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Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes

Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes JCB: REPORT Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes Daniel P. Nickerson, Matthew West, and Greg Odorizzi Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309 he sorting of transmembrane cargo proteins into ESCRT-I or -II. Surprisingly, vesicle budding into the endo- the lumenal vesicles of multivesicular bodies (MVBs) some lumen occurs in the absence of Did2 function even T depends on the recruitment of endosomal sorting though Did2 is required for the effi cient sorting of MVB complexes required for transport (ESCRTs) to the cytosolic cargo proteins into lumenal vesicles. This uncoupling of face of endosomal membranes. The subsequent dissocia- MVB cargo sorting and lumenal vesicle formation sug- tion of ESCRT complexes from endosomes requires Vps4, gests that the Vps4-mediated dissociation of ESCRT-III is a member of the AAA family of adenosine triphospha- an essential step in the sorting of cargo proteins into MVB tases. We show that Did2 directs Vps4 activity to the dis- vesicles but is not a prerequisite for the budding of vesi- sociation of ESCRT-III but has no role in the dissociation of cles into the endosome lumen. Introduction Transmembrane proteins monoubiquitinated on their cytosolic Doa4, which deubiquitinates cargoes before their enclosure domains are sorted into the lumenal vesicles of late endosomal within MVB vesicles (Luhtala and Odorizzi, 2004). Vps4 is an multivesicular bodies (MVBs; for review see Babst, 2005). ATPase that catalyzes the dissociation of class E Vps proteins MVB vesicles and their cargoes are exposed to the hydrolytic from endosomal membranes, and, in the absence of Vps4 activ- interior of the lysosome upon fusion of the limiting endosomal ity, ESCRT complexes accumulate on endosomes (Katzmann membrane with the lysosomal membrane. The mechanism of et al., 2001; Babst et al., 2002a,b). MVB cargo sorting is conserved and mediated by class E Vps A central question of Vps4 function concerns how its ac- proteins originally identifi ed in Saccharomyces cerevisiae. tivity is coordinated to dissociate multiple protein complexes. Most class E VPS genes encode soluble cytosolic proteins re- We report that Did2, a protein related to ESCRT-III subunits cruited transiently to endosomes. Genetic and biochemical data (Amerik et al., 2000), directs Vps4 activity to the dissociation of suggest a sequence that begins with recruitment of the Vps27– ESCRT-III. In the absence of Did2, ESCRT-I and -II dissocia- Hse1 complex, which recognizes monoubiquitinated cargoes, tion occurs, whereas ESCRT-III and downstream components followed by recruitment of three distinct endosomal sorting accumulate on endosomes. Surprisingly, MVB vesicle budding complexes required for transport (ESCRTs; for review see proceeds in the absence of Did2 despite the requirement for Hurley and Emr, 2006). Like the Vps27–Hse1 complex, ESCRT-I Did2 in sorting cargoes, demonstrating that vesicle formation and -II bind monoubiquitinated cargoes, whereas ESCRT-III and MVB cargo sorting can be uncoupled. lacks ubiquitin-binding subunits and functions downstream of cargo recognition. Results and discussion ESCRT-III is comprised of the Vps20–Snf7 and Vps2– Vps24 subcomplexes (Babst et al., 2002a). Although its molec- The C terminus of Did2 binds the MIT ular function is not fully understood, one role for ESCRT-III is domain of Vps4 the recruitment of late-acting components of the sorting ma- The N terminus of Did2 is predominantly comprised of basic chinery. Snf7 recruits Bro1 (Kim et al., 2005), and Bro1 recruits amino acids, whereas its C terminus predominantly contains acidic residues (Fig. 1 A). As shown in Fig. 1 B, bacterially Correspondence to Greg Odorizzi: [email protected] expressed His -Vps4 copurifi ed with GST-Did2 but not GST Abbreviations used in this paper: CPS, carboxypeptidase S; DIC, differential alone. This interaction occurred regardless of whether Vps4 interference contrast; ESCRT, endosomal sorting complex required for transport; E233Q was locked in its ATP-bound state (His -Vps4 ) or was MIT, microtubule interaction and traffi cking; MVB, multivesicular body; VTE, K179A vesicular tubular endosome. disabled from binding ATP (His -Vps4 ; Fig. 1 B). In The online version of this article contains supplemental material. contrast, GST-Vta1 showed a strong preference for binding © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 175, No. 5, December 4, 2006 715–720 http://www.jcb.org/cgi/doi/10.1083/jcb.200606113 JCB 715 THE JOURNAL OF CELL BIOLOGY E233Q His -Vps4 (Fig. 1 B), which is consistent with Vta1 inter acting with ATP-bound Vps4 to stimulate its oligomer- ization (Azmi et al., 2006). Studies of CHMP1b and Vps4a, the mammalian ortho- logues of Did2 and Vps4, respectively, demonstrated that the C terminus of CHMP1b binds Vps4a and that this interaction is disrupted by the mutation of leucine-64 in the microtubule inter- action and traf cking (MIT) domain of fi Vps4a (Scott et al., 2005). This leucine is conserved in the MIT domain of yeast Vps4 (Fig. 1 A), suggesting that it is important for the interaction between L64A Did2 and Vps4. Indeed, His -Vps4 failed to bind GST-Did2 but still bound GST-Vta1 (Fig. 1 B), which is in agreement with Vta1 binding the AAA domain rather than the MIT domain of Vps4 (Yeo et al., 2003). We further observed that His -Vps4 104–204 interacted with the C terminus of Did2 (GST-Did2 ) but 1–103 not its N terminus (GST-Did2 ; Fig. 1 C). Thus, the binding mechanism between Vps4 and Did2 appears conserved. Because the MIT domain of Vps4 is essential for its local- ization to endosomes (Babst et al., 1998), we addressed whether its binding to Did2 mediates the endosomal recruitment of Vps4. E233Q Locked in the ATP-bound state, Vps4 is unable to catalyze the dissociation of itself and its substrate proteins from endo- E233Q somes (Babst et al., 1998). Thus, GFP fused to Vps4 ap- peared concentrated at class E compartments stained with the E233Q lipophilic dye FM 4-64 (Fig. 1 D, arrowheads). GFP-Vps4 also localized to endosomes in did2∆ cells (Fig. 1 D, arrow- heads), indicating that the recruitment of Vps4 does not require E233Q Did2. Localization of Vps4 to the endosomal membrane in Figure 1. The C terminus of Did2 binds Vps4. (A) Domain maps of Did2 the absence of Did2 was also observed by subcellular fraction- and Vps4. (B and C) Western blots of in vitro glutathione-Sepharose pull ation (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/ downs of wild-type or mutant His -Vps4 mixed with GST-Did2 (B) or wild- type His -Vps4 mixed with wild-type or mutant GST-Did2 (C). (D) Fluores- jcb.200606113/DC1), which was not surprising given that Vps4 cence and DIC microscopy of FM 4-64–stained cells expressing binds multiple distinct ESCRT-III components. Indeed, the dele- E233Q GFP-Vps4 . Arrowheads indicate endosomal membranes. tion of DID2 did not affect the ability of GST-Vps4 to pull down Snf7 from yeast lysates (Fig. S1 B), which is consistent with our observation that GST-Vps4 interacts directly with His -Snf7 simplest explanation for the mislocalization of Did2 to the cyto- (Fig. S1 C) and with a previous study showing that Vps4 inter- sol in vps4∆ cells lacking either Vps2 or Vps24 is that the acts directly with Vps20 (Yeo et al., 2003). N terminus of Did2 interacts directly with the Vps2–Vps24 subcomplex. Indeed, recombinant His -Vps24 bound GST- 1–103 104–204 The Vps2–Vps24 subcomplex of ESCRT-III Did2 but not GST-Did2 or GST alone (Fig. 2 C). In recruits Did2 to endosomes contrast, recombinant His -Snf7 failed to bind GST-Did2 Did2 binds Vta1 and is required for the interaction of Vta1 with (unpublished data). the ESCRT-III component Snf7 (Lottridge et al., 2006). There- fore, we examined whether Vta1 or ESCRT-III proteins mediate Endosomal dissociation of ESCRT-III the recruitment of Did2 to endosomes. Genetic and biochemical requires Did2 studies suggest that ESCRT-III consists of a Snf7–Vps20 sub- To address the functional signifi cance of the interaction be- complex and a Vps2–Vps24 subcomplex (Babst et al., 2002a). tween Did2 and Vps4, we examined the ability of Vps4 to me- Did2 accumulated at the class E compartment in vps4∆ cells re- diate the dissociation of ESCRT complexes in the absence of gardless of whether VTA1, SNF7 (Fig. 2 A), or VPS20 (not de- Did2. As shown previously (Katzmann et al., 2001; Babst et al., picted) had been deleted. In contrast, the deletion of either 2002b), Vps23-GFP of ESCRT-I (Fig. 3 A) and Vps36-GFP of VPS24 (Fig. 2 A) or VPS2 (not depicted) caused Did2-GFP to ESCRT-II (Fig. 3 B) in wild-type cells were predominantly cy- remain cytosolic. Similarly, subcellular fractionation showed tosolic in addition to being localized weakly at punctate that Did2-GFP was concentrated in the membrane fraction in structures. As expected, Vps23- and Vps36-GFP in vps4∆ cells vps4∆ cell extracts but was soluble both in vps4∆ vps2∆ and accumulated at class E compartments (Fig. 3, A and B; arrow- vps4∆ vps24∆ extracts (Fig. 2 B). heads). However, the distributions of both proteins in did2∆ Fluorescence microscopy (Fig. 2 A) and subcellular frac- cells appeared to be similar to their distributions in wild-type tionation (Fig. 2 B) indicated that the N terminus of Did2 is nec- cells (Fig. 3, A and B). Thus, Did2 is not required for the disso- essary and suffi cient for endosomal localization. Therefore, the ciation of either ESCRT-I or -II. Moreover, Did2 has no role in 716 JCB • VOLUME 175 • NUMBER 5 • 2006 Figure 2. The N terminus of Did2 requires the Vps2–Vps24 subcomplex for recruitment to endosomes. (A and B) Fluorescence and DIC microscopy of FM 4-64–stained cells expressing GFP-tagged wild-type (A) or mutant (B) Did2 proteins. Arrowheads indicate endosomal membranes. (C and D) Subcellular fractionation and Western blot analysis of cells expressing GFP- tagged wild-type (C) or mutant (D) Did2 proteins. Cell lysates (T, total) were Figure 3. Did2 is required for the endosomal dissociation of ESCRT-III. separated into membrane-associated pellet (P13) and soluble cytosolic (A and B) Fluorescence and DIC microscopy of FM 4-64–stained cells (S13) fractions. PGK, 3-phosphoglycerate kinase. (E) In vitro glutathione- expressing Vps23- (A) or Vps36-GFP (B). Arrowheads indicate class E com- Sepharose pull downs of His -Vps24 with GST-tagged wild-type or mutant partments. (C) Subcellular fractionation and Western blot analysis of Did2 proteins. endogenous Snf7 and Vps24. T, total; PGK, 3-phosphoglycerate kinase. dissociation of both proteins. Likewise, Did2 was required for the endosomal recruitment of ESCRT-I and -II because both the endosomal dissociation of Bro1 and Doa4 (Table I), com- Vps23- (Fig. 3 A) and Vps36-GFP (Fig. 3 B) accumulated at ponents that function downstream in the MVB pathway but class E compartments in vps4∆ did2∆ cells. depend on ESCRT-III for recruitment to endosomes (Luhtala Fusion of GFP to ESCRT-III proteins disrupts their func- and Odorizzi, 2004; Kim et al., 2005). The ability of Bro1 and tion in MVB sorting (unpublished data). Thus, we assessed the Doa4 to dissociate from endosomes may require Did2 to coor- distributions of endogenous Snf7 and Vps24 by subcellular dinate the Vps4-mediated dissociation of ESCRT-III. Although fractionation and Western blotting. As shown previously (Babst Vta1 binds Did2 and requires Vps4 to dissociate from endo- et al., 1998), Snf7 was predominantly soluble, and Vps24 was somes (Shifl ett et al., 2004), Vta1 appeared predominantly cyto- evenly distributed between membrane and soluble fractions in solic in the absence of Did2 (Table I), indicating that it does wild-type cells, whereas both proteins shifted entirely to the not need Did2 for dissociation. This quality makes Vta1 unique membrane pellet in vps4∆ cells (Fig. 3 C). Snf7 and Vps24 among ESCRT-III–associated Vps4 substrates acting late in were similarly concentrated in the pellet fraction of did2∆ cells the MVB pathway. (Fig. 3 C), indicating that Did2 is essential for the membrane DID2 DIRECTS DISSOCIATION OF ESCRT-III BY VPS4 • NICKERSON ET AL. 717 Table I. Localization of Vps4 substrates in wild-type, vps4𝚫 , and did2𝚫 mutant cells Substrate Complex Wild type vps4𝚫 did2𝚫 a b a Vps23-GFP ESCRT-I Cytosolic Punctate Cytosolic a b a Vps36-GFP ESCRT-II Cytosolic Punctate Cytosolic c d d Snf7 ESCRT-III Soluble Membrane Membrane c d d Vps24 ESCRT-III Soluble Membrane Membrane a b b Bro1-GFP NA Cytosolic Punctate Punctate a b b Doa4-GFP NA Cytosolic Punctate Punctate a b a Vta1-GFP With Vps60 Cytosolic Punctate Cytosolic NA, not applicable. Predominantly cytosolic GFP signal. Punctate GFP signal adjacent to the vacuole with a reduction in cytosolic signal. Soluble as determined by subcellular fractionation. Membrane bound as determined by subcellular fractionation. MVB vesicle budding in the absence of did2∆ cells appeared electron dense and were uniformly of ESCRT-III dissociation larger (by 38%) than lumenal vesicles of MVBs in wild-type Class E compartments stained by FM 4-64 are a hallmark pheno- cells (P < 0.0001; 23.98 ± 0.23 vs. 33.01 ± 0.56 nm, re- type caused by mutations in VPS4 and other class E VPS genes. spectively; Fig. 4 J), raising the possibility that ESCRT-III, By EM, these abnormal late endosomes appear as fl attened stacks which is unable to dissociate from the membrane, is mis- of cisterna-like structures devoid of lumenal vesicles (Rieder takenly packaged as cargo. However, ESCRT-III was only et al., 1996; Odorizzi et al., 1998). We examined did2∆ cells detected at the limiting membrane of VTEs in thin sections v ersus wild-type and vps4∆ cells using high resolution EM and of did2∆ cells examined by immunogold labeling using anti- tomographic modeling. An example of a typical wild-type MVB bodies against Vps24 (Fig. S2 D, available at http://www.jcb. is shown in the tomogram in Fig. 4 A and is modeled in Fig. 4 org/cgi/content/full/jcb.200606113/DC1). (B and C; and see Videos 1 and 2, available at http://www.jcb. The similarity of the class E compartment and VTE when org/cgi/content/full/jcb.200606113/DC1). The limiting membrane viewed by fl uorescence microscopy (Fig. 1 D) underscores the of this MVB is approximately spherical and surrounds numerous need for EM when reaching any conclusion regarding endosome lumenal vesicles. As expected, no multivesicular endo somes morphology. Moreover, the ultrastructural differences between were detected in vps4∆ cells, which instead contained class class E compartments and VTEs suggest that the class E pheno- E compartments similar to the structures described previously in type warrants subdivision based on endosome morphology. The cells lacking Vps4 function (Odorizzi et al., 1998). An example absence of lumenal vesicles in class E compartments as a result of a class E compartment in vps4∆ cells is shown in the tomo- of the loss of function of ESCRTs, Vps4, or Bro1 has been gram in Fig. 4 D and is modeled in Fig. 4 (E and F; and see thought to signify that these components comprise the core class Videos 3 and 4). Three-dimensional analysis indicated that its E Vps machinery required for vesicle budding (for review see elongated cisternae-like elements did not connect with one an- Babst, 2005). However, the VTEs observed in did2∆ cells con- other. Similar characteristics were observed in serial sections that tradict the view that the dynamic cycling of ESCRT-III, a subset included entire class E compartments (unpublished data), and lu- of this core machinery, is either a pre- or corequisite for MVB menal vesicles were not observed in >300 class E compartments vesicle formation, although it remains likely that the assembly of vps4∆ cells examined by EM (including three structures mod- of ESCRT-III on endosomes is critical to the budding event. eled by tomography), which is consistent with an essential role Like Vps4, Did2 is required for effi cient sorting of MVB for Vps4 function in the biogenesis of MVB vesicles. cargoes, as indicated by the failure of GFP–carboxypeptidase S Surprisingly, multivesicular endosomes were readily (CPS), a biosynthetic protein, to be sorted into the vacuole apparent in did2∆ cells, an example of which is shown in the lumen in did2∆ cells (Fig. 4 K). Sna3-GFP (Fig. S2 A), an- tomogram in Fig. 4 G and modeled in Fig. 4 (H and I; and other biosynthetic protein, as well as Ste3-GFP (Fig. S2 B), Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/ an endocytic protein, are also mislocalized, demonstrating that jcb.200606113/DC1). The limiting membrane, rather than the loss of Did2 function causes a broad cargo-sorting defect. being spherical as seen in wild-type cells, was typically Intriguingly, the sorting of GFP-CPS in did2∆ cells was par- elongated, which is similar to the cisternae-like elements of tially rescued upon in-frame fusion of ubiquitin to its cytosolic class E compartments. In >200 sections examined by EM, domain (Fig. S3 C, available at http://www.jcb.org/cgi/content/ these vesicular tubular endosomes (VTEs) were most of- full/jcb.200606113/DC1). However, the mislocalization of Sna3- ten observed crowded together, which is reminiscent of the GFP suggests that the molecular basis for the cargo-sorting compact organization displayed by class E compartments defect in did2∆ cells is not directly related to ubiquitination in vps4∆ cells (Fig. 4, D–F). Three-dimensional analysis because Sna3 does not require ubiquitin to be sorted into the of tomograms and serial sections that encompassed entire MVB pathway (Reggiori and Pelham, 2001). The ubiquitin- VTEs (unpublished data) indicated that the lumenal vesicles independent localization of Sna3-GFP to the vacuole lumen were not interconnected, nor were they connected to the lim- indicates, albeit indirectly, that cargo sorting can be uncoupled iting membrane. The interior of lumenal vesicles in VTEs from lumenal vesicle formation in yeast. Indeed, MVB vesicles 718 JCB • VOLUME 175 • NUMBER 5 • 2006 Figure 4. Tomographic analysis of endosome morphology. (A–I) Two-dimensional cross sections and three-dimensional models derived from 200-nm–thick section tomograms. V, vacuole. Models depict a wild-type MVB (B and C), a vps4∆ class E compartment (E and F), and a did2∆ VTE (H and I). Models in C, F, and I are rotated 25° relative to the models in B, E, and H. In MVB and VTE models, the endosomal limiting membrane is yellow, and lumenal vesicles are red. Cisternae in the vps4∆ class E compartment model are depicted in various colors to easily discriminate individual membrane structures. (J) Vesicle diameters in wild-type and did2∆ cells (n = 284 and 175, respectively). Mean values ± SEM (error bars; unpaired t test; P < 0.0001). (K) Fluorescence and DIC microscopy of cells expressing GFP-CPS. are observed in cells lacking functional Doa4 or Rsp5, the primary tion downstream of the Vps2–Vps24 subcomplex in order of E3 ubiquitin ligase for MVB cargoes in yeast (unpublished data). assembly because Vps24 can be recruited to the membrane in Similarly, MVB vesicles are observed by EM despite deletion the absence of Did2 but not vice versa. The signifi cance of Did2 of the FAB1 gene (unpublished data), which blocks MVB sort- recruitment is that Vps4 requires Did2 to catalyze the endo- ing of CPS but not Ste2, an endocytic cargo protein (Odorizzi somal dissociation of ESCRT-III as well as factors that function et al., 1998). In mammalian cells, the overexpression of a mu- tant form of Hrs that is defective in ubiquitin binding has no effect on MVB vesicle formation but reduces the effi ciency of cargo sorting, perhaps because of a failure in the concentration of cargoes at the site of vesicle budding (Urbé et al., 2003). Although the nature of the sorting defect in did2∆ is not clear, it might be caused by the trapping of cargoes within an ESCRT-III network that is unable to release from endosomes, in which case the in-frame fusion of ubiquitin could promote sorting by enhancing cargo interactions with ESCRT-I and -II to the exclusion of ESCRT-III. Our fi ndings suggest that Did2 functions to coordinate Figure 5. Model of Did2 function in MVB sorting. The Did2 C terminus interacts with Vps4, and its N terminus interacts with the ESCRT-III subunit Vps4 activity to ESCRT-III dissociation (Fig. 5). The C termi- Vps24. Did2 is required for the endosomal dissociation of ESCRT-III and nus of Did2 binds the MIT domain of Vps4, whereas the N downstream components, which are Did2-dependent Vps4 substrates terminus of Did2 binds Vps24 of ESCRT-III. Did2 has a posi- (green). ESCRT-I and -II (orange) are Did2-independent Vps4 substrates. DID2 DIRECTS DISSOCIATION OF ESCRT-III BY VPS4 • NICKERSON ET AL. 719 shown in Fig. 4. Online supplemental material is available at http://www. downstream. Therefore, this set of Vps4 substrates is Did2 de- jcb.org/cgi/content/full/jcb.200606113/DC1. pendent, which is in contrast with ESCRT-I and -II, which are Did2 independent (Fig. 5). The selective role Did2 plays in We thank Caitlin White-Root for constructing plasmids and the Boulder three-dimensional laboratory for tomography aid. coordinating Vps4 with ESCRT-III dissociation implies that This work was funded by National Institutes of Health (NIH) grant additional f actors couple Vps4 function to the dissociation of GM065505. D.P. Nickerson is supported by NIH Training Grant GM07135, and ESCRT-I and -II. G. Odorizzi is an Arnold and Mabel Beckman Foundation Young Investigator. Submitted: 21 June 2006 Materials and methods Accepted: 31 October 2006 Yeast strains and plasmids Yeast strains and plasmids used in this study are listed in Table S1 (available References at http://www.jcb.org/cgi/content/full/jcb.200606113/DC1). Yeast manipulations were performed using standard protocols. Gene deletions Amerik, A.Y., J. Nowak, S. Swaminathan, and M. Hochstrasser. 2000. 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Emr. 1996. et al., 1995). 200-nm semithick sections were placed on Rhodium-plated Multilamellar endosome-like compartment accumulates in the yeast Formvar-coated copper slot grids and mapped on an electron microscope vps28 vacuolar protein sorting mutant. Mol. Biol. Cell. 7:985–999. (CM10 TEM; Phillips) at 80 kV. Dual tilt series images were collected from Scott, A., J. Gaspar, M.D. Stuchell-Brereton, S.L. Alam, J.J. Skalicky, and W.I. 60 to −60° with 1° increments at 200 kV using an electron microscope Sundquist. 2005. Structure and ESCRT-III protein interactions of the MIT (Tecnai 20 FEG; FEI). Tomograms were imaged at 29,000× with a 0.77-nm domain of human VPS4A. Proc. Natl. Acad. Sci. USA. 102:13813–13818. pixel (binning 2). Sections were coated on both sides with 15-nm fi ducial Shifl ett, S.L., D.M. Ward, D. Huynh, M.B. Vaughn, J.C. Simmons, and J. Kaplan. gold for the reconstruction of back projections using IMOD software (Kremer 2004. Characterization of Vta1p, a class E Vps protein in Saccharomyces et al., 1996). 3dmod software was used for mapping structure surface ar- cerevisiae. J. Biol. Chem. 279:10982–10990. eas. Mean z-scale values for wild-type and did2∆ sections were within 3%. Urbé, S., M. Sachse, P.E. Row, C. Preisinger, F.A. Barr, G. Strous, J. Klumperman, Best fi t sphere models were used to measure vesicle diameters to the outer and M.J. Clague. 2003. The UIM domain of Hrs couples receptor sorting leafl et of membrane bilayers. IMOD calculated limiting membrane surface to vesicle formation. J. Cell Sci. 116:4169–4179. areas using three- dimensional mesh structures derived from closed contours Winey, M., C.L. Mamay, E.T. O’Toole, D.M. Mastronarde, T.H. Giddings, K.L. that were drawn each 3.85 nm using imodmesh software. McDonald, and J.R. McIntosh. 1995. Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle. J. Cell Biol. Online supplemental material 129:1601–1615. Table SI describes strains and plasmids used in this study. Fig. S1 shows Yeo, S.C.L., L. Xu, J. Ren, V.J. Boulton, M.D. Wagle, C. Liu, G. Ren, P. Wong, that Did2 is not required for Vps4 to interact with ESCRT-III. Fig. S2 shows R. Zahn, P. Sasajala, et al. 2003. Vps20p and Vta1p interact with Vps4p MVB cargo localization in did2∆. Videos 1–6 depict the tomograms and and function in multivesicular body sorting and endosomal transport in three-dimensional models of wild-type, did2∆, and vps4∆ endosomes Saccharomyces cerevisiae. J. Cell Sci. 116:3957–3970. 720 JCB • VOLUME 175 • NUMBER 5 • 2006 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Pubmed Central

Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes

Nickerson, Daniel P.; West, Matthew; Odorizzi, Greg
The Journal of Cell Biology , Volume 175 (5) – Dec 4, 2006
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Copyright © 2006, The Rockefeller University Press
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10.1083/jcb.200606113
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Abstract

JCB: REPORT Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes Daniel P. Nickerson, Matthew West, and Greg Odorizzi Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309 he sorting of transmembrane cargo proteins into ESCRT-I or -II. Surprisingly, vesicle budding into the endo- the lumenal vesicles of multivesicular bodies (MVBs) some lumen occurs in the absence of Did2 function even T depends on the recruitment of endosomal sorting though Did2 is required for the effi cient sorting of MVB complexes required for transport (ESCRTs) to the cytosolic cargo proteins into lumenal vesicles. This uncoupling of face of endosomal membranes. The subsequent dissocia- MVB cargo sorting and lumenal vesicle formation sug- tion of ESCRT complexes from endosomes requires Vps4, gests that the Vps4-mediated dissociation of ESCRT-III is a member of the AAA family of adenosine triphospha- an essential step in the sorting of cargo proteins into MVB tases. We show that Did2 directs Vps4 activity to the dis- vesicles but is not a prerequisite for the budding of vesi- sociation of ESCRT-III but has no role in the dissociation of cles into the endosome lumen. Introduction Transmembrane proteins monoubiquitinated on their cytosolic Doa4, which deubiquitinates cargoes before their enclosure domains are sorted into the lumenal vesicles of late endosomal within MVB vesicles (Luhtala and Odorizzi, 2004). Vps4 is an multivesicular bodies (MVBs; for review see Babst, 2005). ATPase that catalyzes the dissociation of class E Vps proteins MVB vesicles and their cargoes are exposed to the hydrolytic from endosomal membranes, and, in the absence of Vps4 activ- interior of the lysosome upon fusion of the limiting endosomal ity, ESCRT complexes accumulate on endosomes (Katzmann membrane with the lysosomal membrane. The mechanism of et al., 2001; Babst et al., 2002a,b). MVB cargo sorting is conserved and mediated by class E Vps A central question of Vps4 function concerns how its ac- proteins originally identifi ed in Saccharomyces cerevisiae. tivity is coordinated to dissociate multiple protein complexes. Most class E VPS genes encode soluble cytosolic proteins re- We report that Did2, a protein related to ESCRT-III subunits cruited transiently to endosomes. Genetic and biochemical data (Amerik et al., 2000), directs Vps4 activity to the dissociation of suggest a sequence that begins with recruitment of the Vps27– ESCRT-III. In the absence of Did2, ESCRT-I and -II dissocia- Hse1 complex, which recognizes monoubiquitinated cargoes, tion occurs, whereas ESCRT-III and downstream components followed by recruitment of three distinct endosomal sorting accumulate on endosomes. Surprisingly, MVB vesicle budding complexes required for transport (ESCRTs; for review see proceeds in the absence of Did2 despite the requirement for Hurley and Emr, 2006). Like the Vps27–Hse1 complex, ESCRT-I Did2 in sorting cargoes, demonstrating that vesicle formation and -II bind monoubiquitinated cargoes, whereas ESCRT-III and MVB cargo sorting can be uncoupled. lacks ubiquitin-binding subunits and functions downstream of cargo recognition. Results and discussion ESCRT-III is comprised of the Vps20–Snf7 and Vps2– Vps24 subcomplexes (Babst et al., 2002a). Although its molec- The C terminus of Did2 binds the MIT ular function is not fully understood, one role for ESCRT-III is domain of Vps4 the recruitment of late-acting components of the sorting ma- The N terminus of Did2 is predominantly comprised of basic chinery. Snf7 recruits Bro1 (Kim et al., 2005), and Bro1 recruits amino acids, whereas its C terminus predominantly contains acidic residues (Fig. 1 A). As shown in Fig. 1 B, bacterially Correspondence to Greg Odorizzi: [email protected] expressed His -Vps4 copurifi ed with GST-Did2 but not GST Abbreviations used in this paper: CPS, carboxypeptidase S; DIC, differential alone. This interaction occurred regardless of whether Vps4 interference contrast; ESCRT, endosomal sorting complex required for transport; E233Q was locked in its ATP-bound state (His -Vps4 ) or was MIT, microtubule interaction and traffi cking; MVB, multivesicular body; VTE, K179A vesicular tubular endosome. disabled from binding ATP (His -Vps4 ; Fig. 1 B). In The online version of this article contains supplemental material. contrast, GST-Vta1 showed a strong preference for binding © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 175, No. 5, December 4, 2006 715–720 http://www.jcb.org/cgi/doi/10.1083/jcb.200606113 JCB 715 THE JOURNAL OF CELL BIOLOGY E233Q His -Vps4 (Fig. 1 B), which is consistent with Vta1 inter acting with ATP-bound Vps4 to stimulate its oligomer- ization (Azmi et al., 2006). Studies of CHMP1b and Vps4a, the mammalian ortho- logues of Did2 and Vps4, respectively, demonstrated that the C terminus of CHMP1b binds Vps4a and that this interaction is disrupted by the mutation of leucine-64 in the microtubule inter- action and traf cking (MIT) domain of fi Vps4a (Scott et al., 2005). This leucine is conserved in the MIT domain of yeast Vps4 (Fig. 1 A), suggesting that it is important for the interaction between L64A Did2 and Vps4. Indeed, His -Vps4 failed to bind GST-Did2 but still bound GST-Vta1 (Fig. 1 B), which is in agreement with Vta1 binding the AAA domain rather than the MIT domain of Vps4 (Yeo et al., 2003). We further observed that His -Vps4 104–204 interacted with the C terminus of Did2 (GST-Did2 ) but 1–103 not its N terminus (GST-Did2 ; Fig. 1 C). Thus, the binding mechanism between Vps4 and Did2 appears conserved. Because the MIT domain of Vps4 is essential for its local- ization to endosomes (Babst et al., 1998), we addressed whether its binding to Did2 mediates the endosomal recruitment of Vps4. E233Q Locked in the ATP-bound state, Vps4 is unable to catalyze the dissociation of itself and its substrate proteins from endo- E233Q somes (Babst et al., 1998). Thus, GFP fused to Vps4 ap- peared concentrated at class E compartments stained with the E233Q lipophilic dye FM 4-64 (Fig. 1 D, arrowheads). GFP-Vps4 also localized to endosomes in did2∆ cells (Fig. 1 D, arrow- heads), indicating that the recruitment of Vps4 does not require E233Q Did2. Localization of Vps4 to the endosomal membrane in Figure 1. The C terminus of Did2 binds Vps4. (A) Domain maps of Did2 the absence of Did2 was also observed by subcellular fraction- and Vps4. (B and C) Western blots of in vitro glutathione-Sepharose pull ation (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/ downs of wild-type or mutant His -Vps4 mixed with GST-Did2 (B) or wild- type His -Vps4 mixed with wild-type or mutant GST-Did2 (C). (D) Fluores- jcb.200606113/DC1), which was not surprising given that Vps4 cence and DIC microscopy of FM 4-64–stained cells expressing binds multiple distinct ESCRT-III components. Indeed, the dele- E233Q GFP-Vps4 . Arrowheads indicate endosomal membranes. tion of DID2 did not affect the ability of GST-Vps4 to pull down Snf7 from yeast lysates (Fig. S1 B), which is consistent with our observation that GST-Vps4 interacts directly with His -Snf7 simplest explanation for the mislocalization of Did2 to the cyto- (Fig. S1 C) and with a previous study showing that Vps4 inter- sol in vps4∆ cells lacking either Vps2 or Vps24 is that the acts directly with Vps20 (Yeo et al., 2003). N terminus of Did2 interacts directly with the Vps2–Vps24 subcomplex. Indeed, recombinant His -Vps24 bound GST- 1–103 104–204 The Vps2–Vps24 subcomplex of ESCRT-III Did2 but not GST-Did2 or GST alone (Fig. 2 C). In recruits Did2 to endosomes contrast, recombinant His -Snf7 failed to bind GST-Did2 Did2 binds Vta1 and is required for the interaction of Vta1 with (unpublished data). the ESCRT-III component Snf7 (Lottridge et al., 2006). There- fore, we examined whether Vta1 or ESCRT-III proteins mediate Endosomal dissociation of ESCRT-III the recruitment of Did2 to endosomes. Genetic and biochemical requires Did2 studies suggest that ESCRT-III consists of a Snf7–Vps20 sub- To address the functional signifi cance of the interaction be- complex and a Vps2–Vps24 subcomplex (Babst et al., 2002a). tween Did2 and Vps4, we examined the ability of Vps4 to me- Did2 accumulated at the class E compartment in vps4∆ cells re- diate the dissociation of ESCRT complexes in the absence of gardless of whether VTA1, SNF7 (Fig. 2 A), or VPS20 (not de- Did2. As shown previously (Katzmann et al., 2001; Babst et al., picted) had been deleted. In contrast, the deletion of either 2002b), Vps23-GFP of ESCRT-I (Fig. 3 A) and Vps36-GFP of VPS24 (Fig. 2 A) or VPS2 (not depicted) caused Did2-GFP to ESCRT-II (Fig. 3 B) in wild-type cells were predominantly cy- remain cytosolic. Similarly, subcellular fractionation showed tosolic in addition to being localized weakly at punctate that Did2-GFP was concentrated in the membrane fraction in structures. As expected, Vps23- and Vps36-GFP in vps4∆ cells vps4∆ cell extracts but was soluble both in vps4∆ vps2∆ and accumulated at class E compartments (Fig. 3, A and B; arrow- vps4∆ vps24∆ extracts (Fig. 2 B). heads). However, the distributions of both proteins in did2∆ Fluorescence microscopy (Fig. 2 A) and subcellular frac- cells appeared to be similar to their distributions in wild-type tionation (Fig. 2 B) indicated that the N terminus of Did2 is nec- cells (Fig. 3, A and B). Thus, Did2 is not required for the disso- essary and suffi cient for endosomal localization. Therefore, the ciation of either ESCRT-I or -II. Moreover, Did2 has no role in 716 JCB • VOLUME 175 • NUMBER 5 • 2006 Figure 2. The N terminus of Did2 requires the Vps2–Vps24 subcomplex for recruitment to endosomes. (A and B) Fluorescence and DIC microscopy of FM 4-64–stained cells expressing GFP-tagged wild-type (A) or mutant (B) Did2 proteins. Arrowheads indicate endosomal membranes. (C and D) Subcellular fractionation and Western blot analysis of cells expressing GFP- tagged wild-type (C) or mutant (D) Did2 proteins. Cell lysates (T, total) were Figure 3. Did2 is required for the endosomal dissociation of ESCRT-III. separated into membrane-associated pellet (P13) and soluble cytosolic (A and B) Fluorescence and DIC microscopy of FM 4-64–stained cells (S13) fractions. PGK, 3-phosphoglycerate kinase. (E) In vitro glutathione- expressing Vps23- (A) or Vps36-GFP (B). Arrowheads indicate class E com- Sepharose pull downs of His -Vps24 with GST-tagged wild-type or mutant partments. (C) Subcellular fractionation and Western blot analysis of Did2 proteins. endogenous Snf7 and Vps24. T, total; PGK, 3-phosphoglycerate kinase. dissociation of both proteins. Likewise, Did2 was required for the endosomal recruitment of ESCRT-I and -II because both the endosomal dissociation of Bro1 and Doa4 (Table I), com- Vps23- (Fig. 3 A) and Vps36-GFP (Fig. 3 B) accumulated at ponents that function downstream in the MVB pathway but class E compartments in vps4∆ did2∆ cells. depend on ESCRT-III for recruitment to endosomes (Luhtala Fusion of GFP to ESCRT-III proteins disrupts their func- and Odorizzi, 2004; Kim et al., 2005). The ability of Bro1 and tion in MVB sorting (unpublished data). Thus, we assessed the Doa4 to dissociate from endosomes may require Did2 to coor- distributions of endogenous Snf7 and Vps24 by subcellular dinate the Vps4-mediated dissociation of ESCRT-III. Although fractionation and Western blotting. As shown previously (Babst Vta1 binds Did2 and requires Vps4 to dissociate from endo- et al., 1998), Snf7 was predominantly soluble, and Vps24 was somes (Shifl ett et al., 2004), Vta1 appeared predominantly cyto- evenly distributed between membrane and soluble fractions in solic in the absence of Did2 (Table I), indicating that it does wild-type cells, whereas both proteins shifted entirely to the not need Did2 for dissociation. This quality makes Vta1 unique membrane pellet in vps4∆ cells (Fig. 3 C). Snf7 and Vps24 among ESCRT-III–associated Vps4 substrates acting late in were similarly concentrated in the pellet fraction of did2∆ cells the MVB pathway. (Fig. 3 C), indicating that Did2 is essential for the membrane DID2 DIRECTS DISSOCIATION OF ESCRT-III BY VPS4 • NICKERSON ET AL. 717 Table I. Localization of Vps4 substrates in wild-type, vps4𝚫 , and did2𝚫 mutant cells Substrate Complex Wild type vps4𝚫 did2𝚫 a b a Vps23-GFP ESCRT-I Cytosolic Punctate Cytosolic a b a Vps36-GFP ESCRT-II Cytosolic Punctate Cytosolic c d d Snf7 ESCRT-III Soluble Membrane Membrane c d d Vps24 ESCRT-III Soluble Membrane Membrane a b b Bro1-GFP NA Cytosolic Punctate Punctate a b b Doa4-GFP NA Cytosolic Punctate Punctate a b a Vta1-GFP With Vps60 Cytosolic Punctate Cytosolic NA, not applicable. Predominantly cytosolic GFP signal. Punctate GFP signal adjacent to the vacuole with a reduction in cytosolic signal. Soluble as determined by subcellular fractionation. Membrane bound as determined by subcellular fractionation. MVB vesicle budding in the absence of did2∆ cells appeared electron dense and were uniformly of ESCRT-III dissociation larger (by 38%) than lumenal vesicles of MVBs in wild-type Class E compartments stained by FM 4-64 are a hallmark pheno- cells (P < 0.0001; 23.98 ± 0.23 vs. 33.01 ± 0.56 nm, re- type caused by mutations in VPS4 and other class E VPS genes. spectively; Fig. 4 J), raising the possibility that ESCRT-III, By EM, these abnormal late endosomes appear as fl attened stacks which is unable to dissociate from the membrane, is mis- of cisterna-like structures devoid of lumenal vesicles (Rieder takenly packaged as cargo. However, ESCRT-III was only et al., 1996; Odorizzi et al., 1998). We examined did2∆ cells detected at the limiting membrane of VTEs in thin sections v ersus wild-type and vps4∆ cells using high resolution EM and of did2∆ cells examined by immunogold labeling using anti- tomographic modeling. An example of a typical wild-type MVB bodies against Vps24 (Fig. S2 D, available at http://www.jcb. is shown in the tomogram in Fig. 4 A and is modeled in Fig. 4 org/cgi/content/full/jcb.200606113/DC1). (B and C; and see Videos 1 and 2, available at http://www.jcb. The similarity of the class E compartment and VTE when org/cgi/content/full/jcb.200606113/DC1). The limiting membrane viewed by fl uorescence microscopy (Fig. 1 D) underscores the of this MVB is approximately spherical and surrounds numerous need for EM when reaching any conclusion regarding endosome lumenal vesicles. As expected, no multivesicular endo somes morphology. Moreover, the ultrastructural differences between were detected in vps4∆ cells, which instead contained class class E compartments and VTEs suggest that the class E pheno- E compartments similar to the structures described previously in type warrants subdivision based on endosome morphology. The cells lacking Vps4 function (Odorizzi et al., 1998). An example absence of lumenal vesicles in class E compartments as a result of a class E compartment in vps4∆ cells is shown in the tomo- of the loss of function of ESCRTs, Vps4, or Bro1 has been gram in Fig. 4 D and is modeled in Fig. 4 (E and F; and see thought to signify that these components comprise the core class Videos 3 and 4). Three-dimensional analysis indicated that its E Vps machinery required for vesicle budding (for review see elongated cisternae-like elements did not connect with one an- Babst, 2005). However, the VTEs observed in did2∆ cells con- other. Similar characteristics were observed in serial sections that tradict the view that the dynamic cycling of ESCRT-III, a subset included entire class E compartments (unpublished data), and lu- of this core machinery, is either a pre- or corequisite for MVB menal vesicles were not observed in >300 class E compartments vesicle formation, although it remains likely that the assembly of vps4∆ cells examined by EM (including three structures mod- of ESCRT-III on endosomes is critical to the budding event. eled by tomography), which is consistent with an essential role Like Vps4, Did2 is required for effi cient sorting of MVB for Vps4 function in the biogenesis of MVB vesicles. cargoes, as indicated by the failure of GFP–carboxypeptidase S Surprisingly, multivesicular endosomes were readily (CPS), a biosynthetic protein, to be sorted into the vacuole apparent in did2∆ cells, an example of which is shown in the lumen in did2∆ cells (Fig. 4 K). Sna3-GFP (Fig. S2 A), an- tomogram in Fig. 4 G and modeled in Fig. 4 (H and I; and other biosynthetic protein, as well as Ste3-GFP (Fig. S2 B), Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/ an endocytic protein, are also mislocalized, demonstrating that jcb.200606113/DC1). The limiting membrane, rather than the loss of Did2 function causes a broad cargo-sorting defect. being spherical as seen in wild-type cells, was typically Intriguingly, the sorting of GFP-CPS in did2∆ cells was par- elongated, which is similar to the cisternae-like elements of tially rescued upon in-frame fusion of ubiquitin to its cytosolic class E compartments. In >200 sections examined by EM, domain (Fig. S3 C, available at http://www.jcb.org/cgi/content/ these vesicular tubular endosomes (VTEs) were most of- full/jcb.200606113/DC1). However, the mislocalization of Sna3- ten observed crowded together, which is reminiscent of the GFP suggests that the molecular basis for the cargo-sorting compact organization displayed by class E compartments defect in did2∆ cells is not directly related to ubiquitination in vps4∆ cells (Fig. 4, D–F). Three-dimensional analysis because Sna3 does not require ubiquitin to be sorted into the of tomograms and serial sections that encompassed entire MVB pathway (Reggiori and Pelham, 2001). The ubiquitin- VTEs (unpublished data) indicated that the lumenal vesicles independent localization of Sna3-GFP to the vacuole lumen were not interconnected, nor were they connected to the lim- indicates, albeit indirectly, that cargo sorting can be uncoupled iting membrane. The interior of lumenal vesicles in VTEs from lumenal vesicle formation in yeast. Indeed, MVB vesicles 718 JCB • VOLUME 175 • NUMBER 5 • 2006 Figure 4. Tomographic analysis of endosome morphology. (A–I) Two-dimensional cross sections and three-dimensional models derived from 200-nm–thick section tomograms. V, vacuole. Models depict a wild-type MVB (B and C), a vps4∆ class E compartment (E and F), and a did2∆ VTE (H and I). Models in C, F, and I are rotated 25° relative to the models in B, E, and H. In MVB and VTE models, the endosomal limiting membrane is yellow, and lumenal vesicles are red. Cisternae in the vps4∆ class E compartment model are depicted in various colors to easily discriminate individual membrane structures. (J) Vesicle diameters in wild-type and did2∆ cells (n = 284 and 175, respectively). Mean values ± SEM (error bars; unpaired t test; P < 0.0001). (K) Fluorescence and DIC microscopy of cells expressing GFP-CPS. are observed in cells lacking functional Doa4 or Rsp5, the primary tion downstream of the Vps2–Vps24 subcomplex in order of E3 ubiquitin ligase for MVB cargoes in yeast (unpublished data). assembly because Vps24 can be recruited to the membrane in Similarly, MVB vesicles are observed by EM despite deletion the absence of Did2 but not vice versa. The signifi cance of Did2 of the FAB1 gene (unpublished data), which blocks MVB sort- recruitment is that Vps4 requires Did2 to catalyze the endo- ing of CPS but not Ste2, an endocytic cargo protein (Odorizzi somal dissociation of ESCRT-III as well as factors that function et al., 1998). In mammalian cells, the overexpression of a mu- tant form of Hrs that is defective in ubiquitin binding has no effect on MVB vesicle formation but reduces the effi ciency of cargo sorting, perhaps because of a failure in the concentration of cargoes at the site of vesicle budding (Urbé et al., 2003). Although the nature of the sorting defect in did2∆ is not clear, it might be caused by the trapping of cargoes within an ESCRT-III network that is unable to release from endosomes, in which case the in-frame fusion of ubiquitin could promote sorting by enhancing cargo interactions with ESCRT-I and -II to the exclusion of ESCRT-III. Our fi ndings suggest that Did2 functions to coordinate Figure 5. Model of Did2 function in MVB sorting. The Did2 C terminus interacts with Vps4, and its N terminus interacts with the ESCRT-III subunit Vps4 activity to ESCRT-III dissociation (Fig. 5). The C termi- Vps24. Did2 is required for the endosomal dissociation of ESCRT-III and nus of Did2 binds the MIT domain of Vps4, whereas the N downstream components, which are Did2-dependent Vps4 substrates terminus of Did2 binds Vps24 of ESCRT-III. Did2 has a posi- (green). ESCRT-I and -II (orange) are Did2-independent Vps4 substrates. DID2 DIRECTS DISSOCIATION OF ESCRT-III BY VPS4 • NICKERSON ET AL. 719 shown in Fig. 4. Online supplemental material is available at http://www. downstream. Therefore, this set of Vps4 substrates is Did2 de- jcb.org/cgi/content/full/jcb.200606113/DC1. pendent, which is in contrast with ESCRT-I and -II, which are Did2 independent (Fig. 5). The selective role Did2 plays in We thank Caitlin White-Root for constructing plasmids and the Boulder three-dimensional laboratory for tomography aid. coordinating Vps4 with ESCRT-III dissociation implies that This work was funded by National Institutes of Health (NIH) grant additional f actors couple Vps4 function to the dissociation of GM065505. D.P. Nickerson is supported by NIH Training Grant GM07135, and ESCRT-I and -II. G. Odorizzi is an Arnold and Mabel Beckman Foundation Young Investigator. Submitted: 21 June 2006 Materials and methods Accepted: 31 October 2006 Yeast strains and plasmids Yeast strains and plasmids used in this study are listed in Table S1 (available References at http://www.jcb.org/cgi/content/full/jcb.200606113/DC1). Yeast manipulations were performed using standard protocols. Gene deletions Amerik, A.Y., J. Nowak, S. Swaminathan, and M. Hochstrasser. 2000. The Doa4 and introduction of epitopes in yeast were constructed by homologous deubiquitinating enzyme is functionally linked to vacuolar protein- sorting and endocytic pathways. Mol. Biol. Cell. 11:3365–3380. recombination of PCR products (Longtine et al., 1998). Genes PCR ampli- fi ed from genomic DNA were TOPO cloned into pCR2.1 (Invitrogen) and Azmi, I., B. Davies, C. Dimaano, J. Payne, D. Eckert, M. Babst, and D.J. subcloned using T4 DNA ligase into an expression vector. Katzmann. 2006. Recycling of ESCRTs by the AAA-ATPase Vps4 is regulated by a conserved VSL region in Vta1. J. Cell Biol. 172:705–717. Protein binding studies Babst, M. 2005. A protein’s fi nal ESCRT. Traf c fi . 6:2–9. BL21(DE3) cells transformed with pGEX4T1 or pET-His PL plasmids were Babst, M., B. Wendland, E.J. Estepa, and S.D. Emr. 1998. The Vps4p AAA grown at 37°C to logarithmic phase and were induced to express recom- ATPase regulates membrane association of a Vps protein complex binant genes by the addition of 0.5 mM isopropyl-β-D-thiogalactoside. required for normal endosome function. EMBO J. 17:2982–2993. Cells were harvested 1–2 h later, lysed under native conditions, and Babst, M., D.J. Katzmann, E.J. Estepa-Sabal, T. Meerloo, and S.D. Emr. 2002a. cleared of cell debris. GST-tagged proteins were purifi ed using GSTrap ESCRT-III: an endosome-associated heterooligomeric protein complex FF columns (GE Healthcare), and His -tagged proteins were purifi ed us- required for MVB sorting. Dev. Cell. 3:271–282. 2+ ing Ni -agarose beads (QIAGEN). 1.2 μg of purifi ed recombinant pro- Babst, M., D.J. Katzmann, W.B. Snyder, B. Wendland, and S.D. Emr. 2002b. teins were tested for stable interactions using glutathione–Sepharose beads Endosome-associated complex, ESCRT-II, recruits transport machinery (GE Healthcare) essentially as described previously (Yeo et al., 2003). for protein sorting at the multivesicular body. Dev. Cell. 3:283–289. Hurley, J.H., and S.D. Emr. 2006. The ESCRT complexes: structure and mecha- Subcellular fractionation nism of a membrane-traffi cking network. Annu. Rev. Biophys. Biomol. Fractionation of proteins into membrane-associated pellet and soluble cyto- Struct. 35:277–298. solic fractions was performed as described previously (Luhtala and Odorizzi, Katzmann, D.J., M. Babst, and S.D. Emr. 2001. Ubiquitin-dependent sorting 2004). One-half of OD unit equivalent of each sample was resolved by 600 into the multivesicular body pathway requires the function of a conserved SDS-PAGE and analyzed by Western blotting. Yeast 3-phosphoglycerate endosomal protein sorting complex, ESCRT-I. 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Additional modules for with an NA 1.40 oil immersion objective (Carl Zeiss MicroImaging, Inc.). versatile and economical PCR-based gene deletion and modifi cation in Differential interference contrast (DIC) and fl uorescence microscopy Saccharomyces cerevisiae. Yeast. 14:953–961. images were acquired with a digital camera (Cooke SensiCam; Applied Lottridge, J.M., A.R. Flannery, J.L. Vincelli, and T.H. Stevens. 2006. Vta1p and Scientifi c Instruments) and processed using Slidebook (Intelligent Imaging Vps46p regulate the membrane association and ATPase activity of Vps4p at Innovations) and Photoshop 7.0 software (Adobe). GFP-CPS was intro- the yeast multivesicular body. Proc. Natl. Acad. Sci. USA. 103:6202–6207. duced by transforming cells with pGO45 (Odorizzi et al., 1998). Pulse- Luhtala, N., and G. Odorizzi. 2004. 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Table SI describes strains and plasmids used in this study. Fig. S1 shows Yeo, S.C.L., L. Xu, J. Ren, V.J. Boulton, M.D. Wagle, C. Liu, G. Ren, P. Wong, that Did2 is not required for Vps4 to interact with ESCRT-III. Fig. S2 shows R. Zahn, P. Sasajala, et al. 2003. Vps20p and Vta1p interact with Vps4p MVB cargo localization in did2∆. Videos 1–6 depict the tomograms and and function in multivesicular body sorting and endosomal transport in three-dimensional models of wild-type, did2∆, and vps4∆ endosomes Saccharomyces cerevisiae. J. Cell Sci. 116:3957–3970. 720 JCB • VOLUME 175 • NUMBER 5 • 2006

Journal

The Journal of Cell Biology – Pubmed Central

Published: Dec 4, 2006

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