required for transport vesicles at endosomes, but its role in membrane scission is (ESCRTs) promote the invagination of vesicles into unknown. We show that overexpression of Bro1 or its E the lumen of endosomes, the budding of enveloped N-terminal Bro1 domain that binds Snf7 enhances the stabil- viruses, and the separation of cells during cytokinesis. ity of ESCRT-III by inhibiting Vps4-mediated disassembly These processes share a topologically similar membrane in vivo and in vitro. This stabilization effect correlates with scission event facilitated by ESCRT-III assembly at the cyto- a reduced frequency in the detachment of intralumenal solic surface of the membrane. The Snf7 subunit of ESCRT-III vesicles as observed by electron tomography, implicating in yeast binds directly to an auxiliary protein, Bro1. Like Bro1 as a regulator of ESCRT-III disassembly and mem- ESCRT-III, Bro1 is required for the formation of intralumenal brane scission activity. Introduction The endosomal sorting complexes required for transport membranes is stimulated upon binding Vps20, which relieves (ESCRTs) are recruited to the cytosolic surface of endosomes, an autoinhibitory intramolecular interaction between the N­ and where they selectively package ubiquitinated transmembrane C­ terminal regions of Snf7 (Teis et al., 2008; Saksena et al., protein cargoes into the intralumenal vesicles (ILVs) of multi­ 2009). Membrane­ associated Snf7 polymers are thought to be vesicular bodies (MVBs). The ILVs and their cargoes are sub­ capped by Vps24, which recruits Vps2 (Babst et al., 2002; Teis sequently degraded upon fusion of MVBs with lysosomes. et al., 2008). In turn, Vps2 promotes activation of the AAA­ ESCRT­ 0, ­ I, and ­ II are stable heteromeric complexes, each ATPase Vps4, which catalyzes disassembly of ESCRT­ III and containing subunits that bind directly to ubiquitin (Ub) linkages recycling of its subunits from membranes to the cytosol (Babst on the cytosolic domains of cargoes (Piper and Katzmann, et al., 1998; Babst et al., 2002). Did2, Ist1, and Vps60 are three 2007; Raiborg and Stenmark, 2009). Recent studies indicate accessory ESCRT­ III subunits that appear to serve primarily as that ESCRT­ I and ­ II can also induce membrane invaginations regulators of Vps4 activity (Nickerson et al., 2006; Azmi et al., of nascent ILVs in vitro (Wollert and Hurley, 2010) and that 2008; Dimaano et al., 2008; Rue et al., 2008). ESCRT­ II can stimulate assembly of ESCRT­ III (Im et al., 2009; In vitro reconstitution of ESCRT­ III assembly on synthetic Saksena et al., 2009). membranes revealed that excess amounts of the yeast core The polymerization of ESCRT­ III subunits is required for ESCRT­ III subunits deform liposomes (Saksena et al., 2009) and completion of ILV budding. Genetic and biochemical studies of drive both the formation and detachment of lumenal membrane ESCRT­ III proteins from Saccharomyces cerevisiae have con­ invaginations (Wollert et al., 2009). In limiting amounts, however, tributed much to our understanding about assembly of the ESCRT­ III subunits lack the ability to deform the membrane complex, principles that are likely to be generally conserved. and, instead, their activity is restricted to facilitating the severing ESCRT­ III in yeast is comprised of four core subunits (Snf7, of membrane invaginations generated by ESCRT­ I and ­ II Vps20, Vps24, and Vps2; Babst et al., 2002), the most abundant (Wollert and Hurley, 2010). These observations suggest that of which is Snf7 (Teis et al., 2008). Polymerization of Snf7 on ESCRT­ III is responsible for execution of the membrane scission © 2011 Wemmer et al. This article is distributed under the terms of an Attribution– Correspondence to Greg Odorizzi: [email protected] Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub - Abbreviations used in this paper: DIC, differential interference contrast; ESCRT, lication date (see http://www.rupress.org/terms). After six months it is available under a endosomal sorting complex required for transport; ILV, intralumenal vesicle; MIT, Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, microtubule interacting and trafc fi king; MVB, multivesicular body; Ub, ubiquitin. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 192 No. 2 295–306 www.jcb.org/cgi/doi/10.1083/jcb.201007018 JCB 295 THE JOURNAL OF CELL BIOLOGY step during ILV formation. Consistent with these in vitro find ­ ings, mammalian ESCRT­III is required during cytokinesis for midbody abscission and for the detachment of fully assembled retroviruses from the plasma membrane (McDonald and Martin­ Serrano, 2009). Both abscission and retroviral budding require membranes to be severed in a manner that is topologically equivalent to ILV formation at MVBs, and all three processes are disabled by inhibiting Vps4 function (Garrus et al., 2001; Carlton et al., 2008). Overexpression of catalytically inactive Vps4 in mammalian cells dramatically increases the frequency of ILV budding profiles and reduces the number of freely de ­ tached ILVs at MVBs (Sachse et al., 2004). However, based on studies of reconstituted ILV budding in vitro (Wollert et al., 2009; Wollert and Hurley, 2010), Vps4 is thought to function indirectly in membrane scission by recycling ESCRT­III sub ­ units for repeated rounds of assembly. In addition to its membrane scission activity, ESCRT­III is required for coordinating the deubiquitination of trans­ membrane protein cargoes before their enclosure within ILVs. In humans, two distinct Ub hydrolases bind directly to ESCRT­ III via microtubule­ interacting and trafficking (MIT) domains (Agromayor and Martin­ Serrano, 2006; Tsang et al., 2006; Row et al., 2007), and in yeast, recruitment of the Ub hydrolase Doa4 to ESCRT­III is promoted by Bro1, which binds both to Snf7 and to the Doa4 catalytic domain (Luhtala and Odorizzi, 2004; Kim et al., 2005; Richter et al., 2007). Bro1 and its closest human or­ thologue, Alix, each have a conserved N­terminal Bro1 domain that binds directly to Snf7 and CHMP4 proteins, respectively (Kim et al., 2005; Fisher et al., 2007). Although this interaction is essential for Alix to promote retrovirus budding (Fisher et al., 2007), the means by which it contributes to ESCRT­III function in membrane scission remain unclear. In yeast, Bro1 likely has a role in ILV budding independent of its activity in regulating deubiquitination because ILV formation is strongly impaired by deletion of BRO1 but not DOA4 (Richter et al., 2007). In this study, we report that binding of Bro1 to Snf7 enhances the stability of ESCRT­III in vivo and inhibits Vps4­mediated dis ­ assembly of the complex in vitro. This effect correlates with a reduced efficiency in detachment of ILVs from the limiting endosomal membrane. Conversely, mutation of the Bro1­ binding site in Snf7 strongly inhibits vesicle formation and cargo sort­ ing. These results implicate Bro1 as a regulator of ESCRT­III disassembly and membrane scission activity. Results The Bro1 domain binds a MIM1-like motif in the Snf7 subunit of ESCRT-III Yeast two­hybrid analysis (unpublished data) was used to map the amino acids in Snf7 required for its interaction with the Figure 1. The Bro1 domain binds a MIM1-like motif in Snf7. (A) Schematic Bro1 domain of yeast Bro1 (Fig. 1 A). This site is centered diagrams of Bro1 and Snf7, including the amino acid sequence align- ment of MIM1-like motifs from yeast Snf7 and human CHMP4a. (B) In vitro around residues Leu­231 and Leu­234 at the C terminus of pull-downs of purie fi d GST or GST-Bro1 domain incubated with bacterial Snf7, which resembles the site in CHMP4 proteins that binds L231A/L234A lysates expressing Snf7-HA or Snf7 -HA. GST and GST-Bro1 the Bro1 domain of Alix (McCullough et al., 2008) and is simi­ domain inputs were loaded at 10% of their total used in each pull-down. IB, immunoblotting. (C) Fluorescence and DIC microscopy of vps4, vps4 lar to the MIM1 motif that was identified in Vps2/CHMP2 and L231/234A snf7, and vps4 snf7 cells expressing GFP-Bro1 domain or GFP- Did2/CHMP1 proteins (Obita et al., 2007; Stuchell­Brereton Bro1. Arrowheads indicate where GFP does (closed) or does not (open) et al., 2007). The MIT domain of Vps4 has relatively strong colocalize with FM4-64–labeled class E compartments. Bars, 2 µm. 296 JCB • VOLUME 192 • NUMBER 2 • 2011 afn fi ity for MIM1 of Vps2/CHMP2 and Did2/CHMP1 but binds poorly to the MIM1­ like site in CHMP4 proteins (Kieffer et al., 2008), which is consistent with the lack of in vitro binding between purified Snf7 and Vps4 (Shestakova et al., 2010; un ­ published data). Leu­ 231 and Leu­ 234 in the MIM1­ like site of Snf7 were essential for its in vitro binding to the Bro1 domain of yeast Bro1 (Fig. 1 B). In vivo, these residues were also required for Snf7 to mediate endosomal localization of the Bro1 domain fused to GFP (Fig. 1 C). However, full­ length Bro1­ GFP weakly localized to endosomes despite disruption of the Bro1 domain– L231A/L234A binding site in Snf7 (snf7 ; Fig. 1 C), which matches our previous observation that full­ length Bro1 weakly localizes to endosomes despite mutation of the Snf7 interaction surface in the Bro1 domain, whereas the same mutation completely dis­ ables endosomal localization of the Bro1 domain alone (Kim et al., 2005). Bro1 binding to Snf7 enhances the stability of ESCRT-III polymers in vivo The position of the Bro1 domain–binding site at the C terminus of Snf7 (Fig. 1) suggested that Bro1 might act like Vps20 to re­ lieve Snf7 autoinhibition (Teis et al., 2008; Saksena et al., 2009), thereby promoting Snf7 polymerization and incorporation into ESCRT­ III. However, it was previously shown that Bro1 is not essential for Snf7 polymerization based on the analysis of cells Figure 2. Overexpression of Bro1 or the Bro1 domain stabilizes ESCRT-III in which ESCRT­ III disassembly by the Vps4 ATPase had been polymers at endosomes. (A) Western blot analysis of detergent-solubilized disabled by deletion of the VPS4 gene (Teis et al. 2008). There­ yeast membranes resolved by isopycnic density gradient centrifugation. Inclusion of 1% Triton X-100 throughout the gradients yielded identical fore, we investigated whether Bro1 might inu fl ence Snf7 poly­ sedimentation profiles. (B) Quantitation of triplicate gradients from A. merization in the context of functional Vps4 by examining the distribution of Snf7 in detergent­ solubilized yeast membranes L231A/L234A separated by isopycnic density gradient centrifugation. In ex­ mutant snf7 that could be detected as a polymer, and tracts from wild­ type cells, the majority of Snf7 was seen in low wild­ type Snf7 was similarly unaffected by overexpression of a density fractions, but a small amount (19%) was detected in mutant Bro1 domain that lacks the Snf7­ binding site (Fig. S1 B), higher density fractions (Fig. 2, A and B). This high density pool indicating that the effect of Bro1 domain overexpression toward of Snf7 likely corresponds to the transient ESCRT­ III complexes stabilizing ESCRT­ III depends on its ability to bind Snf7. Bro1 that were reported to exist at steady­ state (Teis et al., 2008) domain overexpression caused a similar shift toward high because the same gradient fractions contained 90% of Snf7 in density gradient fractions for another core ESCRT­ III subunit, extracts from vps4 cells (Fig. 2, A and B) in which ESCRT­ III Vps24 (Fig. S2), although concomitant shifts in ESCRT­ I or is irreversibly trapped as an assembled complex (Babst et al., ESCRT­ II were not observed (not depicted). However, the total 1998; Teis et al., 2008). The polymeric Snf7 we observed had amounts of membrane­ associated Snf7 and Vps24 were not sig­ the same properties that were reported previously (Teis et al., nic fi antly altered by overexpression of the Bro1 domain ( Fig. S3), 2008) because its stabilization upon deletion of VPS4 was indicating that the membrane recruitment step preceding largely dependent on Vps20 but not Bro1 (Fig. 2, A and B). ESCRT­ III assembly was not enhanced. Moreover, we did not Moreover, recombinant Snf7 that was expressed in bacteria and detect an increase in the amount of Vps4 in high density gradi­ isolated as a soluble polymer by size­ exclusion chromatography ent fractions upon overexpression of the Bro1 domain (unpub­ before being subjected to isopycnic density gradient centrifuga­ lished data), indicating that binding of ESCRT­ III to Vps4 was tion also migrated to high density fractions (Fig. S1 A). not dramatically increased under these conditions. Together, Although a relatively minor percentage of membrane­ these observations suggest that driving the interaction between associated Snf7 was polymeric in extracts from wild­ type cells Bro1 and Snf7 by overexpression of Bro1 or the Bro1 domain (19%), the percentage of polymeric Snf7 increased upon over­ enhances the stability of ESCRT­ III assembled at endosomal expression of either full­ length Bro1 or the Bro1 domain (29% membranes despite Vps4 being present. and 27%, respectively; Fig. 2, A and B). This effect required that Snf7 first undergo activation by Vps20 because overexpres ­ Bro1 binding to Snf7 inhibits Vps4- sion of the Bro1 domain in vps20 cells failed to enhance mediated disassembly of ESCRT-III in vitro detection of polymeric Snf7 (Fig. 2, A and B). Bro1 domain The stabilizing effect exerted by Bro1 toward ESCRT­ III was overexpression also did not cause an increase in the amount of examined further using an in vitro system that reconstitutes Bro1 regulation of ESCRT-III • Wemmer et al. 297 Vps4­ mediated disassembly of the complex. In this assay, abnormal endosomal structures known as class E compart­ membrane­ associated ESCRT­ III generated from extracts of vps4 ments, which consist of stacks of aberrantly attened fl endo ­ cells were incubated with 1 mM ATP and 10 nM purified Vps4 somes that generally lack ILVs (Fig. 4 B and Video 2), which is for 10 min at 30°C, and the extent of ESCRT­III disassembly consistent with ESCRT­ III promoting membrane scission. L231A/L234A was determined by probing the amount of Snf7 that remains Expression of the snf7 allele in place of wild­ type membrane associated after centrifugation. As shown previously SNF7 did not result in class E compartments like those seen in (Davies et al., 2010), this reaction condition resulted in a 50% snf7 cells. Nevertheless, this mutation caused a sharp reduc­ reduction in membrane­associated Snf7 ( Fig. 3 A), indicating tion in the number of ILVs and a gross distortion of endosomes that exogenous Vps4 has potent activity in mediating in vitro in a manner reminiscent of the class E compartment morphol­ disassembly of ESCRT­III that had been preassembled in vivo. ogy (Fig. 4 C, Fig. 5 C [quantic fi ation], and Video 3). This reduc­ However, this activity was inhibited by 50% if ESCRT­III was tion in the amount of ILVs is consistent with the interpretation generated from vps4 cells overexpressing the Bro1 domain and, that Bro1 binding to Snf7 is critical for proper ESCRT­III func ­ to a lesser degree, if the source of ESCRT­ III was vps4 cells over­ tion in vivo. expressing full­length Bro1 (Fig. 3 A). Unexpectedly, we discovered that Bro1 domain over­ The inhibitory effect of Bro1 on in vitro disassembly of expression in wild­type cells increased the frequency of intra­ ESCRT­III (Fig. 3 A) was similar to the in vivo results seen in lumenal invaginations that had not detached from the limiting Fig. 2, wherein the stability of membrane­associated ESCRT ­III endosomal membrane (Fig. 4, D and E; and Videos 4 and 5). was enhanced upon overexpression of Bro1 or the Bro1 domain. Such ILV budding profiles are not common, with a mean of 1.2 Although these observations support a model in which Bro1 observed per MVB in wild­type cells, but 2.4 budding profiles binding to Snf7 interferes with Vps4­mediated disassembly of per MVB on average were seen upon overexpression of the ESCRT­III, an alternative possibility was that excess Bro1 or Bro1 domain (Fig. 5 A). Plotting the frequency distribution Bro1 domain compromises the ESCRT­III assembly process of ILV budding profiles (Fig. 5 B) showed that overexpression and results in aberrant complexes refractory to Vps4. However, of the Bro1 domain resulted in more instances of multiple the addition of purified Bro1 domain (fused to GST) effectively budding profiles at individual MVBs (five budding profiles at negated in vitro Vps4­mediated disassembly of preassembled the MVB shown in Fig. 4 E; Video 5). Overexpression of full­ ESCRT­III generated from vps4 cells (Fig. 3 B). Moreover, length Bro1 had the same effect as the Bro1 domain (Fig. 4 F; this inhibitory effect was observed even if Vps4 was allowed to Fig. 5, A and B; and Video 6), demonstrating that the increase in initiate disassembly of ESCRT­III for 5 min before the addition ILV budding profiles was not simply caused by the absence of of GST­Bro1 domain (Fig. 3 C). Thus, Bro1 inhibition of Vps4­ an activity normally provided by the C­terminal region of Bro1. mediated disassembly of ESCRT­III is manifested downstream A gallery of tomograms highlighting ILV budding profiles of complex assembly. versus detached ILVs at individual MVBs in wild­type cells We also tested whether the inhibitory effect of the Bro1 with and without overexpression of Bro1 or the Bro1 domain is domain relied exclusively on its ability to bind Snf7 by generat­ shown in Fig. 4 (G–L). Vps2MIM1 ing ESCRT­III from vps4 cells in which a Snf7 chi­ The low frequency of ILV budding profiles in wild­type mera was expressed in place of wild­type Snf7. In this chimera, yeast presumably reflects the rapid rate at which membrane the MIM1­like Bro1 domain–binding site is replaced with the scission normally occurs. This process is most likely delayed bona fide MIM1 from Vps2 that binds the MIT domain of Vps4 rather than accelerated in response to Bro1 or Bro1 domain Vps2MIM1 (Obita et al., 2007). The Snf7 chimera rescues in vitro overexpression because quantitation of membrane surface areas Vps4­mediated disassembly of ESCRT ­III formed in the ab ­ showed a decrease in the lumenal membrane content and a cor­ sence of Vps2 expression, demonstrating that it effectively responding increase in the amount of limiting membrane (Fig. 5 C), serves as a substrate for Vps4 (Davies et al., 2010). Similarly, although the mean vesicle diameter did not change (Fig. 5 D). Vps2MIM1 ESCRT­III assembled with Snf7 in place of wild­type In contrast, an acceleration of ILV formation would be expected Snf7 was susceptible to disassembly by Vps4 even upon addi­ to show an increase in the amount of lumenal membrane con­ tion of purified Bro1 domain (Fig. 3 D). We conclude that direct tent within endosomes, as is observed in yeast lacking Ist1 binding of the Bro1 domain to Snf7 inhibits Vps4­mediated dis ­ (Nickerson et al., 2010), an accessory ESCRT­III protein that assembly of ESCRT­III. inhibits Vps4 activity (Dimaano et al., 2008). Compared with wild­type cells, the ratio of lumenal to limiting membrane sur ­ Overexpression of Bro1 or the Bro1 face area was lower upon mutation of the Bro1 domain–binding L231A/L234A domain reduces ILV budding efficiency site in Snf7 (snf7 ; 23.3% of total endosomal membrane at MVBs surface area was lumenal; Fig. 5 C), whereas snf7 cells were To evaluate whether binding of the Bro1 domain to Snf7 almost exclusively devoid of lumenal membranes (0.27% of the influences the membrane scission activity of ESCRT ­ III, we total endosomal membrane surface area; Fig. 5 C), which is examined the morphology of yeast endosomes by EM and 3D consistent with the stronger effects on endosomal morphology tomography. Deletion of SNF7 or genes encoding the other core caused by these mutations (Fig. 4). ESCRT­III subunits inhibits the biogenesis of MVBs like those Measurements of tomograms further revealed that the seen in wild­type cells ( Fig. 4 A and Video 1). Instead, snf7 amount of membrane incorporated into ILV budding profiles cells (and other ESCRT­III deletion strains; not depicted) have in wild­ type cells accounted for only 6.3% of the limiting 298 JCB • VOLUME 192 • NUMBER 2 • 2011 Figure 3. Bro1 binding to Snf7 inhibits in vitro disassembly of ESCRT-III. (A) 0.1 µM purified Vps4, ATP, and an ATP regeneration system were incubated with membranes isolated from vps4 yeast cells; overexpression of full-length Bro1 or the Bro1 domain (BOD) is indicated. The amount of membrane- associated Snf7 remaining after incubation at 30°C for 10 min was determined by quantitative Western blot analysis. P < 0.001 for both samples. (B) In vitro ESCRT-III release performed in the presence or absence of ATP, 0.1 µM Vps4, and 0.1 µM GST-Bro1 domain (GST-BOD). (C) In vitro ESCRT-III release performed as in B except that the reaction was allowed to proceed for 5 min in the absence of GST-BOD or for 20 min in the absence or presence of 1 µM GST-BOD that was added either at the same time as purified Vps4 or 5 min after the addition of Vps4. (D) In vitro ESCRT-III release performed Vps2MIM1 with membranes from yeast expressing wild-type Snf7 or the Snf7 chimera in the presence or absence of ATP, 0.1 µM Vps4, and 0.1 µM GST-BOD. Error bars show the standard deviation. endosomal membrane surface area. In contrast, overexpression observed under these conditions (Fig. 5 A). Despite the increase of Bro1 or the Bro1 domain resulted in the incorporation of in the number of ILV budding profiles, their overall morphol ­ 18.6% and 17.5%, respectively (Fig. 5 E), which is propor­ ogy was not altered significantly, as neither the diameter of tional to the increased frequency of ILV budding profiles the vesicles attached to the limiting endosomal membrane Bro1 regulation of ESCRT-III • Wemmer et al. 299 Figure 4. Endosome morphologies in cells overexpressing Bro1 or the Bro1 domain. (A–F) 2D cross sections and 3D models from 200-nm-thick section electron tomograms. Spherical endosomal limiting membranes are depicted in yellow, whereas flattened class E compartments are depicted in various colors to discriminate discrete membranes. Lumenal vesicles are red. V, vacuole. Bars, 100 nm or 200 nm as noted. (G–L) Gallery of tomographic slices of wild-type cells with and without overexpression of Bro1 or the Bro1 domain. Freely detached ILVs are traced in red, whereas ILV budding profiles and the limiting endosomal membrane are traced in yellow. In some cases, the continuity of ILV budding profiles with the limiting endosomal membrane is out of plane in the tomographic slice but evident in the 3D reconstruction. Bars, 50 nm. 300 JCB • VOLUME 192 • NUMBER 2 • 2011 (Fig. S4 A) nor the diameter of the membrane neck at ILV budding profiles (Fig. S4 B) were statistically different in wild­ type cells with or without overexpression of Bro1 or the Bro1 domain. Cargo sorting and deubiquitination are not affected by Bro1 or Bro1 domain overexpression in wild-type cells Transmembrane proteins sorted into the ILVs of MVBs are ultimately delivered to the vacuole lumen upon MVB–vacuole fusion, but disrupting the activity of any of the ESCRTs results in mislocalization of these cargoes to the vacuole membrane and their accumulation at class E compartments (Odorizzi et al., 1998). Therefore, we evaluated how the effects on ILV budding revealed by EM tomography (Fig. 4) impacted the efficiency of cargo sorting using live cell fluorescence microscopy of yeast expressing GFP fused to the cytoplasmic domain of carboxypeptidase S (GFP­ CPS). We previously reported mislocalization of GFP­ CPS upon mutation of the Snf7­ binding site in the Bro1 domain (Kim et al., 2005). Simi­ larly, mislocalization of GFP­ CPS occurred both in snf7 cells L231A/L234A and in snf7 cells lacking the Bro1­ binding site within Snf7 (Fig. 6 A), which was consistent with the strong reduc­ tion in ILV formation caused by these mutations (Fig. 5). Deliv­ ery of GFP­ CPS into the vacuole lumen in wild­ type cells was only mildly affected upon overexpression of the Bro1 domain (Fig. 6 A), whereas full­ length Bro1 overexpression did not appear to cause significant cargo mislocalization (Luhtala and Odorizzi, 2004). Doa4 is the Ub hydrolase that deubiquitinates CPS and other MVB vesicle cargoes (Dupré and Haguenauer­Tsapis, 2001; Katzmann et al., 2001; Losko et al., 2001), and its activity is stimulated upon binding to Bro1 (Richter et al., 2007). This process occurs efficiently in wild­type cells, resulting in detection of very little ubiquitinated CPS (Ub­CPS; Fig. 6 B). In contrast, Ub­CPS accumulated upon deletion of SNF7 or L231A/L234A upon mutation of its Bro1 domain–binding site (snf7 ; Fig. 6 B), demonstrating that its interaction with Snf7 is im­ portant for Bro1 to promote Doa4 activity at endosomes. Con­ versely, overexpression of Bro1 or the Bro1 domain had no observable effects on CPS deubiquitination (Fig. 6 B), indicat­ ing that the enhancement of ESCRT­III stability and reduced efficiency in membrane scission is not a consequence of altered Doa4 function. of a class E compartment in a single tomogram of snf7 cells (Fig. S5); the rarity of this occurrence is reflected by the observation that the membrane surface area of these ILVs comprised 0.27% of the total endosomal mem- brane surface area in this strain. Tomograms for 12, 14, and 17 MVBs were prepared and modeled for wild-type cells, cells overexpressing the Figure 5. Quantitation of endosome morphology and vesicle budding Bro1 domain, and cells overexpressing full-length Bro1, respectively. Four L231A/L234A profiles. Quantitation of the mean number of ILV budding profiles observed individual tomograms were prepared and modeled for snf7 cells, per MVB (A) versus the frequency distribution of the number of ILV budding which contained a total of 33 MVB, VTE, and cisternal structures. Three profiles observed per MVB (B) observed in tomograms. Quantitation of the individual tomograms were generated and modeled for snf7 cells, each relative percentage of limiting versus lumenal membrane surface areas (C) containing E compartments that were composed of multiple cisternal stacks and the mean ILV diameters (D) observed in tomograms. (E) The percent- and occasionally also spherical endosomal membranes. Methods used for age of the limiting membrane incorporated into ILV budding profiles was the preparation of the tomograms that were used to generate the measure- measured for each strain. Note that no ILV budding profiles were seen for ments shown in this experiment are described in detail in the Materials and snf7 cells, but five freely detached ILVs were observed at the periphery methods section. Bro1 regulation of ESCRT-III • Wemmer et al. 301 S. cerevisiae has been elusive because of the formation of aber­ rant class E compartments rather than MVBs in mutant yeast that generally lack functional ESCRT complexes (Rieder et al., 1996; Odorizzi et al., 1998; Luhtala and Odorizzi, 2004; Nickerson et al., 2006). In this study, we describe a potential role for Bro1 in regulating ESCRT­ III by demonstrating that overexpression of either full­ length Bro1 or its N­ terminal Bro1 domain stabilizes ESCRT­ III polymers and reduces the efcienc fi y of ILV detachment from the limiting endosomal membrane in yeast that contain an otherwise wild­ type ESCRT machinery. ILV budding profiles in yeast are infrequently observed, even in thick sections imaged by 3D EM tomography, presum­ ably because of the rapid rate at which membrane deformation and scission occur. The increased frequency of budding profiles caused by overexpression of Bro1 or the Bro1 domain must be interpreted as a reduction in ILV budding efficiency rather than an acceleration because these conditions also resulted in signifi ­ cantly less lumenal membrane content within MVBs. In the ab­ sence of monitoring this process in real time, it is impossible to exclude whether at least some of the ILV budding profiles are dead­ end structures that fail to complete membrane scission. However, the ILVs seen in cells overexpressing Bro1 or the Bro1 domain appeared structurally normal. Neither their overall mean diameter nor the dimensions at the necks of the budding profiles were significantly different from the same measure ­ ments in wild­ type cells, suggesting that these ILVs will eventu­ ally detach from the limiting endosomal membrane. The specic fi mechanism underlying the reduced efc fi iency of ILV budding resulting from overexpression of Bro1 or the Bro1 domain is unclear, but a clue comes from our n fi ding that this con ­ dition correlates with increased detection of Snf7 and Vps24, both core ESCRT­ III subunits, in a high molecular mass polymer de­ spite wild­ type Vps4 expression. Vps4 is the AAA­ ATPase that catalyzes disassembly of ESCRT­ III (Babst et al., 1998), which accounts for very little of the assembled complex existing at steady­ state (Teis et al., 2008). Based on in vitro reconstitution of ILV budding, Vps4 is thought to function indirectly in membrane scission by recycling ESCRT­ III subunits for repeated rounds of assembly (Wollert et al., 2009; Wollert and Hurley, 2010). Given the intermediate degree to which Snf7 polymers are stabilized upon Bro1 or Bro1 domain overexpression (only 30% of the Figure 6. Cargo sorting and deubiquitination. (A) Fluorescence and DIC total amount of Snf7 that is trapped as a polymer in vps4 cells), microscopy of cells expressing GFP-CPS. Bars, 2 µm. (B) Western blot it seems unlikely that a dec fi iency of free ESCRT ­ III subunits exists analysis of anti-CPS immunoprecipitates. IB, immunoblotting. for complex assembly and subsequent execution of membrane scission. However, we presently cannot determine whether Bro1­ Discussion mediated stabilization of ESCRT­ III is the direct cause of the re­ In vitro studies have pointed to the assembly of ESCRT­ III duced efc fi iency in ILV budding, which would suggest an active being crucial to the membrane scission event that results in de­ role for Vps4 during membrane scission in vivo. Nevertheless, the tachment of ILVs (Wollert et al., 2009; Wollert and Hurley, possibility that membrane scission might be coupled to Vps4 ac­ 2010). Although the specific mechanism by which ESCRT ­ III tivity is consistent with our previous discovery by EM tomogra­ executes this activity is unclear, its role in severing membrane phy that MVBs have signic fi antly more ILVs in the absence of connections is supported by in vivo studies of mammalian cells, Ist1 (Nickerson et al., 2010), an accessory ESCRT­ III protein that showing a requirement for ESCRT­ III in cytokinesis and negatively regulates Vps4 by inhibiting its binding to ESCRT­ III retroviral budding, both of which are membrane scission events (Dimaano et al., 2008). An active role for Vps4 in membrane scis­ that are topologically equivalent to ILV formation at MVBs sion might also explain why overexpression of catalytically in­ (McDonald and Martin­ Serrano, 2009). Genetic dissection of active Vps4 in mammalian cells results in an increased frequency the mechanism of action for ESCRT­ III at endosomes in of ILV budding prol fi es at endosomes (Sachse et al., 2004). 302 JCB • VOLUME 192 • NUMBER 2 • 2011 The membrane scission activity of ESCRT­III in vitro is because complete removal of Bro1 function by deletion of the potently stimulated by ESCRT­II (Im et al., 2009), which is BRO1 gene results in class E compartments rather than MVBs thought to trigger ESCRT­III polymerization through its direct (Luhtala and Odorizzi, 2004; Richter et al., 2007). Bro1 stimu­ binding to Vps20 (Saksena et al., 2009). Unlike ESCRT­III, lates transmembrane protein cargo deubiquitination at endo­ however, ESCRT­II is not required for the budding of human somes by promoting both recruitment and activation of the immunodeficiency virus­1 (HIV ­1; Langelier et al., 2006), rais ­ Doa4 Ub hydrolase (Luhtala and Odorizzi, 2004; Richter et al., ing the question of how ESCRT­III assembly is stimulated at the 2007). However, this activity alone cannot account for the im­ site of viral budding. The p6 domain of the Gag polyprotein of portance of the Bro1–Snf7 interaction during membrane scis­ HIV­1 binds both to Alix (Strack et al., 2003) and to TSG101, a sion because mutations that specifically disable Bro1 from subunit of ESCRT­I (Garrus et al., 2001; Martin­Serrano et al., stimulating Doa4 do not impair ILV formation, nor does re­ 2001; VerPlank et al., 2001). The yeast orthologue of TSG101 placement of wild­ type DOA4 with nonfunctional alleles (Richter (Vps23) also binds Vps20 (Bowers et al., 2004; Teo et al., 2004), et al., 2007). L231A/L234A but an interaction between TSG101 and ESCRT­III has not been The strong reduction in ILV formation seen in snf7 reported. Binding of Alix alone to p6 is sufficient to promote cells is consistent with the failure in delivery of CPS to the HIV­1 budding (Fisher et al., 2007; Usami et al., 2007), but vacuole lumen via the MVB pathway. Given the central role whether this interaction facilitates ESCRT­III assembly is un ­ Bro1 has in regulating deubiquitination at endosomes, it is not known. Our results suggest that Bro1 in yeast lacks this activity surprising that disruption of the Bro1 domain–binding site in because its ability to stabilize ESCRT­III when overexpressed Snf7 caused a strong accumulation of Ub­CPS. However, the was dependent on the presence of Vps20. efficiency of cargo deubiquitination controlled by endogenous Bro1 exerts its stabilization effect toward ESCRT­ III full­length Bro1 in wild­type cells suffered virtually no reduc ­ downstream of complex assembly because the addition of tion upon overexpression of the Bro1 domain. The C terminus exogenous Bro1 domain to preassembled ESCRT­III was suf­ of Bro1 stimulates Doa4 Ub hydrolase activity (Richter et al., c fi ient to protect the complex from Vps4 ­ mediated disassembly 2007), and this region is absent from the Bro1 domain (Fig. 1 A), in vitro. Direct binding to Snf7 was required for Bro1 to inhibit which would compete with endogenous Bro1 for binding to ESCRT­ III disassembly both in vivo and in vitro. The site at Snf7. Although its interaction with Snf7 comprises the primary the C terminus of Snf7 to which the Bro1 domain binds is con­ mechanism for endosomal recruitment of Bro1, a small amount served, as a similar motif was identified at the C terminus of of Bro1 is detectable at endosomes if this interaction has been human CHMP4 proteins and shown to be required for binding disabled (Fig. 1 C; Kim et al., 2005). The fact that cargo deubiq­ to the Bro1 domain of Alix (McCullough et al., 2008). The amino uitination is not significantly compromised by Bro1 domain acid sequence of this motif and its position near the C terminus overexpression suggests that the Snf7­independent mechanism of Snf7 resemble the MIM1 sites in Vps2/CHMP2 and Did2/ of Bro1 recruitment to endosomes (which has yet to be defined) CHMP1 that bind directly to the MIT domain of Vps4, but this might be sufficient to maintain Doa4 function. site in Snf7 and CHMP4 proteins lacks residues critical for the The inhibition of ESCRT­III disassembly resulting from MIT interaction (Obita et al., 2007; Stuchell­Brereton et al., Bro1 binding to Snf7 might be critical for ESCRT­III reaching 2007). Thus, it is unlikely that Bro1 stabilizes ESCRT­III by di ­ a state of assembly necessary to execute membrane scission, rectly masking a site at which Vps4 might bind Snf7. Located explaining the strong reduction in the efficiency of ILV forma ­ upstream of the MIM1­like site in Snf7 and CHMP4 proteins is tion observed upon disruption of the Bro1­binding site in Snf7. a second motif (MIM2) that binds weakly to the Vps4 MIT do­ A similar stabilizing role for Alix would be consistent with ob­ main in vitro (Kieffer et al., 2008). Although it is possible that servations that disruption of its interaction with CHMP4 pro­ the Bro1 domain interaction sterically shields Vps4 recognition teins impairs cytokinesis and reduces the efficiency of retrovirus of the MIM2 site in Snf7, testing this hypothesis has been prob­ budding (Carlton and Martin­Serrano, 2007; Fisher et al., 2007; lematic because the Snf7–Vps4 interaction is so weak that we Morita et al., 2007; Usami et al., 2007; Carlton et al., 2008). At cannot detect binding of either full­length Snf7 or a fragment endosomes, a regulatory function in promoting ESCRT­ III assem­ encompassing MIM2 either to full­length Vps4 or to the Vps4 bly would place Bro1 at the appropriate time and place to coordi­ MIT domain in isolation (unpublished data). nate deubiquitination of transmembrane protein cargoes with the Snf7 is the predominant subunit of the ESCRT­III poly ­ detachment of ILVs. However, a mechanism for coupling these mer (Teis et al., 2008) and is critical for the membrane scission seemingly separate activities of Bro1 has yet to be discovered. activity of the complex in vitro (Wollert et al., 2009). EM tomog­ raphy suggests the membrane scission activity of ESCRT­III Materials and methods at endosomes in yeast relies on Snf7 binding to the Bro1 do­ L231A/L234A main because snf7 cells, which have otherwise wild­ Yeast strains and plasmid constructions Standard protocols were used to construct all yeast strains and plasmids type ESCRT protein activity, exhibit a strong reduction in the described in Tables I and II (Longtine et al., 1998). The bacterial expres- number of ILVs and a concomitant expansion of the limiting sion plasmid encoding SNF7-HA (pGO619) was constructed in vector endosomal membrane, a morphology reminiscent of class E com­ pST39 (Tan, 2001) using a PCR product created from the wild-type SNF7 coding sequence and a reverse primer that includes one copy of the HA partments seen in snf7 cells and other ESCRT mutant strains. epitope. The L231A/L234A point mutations were created by site-directed The residual localization of full­length Bro1 to endosomes in mutagenesis of pGO619, yielding pGO621. The 2-µm plasmid encoding L231A/L234A snf7 cells might account for the few ILVs observed the Bro1 domain (pMWM3) was constructed by homologous recombination Bro1 regulation of ESCRT-III • Wemmer et al. 303 Table I. S. cerevisiae strains used in this study Name Genotype Reference SEY6210 Mata leu2-3,112 ura3-52 his3200 trp1-901 lys2-801 suc2-9 Robinson et al.,1988 MBY3 SEY6210; vps4::TRP1 Babst et al., 1997 TVY1 SEY6210; pep4::LEU2 Gerhardt et al., 1998 MWY22 SEY6210; pep4::LEU2 snf7::HIS3 This study MWY24 SEY6210; snf7::HIS3 This study MWY25 SEY6210; snf7L231A/L234A::KAN This study MWY26 SEY6210; snf7L231A/L234A-KAN vps4::TRP1 This study CRY181 SEY6210; snf7L231A/L234A-KAN vps4::TRP1 pep4::LEU2 This study EEY12 SEY6210; snf7::HIS3 vps4::TRP1 Babst et al., 2002 MBY41 SEY6210; vps20::HIS3 vps4::TRP1 Babst et al., 2002 GOY66 SEY6210; bro1::HIS3 vps4::TRP1 Odorizzi et al., 2003 GOY65 SEY6210; bro1::HIS3 Odorizzi et al., 2003 EEY2-1 SEY6210; vps20::HIS3 Babst et al., 2002 MBY52 SEY6210; vps4::TRP1 pep4::LEU2 prb1::LEU2 Katzmann et al., 2003 JPY140 SEY6210; vps4::TRP1 pep4::LEU2 snf7::HIS3 Davies et al., 2010 CRY39 SEY6210; Vps23-GFP::KAN Nickerson et al., 2010 CRY40 SEY6210; Vps23-GFP::KAN vps4::TRP1 Nickerson et al., 2010 GOY150 SEY6210; Vps36-GFP::HIS3 Nickerson et al., 2010 GOY151 SEY6210; Vps36-GFP::HIS3 vps4::TRP1 Nickerson et al., 2010 GOY74 SEY6210; Doa4-GFP::HIS3 Luhtala and Odorizzi, 2004 GOY75 SEY6210; Doa4-GFP::HIS3 vps4::TRP1 Luhtala and Odorizzi, 2004 of linearized pRS426 with a PCR product containing the Bro1 promoter at 300 kv was used for dual-tilt series images collected from 60 to 60° and Bro1 codons 1–387. with 1° increments. Tilt series were shot at 31,000× magnification with a 0.764-nm working pixel (binning 2) and repeated at a 90° rotation for dual- axis tomography. 15-nm fiducial gold was used to coat sections on both Protein interaction experiments L231A/L234A sides for reconstruction of back projections using IMOD software. Manually Lysates of bacteria expressing Snf7-HA or Snf7 -HA were pre- assigned contours of the endosomal limiting membrane at the inner leaflet pared from cultures of BL21 (DE3; Agilent Technologies) transformed with were used to measure the surface of the bilayers periodically every 3.85 nm pGO619 or pGO621; protein expression was induced at 20°C for 20 h and calculated using imodmesh. Best-fit sphere models were used to mea - after the addition of 0.5 mM isopropyl--d -thiogalactoside. Purified GST- sure the diameters of nearly spherical lumenal vesicles from the outer leaflet Bro1 domain was obtained on glutathione Sepharose resin (GE Health- of the membrane bilayers. Videos 1–6 were made from IMOD images and care) from lysates of BL21 (DE3) that had been transformed with a plasmid completed in QuickTime. Quantitation of ILV budding profiles/MVB were encoding this gene fusion (Kim et al., 2005) and induced to express re- normalized to the mean percent MVB accounted for in the tomograms to combinant protein. For in vitro pull-down experiments, glutathione Sepha- more accurately represent the number of ILV budding profiles per MVB. rose and 5 µg GST-Bro1 domain (or GST alone) were incubated for 1 h at 4°C L231/L234A with lysates prepared from bacteria expressing Snf7-HA or Snf7 -HA. Bound proteins were recovered by centrifugation of the resin, which was Isopycnic density gradient centrifugation washed thrice with ice-cold PBS + 0.5% Triton X-100, then washed twice For the resolution of native ESCRT complexes, 20 A equivalents of cells with ice-cold PBS. Bound proteins were eluted from the resin by boiling were converted to spheroplasts, resuspended in 1 ml ice-cold lysis buffer in Laemmli sample buffer, resolved by SDS-PAGE, and transferred to (200 mM sorbitol, 50 mM potassium acetate, 20 mM Hepes, pH 7.2, and nitrocellulose for Western blot analysis using anti-HA monoclonal antiserum 2 mM EDTA) supplemented with protease inhibitors, and homogenized on (Roche). 10% of the amount of GST or GST-Bro1 used in each pull-down ice. Soluble and membrane fractions were separated by spinning the ly- was resolved by SDS-PAGE and analyzed by Coomassie staining. sates at 13,000 g for 15 min at 4°C. The resulting membrane pellet was resuspended in 950 µl lysis buffer, then solubilized by the addition of 0.5% Microscopy Triton X-100 and rotation at 4°C for 30 min. Solubilized membranes were Fluorescence microscopy of cells labeled with N-[3-triethylammonium- loaded onto the top of a 10–70% sucrose gradient prepared with the Tris pro-pyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) gradient system (Biocomp Instruments) using 10% and 70% sucrose stocks and/or expressing GFP was performed as described previously (Luhtala prepared in 200 mM sorbitol, 50 mM potassium acetate, 20 mM Hepes, and Odorizzi, 2004) using a microscope (Axioplan 2; Carl Zeiss, Inc.) pH 7.2, and 2 mM EDTA. The gradient was subjected to centrifugation in equipped with an NA 1.40 oil immersion 100× objective (Carl Zeiss, Inc.) an SW41 rotor (Beckman Coulter) at 100,000 g for 16–20 h at 4°C, and at room temperature in minimal media. Differential interference contrast fractions were collected from the top. Sodium deoxycholate (0.015% final) (DIC) and fluorescence microscopy images were acquired with a digital and trichloroacetic acid (10% final) were added to each fraction, and pro - camera (Cooke Sensicam; Applied Scientific Instruments, Inc.) and pro - teins were precipitated on ice for at least 30 min, followed by centrifuga- cessed using Slidebook (Intelligent Imaging Innovations) and Photoshop tion at 13,000 g for 10 min. Insoluble material was reprecipitated twice software (version CS2; Adobe). by sonication into ice-cold acetone and centrifugation, then sonicated into For EM, yeast cells were high pressure frozen and freeze substituted Laemmli sample buffer and resolved by SDS-PAGE, transferred to nitro- as previously described (Nickerson et al., 2006; Richter et al., 2007) and cellulose, and examined by Western blotting using antisera against Snf7, embedded at 60°C in Lowicryl HM20 (Polysciences). Plastic blocks were Vps24, or GFP (Roche). Quantitation was performed on triplicate experi- trimmed and cut in 80-nm-thin sections and 250-nm-thick sections with a ments by incubating membranes with Alexa Fluor 680 secondary antibody microtome (Leica) and placed on rhodium-plated copper slot grids. Tomo- (Invitrogen) and visualizing with an infrared imager (Odyssey; LI-COR Bio- graphic samples were stained en bloc with 0.1% uranyl acetate and sciences). Bands were quantitated with Odyssey software (version 2.1). L231A/L234A 0.25% glutaraldehyde as a minimal fixative with no additional after stain - For the snf7 strain, 40 A equivalents were used because of de- ing except for snf7, which required 0.1% uranyl acetate and 2% glutar- creased detection of the mutant version of the protein. The presence of 1% aldehyde for sufficient preservation for tomography. Thick sections were Triton X-100 throughout the sucrose gradient yielded identical results to mapped on a transmission EM (Phillips CM10) at 80 kv, then a Tecnai 30 (FEI) gradients performed where Triton X-100 was omitted from the gradient. 304 JCB • VOLUME 192 • NUMBER 2 • 2011 Table II. Plasmids used in this study Plasmid Protein expressed Description 1-387 R pGO337 GFP-Bro1 1-387 GFP-Bro1 URA3 Ap (pRS416) pGO339 GFP-Bro16 GFP-Bro1 URA3 Ap (pRS41) pGO47 GFP-CPS Odorizzi et al., 1998 pCR180 GFP-Sna3 URA3 Ap (pRS415) GFP-Sna3 pGO547 Snf7 pST39 Shine-Delgarno Snf7 pGO619 Snf7-HA pST39 Shine-Delgarno Snf7-HA L231A/L234A pGO621 Snf7 -HA pST39 Shine-Delgarno Snf7 L231A/L234A-HA pGEX-4T1 GST GE Healthcare 1-387 pGO626 GST-Bro1 -HA pGEX-4T1 GST-Bro1 1-387-HA pMB54 GST-Vps4 Azmi et al., 2006 pGO216 Bro1 URA3 ApR (pRS426) Bro1 pMWM3 Bro11-387 URA3 ApR (pRS426) Bro11-387 pRS426 Snf7 Snf7 Davies et al., 2010 pRS426 Snf7Vps2MIM1 Snf7Vps2MIM1 Davies et al., 2010 In vitro ESCRT-III release assay seen within the tubular endosome. Online supplemental material is avail- The in vitro ESCRT-III release assay was performed as previously described able at http://www.jcb.org/cgi/content/full/jcb.201007018/DC1. (Davies et al., 2010). Purie fi d Vps4 and an ATP regeneration system (10 mM We thank Caleb Richter for yeast strain and plasmid constructions and Sylvie phosphocreatine, 10 U/ml creatine phosphokinase, and 1 mM ATP) were Urbé for helpful discussions. added to yeast membrane fractions containing ESCRT-III complexes (Babst This work was funded by the National Institutes of Health (grants et al.,1997; Azmi et al., 2006). Membranes were generated from shero- GM-065505 to G. Odorizzi and GM-73024 D. Katzmann). M. Wemmer plasted cells that were lysed under native conditions and homogenized, was supported by a National Institutes of Health training grant (GM-07135), then resuspended in ATPase reaction buffer (100 mM KOAc, 20 mM Hepes, I. Azmi was supported by an American Heart Association predoctoral grant pH 7.4, and 5 mM MgOAc) and passed through an 18-G needle three (AHA07-155882), and B. Davies was supported by a grant from the Fraternal times followed by a 30-G needle five times to homogenize the membranes. Order of Eagles. Reactions were allowed to incubate for 10 min at 30°C, then subjected to centrifugation at 13,000 g to separate the membrane-associated and solu- Submitted: 2 July 2010 ble ESCRT-III. SDS-PAGE followed by Western blot analyses was performed with anti-Snf7 antiserum and quantitated using ImageQuant software. Accepted: 11 December 2010 Ub-CPS immunoprecipitation Detection of Ub-CPS was performed as described previously (Katzmann References et al., 2001). The vacuolar protease gene PEP4 was deleted in all strains to reduce the nonspecific cleavage of Ub from CPS after cellular lysis. Agromayor, M., and J. Martin­Serrano. 2006. Interaction of AMSH with ESCRT­III and deubiquitination of endosomal cargo. J. Biol. Chem. 10 A equivalents of cells were harvested and precipitated by the addition 281:23083–23091. doi:10.1074/jbc.M513803200 of trichloroacetic acid (10% final concentration). 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Williams. 2007. Structural basis for selective recognition of ESCRT­ III by the AAA ATPase Vps4. Nature. 449:735–739. doi:10 .1038/nature06171 Odorizzi, G., M. Babst, and S.D. Emr. 1998. Fab1p PtdIns(3)P 5­kinase function essential for protein sorting in the multivesicular body. Cell. 95:847–858. doi:10.1016/S0092­8674(00)81707­ 9 306 JCB • VOLUME 192 • NUMBER 2 • 2011 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Pubmed Central

Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast

Wemmer, Megan; Azmi, Ishara; West, Matthew; Davies, Brian; Katzmann, David; Odorizzi, Greg
The Journal of Cell Biology , Volume 192 (2) – Jan 24, 2011
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Abstract

JCB: Article Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast 1 2 1 2 2 1 Megan Wemmer, Ishara Azmi, Matthew West, Brian Davies, David Katzmann, and Greg Odorizzi Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905 ndosomal sorting complexes required for transport vesicles at endosomes, but its role in membrane scission is (ESCRTs) promote the invagination of vesicles into unknown. We show that overexpression of Bro1 or its E the lumen of endosomes, the budding of enveloped N-terminal Bro1 domain that binds Snf7 enhances the stabil- viruses, and the separation of cells during cytokinesis. ity of ESCRT-III by inhibiting Vps4-mediated disassembly These processes share a topologically similar membrane in vivo and in vitro. This stabilization effect correlates with scission event facilitated by ESCRT-III assembly at the cyto- a reduced frequency in the detachment of intralumenal solic surface of the membrane. The Snf7 subunit of ESCRT-III vesicles as observed by electron tomography, implicating in yeast binds directly to an auxiliary protein, Bro1. Like Bro1 as a regulator of ESCRT-III disassembly and mem- ESCRT-III, Bro1 is required for the formation of intralumenal brane scission activity. Introduction The endosomal sorting complexes required for transport membranes is stimulated upon binding Vps20, which relieves (ESCRTs) are recruited to the cytosolic surface of endosomes, an autoinhibitory intramolecular interaction between the N­ and where they selectively package ubiquitinated transmembrane C­ terminal regions of Snf7 (Teis et al., 2008; Saksena et al., protein cargoes into the intralumenal vesicles (ILVs) of multi­ 2009). Membrane­ associated Snf7 polymers are thought to be vesicular bodies (MVBs). The ILVs and their cargoes are sub­ capped by Vps24, which recruits Vps2 (Babst et al., 2002; Teis sequently degraded upon fusion of MVBs with lysosomes. et al., 2008). In turn, Vps2 promotes activation of the AAA­ ESCRT­ 0, ­ I, and ­ II are stable heteromeric complexes, each ATPase Vps4, which catalyzes disassembly of ESCRT­ III and containing subunits that bind directly to ubiquitin (Ub) linkages recycling of its subunits from membranes to the cytosol (Babst on the cytosolic domains of cargoes (Piper and Katzmann, et al., 1998; Babst et al., 2002). Did2, Ist1, and Vps60 are three 2007; Raiborg and Stenmark, 2009). Recent studies indicate accessory ESCRT­ III subunits that appear to serve primarily as that ESCRT­ I and ­ II can also induce membrane invaginations regulators of Vps4 activity (Nickerson et al., 2006; Azmi et al., of nascent ILVs in vitro (Wollert and Hurley, 2010) and that 2008; Dimaano et al., 2008; Rue et al., 2008). ESCRT­ II can stimulate assembly of ESCRT­ III (Im et al., 2009; In vitro reconstitution of ESCRT­ III assembly on synthetic Saksena et al., 2009). membranes revealed that excess amounts of the yeast core The polymerization of ESCRT­ III subunits is required for ESCRT­ III subunits deform liposomes (Saksena et al., 2009) and completion of ILV budding. Genetic and biochemical studies of drive both the formation and detachment of lumenal membrane ESCRT­ III proteins from Saccharomyces cerevisiae have con­ invaginations (Wollert et al., 2009). In limiting amounts, however, tributed much to our understanding about assembly of the ESCRT­ III subunits lack the ability to deform the membrane complex, principles that are likely to be generally conserved. and, instead, their activity is restricted to facilitating the severing ESCRT­ III in yeast is comprised of four core subunits (Snf7, of membrane invaginations generated by ESCRT­ I and ­ II Vps20, Vps24, and Vps2; Babst et al., 2002), the most abundant (Wollert and Hurley, 2010). These observations suggest that of which is Snf7 (Teis et al., 2008). Polymerization of Snf7 on ESCRT­ III is responsible for execution of the membrane scission © 2011 Wemmer et al. This article is distributed under the terms of an Attribution– Correspondence to Greg Odorizzi: [email protected] Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub - Abbreviations used in this paper: DIC, differential interference contrast; ESCRT, lication date (see http://www.rupress.org/terms). After six months it is available under a endosomal sorting complex required for transport; ILV, intralumenal vesicle; MIT, Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, microtubule interacting and trafc fi king; MVB, multivesicular body; Ub, ubiquitin. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 192 No. 2 295–306 www.jcb.org/cgi/doi/10.1083/jcb.201007018 JCB 295 THE JOURNAL OF CELL BIOLOGY step during ILV formation. Consistent with these in vitro find ­ ings, mammalian ESCRT­III is required during cytokinesis for midbody abscission and for the detachment of fully assembled retroviruses from the plasma membrane (McDonald and Martin­ Serrano, 2009). Both abscission and retroviral budding require membranes to be severed in a manner that is topologically equivalent to ILV formation at MVBs, and all three processes are disabled by inhibiting Vps4 function (Garrus et al., 2001; Carlton et al., 2008). Overexpression of catalytically inactive Vps4 in mammalian cells dramatically increases the frequency of ILV budding profiles and reduces the number of freely de ­ tached ILVs at MVBs (Sachse et al., 2004). However, based on studies of reconstituted ILV budding in vitro (Wollert et al., 2009; Wollert and Hurley, 2010), Vps4 is thought to function indirectly in membrane scission by recycling ESCRT­III sub ­ units for repeated rounds of assembly. In addition to its membrane scission activity, ESCRT­III is required for coordinating the deubiquitination of trans­ membrane protein cargoes before their enclosure within ILVs. In humans, two distinct Ub hydrolases bind directly to ESCRT­ III via microtubule­ interacting and trafficking (MIT) domains (Agromayor and Martin­ Serrano, 2006; Tsang et al., 2006; Row et al., 2007), and in yeast, recruitment of the Ub hydrolase Doa4 to ESCRT­III is promoted by Bro1, which binds both to Snf7 and to the Doa4 catalytic domain (Luhtala and Odorizzi, 2004; Kim et al., 2005; Richter et al., 2007). Bro1 and its closest human or­ thologue, Alix, each have a conserved N­terminal Bro1 domain that binds directly to Snf7 and CHMP4 proteins, respectively (Kim et al., 2005; Fisher et al., 2007). Although this interaction is essential for Alix to promote retrovirus budding (Fisher et al., 2007), the means by which it contributes to ESCRT­III function in membrane scission remain unclear. In yeast, Bro1 likely has a role in ILV budding independent of its activity in regulating deubiquitination because ILV formation is strongly impaired by deletion of BRO1 but not DOA4 (Richter et al., 2007). In this study, we report that binding of Bro1 to Snf7 enhances the stability of ESCRT­III in vivo and inhibits Vps4­mediated dis ­ assembly of the complex in vitro. This effect correlates with a reduced efficiency in detachment of ILVs from the limiting endosomal membrane. Conversely, mutation of the Bro1­ binding site in Snf7 strongly inhibits vesicle formation and cargo sort­ ing. These results implicate Bro1 as a regulator of ESCRT­III disassembly and membrane scission activity. Results The Bro1 domain binds a MIM1-like motif in the Snf7 subunit of ESCRT-III Yeast two­hybrid analysis (unpublished data) was used to map the amino acids in Snf7 required for its interaction with the Figure 1. The Bro1 domain binds a MIM1-like motif in Snf7. (A) Schematic Bro1 domain of yeast Bro1 (Fig. 1 A). This site is centered diagrams of Bro1 and Snf7, including the amino acid sequence align- ment of MIM1-like motifs from yeast Snf7 and human CHMP4a. (B) In vitro around residues Leu­231 and Leu­234 at the C terminus of pull-downs of purie fi d GST or GST-Bro1 domain incubated with bacterial Snf7, which resembles the site in CHMP4 proteins that binds L231A/L234A lysates expressing Snf7-HA or Snf7 -HA. GST and GST-Bro1 the Bro1 domain of Alix (McCullough et al., 2008) and is simi­ domain inputs were loaded at 10% of their total used in each pull-down. IB, immunoblotting. (C) Fluorescence and DIC microscopy of vps4, vps4 lar to the MIM1 motif that was identified in Vps2/CHMP2 and L231/234A snf7, and vps4 snf7 cells expressing GFP-Bro1 domain or GFP- Did2/CHMP1 proteins (Obita et al., 2007; Stuchell­Brereton Bro1. Arrowheads indicate where GFP does (closed) or does not (open) et al., 2007). The MIT domain of Vps4 has relatively strong colocalize with FM4-64–labeled class E compartments. Bars, 2 µm. 296 JCB • VOLUME 192 • NUMBER 2 • 2011 afn fi ity for MIM1 of Vps2/CHMP2 and Did2/CHMP1 but binds poorly to the MIM1­ like site in CHMP4 proteins (Kieffer et al., 2008), which is consistent with the lack of in vitro binding between purified Snf7 and Vps4 (Shestakova et al., 2010; un ­ published data). Leu­ 231 and Leu­ 234 in the MIM1­ like site of Snf7 were essential for its in vitro binding to the Bro1 domain of yeast Bro1 (Fig. 1 B). In vivo, these residues were also required for Snf7 to mediate endosomal localization of the Bro1 domain fused to GFP (Fig. 1 C). However, full­ length Bro1­ GFP weakly localized to endosomes despite disruption of the Bro1 domain– L231A/L234A binding site in Snf7 (snf7 ; Fig. 1 C), which matches our previous observation that full­ length Bro1 weakly localizes to endosomes despite mutation of the Snf7 interaction surface in the Bro1 domain, whereas the same mutation completely dis­ ables endosomal localization of the Bro1 domain alone (Kim et al., 2005). Bro1 binding to Snf7 enhances the stability of ESCRT-III polymers in vivo The position of the Bro1 domain–binding site at the C terminus of Snf7 (Fig. 1) suggested that Bro1 might act like Vps20 to re­ lieve Snf7 autoinhibition (Teis et al., 2008; Saksena et al., 2009), thereby promoting Snf7 polymerization and incorporation into ESCRT­ III. However, it was previously shown that Bro1 is not essential for Snf7 polymerization based on the analysis of cells Figure 2. Overexpression of Bro1 or the Bro1 domain stabilizes ESCRT-III in which ESCRT­ III disassembly by the Vps4 ATPase had been polymers at endosomes. (A) Western blot analysis of detergent-solubilized disabled by deletion of the VPS4 gene (Teis et al. 2008). There­ yeast membranes resolved by isopycnic density gradient centrifugation. Inclusion of 1% Triton X-100 throughout the gradients yielded identical fore, we investigated whether Bro1 might inu fl ence Snf7 poly­ sedimentation profiles. (B) Quantitation of triplicate gradients from A. merization in the context of functional Vps4 by examining the distribution of Snf7 in detergent­ solubilized yeast membranes L231A/L234A separated by isopycnic density gradient centrifugation. In ex­ mutant snf7 that could be detected as a polymer, and tracts from wild­ type cells, the majority of Snf7 was seen in low wild­ type Snf7 was similarly unaffected by overexpression of a density fractions, but a small amount (19%) was detected in mutant Bro1 domain that lacks the Snf7­ binding site (Fig. S1 B), higher density fractions (Fig. 2, A and B). This high density pool indicating that the effect of Bro1 domain overexpression toward of Snf7 likely corresponds to the transient ESCRT­ III complexes stabilizing ESCRT­ III depends on its ability to bind Snf7. Bro1 that were reported to exist at steady­ state (Teis et al., 2008) domain overexpression caused a similar shift toward high because the same gradient fractions contained 90% of Snf7 in density gradient fractions for another core ESCRT­ III subunit, extracts from vps4 cells (Fig. 2, A and B) in which ESCRT­ III Vps24 (Fig. S2), although concomitant shifts in ESCRT­ I or is irreversibly trapped as an assembled complex (Babst et al., ESCRT­ II were not observed (not depicted). However, the total 1998; Teis et al., 2008). The polymeric Snf7 we observed had amounts of membrane­ associated Snf7 and Vps24 were not sig­ the same properties that were reported previously (Teis et al., nic fi antly altered by overexpression of the Bro1 domain ( Fig. S3), 2008) because its stabilization upon deletion of VPS4 was indicating that the membrane recruitment step preceding largely dependent on Vps20 but not Bro1 (Fig. 2, A and B). ESCRT­ III assembly was not enhanced. Moreover, we did not Moreover, recombinant Snf7 that was expressed in bacteria and detect an increase in the amount of Vps4 in high density gradi­ isolated as a soluble polymer by size­ exclusion chromatography ent fractions upon overexpression of the Bro1 domain (unpub­ before being subjected to isopycnic density gradient centrifuga­ lished data), indicating that binding of ESCRT­ III to Vps4 was tion also migrated to high density fractions (Fig. S1 A). not dramatically increased under these conditions. Together, Although a relatively minor percentage of membrane­ these observations suggest that driving the interaction between associated Snf7 was polymeric in extracts from wild­ type cells Bro1 and Snf7 by overexpression of Bro1 or the Bro1 domain (19%), the percentage of polymeric Snf7 increased upon over­ enhances the stability of ESCRT­ III assembled at endosomal expression of either full­ length Bro1 or the Bro1 domain (29% membranes despite Vps4 being present. and 27%, respectively; Fig. 2, A and B). This effect required that Snf7 first undergo activation by Vps20 because overexpres ­ Bro1 binding to Snf7 inhibits Vps4- sion of the Bro1 domain in vps20 cells failed to enhance mediated disassembly of ESCRT-III in vitro detection of polymeric Snf7 (Fig. 2, A and B). Bro1 domain The stabilizing effect exerted by Bro1 toward ESCRT­ III was overexpression also did not cause an increase in the amount of examined further using an in vitro system that reconstitutes Bro1 regulation of ESCRT-III • Wemmer et al. 297 Vps4­ mediated disassembly of the complex. In this assay, abnormal endosomal structures known as class E compart­ membrane­ associated ESCRT­ III generated from extracts of vps4 ments, which consist of stacks of aberrantly attened fl endo ­ cells were incubated with 1 mM ATP and 10 nM purified Vps4 somes that generally lack ILVs (Fig. 4 B and Video 2), which is for 10 min at 30°C, and the extent of ESCRT­III disassembly consistent with ESCRT­ III promoting membrane scission. L231A/L234A was determined by probing the amount of Snf7 that remains Expression of the snf7 allele in place of wild­ type membrane associated after centrifugation. As shown previously SNF7 did not result in class E compartments like those seen in (Davies et al., 2010), this reaction condition resulted in a 50% snf7 cells. Nevertheless, this mutation caused a sharp reduc­ reduction in membrane­associated Snf7 ( Fig. 3 A), indicating tion in the number of ILVs and a gross distortion of endosomes that exogenous Vps4 has potent activity in mediating in vitro in a manner reminiscent of the class E compartment morphol­ disassembly of ESCRT­III that had been preassembled in vivo. ogy (Fig. 4 C, Fig. 5 C [quantic fi ation], and Video 3). This reduc­ However, this activity was inhibited by 50% if ESCRT­III was tion in the amount of ILVs is consistent with the interpretation generated from vps4 cells overexpressing the Bro1 domain and, that Bro1 binding to Snf7 is critical for proper ESCRT­III func ­ to a lesser degree, if the source of ESCRT­ III was vps4 cells over­ tion in vivo. expressing full­length Bro1 (Fig. 3 A). Unexpectedly, we discovered that Bro1 domain over­ The inhibitory effect of Bro1 on in vitro disassembly of expression in wild­type cells increased the frequency of intra­ ESCRT­III (Fig. 3 A) was similar to the in vivo results seen in lumenal invaginations that had not detached from the limiting Fig. 2, wherein the stability of membrane­associated ESCRT ­III endosomal membrane (Fig. 4, D and E; and Videos 4 and 5). was enhanced upon overexpression of Bro1 or the Bro1 domain. Such ILV budding profiles are not common, with a mean of 1.2 Although these observations support a model in which Bro1 observed per MVB in wild­type cells, but 2.4 budding profiles binding to Snf7 interferes with Vps4­mediated disassembly of per MVB on average were seen upon overexpression of the ESCRT­III, an alternative possibility was that excess Bro1 or Bro1 domain (Fig. 5 A). Plotting the frequency distribution Bro1 domain compromises the ESCRT­III assembly process of ILV budding profiles (Fig. 5 B) showed that overexpression and results in aberrant complexes refractory to Vps4. However, of the Bro1 domain resulted in more instances of multiple the addition of purified Bro1 domain (fused to GST) effectively budding profiles at individual MVBs (five budding profiles at negated in vitro Vps4­mediated disassembly of preassembled the MVB shown in Fig. 4 E; Video 5). Overexpression of full­ ESCRT­III generated from vps4 cells (Fig. 3 B). Moreover, length Bro1 had the same effect as the Bro1 domain (Fig. 4 F; this inhibitory effect was observed even if Vps4 was allowed to Fig. 5, A and B; and Video 6), demonstrating that the increase in initiate disassembly of ESCRT­III for 5 min before the addition ILV budding profiles was not simply caused by the absence of of GST­Bro1 domain (Fig. 3 C). Thus, Bro1 inhibition of Vps4­ an activity normally provided by the C­terminal region of Bro1. mediated disassembly of ESCRT­III is manifested downstream A gallery of tomograms highlighting ILV budding profiles of complex assembly. versus detached ILVs at individual MVBs in wild­type cells We also tested whether the inhibitory effect of the Bro1 with and without overexpression of Bro1 or the Bro1 domain is domain relied exclusively on its ability to bind Snf7 by generat­ shown in Fig. 4 (G–L). Vps2MIM1 ing ESCRT­III from vps4 cells in which a Snf7 chi­ The low frequency of ILV budding profiles in wild­type mera was expressed in place of wild­type Snf7. In this chimera, yeast presumably reflects the rapid rate at which membrane the MIM1­like Bro1 domain–binding site is replaced with the scission normally occurs. This process is most likely delayed bona fide MIM1 from Vps2 that binds the MIT domain of Vps4 rather than accelerated in response to Bro1 or Bro1 domain Vps2MIM1 (Obita et al., 2007). The Snf7 chimera rescues in vitro overexpression because quantitation of membrane surface areas Vps4­mediated disassembly of ESCRT ­III formed in the ab ­ showed a decrease in the lumenal membrane content and a cor­ sence of Vps2 expression, demonstrating that it effectively responding increase in the amount of limiting membrane (Fig. 5 C), serves as a substrate for Vps4 (Davies et al., 2010). Similarly, although the mean vesicle diameter did not change (Fig. 5 D). Vps2MIM1 ESCRT­III assembled with Snf7 in place of wild­type In contrast, an acceleration of ILV formation would be expected Snf7 was susceptible to disassembly by Vps4 even upon addi­ to show an increase in the amount of lumenal membrane con­ tion of purified Bro1 domain (Fig. 3 D). We conclude that direct tent within endosomes, as is observed in yeast lacking Ist1 binding of the Bro1 domain to Snf7 inhibits Vps4­mediated dis ­ (Nickerson et al., 2010), an accessory ESCRT­III protein that assembly of ESCRT­III. inhibits Vps4 activity (Dimaano et al., 2008). Compared with wild­type cells, the ratio of lumenal to limiting membrane sur ­ Overexpression of Bro1 or the Bro1 face area was lower upon mutation of the Bro1 domain–binding L231A/L234A domain reduces ILV budding efficiency site in Snf7 (snf7 ; 23.3% of total endosomal membrane at MVBs surface area was lumenal; Fig. 5 C), whereas snf7 cells were To evaluate whether binding of the Bro1 domain to Snf7 almost exclusively devoid of lumenal membranes (0.27% of the influences the membrane scission activity of ESCRT ­ III, we total endosomal membrane surface area; Fig. 5 C), which is examined the morphology of yeast endosomes by EM and 3D consistent with the stronger effects on endosomal morphology tomography. Deletion of SNF7 or genes encoding the other core caused by these mutations (Fig. 4). ESCRT­III subunits inhibits the biogenesis of MVBs like those Measurements of tomograms further revealed that the seen in wild­type cells ( Fig. 4 A and Video 1). Instead, snf7 amount of membrane incorporated into ILV budding profiles cells (and other ESCRT­III deletion strains; not depicted) have in wild­ type cells accounted for only 6.3% of the limiting 298 JCB • VOLUME 192 • NUMBER 2 • 2011 Figure 3. Bro1 binding to Snf7 inhibits in vitro disassembly of ESCRT-III. (A) 0.1 µM purified Vps4, ATP, and an ATP regeneration system were incubated with membranes isolated from vps4 yeast cells; overexpression of full-length Bro1 or the Bro1 domain (BOD) is indicated. The amount of membrane- associated Snf7 remaining after incubation at 30°C for 10 min was determined by quantitative Western blot analysis. P < 0.001 for both samples. (B) In vitro ESCRT-III release performed in the presence or absence of ATP, 0.1 µM Vps4, and 0.1 µM GST-Bro1 domain (GST-BOD). (C) In vitro ESCRT-III release performed as in B except that the reaction was allowed to proceed for 5 min in the absence of GST-BOD or for 20 min in the absence or presence of 1 µM GST-BOD that was added either at the same time as purified Vps4 or 5 min after the addition of Vps4. (D) In vitro ESCRT-III release performed Vps2MIM1 with membranes from yeast expressing wild-type Snf7 or the Snf7 chimera in the presence or absence of ATP, 0.1 µM Vps4, and 0.1 µM GST-BOD. Error bars show the standard deviation. endosomal membrane surface area. In contrast, overexpression observed under these conditions (Fig. 5 A). Despite the increase of Bro1 or the Bro1 domain resulted in the incorporation of in the number of ILV budding profiles, their overall morphol ­ 18.6% and 17.5%, respectively (Fig. 5 E), which is propor­ ogy was not altered significantly, as neither the diameter of tional to the increased frequency of ILV budding profiles the vesicles attached to the limiting endosomal membrane Bro1 regulation of ESCRT-III • Wemmer et al. 299 Figure 4. Endosome morphologies in cells overexpressing Bro1 or the Bro1 domain. (A–F) 2D cross sections and 3D models from 200-nm-thick section electron tomograms. Spherical endosomal limiting membranes are depicted in yellow, whereas flattened class E compartments are depicted in various colors to discriminate discrete membranes. Lumenal vesicles are red. V, vacuole. Bars, 100 nm or 200 nm as noted. (G–L) Gallery of tomographic slices of wild-type cells with and without overexpression of Bro1 or the Bro1 domain. Freely detached ILVs are traced in red, whereas ILV budding profiles and the limiting endosomal membrane are traced in yellow. In some cases, the continuity of ILV budding profiles with the limiting endosomal membrane is out of plane in the tomographic slice but evident in the 3D reconstruction. Bars, 50 nm. 300 JCB • VOLUME 192 • NUMBER 2 • 2011 (Fig. S4 A) nor the diameter of the membrane neck at ILV budding profiles (Fig. S4 B) were statistically different in wild­ type cells with or without overexpression of Bro1 or the Bro1 domain. Cargo sorting and deubiquitination are not affected by Bro1 or Bro1 domain overexpression in wild-type cells Transmembrane proteins sorted into the ILVs of MVBs are ultimately delivered to the vacuole lumen upon MVB–vacuole fusion, but disrupting the activity of any of the ESCRTs results in mislocalization of these cargoes to the vacuole membrane and their accumulation at class E compartments (Odorizzi et al., 1998). Therefore, we evaluated how the effects on ILV budding revealed by EM tomography (Fig. 4) impacted the efficiency of cargo sorting using live cell fluorescence microscopy of yeast expressing GFP fused to the cytoplasmic domain of carboxypeptidase S (GFP­ CPS). We previously reported mislocalization of GFP­ CPS upon mutation of the Snf7­ binding site in the Bro1 domain (Kim et al., 2005). Simi­ larly, mislocalization of GFP­ CPS occurred both in snf7 cells L231A/L234A and in snf7 cells lacking the Bro1­ binding site within Snf7 (Fig. 6 A), which was consistent with the strong reduc­ tion in ILV formation caused by these mutations (Fig. 5). Deliv­ ery of GFP­ CPS into the vacuole lumen in wild­ type cells was only mildly affected upon overexpression of the Bro1 domain (Fig. 6 A), whereas full­ length Bro1 overexpression did not appear to cause significant cargo mislocalization (Luhtala and Odorizzi, 2004). Doa4 is the Ub hydrolase that deubiquitinates CPS and other MVB vesicle cargoes (Dupré and Haguenauer­Tsapis, 2001; Katzmann et al., 2001; Losko et al., 2001), and its activity is stimulated upon binding to Bro1 (Richter et al., 2007). This process occurs efficiently in wild­type cells, resulting in detection of very little ubiquitinated CPS (Ub­CPS; Fig. 6 B). In contrast, Ub­CPS accumulated upon deletion of SNF7 or L231A/L234A upon mutation of its Bro1 domain–binding site (snf7 ; Fig. 6 B), demonstrating that its interaction with Snf7 is im­ portant for Bro1 to promote Doa4 activity at endosomes. Con­ versely, overexpression of Bro1 or the Bro1 domain had no observable effects on CPS deubiquitination (Fig. 6 B), indicat­ ing that the enhancement of ESCRT­III stability and reduced efficiency in membrane scission is not a consequence of altered Doa4 function. of a class E compartment in a single tomogram of snf7 cells (Fig. S5); the rarity of this occurrence is reflected by the observation that the membrane surface area of these ILVs comprised 0.27% of the total endosomal mem- brane surface area in this strain. Tomograms for 12, 14, and 17 MVBs were prepared and modeled for wild-type cells, cells overexpressing the Figure 5. Quantitation of endosome morphology and vesicle budding Bro1 domain, and cells overexpressing full-length Bro1, respectively. Four L231A/L234A profiles. Quantitation of the mean number of ILV budding profiles observed individual tomograms were prepared and modeled for snf7 cells, per MVB (A) versus the frequency distribution of the number of ILV budding which contained a total of 33 MVB, VTE, and cisternal structures. Three profiles observed per MVB (B) observed in tomograms. Quantitation of the individual tomograms were generated and modeled for snf7 cells, each relative percentage of limiting versus lumenal membrane surface areas (C) containing E compartments that were composed of multiple cisternal stacks and the mean ILV diameters (D) observed in tomograms. (E) The percent- and occasionally also spherical endosomal membranes. Methods used for age of the limiting membrane incorporated into ILV budding profiles was the preparation of the tomograms that were used to generate the measure- measured for each strain. Note that no ILV budding profiles were seen for ments shown in this experiment are described in detail in the Materials and snf7 cells, but five freely detached ILVs were observed at the periphery methods section. Bro1 regulation of ESCRT-III • Wemmer et al. 301 S. cerevisiae has been elusive because of the formation of aber­ rant class E compartments rather than MVBs in mutant yeast that generally lack functional ESCRT complexes (Rieder et al., 1996; Odorizzi et al., 1998; Luhtala and Odorizzi, 2004; Nickerson et al., 2006). In this study, we describe a potential role for Bro1 in regulating ESCRT­ III by demonstrating that overexpression of either full­ length Bro1 or its N­ terminal Bro1 domain stabilizes ESCRT­ III polymers and reduces the efcienc fi y of ILV detachment from the limiting endosomal membrane in yeast that contain an otherwise wild­ type ESCRT machinery. ILV budding profiles in yeast are infrequently observed, even in thick sections imaged by 3D EM tomography, presum­ ably because of the rapid rate at which membrane deformation and scission occur. The increased frequency of budding profiles caused by overexpression of Bro1 or the Bro1 domain must be interpreted as a reduction in ILV budding efficiency rather than an acceleration because these conditions also resulted in signifi ­ cantly less lumenal membrane content within MVBs. In the ab­ sence of monitoring this process in real time, it is impossible to exclude whether at least some of the ILV budding profiles are dead­ end structures that fail to complete membrane scission. However, the ILVs seen in cells overexpressing Bro1 or the Bro1 domain appeared structurally normal. Neither their overall mean diameter nor the dimensions at the necks of the budding profiles were significantly different from the same measure ­ ments in wild­ type cells, suggesting that these ILVs will eventu­ ally detach from the limiting endosomal membrane. The specic fi mechanism underlying the reduced efc fi iency of ILV budding resulting from overexpression of Bro1 or the Bro1 domain is unclear, but a clue comes from our n fi ding that this con ­ dition correlates with increased detection of Snf7 and Vps24, both core ESCRT­ III subunits, in a high molecular mass polymer de­ spite wild­ type Vps4 expression. Vps4 is the AAA­ ATPase that catalyzes disassembly of ESCRT­ III (Babst et al., 1998), which accounts for very little of the assembled complex existing at steady­ state (Teis et al., 2008). Based on in vitro reconstitution of ILV budding, Vps4 is thought to function indirectly in membrane scission by recycling ESCRT­ III subunits for repeated rounds of assembly (Wollert et al., 2009; Wollert and Hurley, 2010). Given the intermediate degree to which Snf7 polymers are stabilized upon Bro1 or Bro1 domain overexpression (only 30% of the Figure 6. Cargo sorting and deubiquitination. (A) Fluorescence and DIC total amount of Snf7 that is trapped as a polymer in vps4 cells), microscopy of cells expressing GFP-CPS. Bars, 2 µm. (B) Western blot it seems unlikely that a dec fi iency of free ESCRT ­ III subunits exists analysis of anti-CPS immunoprecipitates. IB, immunoblotting. for complex assembly and subsequent execution of membrane scission. However, we presently cannot determine whether Bro1­ Discussion mediated stabilization of ESCRT­ III is the direct cause of the re­ In vitro studies have pointed to the assembly of ESCRT­ III duced efc fi iency in ILV budding, which would suggest an active being crucial to the membrane scission event that results in de­ role for Vps4 during membrane scission in vivo. Nevertheless, the tachment of ILVs (Wollert et al., 2009; Wollert and Hurley, possibility that membrane scission might be coupled to Vps4 ac­ 2010). Although the specific mechanism by which ESCRT ­ III tivity is consistent with our previous discovery by EM tomogra­ executes this activity is unclear, its role in severing membrane phy that MVBs have signic fi antly more ILVs in the absence of connections is supported by in vivo studies of mammalian cells, Ist1 (Nickerson et al., 2010), an accessory ESCRT­ III protein that showing a requirement for ESCRT­ III in cytokinesis and negatively regulates Vps4 by inhibiting its binding to ESCRT­ III retroviral budding, both of which are membrane scission events (Dimaano et al., 2008). An active role for Vps4 in membrane scis­ that are topologically equivalent to ILV formation at MVBs sion might also explain why overexpression of catalytically in­ (McDonald and Martin­ Serrano, 2009). Genetic dissection of active Vps4 in mammalian cells results in an increased frequency the mechanism of action for ESCRT­ III at endosomes in of ILV budding prol fi es at endosomes (Sachse et al., 2004). 302 JCB • VOLUME 192 • NUMBER 2 • 2011 The membrane scission activity of ESCRT­III in vitro is because complete removal of Bro1 function by deletion of the potently stimulated by ESCRT­II (Im et al., 2009), which is BRO1 gene results in class E compartments rather than MVBs thought to trigger ESCRT­III polymerization through its direct (Luhtala and Odorizzi, 2004; Richter et al., 2007). Bro1 stimu­ binding to Vps20 (Saksena et al., 2009). Unlike ESCRT­III, lates transmembrane protein cargo deubiquitination at endo­ however, ESCRT­II is not required for the budding of human somes by promoting both recruitment and activation of the immunodeficiency virus­1 (HIV ­1; Langelier et al., 2006), rais ­ Doa4 Ub hydrolase (Luhtala and Odorizzi, 2004; Richter et al., ing the question of how ESCRT­III assembly is stimulated at the 2007). However, this activity alone cannot account for the im­ site of viral budding. The p6 domain of the Gag polyprotein of portance of the Bro1–Snf7 interaction during membrane scis­ HIV­1 binds both to Alix (Strack et al., 2003) and to TSG101, a sion because mutations that specifically disable Bro1 from subunit of ESCRT­I (Garrus et al., 2001; Martin­Serrano et al., stimulating Doa4 do not impair ILV formation, nor does re­ 2001; VerPlank et al., 2001). The yeast orthologue of TSG101 placement of wild­ type DOA4 with nonfunctional alleles (Richter (Vps23) also binds Vps20 (Bowers et al., 2004; Teo et al., 2004), et al., 2007). L231A/L234A but an interaction between TSG101 and ESCRT­III has not been The strong reduction in ILV formation seen in snf7 reported. Binding of Alix alone to p6 is sufficient to promote cells is consistent with the failure in delivery of CPS to the HIV­1 budding (Fisher et al., 2007; Usami et al., 2007), but vacuole lumen via the MVB pathway. Given the central role whether this interaction facilitates ESCRT­III assembly is un ­ Bro1 has in regulating deubiquitination at endosomes, it is not known. Our results suggest that Bro1 in yeast lacks this activity surprising that disruption of the Bro1 domain–binding site in because its ability to stabilize ESCRT­III when overexpressed Snf7 caused a strong accumulation of Ub­CPS. However, the was dependent on the presence of Vps20. efficiency of cargo deubiquitination controlled by endogenous Bro1 exerts its stabilization effect toward ESCRT­ III full­length Bro1 in wild­type cells suffered virtually no reduc ­ downstream of complex assembly because the addition of tion upon overexpression of the Bro1 domain. The C terminus exogenous Bro1 domain to preassembled ESCRT­III was suf­ of Bro1 stimulates Doa4 Ub hydrolase activity (Richter et al., c fi ient to protect the complex from Vps4 ­ mediated disassembly 2007), and this region is absent from the Bro1 domain (Fig. 1 A), in vitro. Direct binding to Snf7 was required for Bro1 to inhibit which would compete with endogenous Bro1 for binding to ESCRT­ III disassembly both in vivo and in vitro. The site at Snf7. Although its interaction with Snf7 comprises the primary the C terminus of Snf7 to which the Bro1 domain binds is con­ mechanism for endosomal recruitment of Bro1, a small amount served, as a similar motif was identified at the C terminus of of Bro1 is detectable at endosomes if this interaction has been human CHMP4 proteins and shown to be required for binding disabled (Fig. 1 C; Kim et al., 2005). The fact that cargo deubiq­ to the Bro1 domain of Alix (McCullough et al., 2008). The amino uitination is not significantly compromised by Bro1 domain acid sequence of this motif and its position near the C terminus overexpression suggests that the Snf7­independent mechanism of Snf7 resemble the MIM1 sites in Vps2/CHMP2 and Did2/ of Bro1 recruitment to endosomes (which has yet to be defined) CHMP1 that bind directly to the MIT domain of Vps4, but this might be sufficient to maintain Doa4 function. site in Snf7 and CHMP4 proteins lacks residues critical for the The inhibition of ESCRT­III disassembly resulting from MIT interaction (Obita et al., 2007; Stuchell­Brereton et al., Bro1 binding to Snf7 might be critical for ESCRT­III reaching 2007). Thus, it is unlikely that Bro1 stabilizes ESCRT­III by di ­ a state of assembly necessary to execute membrane scission, rectly masking a site at which Vps4 might bind Snf7. Located explaining the strong reduction in the efficiency of ILV forma ­ upstream of the MIM1­like site in Snf7 and CHMP4 proteins is tion observed upon disruption of the Bro1­binding site in Snf7. a second motif (MIM2) that binds weakly to the Vps4 MIT do­ A similar stabilizing role for Alix would be consistent with ob­ main in vitro (Kieffer et al., 2008). Although it is possible that servations that disruption of its interaction with CHMP4 pro­ the Bro1 domain interaction sterically shields Vps4 recognition teins impairs cytokinesis and reduces the efficiency of retrovirus of the MIM2 site in Snf7, testing this hypothesis has been prob­ budding (Carlton and Martin­Serrano, 2007; Fisher et al., 2007; lematic because the Snf7–Vps4 interaction is so weak that we Morita et al., 2007; Usami et al., 2007; Carlton et al., 2008). At cannot detect binding of either full­length Snf7 or a fragment endosomes, a regulatory function in promoting ESCRT­ III assem­ encompassing MIM2 either to full­length Vps4 or to the Vps4 bly would place Bro1 at the appropriate time and place to coordi­ MIT domain in isolation (unpublished data). nate deubiquitination of transmembrane protein cargoes with the Snf7 is the predominant subunit of the ESCRT­III poly ­ detachment of ILVs. However, a mechanism for coupling these mer (Teis et al., 2008) and is critical for the membrane scission seemingly separate activities of Bro1 has yet to be discovered. activity of the complex in vitro (Wollert et al., 2009). EM tomog­ raphy suggests the membrane scission activity of ESCRT­III Materials and methods at endosomes in yeast relies on Snf7 binding to the Bro1 do­ L231A/L234A main because snf7 cells, which have otherwise wild­ Yeast strains and plasmid constructions Standard protocols were used to construct all yeast strains and plasmids type ESCRT protein activity, exhibit a strong reduction in the described in Tables I and II (Longtine et al., 1998). The bacterial expres- number of ILVs and a concomitant expansion of the limiting sion plasmid encoding SNF7-HA (pGO619) was constructed in vector endosomal membrane, a morphology reminiscent of class E com­ pST39 (Tan, 2001) using a PCR product created from the wild-type SNF7 coding sequence and a reverse primer that includes one copy of the HA partments seen in snf7 cells and other ESCRT mutant strains. epitope. The L231A/L234A point mutations were created by site-directed The residual localization of full­length Bro1 to endosomes in mutagenesis of pGO619, yielding pGO621. The 2-µm plasmid encoding L231A/L234A snf7 cells might account for the few ILVs observed the Bro1 domain (pMWM3) was constructed by homologous recombination Bro1 regulation of ESCRT-III • Wemmer et al. 303 Table I. S. cerevisiae strains used in this study Name Genotype Reference SEY6210 Mata leu2-3,112 ura3-52 his3200 trp1-901 lys2-801 suc2-9 Robinson et al.,1988 MBY3 SEY6210; vps4::TRP1 Babst et al., 1997 TVY1 SEY6210; pep4::LEU2 Gerhardt et al., 1998 MWY22 SEY6210; pep4::LEU2 snf7::HIS3 This study MWY24 SEY6210; snf7::HIS3 This study MWY25 SEY6210; snf7L231A/L234A::KAN This study MWY26 SEY6210; snf7L231A/L234A-KAN vps4::TRP1 This study CRY181 SEY6210; snf7L231A/L234A-KAN vps4::TRP1 pep4::LEU2 This study EEY12 SEY6210; snf7::HIS3 vps4::TRP1 Babst et al., 2002 MBY41 SEY6210; vps20::HIS3 vps4::TRP1 Babst et al., 2002 GOY66 SEY6210; bro1::HIS3 vps4::TRP1 Odorizzi et al., 2003 GOY65 SEY6210; bro1::HIS3 Odorizzi et al., 2003 EEY2-1 SEY6210; vps20::HIS3 Babst et al., 2002 MBY52 SEY6210; vps4::TRP1 pep4::LEU2 prb1::LEU2 Katzmann et al., 2003 JPY140 SEY6210; vps4::TRP1 pep4::LEU2 snf7::HIS3 Davies et al., 2010 CRY39 SEY6210; Vps23-GFP::KAN Nickerson et al., 2010 CRY40 SEY6210; Vps23-GFP::KAN vps4::TRP1 Nickerson et al., 2010 GOY150 SEY6210; Vps36-GFP::HIS3 Nickerson et al., 2010 GOY151 SEY6210; Vps36-GFP::HIS3 vps4::TRP1 Nickerson et al., 2010 GOY74 SEY6210; Doa4-GFP::HIS3 Luhtala and Odorizzi, 2004 GOY75 SEY6210; Doa4-GFP::HIS3 vps4::TRP1 Luhtala and Odorizzi, 2004 of linearized pRS426 with a PCR product containing the Bro1 promoter at 300 kv was used for dual-tilt series images collected from 60 to 60° and Bro1 codons 1–387. with 1° increments. Tilt series were shot at 31,000× magnification with a 0.764-nm working pixel (binning 2) and repeated at a 90° rotation for dual- axis tomography. 15-nm fiducial gold was used to coat sections on both Protein interaction experiments L231A/L234A sides for reconstruction of back projections using IMOD software. Manually Lysates of bacteria expressing Snf7-HA or Snf7 -HA were pre- assigned contours of the endosomal limiting membrane at the inner leaflet pared from cultures of BL21 (DE3; Agilent Technologies) transformed with were used to measure the surface of the bilayers periodically every 3.85 nm pGO619 or pGO621; protein expression was induced at 20°C for 20 h and calculated using imodmesh. Best-fit sphere models were used to mea - after the addition of 0.5 mM isopropyl--d -thiogalactoside. Purified GST- sure the diameters of nearly spherical lumenal vesicles from the outer leaflet Bro1 domain was obtained on glutathione Sepharose resin (GE Health- of the membrane bilayers. Videos 1–6 were made from IMOD images and care) from lysates of BL21 (DE3) that had been transformed with a plasmid completed in QuickTime. Quantitation of ILV budding profiles/MVB were encoding this gene fusion (Kim et al., 2005) and induced to express re- normalized to the mean percent MVB accounted for in the tomograms to combinant protein. For in vitro pull-down experiments, glutathione Sepha- more accurately represent the number of ILV budding profiles per MVB. rose and 5 µg GST-Bro1 domain (or GST alone) were incubated for 1 h at 4°C L231/L234A with lysates prepared from bacteria expressing Snf7-HA or Snf7 -HA. Bound proteins were recovered by centrifugation of the resin, which was Isopycnic density gradient centrifugation washed thrice with ice-cold PBS + 0.5% Triton X-100, then washed twice For the resolution of native ESCRT complexes, 20 A equivalents of cells with ice-cold PBS. Bound proteins were eluted from the resin by boiling were converted to spheroplasts, resuspended in 1 ml ice-cold lysis buffer in Laemmli sample buffer, resolved by SDS-PAGE, and transferred to (200 mM sorbitol, 50 mM potassium acetate, 20 mM Hepes, pH 7.2, and nitrocellulose for Western blot analysis using anti-HA monoclonal antiserum 2 mM EDTA) supplemented with protease inhibitors, and homogenized on (Roche). 10% of the amount of GST or GST-Bro1 used in each pull-down ice. Soluble and membrane fractions were separated by spinning the ly- was resolved by SDS-PAGE and analyzed by Coomassie staining. sates at 13,000 g for 15 min at 4°C. The resulting membrane pellet was resuspended in 950 µl lysis buffer, then solubilized by the addition of 0.5% Microscopy Triton X-100 and rotation at 4°C for 30 min. Solubilized membranes were Fluorescence microscopy of cells labeled with N-[3-triethylammonium- loaded onto the top of a 10–70% sucrose gradient prepared with the Tris pro-pyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) gradient system (Biocomp Instruments) using 10% and 70% sucrose stocks and/or expressing GFP was performed as described previously (Luhtala prepared in 200 mM sorbitol, 50 mM potassium acetate, 20 mM Hepes, and Odorizzi, 2004) using a microscope (Axioplan 2; Carl Zeiss, Inc.) pH 7.2, and 2 mM EDTA. The gradient was subjected to centrifugation in equipped with an NA 1.40 oil immersion 100× objective (Carl Zeiss, Inc.) an SW41 rotor (Beckman Coulter) at 100,000 g for 16–20 h at 4°C, and at room temperature in minimal media. Differential interference contrast fractions were collected from the top. Sodium deoxycholate (0.015% final) (DIC) and fluorescence microscopy images were acquired with a digital and trichloroacetic acid (10% final) were added to each fraction, and pro - camera (Cooke Sensicam; Applied Scientific Instruments, Inc.) and pro - teins were precipitated on ice for at least 30 min, followed by centrifuga- cessed using Slidebook (Intelligent Imaging Innovations) and Photoshop tion at 13,000 g for 10 min. Insoluble material was reprecipitated twice software (version CS2; Adobe). by sonication into ice-cold acetone and centrifugation, then sonicated into For EM, yeast cells were high pressure frozen and freeze substituted Laemmli sample buffer and resolved by SDS-PAGE, transferred to nitro- as previously described (Nickerson et al., 2006; Richter et al., 2007) and cellulose, and examined by Western blotting using antisera against Snf7, embedded at 60°C in Lowicryl HM20 (Polysciences). Plastic blocks were Vps24, or GFP (Roche). Quantitation was performed on triplicate experi- trimmed and cut in 80-nm-thin sections and 250-nm-thick sections with a ments by incubating membranes with Alexa Fluor 680 secondary antibody microtome (Leica) and placed on rhodium-plated copper slot grids. Tomo- (Invitrogen) and visualizing with an infrared imager (Odyssey; LI-COR Bio- graphic samples were stained en bloc with 0.1% uranyl acetate and sciences). Bands were quantitated with Odyssey software (version 2.1). L231A/L234A 0.25% glutaraldehyde as a minimal fixative with no additional after stain - For the snf7 strain, 40 A equivalents were used because of de- ing except for snf7, which required 0.1% uranyl acetate and 2% glutar- creased detection of the mutant version of the protein. The presence of 1% aldehyde for sufficient preservation for tomography. Thick sections were Triton X-100 throughout the sucrose gradient yielded identical results to mapped on a transmission EM (Phillips CM10) at 80 kv, then a Tecnai 30 (FEI) gradients performed where Triton X-100 was omitted from the gradient. 304 JCB • VOLUME 192 • NUMBER 2 • 2011 Table II. Plasmids used in this study Plasmid Protein expressed Description 1-387 R pGO337 GFP-Bro1 1-387 GFP-Bro1 URA3 Ap (pRS416) pGO339 GFP-Bro16 GFP-Bro1 URA3 Ap (pRS41) pGO47 GFP-CPS Odorizzi et al., 1998 pCR180 GFP-Sna3 URA3 Ap (pRS415) GFP-Sna3 pGO547 Snf7 pST39 Shine-Delgarno Snf7 pGO619 Snf7-HA pST39 Shine-Delgarno Snf7-HA L231A/L234A pGO621 Snf7 -HA pST39 Shine-Delgarno Snf7 L231A/L234A-HA pGEX-4T1 GST GE Healthcare 1-387 pGO626 GST-Bro1 -HA pGEX-4T1 GST-Bro1 1-387-HA pMB54 GST-Vps4 Azmi et al., 2006 pGO216 Bro1 URA3 ApR (pRS426) Bro1 pMWM3 Bro11-387 URA3 ApR (pRS426) Bro11-387 pRS426 Snf7 Snf7 Davies et al., 2010 pRS426 Snf7Vps2MIM1 Snf7Vps2MIM1 Davies et al., 2010 In vitro ESCRT-III release assay seen within the tubular endosome. Online supplemental material is avail- The in vitro ESCRT-III release assay was performed as previously described able at http://www.jcb.org/cgi/content/full/jcb.201007018/DC1. (Davies et al., 2010). Purie fi d Vps4 and an ATP regeneration system (10 mM We thank Caleb Richter for yeast strain and plasmid constructions and Sylvie phosphocreatine, 10 U/ml creatine phosphokinase, and 1 mM ATP) were Urbé for helpful discussions. added to yeast membrane fractions containing ESCRT-III complexes (Babst This work was funded by the National Institutes of Health (grants et al.,1997; Azmi et al., 2006). Membranes were generated from shero- GM-065505 to G. Odorizzi and GM-73024 D. Katzmann). M. Wemmer plasted cells that were lysed under native conditions and homogenized, was supported by a National Institutes of Health training grant (GM-07135), then resuspended in ATPase reaction buffer (100 mM KOAc, 20 mM Hepes, I. Azmi was supported by an American Heart Association predoctoral grant pH 7.4, and 5 mM MgOAc) and passed through an 18-G needle three (AHA07-155882), and B. Davies was supported by a grant from the Fraternal times followed by a 30-G needle five times to homogenize the membranes. Order of Eagles. Reactions were allowed to incubate for 10 min at 30°C, then subjected to centrifugation at 13,000 g to separate the membrane-associated and solu- Submitted: 2 July 2010 ble ESCRT-III. SDS-PAGE followed by Western blot analyses was performed with anti-Snf7 antiserum and quantitated using ImageQuant software. Accepted: 11 December 2010 Ub-CPS immunoprecipitation Detection of Ub-CPS was performed as described previously (Katzmann References et al., 2001). The vacuolar protease gene PEP4 was deleted in all strains to reduce the nonspecific cleavage of Ub from CPS after cellular lysis. Agromayor, M., and J. Martin­Serrano. 2006. Interaction of AMSH with ESCRT­III and deubiquitination of endosomal cargo. J. Biol. Chem. 10 A equivalents of cells were harvested and precipitated by the addition 281:23083–23091. doi:10.1074/jbc.M513803200 of trichloroacetic acid (10% final concentration). 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Emr. 1997. Endosomal transport which was autoclaved twice, blocked in 20% fetal calf serum, and probed function in yeast requires a novel AAA­type ATPase, Vps4p. EMBO J. with antibodies against Ub (Invitrogen) or CPS. 16:1820–1831. doi:10.1093/emboj/16.8.1820 Babst, M., B. Wendland, E.J. Estepa, and S.D. Emr. 1998. The Vps4p AAA Online supplemental material ATPase regulates membrane association of a Vps protein complex re­ Fig. S1 shows that Bro1 binding to Snf7 is required for the stabilization of quired for normal endosome function. EMBO J. 17:2982–2993. doi:10 ESCRT-III polymers at endosomes. Fig. S2 shows that overexpression of the .1093/emboj/17.11.2982 Bro1 domain stabilizes ESCRT-III subunit Vps24 at the endosomal mem- Babst, M., D.J. Katzmann, W.B. Snyder, B. Wendland, and S.D. Emr. 2002. brane. 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Journal

The Journal of Cell Biology – Pubmed Central

Published: Jan 24, 2011

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