Functional and biochemical studies of the Vps4 ATPase in Saccharomyces cerevisiae

The multivesicular body (MVB) is a late endosomal compartment containing intralumenal vesicles enriched with a subset of transmembrane cargoes that form as a result of the inward budding of the endosomal limiting membrane. In Sacchoromyces cerevisiae ESCRT (endosomal sorting complexes required for transport) machinery, consisting of distinct protein complexes ESCRT-0, I, II and III, together with the AAA+ (ATPases associate with the variety of cellular activities) Vps4 (vacuolar protein sorting 4) ATPase are responsible for the MVB sorting. Solubilization of ESCRTs by the active Vps4 oligomer is thought to be the final step in the biogenesis of MVB. Vps4 consists of N-terminal ESCRT-III-interacting MIT (microtubule interaction and trafficking) domain, C-terminal nucleotide ATPase domain and the linker region that connects them. Function of the Vps4 is regulated through its recruitment from the cytoplasm through a complex network of interactions with ESCRT-III-associtaed proteins Did2, Ist1, Vta1 and Vps60 to the endosomeassociated ESCRT-III consisting of Vps20, Snf7, Vps2 and Vps24 where Vps4 assembles into an active oligomer. ATPase domains of Vps4 subunits promote oligomerization into closed rings and two stacked rings form an active Vps4 oligomer that contains a functionally important central cavity. Position of the MIT domains in the oligomer varies, suggesting that function of the active Vps4 oligomer might require flexibility in movement of the MIT domains. In Chapter 2, we have perfomed a detailed in vivo analysis of the interactions that mediate recruitment of Vps4 to ESCRT-III. Our data revealed a high degree of redundancy in the Vps4 interaction network. In our model, we propose that interactions with Did2 and Ist1 recruit Vps4 from cytoplasm to ESCRT-III and subsequent interaction with Vta1 promotes formation of an active Vps4 ATPase. We speculate that two rings that comprise active Vps4 oligomer have different functions - one ring is important for the ATP hydrolysis and substrate processing by Vps4, whereas the second ring is not directly involved in the ATP hydrolysis and serves a regulatory role. In Chapter 3, we have investigated if MIT domains need to be tucked away from the central cavity of the active Vps4 oligomer to allow processing of a substrate. We performed detailed functional and biochemical analysis of the serial deletions within the linker region. In our model, we propose that MIT domains are positioned around the central cavity region and cooperate together in solubilization of a substrate.

FUNCTIONAL AND BIOCHEMICAL STUDIES OF THE VPS4 ATPASE IN SACCHAROMYCES CEREVISIAE by Anna Shestakova A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology The University of Utah December 2011 Copyright Ó Anna Shestakova 2011 All Rights Reserved Th e Un i v e r s i t y o f Ut a h Gr a d u a t e S c h o o l STATEMENT OF DISSERTATION APPROVAL The dissertation of has been approved by the following supervisory committee members: , Chair Date Approved , Member Date Approved , Member Date Approved , Member Date Approved , Member Date Approved and by , Chair of the Department of and by Charles A. Wight, Dean of The Graduate School. Anna Shestakova Markus Babst 10-28-2011 Leslie Sieburth 10-28-2011 Villu Maricq 10-28-2011 Dennis Winge 10-28-2011 Janet Shaw 10-28-2011 Neil Vickers Biology ABSTRACT The multivesicular body (MVB) is a late endosomal compartment containing intralumenal vesicles enriched with a subset of transmembrane cargoes that form as a result of the inward budding of the endosomal limiting membrane. In Sacchoromyces cerevisiae ESCRT (endosomal sorting complexes required for transport) machinery, consisting of distinct protein complexes ESCRT-0, I, II and III, together with the AAA+ (ATPases associate with the variety of cellular activities) Vps4 (vacuolar protein sorting 4) ATPase are responsible for the MVB sorting. Solubilization of ESCRTs by the active Vps4 oligomer is thought to be the final step in the biogenesis of MVB. Vps4 consists of N-terminal ESCRT-III-interacting MIT (microtubule interaction and trafficking) domain, C-terminal nucleotide ATPase domain and the linker region that connects them. Function of the Vps4 is regulated through its recruitment from the cytoplasm through a complex network of interactions with ESCRT-III-associtaed proteins Did2, Ist1, Vta1 and Vps60 to the endosome-associated ESCRT-III consisting of Vps20, Snf7, Vps2 and Vps24 where Vps4 assembles into an active oligomer. ATPase domains of Vps4 subunits promote oligomerization into closed rings and two stacked rings form an active Vps4 oligomer that contains a functionally important central cavity. Position of the MIT domains in the oligomer varies, suggesting that function of the active Vps4 iv oligomer might require flexibility in movement of the MIT domains. In Chapter 2, we have perfomed a detailed in vivo analysis of the interactions that mediate recruitment of Vps4 to ESCRT-III. Our data revealed a high degree of redundancy in the Vps4 interaction network. In our model, we propose that interactions with Did2 and Ist1 recruit Vps4 from cytoplasm to ESCRT-III and subsequent interaction with Vta1 promotes formation of an active Vps4 ATPase. We speculate that two rings that comprise active Vps4 oligomer have different functions - one ring is important for the ATP hydrolysis and substrate processing by Vps4, whereas the second ring is not directly involved in the ATP hydrolysis and serves a regulatory role. In Chapter 3, we have investigated if MIT domains need to be tucked away from the central cavity of the active Vps4 oligomer to allow processing of a substrate. We performed detailed functional and biochemical analysis of the serial deletions within the linker region. In our model, we propose that MIT domains are positioned around the central cavity region and cooperate together in solubilization of a substrate. This thesis is dedicated to my grandparents Bondar Pavel Zakharovich, Bondar Margarita Vartanovna and my mother Shestakova Galina. TABLE OF CONTENTS ABSTRACT………………………………………………………………….. iii LIST OF TABLES………………………………………………………....... viii LIST OF FIGURES……………………………………………………........ ix ACKNOWLEDGMENTS………………………………………….............. xi 1 INTRODUCTION…..………………………………….......................... 1 General principles of the membrane trafficking........................... 2 Overview of intracellular membrane trafficking pathways………. 4 Characterization of the vps mutants………………………………. 13 ESCRT machinery promotes MVB biogenesis…………………… 17 ESCRT-III…………………………………………………………….. 23 Vps4 AAA+ ATPase ………………………………………………… 28 References................................................................................... 37 2 ASSEMBLY OF THE AAA ATPASE VPS4 ON ESCRT-III…............ 46 Abstract….. …………………………………………………………... 47 Introduction …………………………………………………………... 47 Materials and methods……………………………………………… 48 Results………………...……………………………………………… 48 Discussion .…………...……………………………………………… 57 Acknowledgments…………………………………………………… 58 References................................................................................... 58 3 FUNCTIONAL AND BIOCHEMICAL STUDIES OF THE LINKER REGION OF THE VPS4 PROTEIN ……………………………………… 60 Abstract….. …………………………………………………………... 61 Introduction …………………………………………………………... 62 Materials and methods……………………………………………… 68 Results and discussion……………...………………………………. 75 Role of the Vps4 in the cytokinesis of budding yeast……………. 96 vii References……………………………………………………………. 106 4 CONCLUSIONS AND FUTURE DIRECTIONS……………………….. 111 Summary for Chapter 2: Functional importance of Vps4- ESCRT-III-mediated interactions in vivo……………………….….. 112 Summary for Chapter 3: Functional and biochemical studies of the linker region of Vps4 ….………………………………………... 114 Future directions ……..……………………………………………… 116 References …………...……………...………………………………. 118 LIST OF TABLES Table Page 1-1. VPS genes required for the transport to the vacuole…………….. 16 1-2. Proteins that regulate MVB sorting………………………………… 18 2-1. Strains and plasmids used in this study…………………………… 49 2-2. Mutations used in this study………………………………………… 51 2-3. CPY-invertase secretion…..………………………………………… 53 3-1. Plasmids, yeast strains and E. coli used in this study…………… 70 3-2. Fluorescence microscopy of the GFP-CPS sorting in yeast expressing mutant Vps4 proteins……………………………………….. 77 3-3. Fluorescence microscopy of the GFP-CPS sorting in yeast lacking Vps4-regulatory genes expressing different Vps4(linker mutations) …………………………………………………………………. 84 3-4. Fluorescence microscopy of the GFP-CPS sorting in yeast expressing Vps4(pore mutants)…………………………………………. 96 3-5. Cell cycle progression of yeast cells synchronized with -factor after release……………………………………………………………….. 103 LIST OF FIGURES Figure Page 1-1. Three steps of membrane trafficking ………………………………… 3 1-2. Intracellular membrane trafficking pathways……………………….... 5 1-3. ESCRT-dependent sorting into MVBs…………………. ……………. 11 1-4. Interactions of ESCRTs with ubiquitin, lipids and each other……… 20 1-5. Structure of the Vps4 AAA+ ATPase…………………………………. 29 1-6. Network of interactions between Vps4 and ESCRT-III proteins…… 35 2-1. Interactions between Vps4 and its substrate and regulators….…… 51 2-2. MIM1 and MIM2 interactions contribute to the recruitment of Vps4 to ESCRT-III.…………………………………………………………………. 52 2-3. Phenotypic analysis of mutations affecting Vps4 interactions…….. 53 2-4. Localization of mutant Vps4 proteins ……………………………….. 55 2-5. Ist1 and Did2 form a stable complex ………………………………… 56 3-1. Linker region of Vps4 .…………………………………………………. 64 3-2. Vps4(linker mutations) have defect in solubilization of ESCRT-III (Snf7)………………………………………………………………………….. 81 3-3. Functional Vps4(linker deletions) are more sensitive to the canavanine in the background of did2 or vps60………………………. 87 3-4. Vps4( 79-118) has higher ATPase activity than wild-type Vps4… 90 x 3-5. Mutant Vps4( 85-115+GT) induces formation of larger intralumenal MVB vesicles………………………………………………….. 92 3-6. Abnormal elongated cellular morphology of elm1 is not exacerbated in elm1 vps4 grown at permissive room temperature (~21°C) ……………………………………………………………………….. 100 3-7. Functional Vps4 rescues temperature-sensitive growth defect of vps4 elm1…………………………………………………………………... 101 ACKNOWLEDGMENTS This thesis is dedicated to all of my colleagues and family, who have made this work possible. I thank all members of the Babst laboratory, especially Shrawan for continuous help with my work. I thank all my committee members, Leslie Sieburth, Dennis Winge, Janet Shaw and Villu Maricq, for the advice they have given me to help my work progress. I am particularly thankful for the support and encouragement I have received from my mentor Markus Babst. I want to thank my close family, especially my grand parents and my mother for their encouragement. I am grateful to my husband Anton Burtsev for his patience and help in everything including my graduate career. Thank you to my little daughter Sophia - joy and happiness of my life. CHAPTER 1 INTRODUCTION General principles of the membrane trafficking Vesicular transport mediates exchange of material between subcellular organelles 1. Vesicular traffic can be subdivided into three essential steps i) vesicle budding, ii) vesicle tethering and iii) vesicle fusion. Steps of the vesicular transport are regulated by the concerted function of intracellular trafficking machinery including coat proteins, small Rab GTPases, tethering molecules and SNARES (Soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein (SNAP) receptors) (reviewed in 2 3 4 5 6). Formation of the vesicle (Figure 1-1) is promoted by the transient assembly of coat proteins (reviewed in 2). In addition to physically sculpting vesicles, coat proteins actively sort cargo proteins and impact transport specificity through interacting with organelle-specific Rabs, tethers and SNAREs. The tethering step is mediated by the activated Rabs 3 that recruit tethering factors 4 7. Tethering factors function through establishing initial loose contact between the vesicle and acceptor membrane and through interaction with SNAREs (Figure 1-1). SNAREs localize to opposite membranes, v-SNAREs localize to vesicles and t-SNAREs to target membranes, and promote membrane fusion by forming trans-SNARE-complexes 8 9 10 11 (Figure 1-1). Trans-SNARE complexes are disassembled by the function of the NSF ATPase and -SNAP (Soluble NSF attachment protein) 12. Figure 1-1. Three steps of membrane trafficking. Coat proteins promote budding of the vesicle from the donor membrane. Tethering is mediated by the interactions between tethers, Rabs and SNAREs. Trans-SNARE complex drives membrane fusion (modified from Brocker et al. 2010). Overview of intracellular membrane trafficking pathways Newly synthesized luminal and transmembrane proteins are transported between ER and Golgi in vesicles. At the trans-Golgi network (TGN), proteins are sorted to their destination compartments. For instance, cell surface proteins are packaged into exocytic vesicles that are delivered to the plasma membrane 13. Vacuolar resident proteins, vacuolar hydrolases and SNAREs, are actively sorted at the TGN into the endosomal system en route to the vacuole. A variety of cellular activities, including downregulation of the cell surface transmembrane proteins and uptake of essential nutrients, are based on endocytosis 14. Endocytosis regulates expression of the transmembrane cell surface proteins by either recycling them to the plasma membrane or sorting into the endosomal system en route to the vacuole for degradation (Figure 1-2). Secretory pathway. Proteins are synthesized by ribosomes that associate with pore complexes on the ER membrane, thereby releasing soluble proteins into the ER lumen or incorporating transmembrane proteins into the ER membrane. Proteins that are folded and posttranslationaly modified are transported to the Golgi in COPII (Coat Protein Complex II) vesicles 2. The small GTPase Sar1 bound to GTP activates COPII components that directly sculpt vesicles and help sort cargo proteins 15 16. Tethering and fusion of the anterograde COPII vesicles are mediated by the small Rab GTPase Ypt1 and tethering factors Uso1 and TRAPPI (Transport protein particle I). Ypt1 and tethers function through establishing initial interactions between vesicles and the Golgi membrane and priming SNAREs Sed5, Bos1, Sec22 and Bet1 for the Figure 1-2. Intracellular membrane trafficking pathways. Secretory and biosynthetic cargoes are transported through ER to Golgi. At the trans-Golgi network (TGN), cargo destined to the cell surface is packaged into exocytic vesicles that traffic to the plasma membrane; vacuolar resident proteins that belong to the biosynthetic pathway are sorted into endosomal system. The endocytic pathway sorts transmembrane cell surface cargo into endosomal system en route to the vacuole. membrane fusion. Retrograde intra-Golgi and Golgi-to-ER traffic is important in maintaining compartment-specific distribution of proteins that continuously traffic in vesicle carriers 17. COPI (Coatomer Complex I) vesicles mediate retrograde intra-Golgi and Golgi to ER traffic, which is important to retain ER- and Golgi-resident proteins. Activated Golgi-localized ARF (ADP-ribosylation factor) GTPase recruits pre-assembled COPI (Coatomer Complex I) 18. COPI retrograde traffic depends on the ER-localized tethering complex Dsl1 19 Ufe1, Sec22, Sec20 and Use1 SNAREs 20. Proteins of the secretory pathway are sorted at TGN into exocytic vesicles that are transported to the plasma membrane. Exomer, a yeast coat protein complex (Chs5 and Chs6), mediates transport from TGN to the plasma membrane 21. The Rab GTPase Sec4 together with tethering complex exocyst and SNAREs Sec9, Snc1, Snc2, Sso1 and Sso2 promote the fusion step of the exocytosis 22 23. Endocytic pathway. Endocytosis is involved in a plethora of cellular functions including regulation of intracellular signaling cascades by downregulation of cell surface receptors, degradation of misfolded cell surface proteins, and degradation of plasma membrane proteins in response to the starvation 24. Endocytosed transmembrane proteins can be either recycled back to the plasma membrane or sent for the degradation to the vacuole. Sorting of the endocytosed cargo into the late endosomal compartment termed the Multivesicular body (MVB) ensures delivery to the vacuole. A highly organized network of proteins including endocytic adaptors, clathrin and actin cytoskeleton regulate endocytosis. AP-2 is a heteromeric complex composed of four subunits ( 2, 2, 2 and 2) that recruits clathrin to the site of endocytosis through interaction with plasma membrane phosphoinositides and dileucine and tyrosine-based motifs in cytosolic tails of the cargo proteins 25. Ent1 and Ent2 are monomeric adaptor proteins that regulate endocytosis through binding to clathrin, plasma membrane lipids and ubiquitinated cargoes 26 27 28. Attachment of ubiquitin, a highly conserved 76 amino acid protein, marks proteins not only for internalization but also for the sorting of these proteins into the MVB sorting pathway 14 29. Rsp5 is an E3 ubiquitin ligase that is required for the efficient ubiquitination of endocytic cargo 30. A single ubiquitin attached to the lysine residue of a plasma membrane protein appears to be sufficient to signal receptor internalization 31. On the other hand, endocytic cargo can be deubiquitinated at the endosome which results in the recycling of the cargo back to the plasma membrane 32. Cargo proteins that remain ubiquitinated are sorted into MVBs which mediates their delivery into the vacuolar lumen for degradation (mechanism of the MVB sorting is described below). Biosynthetic pathway. The biosynthetic pathway delivers soluble (CPY - Carboxypeptidase Y) and transmembrane (CPS - Carboxypeptidase S) vacuolar hydrolases as well as vacuolar SNAREs to the vacuole. There are two major routes that transport cargo to the vacuole - i) AP-3-dependent; and ii) AP3- independent sorting including sorting into the MVB pathway. AP-3-dependent pathway. AP-3-dependent transport delivers the transmembrane vacuolar proteins ALP (alkaline phosphatase) and Vam3 (vacuolar t-SNARE) to the vacuolar limiting membrane 33. Membrane association of AP-3 heteromeric adaptor complex is regulated by the ARF GTPase 34. The cytosolic N-terminus of ALP and Vam3 contains an acidic dileucine sorting signal (EQTRLV) that mediates interaction with AP-3, thereby redirecting sorting of this protein at the TGN into the AP-3 pathway, away from the delivery to the MVB pathway 33 35. AP-3-independent sorting pathway. The GGA proteins (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) are sorting adaptors that are major determinants of the transport from TGN to endosome 36. GGAs are monomeric and they have a unique four-domain structure consisting of a VHS (Vps27, Hrs, STAM) domain, a GAT (GGA and TOM1) domain, a hinge-like domain and an ear domain. GGA proteins are recruited to late Golgi membranes by the action of ARF GTPase and bind to clathrin through a hinge-like region and to acidic dileucine cargo sequence through VHS domain 37. In addition, GGAs were shown to bind ubiquitin through the GAT domain, indicating their role in ubiquitin-sorting at the TGN 38 36. Defects in TGN to endosome trafficking in AP-1 mutant, heteromeric adaptor complex, are exacerbated by deleting GGAs, suggesting that AP-1 and GGAs functions might overlap 25. Retrieval from endosomes to TGN. Resident TGN proteins that contain specific retention signals, for instance CPY receptor Vps10 and Kex2 protease that processes -factor, are prevented from entering the vacuole by an active retrieval mechanism from the endosomal system to the TGN. Cytosolic tails of the proteins Kex2 and Vp10 were demonstrated to contain aromatic sequences that serve as a retention signal for the retromer complex. This protein complex consists of five Snx proteins (sorting nexins), a Vps5-Vps17 dimer and Vps26- Vps29-Vps35 trimer. Snx proteins contain PX domains (phox homology) that allow binding to the endosome-specific lipid phosphatidylinositol 3-phosphate PI(3)P. Furthermore, the Snx proteins contain BAR domains that are predicted to induce membrane curvature 39 40. MVB-dependent sorting. The "multivesicular body" (MVB) was first described in the 1960s by George Palade 1 as a special organelle that consists of a single limiting membrane enclosing multiple vesicles. MVBs represent late endosomes that are packed with intralumenal vesicles enriched with cargo proteins. MVBs play a key role in delivering endocytic cargo and AP-3- independent biosynthetic cargo (for instance transmembrane CPS) to the vacuolar lumen by sorting cargo into intralumenal MVB vesicles. Upon fusion of the MVB with the vacuole, the MVB vesicles are delivered into the vacuolar lumen. However, cargo that was retained on the limiting membrane of the MVB will not reach the vacuolar lumen, but rather be delivered to the vacuolar limiting membrane. ESCRTs form MVBs. MVB biogenesis, or formation of intralumenal vesicles, depends on the function of ESCRT (Endosomal Sorting Complex Required for Transport) machinery that directly sorts cargo into MVB vesicles and promotes formation of MVB vesicles 41. Sequential recruitment of four different ESCRTs (0-, I-, II-, III) to the endosomal membrane, assembly and their subsequent solubilization by the Vps4 ATPase are essential steps for cargo sorting and formation of intralumenal MVB vesicles. Specificity of ESCRT recruitment to the endosomal membrane is achieved through interactions with ubiquinated cargo and endosome-enriched lipids and by binding of ESCRTs to other ESCRT proteins. Although the mechanism by which ESCRTs promote vesicle formation remains not well understood, it is clear that ESCRTs do not physically sculpt vesicles similar to coat proteins such as COP-II and clathrin, but rather force vesicles to form away from the cytosol. The current model (Figure 1-3) of ESCRT-mediated sorting of cargo into MVB vesicles suggests that ESCRT-0 is recruited to endosomes via interaction with lipids and ubiquitinated cargo and can potentially interact with clathrin and GGA proteins. ESCRT-0 recruits ESCRT-I that also interacts with ubiquitinated cargo and lipids. ESCRT-I induces endosomal localization of ESCRT-II that also bind ubiquitin and lipids. ESCRT-II mediates membrane recruitment of ESCRT-III subunits Vps20, Snf7, Vps2 and Vps24. Polymerization of ESCRT-III subunits is believed to drive cargo concentration and deformation of the endosomal membrane resulting in formation of vesicles. Vps4 ATPase is the only known enzyme that is involved in the MVB formation. Energy provided by the ATP hydrolysis by Vps4 is used to remove the ESCRT-III and possibly ESCRT-0, I and II from the endosomal membrane. Vps4-dependent solubilization of ESCRTs is believed to be the final step in the formation of intralumenal MVB vesicles. Figure 1-3. ESCRT-dependent sorting into MVBs. A. General model of the ESCRT-driven MVB biogenesis. ESCRT-0, I, II and III are recruited sequentially to the endosomal membrane where they promote cargo sorting into intralumenal vesicles. ESCRTs are solubilized by the Vps4 ATPase. B. Sorting of the GFP-CPS. Functional MVB pathway sorts GFP-CPS in MVB vesicles that delivers GFP-CPS into the vacuolar lumen (upper panel). Block in the MVB pathway results in the accumulation of GFP-CPS in the aberrant endosomal compartment (lower panel). Activity of Vps4 is regulated by its recruitment to the ESCRT-III complex by ESCRT-III-associated proteins Did2, Ist1, Vta1 and Vps60. Ubiquitin is a signal for sorting into MVBs. The major sorting signal that is recognized by the ESCRTs is ubiquitin. Interaction of ESCRTs with ubiquitinated cargo promotes sorting of that cargo into MVB vesicles 30. Single ubiquitin was demonstrated to be sufficient for re-routing transmembrane cargo to the sorting into MVBs, indicating that monoubiqutination is a sufficient signal that sends cargo for sorting into MVBs 31. Mutations that abolish ubiquitination of the CPS block sorting of the CPS into intralumenal MVB vesicles 29. Intracellular ubiquitin levels are in part maintained by deubiquinating enzymes that recycle ubiquitin from cargo proteins before these proteins are sorted into the MVB vesicles 42. Doa4, a member of a large family of ubiquitin-specific processing proteases (UBP), is the primary UBP that functions on MVBs to remove ubiquitin from ubiquitin-protein conjugates 43. The sorting of the MVB cargo CPS fused to GFP is depicted in the Figure 1-3-B. GFP-CPS is sorted into MVB vesicles and is delivered into the vacuolar lumen, which is observed as homogeneous fluorescence in the vacuolar lumen (Figure1-3-B). When MVB formation is blocked, GFP-CPS is retained on the limiting membrane of the endosome and thus is delivered to the limiting membrane of the vacuole (Figure1-3-B). There is also accumulation of GFP-CPS in the aberrant late endosome, termed the class E compartment, which accumulates adjacent to the vacuole in MVB mutant strains. Fusion of MVBs/endosomes with vacuole. Fusion of endosomes/MVBs with the vacuole delivers cargo proteins into the vacuolar lumen. The yeast vacuole is an acidic compartment that is involved in a variety of functions including amino acid storage, inorganic ion storage and the degradation of macromolecules. Vacuolar resident proteins are hydrolases that function in the proteolytic cleavage of proteins. Amino acids that accumulate in the vacuolar lumen as a result of proteolysis are being pumped back to the cytosol where they can be reused for further rounds of protein translation. Yeast cells contain one to four vacuoles of ~0.5 μm in diameter that are seen under DIC imaging as round organelles 44. Heterotypic fusion of endosomes with the vacuole is promoted by the activated Rab Ypt7, which interacts with the HOPS (homotypic fusion and vacuole protein sorting) tethering complex (Seals et al. 2000). The membrane-associated HOPS complex promotes the formation of a trans-SNARE complex containing Vam7, Vam3, Vti1 and Nyv1 45. Characterization of the vps mutants The observation that the sorting of vacuolar proteins is an active process came from experiments which showed that mutations in the N-terminus of the soluble vacuolar hydrolase CPY or its overproduction resulted in the secretion of CPY 46. Over thirty years ago, several genetic screens in Saccharomyces cerevisiae were performed in an attempt to identify the cellular protein machinery that mediates delivery of the proteins to the vacuole (summarized in 47). As a result of combined efforts of Tom Stevens and Scott Emr working in Randy Schekman's group over 45 genes termed VPS (Vacuolar Protein Sorting) were identified as involved in the transport of newly synthesized hydrolases to the vacuole. A majority of the genetic screens identified mutants that secret CPY as an indication of the perturbed transport to the vacuole. For instance, a screen performed by the Tom Steven's group selected for the acquired auxotrophic growth as a result of CPY secretion in mutagenized yeast. Secreted CPY catalyzes peptide bond cleavage of the N-blocked dipeptide N-CBZ-L-phenylalanyl- L-leucine (CBZ-pheleu) and therefore allows leucine auxotrophs to grow on medium supplemented with CBZ-pheleu as a sole leucine source 48. Scott Emr's group performed a genetic screen that selected for the spontaneous sucrose-fermenting mutants as a result of the secretion of CPY-invertase fusion protein. Invertase is a secretory enzyme that catalyzes sucrose hydrolysis allowing yeast to utilize sucrose as a carbon source. Fusion of CPY to Invertase targets the fusion protein to the vacuole and hence the cells are unable to utilize sucrose as carbon source. Therefore, mutations in genes that are required for transport to the vacuole allow growth on sucrose-rich medium 49. Vacuolar morphology of vps mutants was characterized using fluorescent dyes that label vacuoles. These experiments identified three classes: class A containing large vacuoles, class B containing fragmented vacuoles and class C that lacked structured vacuoles 50. Tom Stevens's group continued to classify vps mutants based on the vacuolar morphology using immunofluorescence microscopy and an antibody against transmembrane vacuolar hydrolase ALP. Assembly of the vacuolar H+-ATPase (v-ATPase) was assayed using antibody against 60 kDa subunit of the v-ATPase. Vacuolar acidification was judged by quinacrine staining 51. The result of this analysis was a classification of the ~45 vps mutants into six groups (Class A to F; see Table 1-1) 52. Class A: Wild-type vacuoles, with properly assembled V-ATPase Class B: Fragmented vacuole with properly assembled V-ATPase; Class C: Absence of coherent vacuoles; Class D: Defect in vacuole inheritance and acidification; Class E: Aberrant prevacuolar compartment termed "class E" found adjacent to the vacuole; Class F: Large central vacuole with one or two vacuoles adjacent. The class E VPS group. Thirteen VPS genes comprise the class E group. Transmission electron microscopy (TEM) demonstrated that the defining feature, the class E compartment, consists of aberrant elongated endosomal membranes that accumulate adjacent to the vacuole 53. The class E compartment is an aberrant late endosome that is devoid of intralumenal vesicles. As a result of the block in the intralumenal MVB vesicle formation, endocytic (for instance Ste3) 54 and biosynthetic (for instance CPS) 53 cargoes that normally undergo sorting into MVB vesicles accumulate on the limiting membrane of the class E compartment. Extensive biochemical and cell biological studies identified that most of the class E proteins comprise subunits of the ESCRT proteins that mediate the formation of MVB vesicles. The function of the ESCRTs, together with the Vps4 ATPase, is described in detail in the next sections. Table 1-1. VPS genes required for the transport to the vacuole. Class Gene names Vacuolar phenotype A VPS8 (CORVET), VPS10, VPS13, VPS29 and VPS35 (retromer), VPS30 (Atg6, Beclin), VPS38 (PtdIn3 kinase complex), VPS44 (N+/K=/H+ exchanger), VPS46 (Did2, Doa4-independent degradation) Wild-type vacuoles, with properly assembled V-ATPase B VPS5 and VPS17 (retromer); VPS39 and VPS41 (HOPS), VPS43 (Vam7) Fragmented vacuole with proper V-ATPase C VPS11, VPS16, VPS18, VPS33 (CORVET)/HOPS Absence of coherent vacuoles D VPS3 (CORVET), VPS6 (SNARE), VPS9 (GEF for Rab5), VPS15, VPS19, VPS21 VPS34 (kinase, synthesizes PI(3)P), VPS45 (SEC1 homolog) Defect in vacuole inheritance and acidification E VPS2, VPS4, VPS20, VPS22, VPS23, VPS24, VPS25, VPS27, VPS28, VPS31 (Bro1), VPS32 (Snf7), VPS36, VPS37. Aberrant prevacuolar compartment Class E found adjacent to the vacuole F VPS1 (Dynamin homolog), VPS26 (Pep8 - retromer) Large central vacuole with one or two vacuoles adjacent ESCRT machinery promotes MVB biogenesis ESCRT-dependent cargo sorting into MVBs. Genetic screens in yeast identified 13 class E vps mutants that have defects in sorting of proteins into MVBs. Twelve of the class E Vps proteins comprise four ESCRT protein complexes (0-, I-, II- and III) and enzyme Vps4 ATPase 55 29 56 57. Each of these complexes has human orthologs that are required for trafficking into lysosome, the mammalian equivalent of the yeast vacuole (Table 1-2). There is genetic and biochemical data to suggest that the ESCRT complexes are recruited sequentially to the endosomal membranes where they interact with endosomal lipids, ubiquitinated cargo and each other in promoting MVB sorting. Vps4 ATPase belongs to the class E vps proteins and is thought to function in the final step of the MVB formation by disassembling the ESCRT-III complex from endosomes. The function of Vps4 is regulated through its recruitment to ESCRT-III. Regulation of Vps4 recruitment to ESCRT-III is a focus of current research and is therefore described in detail in the following chapter. It is important to note that the ability of the ESCRTs to interact with cargo molecules and deform membranes placed them as key components in other cellular processes besides formation of MVBs. ESCRTs promote the abscission step of cytokinesis reviewed in 58, viral budding, including HIV-1 reviewed in 59, autophagy 60 and melanosome biogenesis 61. ESCRTs function on the cytosolic side of the membrane and promote deformation and scission of membranes "away" from the cytosol and ESCRTs, therefore unifying seeming unrelated processes of MVB formation, HIV-1 budding and cytokinesis. Table 1-2. Proteins that regulate MVB sorting Complex name Yeast Mammalian ESCRT-0 Vps27 Hse1 Hrs STAM1/2 ESCRT-I Vps23 Vps28 Vps37 Mvb12 Tsg101 Vps28 Vps37 A, B, C Mvb12 ESCRT-II Vps36 Vps22 Vps25 EAP45 EAP30 EAP22 ESCRT-III Vps20 Snf7 Vps24 Vps2 CHMP6 CHMP4A, B, C CHMP3 CHMP2A,B AAA+ ATPase Vps4 VPS4A, B Bro1 Bro1 Alix Regulatory proteins Ist1 Ist1 Ist1 Did2 Did2 CHMP1A,B Vta1 Vta1 Lip5 Vps60 Vps60 CHMP5 ESCRT-0. Vps27 and Hse1 comprise the ESCRT-0 complex required for the MVB sorting of endocytic and biosynthetic ubiquitinated cargo molecule 62. ESCRT-0 functions in engaging ubiquitinated cargo from early endosomes to sorting into MVBs. ESCRT-0 functions through interaction with ubiquitinated cargo, endosome-enriched lipids, interaction with clathrin and GGAs proteins. Endocytic cargo that does not interact with ESCRT-0 recycles back to the plasma membrane 63 whereas biosynthetic cargo that does not associate with ESCRT-0 is missorted to the vacuolar membrane 64 65. Vps27 contains an amino-terminal VHS (Vps27, Hrs and STAM) domain, FYVE FYVE (Fab-1, YGL023, Vps27 and EEA1) 66, two UIM (Ubiquitin Interaction Motifs) binding domains, GAT (GGA and TOM) and clathrin-binding domains (Figure 1-4). The domain structure of Hse1 includes an N-terminal VHS domain, an UIM domain, an SH3 domain (Src homology 3 domain) and a GAT domain. Vps27 and Hse1 interact with each other through the GAT domains 67. The FYVE domain of Vps27 specifically interacts with endosome-enriched PI(3)P 65 62. Interaction of ESCRT-0 with the ubiquitin-tag of MVB cargo is essential for the vacuolar delivery of these cargo proteins, as exemplified by the endocytic cargo Ste3 and the biosynthetic cargo CPS 68. Two UIM domains in Vps27 and one UIM domain in Hse1 are the major determinants that mediate ubiquitin binding 69. ESCRT-I. Endosome-localized ESCRT-0 recruits ESCRT-I via interaction of the C-terminal PSDP motif of Vps27 with the N-terminal domain of Vps23 65 70. ESCRT-I is recruited to endosomes by an interaction with the ESCRT-0 and this Figure 1-4. Interactions of ESCRTs with ubiquitin, lipids and each other. ESCRT- 0, I and II interact with ubiquitinated cargo and lipids. Structure of ESCRT-III and ESCRT-III-like proteins is similar - coiled-coil, N-terminus is positively charged, and C-terminus is negatively charged. C-terminus of ESCRT-III proteins mediates interaction with Vps4 ATPase. 0 I-I Hse1 a::: CD ~ () CLlIJ) + Vps27 C~thrin CD + Vps23 I Ia:-:: Vps37 i I () r+od CD CLlIJ) Mvb12 Ipl S + Vps28 Vps36 NZF GLUE NZF I I- + + a::: PI(3)P CD () CI) Vps22 LlJ Vps25 Vps20 I Ia:-:: Snfl () Vps4 ATPase CI) LlJ Vps24 Vps2 Q) + + + ..:.:: I I [ I-a::: Ist1 () Did2 CI) LlJ Vps60 recruitment is required for downstream cargo sorting 29. The main role of ESCRT-I is to recognize ubiquitinated cargo via the UEV (enzyme E2 variant) domain of the Vps23 71 64 and ESCRT-I exists as a preassembled complex that consists of four subunits in a 1:1:1:1 ratio - Vps23 Vps28, Vps37 and Mvb12 (Multivesicular body 12) 72. The crystal structure of the yeast ESCRT-I complex revealed an elongated heterotetramer of 􀒢20 nm in length consisting of stalk and headpiece 73. The N-terminal UEV domain of Vps23, which projects from the ESCRT-I stalk, mediates interactions with ESCRT-0. The C-terminus of Vps28, which projects from the head piece, mediates interaction with the N-terminal GLUE (GRAM-like ubiquitin-binding in EAP45) domain of Vps36. The N-terminal UEV domain of Vps23 together with the C-terminal domain of Mvb12 74 mediate binding to ubiquinated cargo (Figure 1-4). Endosome localization of ESCRT-I is achieved in part by weak binding of lipids through the positively charged N-terminus of Vps37 73. Moreover, association with ESCRT-II results in the increased affinity of ESCRT-I to lipids, in particular PI(3)P, suggesting that ESCRT-II helps in recruiting ESCRT-I. ESCRT-I acts upstream of ESCRT-II as demonstrated by the rescue of MVB sorting defect by the overexpression of ESCRT-II in cells lacking ESCRT-I 56. ESCRT-II. ESCRT-II is a stable cytosolic complex that contains Vps22, Vps36 and two subunits of Vps25. Dissection of the ESCRT-II assembly in vivo demonstrated that the "stem" of the "Y" shape of the ESCRT-II complex is contributed by Vps36 and Vps22, whereas "ears" are contributed by two subunits of Vps25 75. ESCRT-II functions in binding to ESCRT-I, PI(3)P and ubiquitinated cargo through the GLUE domain of Vps36. Binding of the GLUE domain together with the FYVE domain of Vps27 provide specificity of interaction with PI(3)P that is enriched in endosomes. 72. Two NZF (Npl4-type zinc finger) domains are inserted into the GLUE domain of Vps36 - the first NZF (110-151 amino acids) binds to Vps28, and the second NZF (176-205) binds ubiquitin (Figure 1-4) 72. Vps25 mediates recruitment of ESCRT-III through interaction with the N-terminus of Vps20 76. ESCRT-III ESCRT-III is functionally the most downstream of the ESCRTs. The core of ESCRT-III consists of the four subunits Vps20, Snf7, Vps2 and Vps24. In contrast to ESCRT-0, -I and -II, ESCRT-III subunits do not interact with ubiquitin and do not exist as a preformed cytosolic complex 57. Current models of ESCRT-III organization propose that the ESCRT-III subunits assemble into a large molecular weight complex. This oligomerization is initiated by the membrane-localization of Vps20 which nucleates polymerization of Snf7 into spiral-like structures. Recruitment of Vps24 and Vps2 regulates size of the Snf7 oligomer 77. After ESCRT-III is assembled on the membrane, the Vps4 ATPase is recruited. Vps4 is essential for the disassembly of ESCRT-III which is believed to promote vesicle formation. ESCRT-III-associated proteins Did2, Ist1, Vta1 and Vps60 aid in the recruitment of Vps4 by direct binding to ESCRT-III core subunits and Vps4. ESCRT-III proteins are structurally similar. ESCRT-III proteins (Vps20, Snf7, Vps24, Vps2, Ist1, Did2 and Vps60) are soluble small ~200 amino acids proteins that have a similar secondary structure. They are composed of a common set of six predicted alpha-helices, of which the N-terminally localized 1- 3 are basic (positively-charged) and 4- 6 are acidic (negatively-charged) 78 (Figure 1-4). The N-terminal region of ESCRT-III proteins is required for the membrane association, as deletion of the part of alpha-1 shifted Snf7 to the soluble fraction 79. ESCRT-III subunits interact with each other through their N-termini as demonstrated by the crystal structure of the N-terminal part of CHMP3, mammalian homolog of Vps24 that formed antiparallel dimers mediated by the helix 2 regions of two subunits 80. C-terminal regions of ESCRT-III subunits mediate interaction with the N-terminus of the Vps4 ATPase MIT (Microtubule Interaction and Trafficking) domain 79 81. ESCRT-III core complex assembly in vivo. Core components of the ESCRT-III complex, Vps20, Snf7, Vps2 and Vps24, were first characterized by Markus Babst as one of the class E proteins that accumulated on the endosomal membranes in vps4 mutant strains 82. Initial biochemical analysis demonstrated that ESCRT-III subunits exist as monomers or dimers in the cytosol, as demonstrated by the size exclusion chromatography (SEC) 82. In contrast, the endosome-associated ESCRT-III complex formed a large structure that was sedimentable at 100,000 X g 82. The membrane-associated ESCRT-III complex was demonstrated to be partially resistant to the treatment with detergent 81. The core of ESCRT-III does not exist in a stoichiometric 1:1:1:1 ratio 77. Snf7 is represented by ~7000 molecules/cell and is ten times more abundant than Vps20 and twice more abundant than Vps2 and Vps24. The proposed stoichiometry of ESCRT-III core complex is Vps20:Snf7:Vps24:Vps2 = 1:10:5:5 77. Membrane localization of ESCRT-III is achieved through interaction of Vp20 with Vps25 and through lipid interaction by the myristyl group attached to the N-terminus of Vps20 82. ESCRT-III subunits were demonstrated to be recruited in the sequential manner. Membrane-localization of Vps20 nucleates recruitment and polymerization of Snf7. Deletion of either VPS20 or SNF7 did not abolish formation of the ESCRT-III complex, but rather result in the formation of two ESCRT-III subcomplexes, Vps20/Vps2/Vps24 and Snf7/Vps2/Vps24. Those ESCRT-III subcomplexes accumulate on endosomes in a Vps4-dependent manner, suggesting that they are recycled by the Vps4 ATPase 82. Deletion of both VPS20 and SNF7 abolished recruitment of Vps2 and Vps24 to membranes, suggesting that Vps20 and Snf7 are recruiting determinants for Vps2 and Vps24. Furthermore, Vps2 and Vps24 require each other for the proper recruitment to the ESCRT-III subunits and for the recruitment of Vps4. In vivo recruitment of the ESCRT-III-associated proteins. Recruitment and assembly of the Vps4 ATPase is aided by ESCRT-III-associated proteins Did2, Ist1, Vta1 and Vps60. Did2 binds Vps2 and Vps24 through its N-terminus, as deletion of either Vps2 or Vps24 caused Did2 to remain cytosolic 83. The N-terminus of Did2 (similar to other ESCRT-III core proteins) is sufficient to interact with Vps2 and Vps24 as demonstrated by endosomal localization of Did2(N)-GFP and in vitro binding of Did2(N-terminus) to Vps24 83. Furthermore, Did2 mediates solubilization of ESCRT-III through recruitment of the Vps4 ATPase as deletion of Did2 results in the enhanced membrane association of Snf7 and Vps2 84. Ist1 (Increased salt tolerance 1) is a 298- amino acid soluble protein containing a coiled-coil C-terminal region and a conserved N-terminal ELYC domain 85. Full-length Ist1 and the C-terminus of Ist1 were identified through yeast two-hybrid assays as a potential interactor with Vps4 85. Although studies have shown that Ist1 is a regulator of Vps4, the deletion of IST1 does not have an obvious defect in MVB sorting 85. The current model proposes that Did2 and Ist1 function together in regulating the recruitment of Vps4 to ESCRT-III 86. Vta1 (Vps20 associated 1) is a soluble 330 amino acid protein. Vta1 was identified as an interactor with Vps4 and by yeast two-hybrid and GST-pulldown assays 87. Vta1 is composed of two N-terminal MIT (Microtubule Interacting and Trafficking) domains (MIT1 and MIT2) and a C-terminal conserved VSL (Vta1/BP1/Lip5) domain of 30 amino acids. Structural analysis revealed that Vta1 exists as an elongated homodimer with VSL regions in the middle and MIT domains pointing in opposite directions 88. Vta1 binds to the Vps4 ATPase domain through its VSL region. Vta1 binds to the ß-domain of Vps4 in the ATP-dependent manner, in a 1:2 ratio (12 Vps4/ 3 dimers Vta1 per oligomer) 89 86. The second MIT domain of Vta1 binds to the C-terminus of Did2 and Vps60 88. Vta1 requires Vps4 and Did2 for endosomal localization, as demonstrated by cytosolic distribution of Vta1-GFP in vps4 did2 cells 84. Vps60 is a 229 amino acid protein homologous to Snf7 and other subunits of the ESCRT-III complex. The first three helices are basic (1-120) and 4- 5 (127-174) are acidic. 4- 5 (127-174) of Vps60 are sufficient for binding to MIT2 of Vta1 88. ESCRT-III subunits deform membranes and polymerize into tubular structures. ESCRT-III core subunits were demonstrated to have an intrinsic ability to polymerize into tubular structures in vitro 80 90 91 92 93. ESCRT-III was demonstrated to polymerize into spiral-shape structures in vivo 94 that is believed to promote concentration of cargo and deform membranes. Overexpression of the mammalian homologs of Snf7, CHMP4A and CHMP4B, in mammalian cells results in their targeting to the plasma membrane where they self-associate into a circular array consisting of ~5 nm-long curved filaments, structures resembling spirals. Human Vps4B hydrolytic mutant, which is incapable of solubilizing CHMP4, promoted bending of membrane associated Snf7 spirals which resulted in the formation of membrane-covered tubes that protruded outwards from the cell. This observation suggested that Snf7 polymers are able to induce membrane deformation 94. In vitro studies done by Dr. Hurley's group reconstituted ESCRT-driven formation of vesicles using Giant Unilamellar Vesicles (GUVs). ESCRT-0 was demonstrated to concentrate ubiquitinated cargo and ESCRT-I and II deformed the membrane into buds, in which cargo was confined. ESCRT-III localized to the bud neck and promoted membrane scission resulting in the formation of intralumenal vesicles devoid of ESCRT components 95 96. Current model of ESCRT-III polymerization. The endosome-associated ESCRT-III core complex is thought to polymerize into a large spiral-shape structure 94 that is believed to promote concentration of cargo and promote membrane deformation and scission. Once assembled on the endosomal membrane, ESCRT-III requires energy provided by the Vps4 ATPase to be solubilized from the membranes. Solubilization of ESCRT-III by the Vps4 ATPase is believed to be the final step that results in vesicle formation 57. Function of the Vps4 ATPase is regulated by its recruitment to ESCRT-III by the four ESCRT-III-associated proteins Did2, Ist1, Vta1 and Vps60. Vps4 AAA+ ATPase Vps4 belongs to a large family of AAA+ (ATPase associated with a variety of cellular activities) ATPases 97. Vps4 is a soluble, nonessential protein that consists of 437 amino acids containing three functionally and structurally separate regions: the N-terminal substrate interacting or MIT (Microtubule Interacting and Trafficking) domain (amino acids 1-79) 98, the unstructured linker domain (79-123) and the C-terminal ATPase domain, which is composed of a large and a small subdomain (amino acids 123-437) (Figure 1-5-A) 99). The AAA+ family comprises functionally diverse group of enzymes that function in a variety of cellular processes by inducing conformational changes in substrate proteins 100. The defining feature of AAA+ proteins is a structurally conserved ATPase domain containing ATP-binding pockets at the interface of neighboring subunits that promote assembly into oligomeric rings 101. Various Figure 1-5. Structure of the Vps4 AAA+ ATPase. A. Schematic of the domain organization. B. Model of the hexameric ring composed of ATPase domains, top view and side view including linker region and MIT domains. C. Hexameric ring positioned in the oligomeric structure. D. MIM1 and MIM2 binding sites on the MIT domain. domains that are attached to the ATPase domains provide functional specificity to AAA+ proteins by interacting with adaptor proteins or substrates 102. For instance, the N-terminal MIT domain provides substrate specificity for Vps4 through interaction with ESCRT-III proteins. Walker A and Walker B motifs are integral parts of the AAA+ ATP-binding pocket. A lysine residue within the Walker-A motif GXXGXGKT/S directly interacts with the phosphates of ATP. Therefore, mutation of this lysine residue of the Walker A domain eliminates nucleotide binding and inactivates the AAA+ protein 102. A glutamate residue within the Walker-B D(D/E) motif promotes ATP hydrolysis by activating a water molecule. Therefore, mutation of this glutamate blocks nucleotide hydrolysis and produces a nonfunctional ATP-locked form of the AAA+ ATPase. Conserved arginine residue from the neighboring subunit functions together with Walker A and B motifs and completes the functional bi-partite nucleotide binding pocket. Therefore, binding of ATP to the bi-partite nucleotide-binding pocket promotes oligomerization of AAA+ ATPases that function as oligomeric rings 101. Class I and Class II. Class I AAA ATPases contain a single ATPase cassette, for instance Katanin and Vps4. Class II AAA ATPases contain two ATPase cassettes termed D1 and D2, for instance mammalian ATPases p97 and NSF 101. Class II ATPases assemble into ring structures where both ATPase domains stack in two separate rings on top of each other. In the case of the NSF, the D1 ATPase domain was demonstrated to be the major ATPase, responsible for the ATP-dependent activity of this enzyme. In contrast, the D2 domain has only minor ATPase activity but seems to play an important structural role in stabilizing the oligomer. Unlike NSF, the D1 domain of p97 is important for oligomerization while the p97-D2 domain is the major ATPase domain 103. Class I ATPases usually assemble into a single hexameric or heptameric ring. For instance, Katanin assembles into a single hexameric ring and this assembly is promoted by binding to microtubules, the substrate of katanin. ATP hydrolysis by katanin severs the bound microtubule 104. In contrast to katanin, the Vps4 oligomer was reported to assemble into a double ring structure. Similar to Class II ATPases, one ring in the Vps4 oligomer is proposed to provide ATP hydrolysis whereas the second ring contributes to a stable assembly of the Vps4 oligomer. Structure of the Vps4 oligomer. The crystal structures of human Vps4B and yeast Vps4 ATPase domains revealed small and large subdomains with a distinct beta-domain inserted within the small ATPase domain 105 106 (Figure 1-5-B). Assembly of the Vps4 hexamer was modeled using the previously solved structure of related AAA+ ATPase p97 107, predicting diameter of the Vps4 hexamer ring ~110 Å (145 including beta-domains) and height 35 Å 108. Three different structures of the assembled Vps4 oligomer revealed a double-stacked assembly of hexameric rings 89 109 or heptameric rings 108. The dodecameric model of the Vps4 oligomer composed of two hexameric rings assembled back-to-back with MIT domains projected outside from both rings fits into the data obtained from modeling of the Vps4 assembly using p97 crystal structure 89. Yu and colleges reported a double-stacked ring structure is ~ 9 nm tall. The bottom ring is ~10 nm in diameter and is similar to the p97 ring conformation. The top ring contains a large cavity of ~ 5 nm in diameter. MIT domains of the top ring form a dense structure of ~ 3.5 nm long above the cavity. MIT domains in the bottom ring are found at the periphery (Figure 1-5-B and C). Function of the Vps4 ATPase in the MVB sorting. Higher metazoans express two alleles, VPS4A and VPS4B, whereas fungi, including S. cerevisiae, express a single VPS4 allele. Human Vps4A and Vps4B share 80% identity between each other and share ~60% identity with yeast Vps4 (for sequence alignment see 105). Deletion of VPS4 in S. cerevisiae by was rescued by murine Vps4B (and Vps4A to a lesser extent), indicating conserved function of Vps4 proteins 110. The function of Vps4 in the formation of MVBs was first described by Markus Babst 97. Deletion of VPS4 displayed phenotypes similar to those of other class E vps mutants. Strains mutated for VPS4 secreted CPY and stabilized the plasma membrane protein Ste6 111. Furthermore, ESCRT-III core proteins where observed to accumulate on endosomes in vps4 cells, suggesting that ESCRT-III might be a substrate for the Vps4 ATPase 57. Further proof that ESCRT-III proteins are a substrate for the Vps4 ATPase was obtained from studies that solved the crystal structure of the C-terminus of Vps2 in complex with the Vps4 MIT domain 112 and the C-terminus of CHMP1A, homolog of Did2, with the human Vps4A MIT domain 113. In vitro biochemical characterization demonstrated that ATPase activity of Vps4 was dependent upon protein concentration. Titration experiments with increasing concentration of Vps4 measured an ATPase activity at 0.3 μM of ~10 ADP/min/Vps4 which increased to ~25 ADP/min/Vps4 at ~1 μM Vps4, suggesting that Vps4 oligomerizes in the concentration-dependent manner 82. Introducing point mutations in the ATPase cassette (K179A or E233Q) abolished Vps4 ATPase activity. The K179A mutation in Vps4 renders the protein defective in ATP binding 82. The E233Q mutation impairs hydrolysis of the bound ATP and thus interferes with the function of Vps4. MIT domains interact with ESCRT-III C-terminal regions. The MIT domain of Vps4 was identified by homology to other MIT domain-containing proteins, including katanin and spastin 98. The MIT domain of Vps4 directly interacts with ESCRT-III and therefore mediates recruitment of Vps4 to MVBs. The MIT domain binds similar motifs at the C-termini of ESCRT-III subunits that are termed MIMs (MIT Interaction Motifs). Two distinct MIMs have been identified in ESCRT-III subunits, called MIM1 and MIM2 [110]. The MIT domain of Vps4A forms an antiparallel three helix bundle 114. Helices two and three form coiled-coil structure and interact through canonical "knob in holes" side chain interaction. The MIM1- and MIM2-binding sites are localized to different groves of the MIT domain, enabling simultaneous binding to both types of MIMs (Figure 1-5-D) 115. The MIM1 consensus motif (- - - LXX+LAAL+) is found at the very C-terminus of Vps2, Vps24, Did2 and Ist1 116. Structural studies of the Vps4 MIT domain bound to the C-terminus of CHMP1A, human homolog of Did2, and Vps2 revealed that the MIM1 motif binds parallel to helix three in the groove between helix two and three of the Vps4 MIT domain. The MIM2 consensus motif ( - LP - VPS- x LP) lies in between helices four and five of Vps20 and Snf7. The NMR structure of the Vps4A MIT domain with the mammalian homolog of Vps20, CHMP6, demonstrated that the MIM2 motif binds parallel to helix three of the MIT domain in the groove between helices one and three 115. As a result of binding to different surface areas of the MIT domain, MIM1 and MIM2 motifs can bind simultaneously to the MIT domain. Ist1 is the only ESCRT-III-type protein that has both MIM1 and MIM2 motifs. As a consequence, Ist1 binds the Vps4 MIT domain with a Kd of ~3 μM, lower than any Kd found for other MIM-Vps4 MIT interactions 91. Interactions of the Vps4 ATPase with ESCRT-III proteins. Interactions with ESCRT-III proteins mediate recruitment of Vps4 to endosome-associated ESCRT-III (Figure 1-6) (detailed network of interactions is summarized in Chapter 2 86) In addition, interaction with ESCRT-III proteins regulates ATPase activity of Vps4 117. Ist1 negatively regulates Vps4 ATPase activity by disrupting oligomerization of Vps4 producing Ist1-Vps4 dimers 85. The C-terminal domain of Ist1 binds to the MIT domain of Vps4 and the N-terminus of Ist1 binds the ATPase cassette Vps4 85. Binding to Vta1 promotes oligomerization of Vps4 and therefore stimulates Vps4 ATPase activity. Vta1 binds to the Vps4 ATPase domain through its C-t rminal VSL region 83 that is sufficient to stimulation ATPase activity of Vps4. Vta1 binds to Vps4 in an ATP-dependent manner, forming a protein complex of 12 Vps4 subunits together with 3 Vta1 dimers 89. Recent studies from Dr. Katzmann's group revealed that binding of Vps2, Did2 and ESCRT-III proteins to the MIT domain of Vps4 stimulates Vps4 ATPase Figure 1-6. Network of interactions between Vps4 and ESCRT-III proteins based on previously published in vitro and in vivo studies (Chapter 2 and 86). activity 117. In addition, binding of the C-terminal region of Ist1 to MIT domains also stimulated ATPase activity of Vps4 85. This increase in ATPase activity as a result of ESCRT-III-binding to MIT domains 118 could be explained by three possible mechanisms - stabilization of the Vps4 oligomer, direct relief of the autoinhibitory interactions through the MIT domain/linker region 119 with the ATPase cassette and conformational changes in the ATPase cassette 117. The fully assembled Vps4 oligomer contains at least twelve MIT domains (dodecamer model) contributed by Vps4 and twelve MIT domains contributed by Vta1. Potentially, all twenty-four MIT domains can interact with ESCRT-III proteins Ist1, Did2, Vps60, Vps20, Snf7, Vps2 and Vps24. Therefore, recruitment of the Vps4 to ESCRT-III, assembly into a functional oligomer, including regulation of its ATPase activity, is a result of a large protein network consisting of ESCRT-III core proteins and ESCRT-III associated proteins. Functional analysis of the ESCRT-III interactions with Vps4 is described in Chapter 2. References 1. Palade, G. 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CHAPTER 2 ASSEMBLY OF THE AAA ATPASE VPS4 ON ESCRT-III Reprinted with permission from Ó Molecular Biology of the Cell Mol Biol Cell. 2010 Mar 15;21(6):1059-71. Epub 2010 Jan 28. Assembly of the AAA ATPase Vps4 on ESCRT-III. Shestakova A, Hanono A, Drosner S, Curtiss M, Davies BA, Katzmann DJ, Babst M. Assembly of the AAA ATPase Vps4 on ESCRT-III Anna Shestakova, * Abraham Hanono, * Stacey Drosner, *t Matt Curtiss, * Brian A. Davies,:!: David J. Katzmann,:!: and Markus Babst* *Department of Biology, University of Utah, Salt Lake City, UT 84112-9202; and +Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905 Submitted July 15, 2009; Revised January 6, 2010; Accepted January 19, 2010 Monitoring Editor: Sandra Lemmon Vps4 is a key enzyme that functions in endosomal protein trafficking, cytokinesis, and retroviral budding. Vps4 activity is regulated by its recruitment from the cytoplasm to ESCRT-III, where the protein oligomerizes into an active ATPase. The recruitment and oligomerization steps are mediated by a complex network of at least 12 distinct interactions between Vps4, ESCRT-III, Istl, Vta1, and Did2. The order of events leading to active, ESCRT-Ill-associated Vps4 is poorly understood. In this study we present a systematic in vivo analysis of the Vps4 interaction network. The data demonstrated a high degree of redundancy in the network. Although no single interaction was found to be essential for the localization or activity of Vps4, certain interactions proved more important than others. The most significant among these were the binding of Vps4 to Vta1 and to the ESCRT-III subunits Vps2 and Snf7. In our model we propose the formation of a recruitment complex in the cytoplasm that is composed of Did2-Istl-Vps4, which upon binding to ESCRT-III recruits Vta1. Vta1 in tum is predicted to cause a rearrangement of the Vps4 interactions that initiates the assembly of the active Vps4 oligomer. INTRODUCTION Plasma membrane proteins are continuously endocytosed and either sorted into the multivesicular body (MVB) path­way for eventual degradation in the lysosome (vacuole in yeast) or recycled to the plasma membrane. Thus, modula­tion of surface protein trafficking is an important regulatory element for numerous cellular responses, such as growth factor receptor function, nutrient uptake, cell-cell commu­nication and the immune response (for review, see Piper and Katzmann, 2007; Davies et aZ., 2009; Raiborg and Stenmark, 2009; Saksena and Emr, 2009). Ubiquitination of endosomal cargo proteins initiates their sorting into the MVB pathway where they are sequestered into vesicles formed when the outer/limiting endosomal membrane invaginates into the lumen of the compartment, giving the structure a multive­sicular appearance. MVBs then fuse with Iysosomes, deliv­ering the vesicles into the lumen of the hydrolytic compart­ment for degradation. The proper recognition and sorting of ubiquitinated cargoes requires the coordinated interaction of the Vps4 ATPase and the ESCRT (endosomal-sorting com­plex required for transport) protein complexes that work in sequence to mediate MVB protein sorting and vesicle for­mation events (for review, see Babst, 2005; Hurley and Emr, 2006; Williams and Urbe, 2007). Current models suggest that This article was published online ahead of print in MBC il1 Press (http://www. molbiolcell.orgl cgi I doi 110.1091 I mbc.E09-07-0572) on January 28, 2010. t Present address: Nelson Laboratories, 6280 S. Redwood Road, Murray, UT 84123. Address correspondence to: Markus Babst (babst®biology.utah.edu). Abbreviations used: AAA, ATPase associated with various cellular activities; ESCRT, endosomal-sorting complex required for trans­port; MlM1/2, MIT-interacting motif 1/2; MIT, microtubule-inter­acting and transport; MVB, multivesicular body. an initial cargo-sorting event occurs when the ubiquitinated cargo is recognized by the ESCRT-O and ESCRT-I complexes on the cytoplasmic side of the MVB membrane. ESCRT-I subsequently activates ESCRT-II, which, in tum, initiates the formation of ESCRT-IIL This latter step is thought to concen­trate cargo and recruit additional factors, including Vps4. Vps4 is an AAA (ATPase associated with various cellular activities)­type ATPase that releases ESCRT-III from the MVB membrane for additional sorting events, which is the final discemable step in the MVB-sorting process. In the absence of Vps4 function, the ESCRT machinery accumulates on the endosome, and ves­icle formation is inhibited. The formation of MVB vesicles requires membrane defor­mation and fusion steps that utilize a reversed topology compared with other vesicle formation events in the cell (e.g., clathrin- and COP-mediated vesicle formation). Inter­estingly, the ESCRT machinery has been implicated in two other membrane fusion events with reverse topology. Ret­roviruses such as HIV -1 form new virus particles a t the plasma membrane by an ESCRT-dependent budding event, and the final step in cytokinesis requires the ESCRT-depen­dent abscission of the plasma membrane in order to form two separate cells (Garrus et aI., 2001; VerPlank et aI., 2001; Spitzer et aZ., 2006; Carlton and Martin-Serrano, 2007; Morita et aZ., 2007; McDonald and Martin-Serrano, 2009). The assembly of ESCRT-III on endosomal membranes has been suggested to both concentrate cargo and deform the membrane, two essential steps in formation of MVB vesicles (Babst et al., 2002; Hanson et aZ., 2008; Wollert et al., 2009). Yeast ESCRT-III is composed of four subunits (Vps2, Vps20, Vps24, Sn£7), each of which has at least one homologue in mammalian cells. These four subunits are predicted to have similar three-dimensional structures (Muziol et aI., 2006). In the cytoplasm the ESCRT-III subunits are in a "closed" inactive conformation that inhibits complex formation. On the endosomal membrane, however, the ESCRT-III subunits seem to switch to an "open" conformation that promotes the formation of ESCRT-III and allows interactions with other ESCRT-III-associated factors (Shim et al., 2007; Lata et aI., 2008; Bajorek et aI., 2009b). Within ESCRT-III the four sub­units form two functionally distinct subcomplexes (Babst et aI., 2002). The Vps20-Snf7 subcomplex interacts with the endoso­mal membrane and with ESCRT-U, the complex that initiates ESCRT-IIl formation. The second subcomplex, Vps2-Vps24, is recruited by Vps20-Snf7 and thus functions downstream of the Vps20-Snf7 subcomplex. Although recent studies pro­vided new insights into the arrangement of the subunits within ESCRT-III (Saksena et al., 2009), the overall structure of ESCRT­III remains to be determined. Vps4 belongs to the large protein family of AAA-type ATPases (for review, see Lupas and Martin, 2002). These proteins function as mechano-enzymes that, in most studied cases, use the energy of ATP hydrolysis to induce confor­mational changes in the bound substrate. Vps4 is composed of an N-terminal substrate-binding domain, called the MIT (microtubule interacting and transport) domain, one central AAA domain (hallmark of type-l AAA ATPases), and a C-terminal region that is involved in Vps4 dimerization (Babst et aI., 1998; Scott et aI., 2005a; Gonciarz et aI., 2008; Vajjhala et al., 2008). Vps4 uses energy from ATP hydrolysis to disassemble ESCRT-lll, thereby recycling the ESCRT-Ill subunits for additional rounds of MVB cargo sorting. The disassembly reaction is initiated by the recruitment of Vps4 monomers or dimers from the cytoplasm to ESCRT-UI, where they assemble into the active oligomeric ATPase (Babst et al., 1998). Because of its dynamic nature the struc­tural analysis of the Vps4 oligomer has been problematic and controversial. However recent studies indicate that the Vps4 oligomer is composed of 12 subunits that assemble into two hexameric rings in a tail-to-tail (antiparallel) orientation (Yu et al., 2008; Landsberg et al., 2009). Both the recruitment of Vps4 and the consequent disassembly reaction require the interaction of the Vps4 MIT domain with the ESCRT-III substrate. This interaction is mediated by two distinct mo­tifs, termed MIMs (MIT-interacting motifs), found in the ESCRT-III subunits. MIMI motifs are located in the very C-terminus of subunits Vps2 and Vps24, whereas MIM2 motifs are found in the C-terminal regions of the Vps20 and Snf7 subunits (Obita et al., 2007; Stuchell-Brereton et al., 2007; Kieffer et aI., 2008; Shim et al., 2008). These two motifs bind to distinct surfaces of the MIT domain, allowing both types of interactions to occur simultaneously. Phenotypic analyses have indicated that both types of MIM motifs are important for proper Vps4 activity, but it remains unknown if all four potential yeast ESCRT-lll MIM sites are functional. Both recruitment and assembly of Vps4 are aided by additional factors that seem to ensure proper localization and timing of the disassembly reaction. Istl and Did2 have been implicated in the recruitment of Vps4 to ESCRT-III, whereas Vtal has been shown to support the assembly and ATPase activity of Vps4 (Yeo et al., 2003; Shiflett et al., 2004; Azmi et aI., 2006; Lottridge et aI., 2006; Nickerson et al., 2006; Azmi et al., 2008; Dimaano et al., 2008; Rue et nl., 2008). A fourth factor, Vps60, seems to function together with Vtal at late stage of Vps4 activation; however, its precise role re­mains unknown (Ward et aI., 2005; Azmi et aI., 2008; Rue et aI., 2008; Shim et nl., 2008). Together, Vps4, ESCRT-III, Did2, Istl, and Vtal form a complex network of interactions that leads to the formation of an active ATPase complex and the disassembly of ESCRT-III. Although much is known about each of these interactions individually, the temporal ar­rangement of the interactions and how they work together to achieve the disassembly reaction is poorly understood. We present a detailed in vivo analysis of the interactions known to be involved in the recruitment and assembly steps of Vps4. MATERIALS AND METHODS Antibodies The anti-HA (hemagglutinin) mAb used for immunoprecipitations and Western blotting was purchased from Covance (princeton, NJ). The antisera against Vps4, 5nf7, and Vps24 were previously described (Babst et aI., 1998). The antiserum against Vps20 was a gift from Scott D. Emr (Cornell University, itl1aca, NY). Strains and Media Saccharomyces cerevisiae strains llsed in this work are listed in Table 1. To maintain plasmids, yeast strains were grown in corresponding complete synthetic dropout medium (Sherman et ai., 1979). Wild-type, integrated, and knockout strains were grown in rich YPD medium (yeast extract-peptone­dextrose). Yeast gene knockouts were constructed as previously described (Baud in et al., 1993). DNA Manipulations Plasmids used in this study are listed in Table 1. All plasmids were con­structed using standard cloning techniques. Plasmids obtained by PeR-based cloning techniques were confirmed by DNA sequencing. Point mutations were introduced using Stratagene QuikChange Site-Directed Mutagenesis Kit (Agilent Teclu10logies, La Jolla, CAl. The pRS4XX shuttle vectors used in this study have been described previously (Christianson et al., 1992). pEGFP-C1 was from Clontech Laboratories (Palo Alto, CA). MIT-green fluorescent pro­tein (GFP) was constructed by fusing EGFP to the Ec047III site of VPS4. GFP-VPS4'MIT-type constructs were obtained by fusing a fragment contain­ing the PRCl promoter and GFP of pG036 into the Ec0471T1 site of a VPS4- containing plasmid. Procedures Fluorescence microscopy was perfonned on a deconvolution microscope (Delta Vision, Applied Precision, Issaquah, WA). The distribution of MIT-GFP was quantified from deconvoluted Z-stack projections of cells using an open­source GlMP 2.6.6 program (www.gimp.org). To achieve a more uniform level of protein expression in cells MIT -GFP was expressed from a CPSl promoter (pMC48 and pMCSO). The percent signal on endosomes was calcu­lated by dividing endosomal fluorescence intensity by total cellular fluores­cence intensity. Using an unpaired t test, p values were calculated using Instat softvvare. Subcellular fractionation experiments were performed as described previously (Dimaano et ai., 2008), except for subcellular fractionation exper­iments localizing Vps4, where spheroplasted cells were lysed by osmotic stress in Pop buffer (100 mM KCI, 50 mM KAc, 20 mM PIPES, pH 6.8, 5 mM MgAcz, 100 mM sorbitol) containing 0.1 mM AEBSF and Complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN). When localizing ESCRT-III components, spheroplasts were lysed by douncing with 15-20 strokes in a glass homogenizer in PBS containing Complete protease inhibitor cocktail. lmmunoprecipitabon experiments were performed as de­scribed previously (Babst et al., 2002). For in vitro binding experiments glutathione S-transferase (GST) fusion proteins were expressed in Escherichia coli and purified by affinity purification using standard methods on glutathi­one- Sepharose 4 Fast Flow resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The purification of Vps4E233Q, Istl, and Vta1 were previously de­scribed (Babst et ai., 1998; Azmi et ai., 2006; Dimaano et ai., 2008). To test the interaction between proteins in vitro, 40 Ilg of purified GST fusion protein was prebound to - IS-ILl bead volume of glutathione-Sepharose 4 Fast Flow resin. Equimolar amounts of purified test proteins were then added to the resin in GST pulldown buffer (100 mM KAc, 5 mM MgAc20 20 mM HEPES, pH 7.4) to a final volume of 150 III and incubated for 10 min at room temperature with gentle mixing; 1 mM ATP or ADP was included as indicated. An unbound sample was taken, and the bound protein was washed extensively in GST pulldown buffer. The bound protein was eluted by boiling for 5 min in SDS-PAGE sample buffer (2% SDS, 0.1 M Tris, pH 6.8, 10% glycerol, 0.01% bromophenol blue, 5% is-mercaptoethanol), and 10 JLl of each sample was separated by SDS-PAGE and stained by Coomassie Brilliant Blue. The liquid overlay assay was performed as previously described (Darsow et al., 2000). RESULTS The recruitment of Vps4 subunits from the cytoplasm to ESCRT-III and the consequent oligomerization of Vps4 into the active dodecameric state are not well understood. The study of this process is complicated by the number of Vps4 interactions that have been reported, some of which overlap and compete with each other (Figure lA). Furthermore, many Vps4 interaction studies have been performed in vitro Table 1. Strains and plasmids used in this study Strain or plasmid Straina SEY6210 BHY10 MBY2 MBY3 MBY16 MBY28 BWY102 MBY37 JPY50 MCY3 EEYl-3 EEY2-1 EEY8 EEY9 EEY12 EEY26-1 ASY4 ASY5 ASY8 ASY9 ASY12 ASY16 ASY19 ASY20 E. coli: XU-blue Plasm ids pAS22 pAS23 pAS28 pAS42 pAS44 pAS47 pAS51 pAS58 pAS59 pAS62 pAS72 pAS74 pAS76 pAS79 pMB4 pMB66 pMB168 pMB341 pMB343 pMB370 pMB380 Descriptive nan1e WT WT CPY-I vps4Ll CPY -I vps4Ll vps4Ll vps36Ll vps2Ll vps24Ll vps4Ll vps20Ll vps4Ll vlalLl vps4Ll isllLl vps20Ll CPY-T vps20Ll snj7Ll CPY-I snj7Ll vps4Ll snj7 Ll vps4Ll did2Ll vps4Ll vps2 Ll vps24 vps2Ll vps24Ll vps2 Ll CPY-I vps24Ll CPY-I vps2Ll vps24Ll CPY-I vps4Ll vps20Ll snj7 Ll snj7Ll vps20Ll CPY-I vps4Ll vps20Ll snj7 Ll isllLl vps2(LlC)-HA vps24(LlC)-HA vps2(LlC)-HA vps2(LlC)-HA, vps24(LlC)-HA MlT-GFP vps2(LlC)-HA, vps24(LlC)-HA GFP-vps4(LlMIT(E233Q, S377 A)) vps2(LlC)-HA, vps24(LlC)-HA MIT(L64D )-GFP vps4 (LlMlT(E233Q)) snj7(L199D) vps20(L188D) vps20(L188D)-HA vps4(S377 A) VPS4 vps4(E233Q) VPS20-HA GFP-vps4( LlMlT(E233Q)) vps4(E233Q)-GFP vps4(L64D, E233Q)-GFP vps4(T18D, E233Q)-GFP Genotype or description MAT", feu2-3,112 ura3-52 his3-Ll200 Irpl-Ll90I fys2-80I sllc2-!l9 SEY6210, CPY-INVERTASE::LEU2 ura3-52 his3-Ll200 Irpl-Ll90I fys2-80I suc2-Ll9 BHY10, VPS4::TRPI SEY6210, VPS4::TRPI SEY6201O.1, VPS4::TRPl, VPS36::HIS3 SEY6210, VPS2::HTS3 SEY6210, VPS24::HIS3 MBY3, VPS20::HIS3 MBY4, VTAI::HIS3 MBY3, IST1::HIS3 BHY10, VPS20::HTS3 6210, VPS20::HfS3 BHY10, SNF7::HIS3 SEY6210, SNF7::HIS3 MBY3, SNF7::HTS3 MBY3, DID2::HIS3 6210.1, VPS4::TRPI, VPS2::HIS3, VPS24::HIS3 SEY6210, VPS24::HIS3, VPS2::HIS3 BHY10, VPS2::G4I8 BHY10, VPS24::G4I8 ASY8, VPS24::HIS3 MBY37, SNF7::G418 EEYl-3, SNF7::G4I8 ASY16, IST1::URA3 recAl endAl gyrA96 thi-1 hsdR17 supE44 relAl lac [F' proAB ladqZLlM15 TnlO(tetr)] URA3 (pRS416) vps2(LlC)-HA URA3 (pRS416) vps24(LlC)-HA LEW (pRS415) vps2(!1C)-HA TRPI (pRS414) vps2(LlC)-HA, vps24(LlC)-HA URA3 (pRS426) vps4(MIT)-GFP URA3 (pRS416) vps2(LlC)-HA, vps24(LlC)-HA URA3 (pRS416) GFP-vps4(LlMIT(E233Q, S377 A)) LEW (pRS415) vps2(LlC)-HA, vps24(LlC)-HA URA3 (pRS426) vps4(M1T (L64D))-GFP URA3 (pRS416) vps4(LlMIT(E233Q)) LEW (pRS415) snj(L199D) LEW (pRS415) vps20(L188D) URA3 (pRS416) vps20(L188D)-HA Ul~3 (pRS416) vps4(S377A) HIS3 (pRS413) VPS4 HIS3 (pRS413) vps4(E233Q) URA3 (pRS416) VPS20-HA URA3 (pRS416) P(CPY)-GFP-vps4(LlMIT(E233Q)) URA3 (pRS416) vps4(E233Q)-HA-GFP URA3 (pRS416) vps4(L64D, E233Q)-HA-GFP URA3 (pRS416) vps4(T18D, E233Q)-HA-GFP Reference or source Robinson el af. (1988) Horazdovsky el af. (1994) Babst el af. (1997) Babst el af. (1997) Babst et al. (2002) Babst el al. (2002) Babst et al. (2002) Babst et af. (2002) Azmi el af. (2006) Dimaano el af. (2008) This study Babst et al. (2002) This study Babst et af. (2002) Babst et af. (2002) Dimaano et al. (2008) This study This study This study This study This study This study This study This study Stratagene (La Jolla, CAl This study This study This study This study This study This study This study This study This study This study This study This study This study This study Babst et af. (1997) Babst et af. (1998) Babst et al. (2002) This study This study This study This study Conlinued Table 1. Continued Reference or Strain or plasmid Descriptive nan1e Genotype or description source pMB393 snp(Ll99D), vps20(Ll88D) URA3 (pRS416) snf7(Ll99D), vps20(Ll88D) This study pMB394 snp(Ll99D), vps20(LI88D) LEU2 (pRS415) snp(LI99D), vps20(LI88D) This study pPN3 VPS20 LEU2 (pRS415) VPS20 This study pVPS4(I180) vps4(118D) URA3 (pRS416) vps4(l18D) This study pVPS4(L640) vps4(L64D) URA3 (pRS416) vps4(L64D) This study pVPS4(I180, vps4(l18D, E233Q) URA3 (pRS416) vps4(l18D, E233Q) This study E233Q) pVPS4(L640, vps4(L64D, E233Q) URA3 (pRS416) vps4(L64D, E233Q) This study E233Q) pG045 GFP-CPS URA3(pRS426) GFP-CPSI Odorizzi et at. (1998) pMC48 MIT-GFP URA3(pRS416) P(CPSl)-vps4(MlT)-GFP This study pMC50 MlT(118D)-GFP URA3 (pRS416) P(CPSl)-vps4(M1T(l18D))-GFP This study pAH31 GST-DID2 (pGEX-KG) GST-DID2 This study pAH32 GST-CT(DID2) (pGEX-KG) GST-DID2(113-204) This study pMB411 GST-vps20IC) (pGEX-KG) GST-vps20(101-221) This study pMB412 GST-snp(C, LI99D) (pGEX-KG) GST-snp(101-240)(LI99D) This study pMB413 GST-snp(C) (pGEX-KG) GST-snpOOI-240) This study pMB414 GST-vps20(C, LI88D) (pGEX-KG) GST-snpOOI-221)(LI88D) This study pAS85 snp(LI99D), vps20(L188D), MlT-GFP LEU2 (pRS415) snp(LI99D), vps20(LI88D), P(CPSl)-MlT-GFP This study a All strains are Saccharomyces cereuisiae are except the one marked E. coli. in the presence of only one or a few of the ESCRT factors, which raises questions of the relevance of these observations for the in vivo situation. Therefore we dissected the Vps4 interactions in vivo by expressing different truncated or mutated forms of Vps4 in a variety of ESCRT deletion strains (see Table 2). These data resulted in a model for the recruit­ment and assembly of Vps4 on MVBs (Figure 1B). Analysis of the Vps4 MIT Interactions The MIT domain of Vps4 has been shown to interact with six different proteins of the ESCRT machinery: the four subunits of ESCRT-Ill (Vps2, Vps20, Vps24, Snf7) and two factors that are related to ESCRT-III subunits and have been implicated in the endosomal recruitment of Vps4, Did2, and Istl (Figure lA; Shim et al., 2007; Azmi et al., 2008; Dimaano et aI., 2008; Kieffer et aI., 2008; Xiao et aI., 2008). These six factors bind via two distinct binding motifs, MIMI and MIM2, to two differ­ent surface areas of the MIT domain (Figure 1C). This ar­rangement allows the MIT domain to simultaneous bind to MIMI and MIM2. To test which of the MIT interactions are involved in the recruitment of Vps4 to ESCRT-IIl, we ex­pressed a MIT-GFP fusion construct in yeast strains deleted for VPS4 alone or in combination with other ESCRT muta­tions. These yeast cells were analyzed by fluorescence microscopy, and the resulting pictures were judged by the ratio of MIT-GFP signal localized either to the cytoplasm or to the aberrant endosomes formed in these mutant strains (class E compartments; Figure 2A). Enlarged exam­ples of the microscopy pictures are shown in Supplemental Figure 1. Furthermore, for a subset of MIT-GFP localization experiments, the ratio of endosomal-to-cytoplasmic localiza­tion was quantified (Figure 2B). Previous studies have shown that vps4/l cells accumulate ESCRT-III and its associated proteins Did2 and Istl on en­dosomal membranes (Babst et al., 2002; Nickerson et al., 2006; Dimaano et al., 2008). Thus the majority of MIT-GFP local­ized to class E compartments in cells deleted for VPS4 and to cytoplasm in wild-type cells (Figure 2, A and B, 1 and 2). Additional deletions of OID2 or ISTl resulted in a partial redistribution of MIT -GFP to the cytoplasm (Figure 2, A and B, 7 [p < 0.0001] and 8 [p < 0.0001]). This result is consistent with previously published models in which Did2 and Istl function together in the recruitment of Vps4 (Dimaano et al., 2008; Rue et aI., 2008). In contrast, deletion of VTAI did not interfere with recruitment of the MIT domain (Figure 2, A and B, 9) supporting a later role for this Vps4-interacting protein. The ESCRT-III subunits Vps2 and Vps24 both contain C-terminal MIMI interaction sites (Obita et al., 2007; Stuch­ell- Brereton et aI., 2007). To study the effect of loss of MIMI, 3' truncations of VPS2 and VPS24 were constructed that lacked the co dons for the last 11 amino acids (vps2"'c, vps24"'c, Figure Ie. Table 2). These mutations did not affect stability or MVB recruitment of Vps2 and Vps24 (Figure 2C, lanes 3-6). A partial redistribution of MIT-GFP to the cyto­plasm was observed in vps4/l strains that lacked the Vps2 MIMI motif (Figure 2, A and B, 5; P < 0.0001). The loss of Vps24 MIMI had no effect on MIT-GFP localization (Figure 2A, 6). However, when combined with vps2"'c the deletion of Vps24 MIMI showed a small but significant increase in MIT-GFP relocalization (Figure 2, A and B, 4; P < 0.0048). These data indicated that, although both MIMI sites are functional, the Vps2 MIMI site is more important for Vps4 recruitment than the Vps24 MIMI motif. Deletion of both VPS2 and VPS24 abolished MIT-GFP recruitment (Figure 2A, 3), which can be explained by the fact that this strain not only lacks the ESCRT-III MIMI sites but in addition does not properly localize the other two MIMI-containing proteins Did2 and Istl (Nickerson et al., 2006; Dimaano et al., 2008). Similarly, a mutation in the MIT domain shown in mammalian Vps4 to block the MIT- MIMI interaction (MITL64D; Figure 1C; Stuchell-Brereton et aI., 2007) did not localize to class E compartments of vps4/l cells, supporting the idea that loss of all MIMI sites both in ESCRT-III as well as in the recruitment factors Did2 and Istl abolishes recruitment of MTT-GFP (Figure 2A, 14). A C hlST1 352FDDLSRRFEE LKKKT Vps20, Snf7 _ Mn~2 Ist1 285LDELKKRFDALRRK I (D ® .. i CHMP2A 209DAD LEE RLKNLRRD MIM1 ® ~ Vps2 218DDDLQARLNTLKKQT Vps2, Vps24 _~ Vps4 I @ I AAA ® M)M7 ®! I® ~ .... I® I@ CHMP3 210LEAMQSRLATLRS Vps24 ~2VNEMRE RL RA L QN ~ c hlST1 323FVLPELPSVPDTLP336 Did2_o~ ~~ Vta1 (dime,) ~IM1+2 N CHMP6 168IELPEVPSEPLPEK179 ~ Vps20 186EGLPSLPQGEQTEQ199 :i!: CHMP4B 189VPLPNVPSIALPSK202 B Vps2, Vps20, Vps4 Ist1 Did2 (closed) New Interactions: 6,7,9,10 Lost Interactions: j 4 1,2,8 9, 10 IIst1 Snf7 197VSLPSVPSNKI KQS21O * 11 12 Figure 1. Interactions between Vps4 and its substrate and regulators. (A) Vps4 interaction network based on previous studies (Scott et al., 2005b; Azmi et al., 2008; Kieffer et aI., 2008; Bajorek et al., 2009a; Xiao et al., 2009). (B) Model for the recruitment and assembly of Vps4. The numbers indicating New Interactions or Lost Interactions refer to the numbers in A. (C) Alignments of the putative MIMI and MIM2 motifs of yeast and mammalian ESCRT-III subunits (yeast Isll does not contain an obvious MIM2 consensus sequence). Mutations used in this study are marked in red. To test the effect of loss of Vps2 and Vps24 MIMI motifs on the recruitment of full-length Vps4, we fractionated cell extracts by centrifugation, separating the soluble, cy­toplasmic pool of Vps4 (S) from the endosomal fraction found in the pellet (P). As previously observed, in wild­type cells the ATP-Iocked mutant VpS4E233Q accumulated in the pellet fraction, indicating efficient recruitment of VpS4E233Q to ESCRT-lll (Figure 2C, lanes 1 and 2; Babst et aI., Table 2. Mutations used in this study Name VpS4"8D VpS4L64D VpS4dMIT VpS4S377A VpS4E233Q Vps2dC Vps24d C VpS20L1 88D Snf7L1 99D Mutation Ile(18) to Asp Leu(64) to Asp aa 2-87 deleted Ser(377) to Ala Glu(233) to Gin aa 222-end deleted aa 214-end deleted Leu(188) to Asp Leu(199) to Asp Affected interactions (see Figure lA) 1,10 2,10 1,2,10 8 None (ATP locked) 2 2 1 1998). Similarly, mutant strains that lacked the MIMI motif of either Vps2 or Vps24 efficiently recruited VpS4E233Q to membranes (Figure 2C, lanes 3-6). The lack of both MIMI sites caused a partial redistribution of VpS4E233Q to the soluble fraction, whereas Vps2"c or Vps24"c remained in the membrane-bound pellet fraction (Figure 2C, lanes 7 and 8). These results supported the notion that the ESCRT-III MIMI motifs are functionally redundant. However, the re­cruitment defects observed by cell fractionation were less severe than the MIT-GFP localization phenotypes, which can be explained by the presence of Vps4-Vps4 and Vps4 - Vtal interactions that stabilize ESCRT-III-associated Vps4 oligomers (see below). We tested the importance of the MIM2 interaction mo­tifs in Vps4 recruitment by mutating a conserved leucine residue of the Vps20 and Snf7 MIM2 sites (L188D in Vps20 and L199D in Snf7) that has been previously shown to be important for the MTM2-MTT interaction of the mammalian ESCRT system (Figure IC; Kieffer et al., 2008). The effect of these mutations on the interaction with yeast Vps4 was first analyzed in vitro using immobilized GST-fusion proteins containing the C-terminal half of either SnO or Vps20. Sepharose beads presenting either wild-type or mutant ver­sions of the fusion proteins were incubated with recombi­nant VpS4E233Q in the presence of either A TP or ADP. The A B c • Strain MIT -GFP L~. InL Strain MIT -GFP L~. Int. WT 2 vps4/3, vps4/3, 3 vpsZt. vps2411 vps46 4 vps211C vps24uC vps46 vpsZ",C 6 vps46 vps24°C vpS4A did2c. vps4/3, ist1A vps4ll. vta16 1.2 1.0 0.6 0.6 0.4 0.2 1 0.0 WT Int.: WT 10 vps4!J. 1180 +/- 1,(10) vps4t:. 1,2, WT + 11 vps2JiC 1180 (10) vps24f.C WT - 2,10 12 vps411 1180 1, 10 did26 +/- vps4A • WT 2 13 ist1 tt. 1180 1,10 WT +/- (2) ,. vps411 L54D ,p 2, (10) vps4!J. WT + (2) 15 vps206 WT 1,2, snf1tJ. 10 WT +/- 10 16 vpS4fl WT +/- (1) vps20L1811D WT +/- 10 17 vpS411 WT +/ - (1) snf7L1990 vps4tJ. WT + 18 vpS20L188D WT +/- snfTL199D vps46 ist16 19 vps20L18S0 snfTL199D WT +/- 1,10 I I ~. vps46 vps41l vps46 vps4a. vps41l vps46 vps2JiC did2tJ. vta1lt. snf7Lf99D MIT (118D) vps24JiC_ L- - L- - - - ~ vps411 vps4!J. vps4tJ. vps4!J. vps2 '"'C ist1t. vps20L188D vps20L188D snf7L1990 (2) (21 (1) 1, 2,8, (1) 9, 10 Q~ ~~ u~ ~~~ il i ~ ~ - ~ vps461st1 11 vps20L188D snf7L1990 ~ ~ ~~ ~ ~~ ~~ ~ vpS4E233Q: _~ ___~_ __~ _ ~ _._...!!...~-.L PSPSPSPS PSPSPSPS VpS4E211Q _ ..... _ ____ ____ _ ___ _ _ -VpS4E233Q VPS2 ~-HA,--­VPS24M:- HA/ - -- ~ __ VpS20l1880_HA _Snf7l1 '190 1 2 3 4 5 6 7 8 910111213141516 o bound unbound GST-Snf7(C) GST-Vps20(C) GST-Snf7(C) GST-Vps20(C) ~ l199D~ l188D ~ l199D ~ L188D ATP AOP ATP ADP ATP ADP ATP ADP ATP ADP ATP ADP ATP ADP ATP ADP VPS4E25~ ____ ___ ~. - -- -~ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Figure 2. MIMI and MIM2 interactions contribute to the recruit­ment of Vps4 to ESCRT-lIl. (A) Fluorescence microscopy analysis of the Vps4 MIT domain fused to GFP (MIT-GFP). The wild-type, MIMI mutant (L640), or MIM2 mutant (1180) version of MIT-GFP was expressed in different yeast strains (see Table 1), and the extent of endosomallocalization was determined (Loc.). For better visual­ization the fluorescence microscopy pictures were inverted and the intensity was adjusted to the individual brighhless range (black is the brightest Signa l). Vps4 interactions affected by the different mutations are listed ([nt., numbers are based on the interactions in Figure lA). Numbers in parentheses indicate partially disrupted resulting bound and unbound fractions were then analyzed by SDS-PAGE (Figure 2D). Wild-type, but not L188D Vps20 was able to bind to Vps4 in presence of ATP (lane 5), demonstrating that the mutation indeed inhibited the Vps20 MIM2-MIT interaction. In contrast, neither wild-type nor mutant Snf7 bound to Vps4 under the conditions tested. These results are consistent with previously published data from both yeast and mammalian systems that suggested a preferred interaction of Vps4 with Vps20 rather than Snf7 (Azmi et al., 2008; Kieffer et al., 2008) . When expressed in yeast, the mutant proteins VpS20L188D and Snf7L199D were stable and efficiently recruited to endo­somal membranes, indicating that the mutations did not interfere with normal protein folding (Figure 2C, lanes 9-10 and 15- 16). However, when analyzed by SDS-PAGE the L199D mutation in Snf7 resulted in an apparent size shift, possibly because of the additional negative charge (Figure 3B, lanes 19-22). Mutation of each of the MIM2 motifs resulted in partial loss of MIT-GFP recruitment to ESCRT-lll (Figure 2, A and B, 16 [p < 0.0001] and 17 [p < 0.0001]). Similarly, the combination of both mutations impaired en­dosomallocalization of MIT-GFP (Figure 2, A and B, 18; P < 0.0001). Together, these data indicated that both ESCRT-ITI MIM2 motifs are important for MIT recruitment even though the in vitro data suggested only a minor role of Snf7 in the interaction of ESCRT-III with Vps4 (Figure 2D). To test the effect of Snf7 and Vps20 MIM2 mutations on the recruitment of full-length Vps4, we performed subcellu­lar fractionation experiments. MTM2 mutations in either Snf7 or Vps20 or the combination of both did not significantly redistribute Vps4 into the cytoplasmic fraction (Figure 2C, lanes 9-10 and 13-16). This result suggested, as observed with MIT-GFP, that MIMI interactions playa more impor­tant role in the recruitment of Vps4 than the MIM2 motifs. Deletion of both SNF7 and VPS20 resulted in a relocalization of VpS4E233Q to the cytoplasm, which is likely caused by the loss of endosomal ESCRT-III and its associated proteins in this strain (Figure 2C, lanes 11 and 12). The function of the MIM2 interaction was further ana­lyzed by mutating the MIM2-binding site in the MIT domain of Vps4. A previous study indentified the valine at position 13 of human VPS4A as a crucial amino acid for the interac­tion between MlM2 and the MIT domain (Kieffer et aI., 2008). However, the corresponding mutation in yeast Vps4 (V14D) resulted in an unstable protein, suggesting that this muta­tion might interfere with protein folding (Supplemental Fig­ure 2). Therefore we changed isoleucine at position 18 to aspartate, a mutation that based on published NMR analysis is predicted to interfere with the MIM2 interaction (Kieffer et interactions. (B) Quantification of endosome-localized MIT-GFP rela­tive to wild type (0.0) and vps4t. (1.0). The data shown represent the results of at least 15 individually analyzed cells. Nwnbers refer to the experiment number in A. (C) Subcellular fractionation of different yeast strains expressing VpS4E233Q into soluble, cytoplasmic fraction (S) and pelletable, membrane-associated fraction (P). Fractions were analyzed by Western blot using antibodies specific for Vps4 (top panels), Snf7 (bottom panel, lanes 9-14) and the HA-tag (bottom panels, lanes 1-8 and 15-16). Vps4 interactions affected by the different mutations are listed (Int., numbers are based on the interactions in Figure lA). Num­bers in parentheses indicate partially disrupted interactions. (0) In vitro Vps4 interaction studies using wild-type and MIM2 mutant forms of GST-Vps20(C) (fusion of GST with C-tenninal half of Vps20) or GST-Snf7(C) immobilized on GSH-Sepharose. Vps4E233Q was added in the presence of ATP or AOP to immobilized proteins; bOWld and unbound fractions were analyzed by SOS-PAGE and Coomassie staining. A # Strain GFP-CPS Sorting Int. "Strain GFP-CPS Sorting Int. 1 2 3 • 5 • 7 8 B Vps4 complementation ESCRTIII MIM1/MIM2 complementation VP$4 • • + vps26 vps246 vps46 10 vps~C C'Q) +/ - ..... .. .' vps4 J1aO 0 D " (1 0) 11 vps246c - ./-. vps4 L&40 .r 2, (10) 12 vvppssU~c6 C q \ ~ : .. VpS4 S377A ~ +/- 13 vps206 :Aj;;; snf76 Vps4 dominant-negative VpS4'110,E233Q .. +/ - 1, (10) ,. snf7 l1g90 VPS4 'i" vps4 LUD,E23JQ .'-+ 2, (10) 15 VpS20 L111D 'f ::( .. !~ VP$4 vps4 MlIT,E23JQ ....... lit ) + 1,2, ,. snf7 l1g90 (~\~ VP$4 :.- lD VpS20 L111O Int. : 2,8, (2) 2,8, 2, (10) 1, (10) 9, 10 j 9, 10 o~~~ <:j ~ 'R. ~~ 'R.~ _",.". _ __~ _ ---.L ---.L ~ ~ ~ ~ PS P S P S P S P SPS P SPS Vps24- _. __ --__ -----....... - Snf7-~ - -_- ---.- __ - . _____ + Int.: VpS24 - Snf7:...... Snf7LIli19D_ --- ---.= .... _,...,- -- ~-- - ------ 2,8, 9, 10 +/- (2) + (2) +/ - 1,2,8, 9,10 + (1) + (1) + Figure 3. Phenotypic analysis of mutations affecting Vps4 interac­tions. (A) Fluorescence microscopy analysis of yeast strains expressing GFP-CPS. The efficiency of GFP-CPS sorting into the lumen of the vacuole is indicated (+, + / - , - ). Vps4 interactions affected by the different mutations are listed. (B) Subcellular fractionation of yeast strains into soluble (S) and membrane-associated pellet fractions (P). The samples were analyzed by Westem blot using antibodies specific for Vps24 and Snf7. Vps4 interactions affected by the different muta­tions are listed. (A and B) lnt., numbers are based on the interactions in Figure lA, and numbers in parentheses indicate partially disrupted interactions. aI., 200S). The I1SD mutation impaired Vps4 function with­out affecting in vivo protein stability (Figure 3, 3, and Table 3; Supplemental Figure 2). The mutation strongly inhibited the recruitment of MIT-GFP to class E compartments of vps4/l cells (Figure 2, A and B, 10; P < 0.0001), which is a more dramatic MIT recruitment phenotype than observed in the double MIM2 mutant strain (vps4/l vpS20L188D snf7L199D, Figure 2A, IS). A likely explanation for this result is that the I1SD MIT mutation not only affected the interaction with ESCRT-III but also the binding to the recruitment factor Istl (interactions 1 and 10, Figure 1A). Consistent with this idea we observed an increased recruitment defect when the vpS20L188D snf7L199D mutations were combined with a dele­tion of ISTl (Figure 2, A and B, 19; P < 0.0001). However, deletion of ISTl caused a complete loss of MIT"8D-GFP Table 3. CPY -invertase secretion Affected Vps4 interactions Strain Secretion ('Yo) (Figure 1A) Complementation Wild type 0 :': 0 None vps4tl 100 :': 8 All VpS4118D 59 :': 8 1,10 VpS4L64D 73 :': 4 2,10 VpS4S377A 12:': 0 8 vps2~ c 30 :': 10 2 vps24~c 27 :': 6 2 vps2~c, vps24tJ.c 48 :': 0 2 s nf1L199D 3 :': 1 1 vpS20L188D 3 :': 2 1 snj7L199D, VpS20L188D - 1 :': 1 1 Dominant-negative VPS4 0 :': 0 None VpS4E233Q, VPS4 100 :': 3 None VpS4/J80, E233Q, VPS4 68 :': 5 1,10 VpS4L64D, E233Q, VPS4 10 :': 0 2,10 VpS4tJ.MIT. E233Q, VPS4 - 7 :': 0 1,2,10 recruitment to the endosome, which is a more severe phe­notype than observed in the vpS20L188D snf7L199D isti/l strain (Figure 2A, 13 and 19). This result suggested either the existence of an additional unknown MIM2 motif or the possibility that the I1SD mutation might affect the MIM1- interaction site of the MIT domain. Combining the I1SD MIT mutation with strains lacking Did2 or the MIMI-binding sites in ESCRT-III resulted in loss of MIT-GFP recruitment (Figure 2A, 11 and 12), which suggested that MIMI and MIM2 interactions act synergistically in the recruitment of Vps4. The in vivo analysis of MIT domain localization to ESCRT-III has demonstrated a high level of redundancy in the Vps4 recruitment system. Not one of the tested MIT interactions has been found to be essential for recruitment. Only mutations that interfere with at least two of the known MIT interactions are able to disrupt binding of the MIT domain to ESCRT-Ill. Furthermore, the MIMI and MIM2 interactions act cooperatively in the recruitment of the MIT domain, suggesting that both interactions occur simulta­neously on ESCRT-III. In contrast to the microscopy analy­sis, subcellular fractionations have shown that in vps4/l MIT­GFP localizes to the soluble, cytoplasmic fraction (data not shown). This result suggested that binding of the MIT do­main to ESCRT-III is too weak to maintain the MIT-ESCRT­III interaction during the fractionation procedure, an obser­vation that fits well with the micromolar affinities found in vitro for MIT -MIM interactions (Obita et aI., 2007; Stuchell­Brereton et al., 2007; Kieffer et al., 200S). Functional Redundancy among the ESCRT-III MIM Motifs The MIT-GFP localization studies revealed redundancy in the function of the different MIT interaction motifs in Vps4 recruitment. To test if the ESCRT-III MIM sites are also functionally redundant in regard to MVB cargo sorting, we analyzed potential trafficking phenotypes in strains that lack either one or both of the ESCRT-III MIMI or MIM2 motifs. Newly synthesized carboxypeptidase S (CPS) is a type I transmembrane protein that traffics via the MVB pathway to the vacuolar lumen where it functions as a vacuolar hydro- lase (Odorizzi et al., 1998). Fluorescence microscopy of wild­type cells expressing GFP-tagged CPS (GFP-CPS) showed mainly staining of the vacuolar lumen (Figure 3A, 1). In contrast, ESCRT mutants lacked luminal staining of the vac­uole but instead accumulated GFP-CPS in aberrant endo­somes, the class E compartments, and the limiting mem­brane of the vacuole (Figure 3A, 2). Cells that expressed the MIMI mutant form of VPS2, vps2"c, exhibited a partial GFP-CPS sorting phenotype, whereas the loss of the VPS24 MIMI motif showed no obvious MVB trafficking defect (Fig­ure 3A, 10 and 11). Loss of both MIMI motifs resulted in a sorting defect similar than that of the vps2"c mutant strain, consistent with the MIT-GFP localization studies that indi­cated the Vps2 MIMI motif plays a more important role in Vps4 recruitment (Figure 3A, 12). The Vps4 L64D mutation caused a severe GFP-CPS sort­ing defect indicating that the loss of all MIMI interactions blocked Vps4 function (Figure 3A, 4). In contrast, the MIM2- interaction mutant VpS4"8D only partially inhibited the ac­tivity of Vps4 (Figure 3A, 3), which is consistent with the observed partial loss of MITI18D-GFP recruitment to endo­somes (Figure 2, A and B, 10). However, single and double mutants of the ESCRT-III MIM2 motifs resulted in normal GFP-CPS trafficking, further supporting the notion that mutants with weak MIT-localization defects show corre­spondingly weak (or no) MVB-trafficking phenotypes (Figure 3A, 14 -16). The soluble fusion protein CPY-invertase is synthesized and translocated at the endoplasmic reticulum, transported by the sorting receptor Vpsl0 from the trans-Golgi to a MVB and finally delivered to the vacuole. ESCRT mutants impair the recycling of Vpsl0 back to the trans-Golgi, thereby lim­iting the transport function of this receptor. As a conse­quence, ESCRT mutants secrete a portion of newly synthe­sized CPY-invertase, a phenotype that can be detected by a colorimetric assay (Table 3; Paravicini et al., 1992). The CPY-invertase assay revealed similar functional re­dundancies of the ESCRT-III MIMI motifs as observed by the analysis of GFP-CPS trafficking, although there was no clear difference in phenotypic severity between the vps2"c and vps24"c mutants. Loss of the Vps2 or Vps24 MIMI motif resulted in a modest secretion phenotype (vps2"C and vps24"c, -30% secretion relative to the secretion of vps4!l), whereas loss of both motifs caused a more severe phenotype (- 50% secretion relative to vps4!l). Mutation of the Vps4 MIMI interaction site (Vps4L64D) further enhanced CPY­invertase secretion (- 70% secretion), consistent with the idea that this mutation interferes with all potential Vps4 MIMI interactions, including those with Did2 and Istl. In contrast to the MIMI mutants, the ESCRT-III MIM2 mutant strains did not secrete CPY-invertase (Table 3). How­ever, mutating the Vps4 MIM2 interaction site (VpS4118D) caused detectable secretion (-60% secretion, Table 3), sug­gesting that loss of MlM2 interactions both on ESCRT-lll and Istl together resulted in a synthetic phenotype (interac­tions 1 and 10, Figure lA). Together, the phenotypic char­acterization of the different MIM mutants suggested that MIMI interactions are more important for Vps4 function than the MIM2 interactions. However, we cannot exclude the possibility that the mutations in the MIM2 motifs retain some functionality. The immediate consequence of the loss of Vps4 function is the accumulation of ESCRT-III on endosomes (Babst et aI., 1997). Therefore, we analyzed the effect of the MIM muta­tions on the localization of ESCRT-III subunits by fraction­ating cell extracts into soluble and membrane-associated pellet. In wild-type cells the majority of Vps24 and Snf7 localized to the soluble cytoplasmic pool (S), whereas dele­tion of VPS4 resulted in the accumulation of both proteins in the pellet fraction (P; Figure 3B, lanes 1-4). Similarly, delet­ing VPS2 alone or in combination with VPS24 caused a shift of Snf7 pool to the pellet fraction, consistent with the pub­lished function of Vps2/Vps24 in Vps4 activity (Figure 3B, lanes 5-6 and 9-10; Babst et al., 2002). In contrast, the ma­jority of Vps24 was found in the soluble fraction in vps2!l cells, which can be explained by the observation that Vps2 and Vps24 required each other for proper ESCRT-III assem­bly (Figure 3B, lanes 5 and 6; Babst et aI., 2002). Deletion of the VPS2 MIMI motif resulted in partial accumulation of both Vps24 and Snf7 (vps2"C-HA; Figure 3B, lanes 7 and 8), consistent with the partial trafficking phenotype associated with this strain. The additional deletion of the VPS24 MIMI motif further impaired but did not block the Vps4-depen­dent disassembly of ESCRT-llI (d. vps2"c-HA vps24"c-HA with vps4!l in Figure 3B, lanes 1-2 and 11-12), suggesting that the remaining MIM2 sites were sufficient to maintain some Vps4 activity. Consistent with the observed redun­dancy among the MIMI/2 interactions we found that mu­tating either the MIMI or the MIM2 interaction site in Vps4 only partially inhibited ESCRT-III disassembly (Figure 3B, lanes 13-16). Loss of the Vps20-Snf7 subcomplex in vps4!l caused the redistribution of Vps24 to the soluble, cytoplasmic fraction, an expected result based on previous publications (lanes 17 and 18, Figure 3B; Babst et al., 2002). Additional expression of vpS20L188D and snj7L199D in this strain restored ESCRT-III accumulation on endosomal membranes, indicating that the mutations in MIM2 did not interfere with the assembly of this protein complex (lanes 19 and 20, Figure 3B). However the mutation of the Snf7 MIM2 motif partially impaired the recycling of ESCRT-III from membranes whereas the corre­sponding mutation in Vps20 did not interfere with the Vps4- dependent disassembly reaction (lanes 21-26, Figure 3B). These observations suggested that although both MIM2 mo­tifs of ESCRT-III are involved in the binding of the Vps4 MIT domain only Snf7 MIM2 seems to play an important role in the ESCRT-III disassembly reaction. In summary the fractionation experiments indicated that MIMI and MIM2 motifs of ESCRT-III act together not only in the recruitment of Vps4 but also in the subsequent ESCRT-lIl disassembly reaction. Both type of MIT interac­tions are important for Vps4 activity and loss of MIMI or MIM2 affect recycling of both ESCRT-III subcomplexes, Vps20/Snf7 and Vps2/Vps24. Characterization of the Vps4 AAA Domain Interactions We tested the role of the Vps4 AAA domain interactions on the recruitment of the ATPase to the endosome in vivo by analyzing the localization of an ATP-Iocked Vps4 protein in which the MIT domain had been exchanged with GFP (GFP­VpS4" MIT.E233Q ). The only known binding partners of this protein are Vtal, Istl, and Vps4 itself (interactions 8, 9, 11, and 12 in Figure lA). In vps4!l cells GFP_Vps4"MIT.E233Q localized mainly to class E compartments (Figure 4A, 1), indicating that the MIT domain is not essential for the local­ization of Vps4 to endosomes. Loss of the AAA-Istl interac­tion did not affect the recruitment of GFP_Vps4"MlT.E233Q to ESCRT-III (vps4!listI!l, Figure 4A, 4), which is consistent with previous reports that described a rather weak interac­tion (Dimaano et aI., 2008). In contrast, cells that lacked Vtal were not able to recruit GFP_Vps4"MIT.E233Q to ESCRT-III (Figure 4A, 3). Similarly, lack of Did2 abolished endosomal recruitment of GFP_Vps4"MlT,E233Q (Figure 4A, 2). Because Did2 has been shown to be important for endosomallocal- A GFP-Vps4 <.\MIT,E233Q B vps4t> vps4t>vta1t> # Strain FM Loc. Int. + + vps4t> fJ + 12 4 E233Q VpS GFP_Vps4"MIT,E2330 _ VpS4E2330 _ -------- PSPSPSPS - .... - ~~ - -"'- Figure 4. Localization of mutant Vps4 pro­teins. (A) Localization of MIT -deleted Vps4 (GFP­Vps4" MIT,E233Q and GFP_Vps4"MIT,E233Q,S377A) 2 4 vps4t> did211 vps4t> vta1d vps4t> ist1t> VpS4E233Q 5 vta1 t> 8, 9, 12 - 8, 12 + 9, 12 +/- 8 c D - - - IP: a HA ~ unbound ~ ~ ~ ~ ~ ~ ~ ~ ~Q.~~~Q.~~~ :<> ~ ~ :<> ~ ~ VPS20-HA- _ _ _ 1 _ --- Vps 20- _ . __ # Strain FM Loc. Int. in different yeast mutant strains (see Table 1) determined by fluorescence microscopy (FM). (A and D) The extent of observed endosomal localization is indicated (Loc.), Vps4 interac­tions affected by the different mutations are listed. Int., numbers are based on the interac­tions in Figure 1A. (8) Endosomal recruitment of MIT-deleted Vps4 in the presence or absence of full-length Vps4 protein determined by sub­cellular fractionation and Westem blot analysis (5, soluble; P, pellet). (C) Immunoprecipitation of Vps20-HA from detergent-solubilized mem­brane fractions. The resulting bound and un­bound samples were analyzed by Westem blot using anti-Vps20 antiserum. (D) Fluorescence microscopy (FM) analysis of GFP-tagged full­length Vps4 protein in different mutant strains (Table 1). Numbers in parentheses indicate par­tially disrupted interactions, GFP-Vps4 <.\MIT,E233Q,S377A vps4t> vta1 t> 1180 + 1, (10) # Strain Loc. Int. 6 vps4t> 8, 12 vps411 vta1 11 L640 + 2, 8, (10) 7 VpS4 E233Q ~ + vps411 3 vps2011 snf711 L640 1, 2, 8, 9, (10) ization of Vtal, the mislocalization of GFP-VpS4"M IT,E233Q in vps4/l did2/l is likely due to a lack of ESCRT-III-associated Vtal in this strain (Azmi et aI., 2008). To further corroborate the role of Vtal in GFP­VpS4" MJT,E233Q localization, the previously published muta­tion S377 A was introduced into the beta-loop of Vps4, a mutation predicted to interfere with Vtal binding (Scott et aI., 2005b). When expressed in yeast, the VpS4S377A mutant protein was stable and present at normal levels, indicating that the mutation did not interfere with protein folding (Supplemental Figure 2). As expected, the mutation resulted in a partial GFP-CPS and a weak CPY-invertase trafficking phenotype (Figure 3A, 5, and Table 3), similar to the phenotype observed in cells deleted for VTAI (Azmi et aI" 2006; Saksena et aI" 2009). When introduced into GFP­VpS4" MIT,E233Q, the resulting mutant construct GFP­Vps4" MIT,E233Q,S377A did not localize to endosomes in vps4/l cells (Figure 4A, 6), which is consistent with the loss of Vtal interaction. Together, the data supported the model that Vtal localizes to ESCRT-III via Did2 and that the ESCRT­III- associated pool of Vtal is sufficient to recruit MIT-de­leted Vps4. Introducing full-length VpS4E233Q into vps4/lvtal/l cells partially restored endosomal localization of GFP­VpS4" MIT,E233Q (VpS4 E233Q vtal!l; Figure 4A, 5). Similarly the presence of Vps4E233Q suppressed the localization defect of VpS4"MJT,E233Q,S377A (Figure 4A, 7). These results indicated that Vps4-Vps4 interactions were sufficient to recruit the ATPase to ESCRT-III. In contrast to the microscopy studies, subcellular fraction­ation experiments GFP_Vps4"MIT,E233Q localized almost ex­clusively to the soluble, cytoplasmic fraction in presence or absence of either VpS4E233Q or Vta1 (Figure 4B). This dis-crepancy is likely caused by the dissociation of GFP­VpS4" MIT,E233Q during the fractionation procedure, suggesting that the lack of the MIT domain weakened the interaction not only between Vps4 and ESCRT-III but also the interactions within the Vps4 oligomer. In summary, the localization studies using MIT-deleted Vps4 suggested that Vps4 could be recruited to ESCRT-III either via binding to Vtal or via interactions with other ESCRT-lll-associated Vps4 subunits. However, loss of the MIT domain resulted in reduced stability of the ESCRT-lIl­associated Vps4 oligomer. Vps4 Oligomer and ESCRT-III Make Multiple Contacts Recently, a model for the ESCRT-lll structure has been proposed that suggested a single linear polymer consist­ing of the ordered assembly of one subunit of Vps20, followed by - 10 subunits of Sn£7, followed by ab