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• W2893281237 abstract "•Coordination of Rab, kinase, and SNARE cycles fine-tunes ILF formation during fusion•A D44N mutation in the Rab Ypt7 uncouples activation from effector binding•Phosphorylation of HOPS by Yck3 targets pore formation to reduce ILF production•ILF formation protects against vacuole membrane permeability and cell death Upon vacuolar lysosome (or vacuole) fusion in S. cerevisiae, a portion of membrane is internalized and catabolized. Formation of this intralumenal fragment (ILF) is important for organelle protein and lipid homeostasis and remodeling. But how ILF formation is optimized for membrane turnover is not understood. Here, we show that fewer ILFs form when the interaction between the Rab-GTPase Ypt7 and its effector Vps41 (a subunit of the tethering complex HOPS) is interrupted by a point mutation (Ypt7-D44N). Subsequent phosphorylation of Vps41 by the casein kinase Yck3 prevents stabilization of trans-SNARE complexes needed for lipid bilayer pore formation. Impairing ILF formation prevents clearance of misfolded proteins from vacuole membranes and promotes organelle permeability and cell death. We propose that HOPS coordinates Rab, kinase, and SNARE cycles to modulate ILF size during vacuole fusion, regulating lipid and protein turnover important for quality control and membrane integrity. Upon vacuolar lysosome (or vacuole) fusion in S. cerevisiae, a portion of membrane is internalized and catabolized. Formation of this intralumenal fragment (ILF) is important for organelle protein and lipid homeostasis and remodeling. But how ILF formation is optimized for membrane turnover is not understood. Here, we show that fewer ILFs form when the interaction between the Rab-GTPase Ypt7 and its effector Vps41 (a subunit of the tethering complex HOPS) is interrupted by a point mutation (Ypt7-D44N). Subsequent phosphorylation of Vps41 by the casein kinase Yck3 prevents stabilization of trans-SNARE complexes needed for lipid bilayer pore formation. Impairing ILF formation prevents clearance of misfolded proteins from vacuole membranes and promotes organelle permeability and cell death. We propose that HOPS coordinates Rab, kinase, and SNARE cycles to modulate ILF size during vacuole fusion, regulating lipid and protein turnover important for quality control and membrane integrity. Biomaterials are recycled by lysosomes providing an important source of nutrients for eukaryotic cells (Perera and Zoncu, 2016Perera R.M. Zoncu R. The lysosome as a regulatory hub.Annu. Rev. Cell Dev. Biol. 2016; 32: 223-253Crossref PubMed Scopus (284) Google Scholar). They require three fundamental processes to perform this function: membrane fusion—to deliver membrane-encapsulated biomaterials to the lysosomal lumen (Huber and Teis, 2016Huber L.A. Teis D. Lysosomal signaling in control of degradation pathways.Curr. Opin. Cell Biol. 2016; 39: 8-14Crossref PubMed Scopus (82) Google Scholar); biomaterial catabolism—within the lumen, biomaterials encounter acid hydrolases that break them down into their constituents (e.g., lipids, amino acids, and sugars); and nutrient transport—nutrient transporter proteins return products of catabolism to the cytoplasm for reuse by the cell (Xu and Ren, 2015Xu H. Ren D. Lysosomal physiology.Annu. Rev. Physiol. 2015; 77: 57-80Crossref PubMed Scopus (587) Google Scholar). Besides being critical for lysosome function, membrane fusion is also essential for organelle homeostasis, mediating delivery of biosynthetic cargoes (Odorizzi et al., 1998Odorizzi G. Cowles C.R. Emr S.D. The AP-3 complex: a coat of many colours.Trends Cell Biol. 1998; 8: 282-288Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, Luzio et al., 2014Luzio J.P. Hackmann Y. Dieckmann N.M. Griffiths G.M. The biogenesis of lysosomes and lysosome-related organelles.Cold Spring Harb. Perspect. Biol. 2014; 6: a016840Crossref PubMed Scopus (208) Google Scholar), driving organelle inheritance (Weisman, 2003Weisman L.S. Yeast vacuole inheritance and dynamics.Annu. Rev. Genet. 2003; 37: 435-460Crossref PubMed Scopus (100) Google Scholar), and controlling organelle morphology (Brett and Merz, 2008Brett C.L. Merz A.J. Osmotic regulation of Rab-mediated organelle docking.Curr. Biol. 2008; 18: 1072-1077Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, Klumperman and Raposo, 2014Klumperman J. Raposo G. The complex ultrastructure of the endolysosomal system.Cold Spring Harb. Perspect. Biol. 2014; 6: a016857Crossref PubMed Scopus (207) Google Scholar). Thus, the molecular underpinnings of lysosome membrane fusion have been under investigation since the discovery of the organelle (de Duve, 2005de Duve C. The lysosome turns fifty.Nat. Cell Biol. 2005; 7: 847-849Crossref PubMed Scopus (262) Google Scholar). Greatest insight into this process has been gleaned from use of the yeast Saccharomyces cerevisiae and its vacuolar lysosome (or “vacuole”) as models (Li and Kane, 2009Li S.C. Kane P.M. The yeast lysosome-like vacuole: endpoint and crossroads.Biochim. Biophys. Acta. 2009; 1793: 650-663Crossref PubMed Scopus (281) Google Scholar, Nickerson et al., 2009Nickerson D.P. Brett C.L. Merz A.J. Vps-C complexes: gatekeepers of endolysosomal traffic.Curr. Opin. Cell Biol. 2009; 21: 543-551Crossref PubMed Scopus (179) Google Scholar, Wickner, 2010Wickner W. Membrane fusion: Five lipids, four SNAREs, three chaperones, two nucleotides, and a Rab, all dancing in a ring on yeast vacuoles.Annu. Rev. Cell Dev. Biol. 2010; 26: 115-136Crossref PubMed Scopus (215) Google Scholar), whereby homotypic vacuole membrane fusion requires four biochemically distinct, sequential stages: priming, tethering, docking, and fusion. Priming involves the ATP-dependent unraveling of cis-SNARE (soluble NSF attachment protein receptor) complexes by the αSNAP Sec17 and NSF ortholog Sec18 (Mayer et al., 1996Mayer A. Wickner W. Haas A. Sec18p (NSF)-driven release of Sec17p (alpha-SNAP) can precede docking and fusion of yeast vacuoles.Cell. 1996; 85: 83-94Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). Tethering is when apposing organelle membranes make first contact. This requires the Rab-GTPase Ypt7 and its cognate multisubunit tethering complex homotypic vacuole fusion and protein sorting (HOPS) (Mayer and Wickner, 1997Mayer A. Wickner W. Docking of yeast vacuoles is catalyzed by the Ras-like GTPase Ypt7p after symmetric priming by Sec18p (NSF).J. Cell Biol. 1997; 136: 307-317Crossref PubMed Scopus (202) Google Scholar, Hickey and Wickner, 2010Hickey C.M. Wickner W. HOPS initiates vacuole docking by tethering membranes before trans-SNARE complex assembly.Mol. Biol. Cell. 2010; 21: 2297-2305Crossref PubMed Scopus (103) Google Scholar, Orr et al., 2015Orr A. Wickner W. Rusin S.F. Kettenbach A.N. Zick M. Yeast vacuolar HOPS, regulated by its kinase, exploits affinities for acidic lipids and Rab:GTP for membrane binding and to catalyze tethering and fusion.Mol. Biol. Cell. 2015; 26: 305-315Crossref PubMed Scopus (23) Google Scholar). HOPS contains six protein subunits, two of which have Ypt7-binding sites that mediate intermembrane attachment: Vps39 and Vps41 (Seals et al., 2000Seals D.F. Eitzen G. Margolis N. Wickner W.T. Price A. A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion.Proc. Natl. Acad. Sci. USA. 2000; 97: 9402-9407Crossref PubMed Scopus (362) Google Scholar, Brett et al., 2008Brett C.L. Plemel R.L. Lobingier B.T. Vignali M. Fields S. Merz A.J. Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase.J. Cell Biol. 2008; 182: 1141-1151Crossref PubMed Scopus (101) Google Scholar, Nickerson et al., 2009Nickerson D.P. Brett C.L. Merz A.J. Vps-C complexes: gatekeepers of endolysosomal traffic.Curr. Opin. Cell Biol. 2009; 21: 543-551Crossref PubMed Scopus (179) Google Scholar, Bröcker et al., 2012Bröcker C. Kuhlee A. Gatsogiannis C. Balderhaar H.J. Hönscher C. Engelbrecht-Vandré S. Ungermann C. Raunser S. Molecular architecture of the multisubunit homotypic fusion and vacuole protein sorting (HOPS) tethering complex.Proc. Natl. Acad. Sci. USA. 2012; 109: 1991-1996Crossref PubMed Scopus (184) Google Scholar). Vps39 binds Ypt7 independent of its nucleotide-bound state to accommodate activation by its guanine nucleotide exchange factor (GEF) composed of Mon1 and Ccz1 (Nordmann et al., 2010Nordmann M. Cabrera M. Perz A. Bröcker C. Ostrowicz C. Engelbrecht-Vandré S. Ungermann C. .The Mon1-Ccz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7.Curr. Biol. 2010; 20: 1654-1659Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), whereas Vps41 is an effector of Ypt7 and thus only binds the Rab when it is active (GTP-bound; Brett et al., 2008Brett C.L. Plemel R.L. Lobingier B.T. Vignali M. Fields S. Merz A.J. Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase.J. Cell Biol. 2008; 182: 1141-1151Crossref PubMed Scopus (101) Google Scholar). Vps41 may also associate to membranes through its amphipathic lipid packing sensor (ALPS) domain (Cabrera et al., 2010Cabrera M. Langemeyer L. Mari M. Rethmeier R. Orban I. Perz A. Bröcker C. Griffith J. Klose D. Steinhoff H.J. et al.Phosphorylation of a membrane curvature-sensing motif switches function of the HOPS subunit Vps41 in membrane tethering.J. Cell Biol. 2010; 191: 845-859Crossref PubMed Scopus (88) Google Scholar), which may directly contribute to organelle tethering (Ho and Stroupe, 2016Ho R. Stroupe C. The HOPS/Class C Vps complex tethers high-curvature membranes via a direct protein-membrane interaction.Traffic. 2016; 17: 1078-1090Crossref PubMed Scopus (17) Google Scholar). Docking involves the enrichment of fusogenic lipids and proteins in a ring formed at the vertex between apposed membranes (Wang et al., 2003Wang L. Merz A.J. Collins K.M. Wickner W. Hierarchy of protein assembly at the vertex ring domain for yeast vacuole docking and fusion.J. Cell Biol. 2003; 160: 365-374Crossref PubMed Scopus (103) Google Scholar, Fratti et al., 2004Fratti R.A. Jun Y. Merz A.J. Margolis N. Wickner W. Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles.J. Cell Biol. 2004; 167: 1087-1098Crossref PubMed Scopus (169) Google Scholar). Here, Vps33, an SM-protein found within HOPS, mediates trans-SNARE complex formation between the R-SNARE Nyv1 on one membrane with the Qa-SNARE Vam3, Qb-SNARE Vti1, and soluble Qc-SNARE Vam7 on the apposing membrane (Seals et al., 2000Seals D.F. Eitzen G. Margolis N. Wickner W.T. Price A. A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion.Proc. Natl. Acad. Sci. USA. 2000; 97: 9402-9407Crossref PubMed Scopus (362) Google Scholar, Lobingier and Merz, 2012Lobingier B.T. Merz A.J. Sec1/Munc18 protein Vps33 binds to SNARE domains and the quaternary SNARE complex.Mol. Biol. Cell. 2012; 23: 4611-4622Crossref PubMed Scopus (86) Google Scholar, Baker et al., 2015Baker R.W. Jeffrey P.D. Zick M. Phillips B.P. Wickner W.T. Hughson F.M. A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly.Science. 2015; 349: 1111-1114Crossref PubMed Scopus (185) Google Scholar). Partially zippered SNARE complexes are proofread when bound by HOPS (Starai et al., 2008Starai V.J. Hickey C.M. Wickner W. HOPS proofreads the trans-SNARE complex for yeast vacuole fusion.Mol. Biol. Cell. 2008; 19: 2500-2508Crossref PubMed Scopus (99) Google Scholar), which also protects them from disassembly by Sec18 (Xu et al., 2010Xu H. Jun Y. Thompson J. Yates J. Wickner W. HOPS prevents the disassembly of trans-SNARE complexes by Sec17p/Sec18p during membrane fusion.EMBO J. 2010; 29: 1948-1960Crossref PubMed Scopus (80) Google Scholar, Lobingier et al., 2014Lobingier B.T. Nickerson D.P. Lo S.Y. Merz A.J. SM proteins Sly1 and Vps33 co-assemble with Sec17 and SNARE complexes to oppose SNARE disassembly by Sec18.Elife. 2014; 3: e02272Crossref PubMed Scopus (56) Google Scholar). Orchestration of fusion protein assembly is further regulated by the casein kinase Yck3 that responds to Ypt7 inactivation to phosphorylate and inhibits activities of Vps41 and Vam3 (LaGrassa and Ungermann, 2005LaGrassa T.J. Ungermann C. The vacuolar kinase Yck3 maintains organelle fragmentation by regulating the HOPS tethering complex.J. Cell Biol. 2005; 168: 401-414Crossref PubMed Scopus (116) Google Scholar, Brett et al., 2008Brett C.L. Plemel R.L. Lobingier B.T. Vignali M. Fields S. Merz A.J. Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase.J. Cell Biol. 2008; 182: 1141-1151Crossref PubMed Scopus (101) Google Scholar). Ultimately, successful trans-SNARE pairing elicits Ca2+ efflux from the vacuole lumen (Merz and Wickner, 2004Merz A.J. Wickner W.T. Trans-SNARE interactions elicit Ca2+ efflux from the yeast vacuole lumen.J. Cell Biol. 2004; 164: 195-206Crossref PubMed Scopus (67) Google Scholar), which is thought to trigger downstream membrane fusion. Fusion is a lipid bilayer merger accomplished by full zippering of trans-SNARE-complexes to drive membranes together and form pores necessary for lumenal content mixing (Nichols et al., 1997Nichols B.J. Ungermann C. Pelham H.R. Wickner W.T. Haas A. Homotypic vacuolar fusion mediated by t- and v-SNAREs.Nature. 1997; 387: 199-202Crossref PubMed Scopus (379) Google Scholar, Schwartz and Merz, 2009Schwartz M.L. Merz A.J. Capture and release of partially zipped trans-SNARE complexes on intact organelles.J. Cell Biol. 2009; 185: 535-549Crossref PubMed Scopus (77) Google Scholar). As the lipid bilayer is severed by the fusion machinery at the vertex ring, the encircled area of membrane at the interface between apposing organelles (called the boundary) is entrapped within the lumen as a membrane fragment (Wang et al., 2002Wang L. Seeley E.S. Wickner W. Merz A.J. Vacuole fusion at a ring of vertex docking sites leaves membrane fragments within the organelle.Cell. 2002; 108: 357-369Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, Mattie et al., 2017Mattie S. McNally E.K. Karim M.A. Vali H. Brett C.L. How and why intralumenal membrane fragments form during vacuolar lysosome fusion.Mol. Biol. Cell. 2017; 28: 309-321Crossref PubMed Google Scholar). This intralumenal fragment (ILF) is then degraded by lumenal hydrolases and recycled. Recently, we discovered that vacuolar nutrient transporter proteins are selectively sorted into the boundary, internalized, and degraded in response to substrate levels, protein misfolding or TOR-signaling (McNally et al., 2017McNally E.K. Karim M.A. Brett C.L. Selective lysosomal transporter degradation by organelle membrane fusion.Dev. Cell. 2017; 40: 151-167Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Thus, ILF formation during fusion is important for vacuole lipid and protein turnover necessary for organelle homeostasis and remodeling. Despite being critical for vacuole biology, relatively little is known about how ILF formation is regulated. However, we do know that ILF formation is not required for vacuole fusion, as not all fusion events produce visible ILFs (Wang et al., 2002Wang L. Seeley E.S. Wickner W. Merz A.J. Vacuole fusion at a ring of vertex docking sites leaves membrane fragments within the organelle.Cell. 2002; 108: 357-369Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, Mattie et al., 2017Mattie S. McNally E.K. Karim M.A. Vali H. Brett C.L. How and why intralumenal membrane fragments form during vacuolar lysosome fusion.Mol. Biol. Cell. 2017; 28: 309-321Crossref PubMed Google Scholar). This also suggests ILF formation can be modulated. We also know that ILF formation relies on hemifusion intermediate formation during lipid bilayer fusion (Wang et al., 2002Wang L. Seeley E.S. Wickner W. Merz A.J. Vacuole fusion at a ring of vertex docking sites leaves membrane fragments within the organelle.Cell. 2002; 108: 357-369Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar; Jun and Wickner, 2007; Mattie et al., 2017Mattie S. McNally E.K. Karim M.A. Vali H. Brett C.L. How and why intralumenal membrane fragments form during vacuolar lysosome fusion.Mol. Biol. Cell. 2017; 28: 309-321Crossref PubMed Google Scholar). Termed “ring fusion-by-hemifusion,” vacuole lipid bilayer fusion is initiated by “stalk formation,” whereby lipids within closely apposed outer leaflets mix, causing merger (Reese et al., 2005Reese C. Heise F. Mayer A. Trans-SNARE pairing can precede a hemifusion intermediate in intracellular membrane fusion.Nature. 2005; 436: 410-414Crossref PubMed Scopus (101) Google Scholar, Jun and Wickner, 2007Jun Y. Wickner W. Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing.Proc. Natl. Acad. Sci. USA. 2007; 104: 13010-13015Crossref PubMed Scopus (63) Google Scholar, Wickner, 2010Wickner W. Membrane fusion: Five lipids, four SNAREs, three chaperones, two nucleotides, and a Rab, all dancing in a ring on yeast vacuoles.Annu. Rev. Cell Dev. Biol. 2010; 26: 115-136Crossref PubMed Scopus (215) Google Scholar). This lipid stalk expands, forming a hemifusion diaphragm, a hybrid lipid bilayer consisting of lumenal facing inner leaflets donated from each organelle (Mattie et al., 2017Mattie S. McNally E.K. Karim M.A. Vali H. Brett C.L. How and why intralumenal membrane fragments form during vacuolar lysosome fusion.Mol. Biol. Cell. 2017; 28: 309-321Crossref PubMed Google Scholar). Next, trans-SNARE-pin zippering drives “pore formation,” the rate-limiting step of the fusion reaction (Reese and Mayer, 2005Reese C. Mayer A. Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion.J. Cell Biol. 2005; 171: 981-990Crossref PubMed Scopus (53) Google Scholar), whereby the hemifusion diaphragm is ruptured, allowing lumenal contents to mix (Schwartz and Merz, 2009Schwartz M.L. Merz A.J. Capture and release of partially zipped trans-SNARE complexes on intact organelles.J. Cell Biol. 2009; 185: 535-549Crossref PubMed Scopus (77) Google Scholar, D'Agostino et al., 2016D'Agostino M. Risselada H.J. Mayer A. Steric hindrance of SNARE transmembrane domain organization impairs the hemifusion-to-fusion transition.EMBO Rep. 2016; 17: 1590-1608Crossref PubMed Scopus (14) Google Scholar). This pore expands to complete full membrane merger resulting in a single organelle. The ILF formation during vacuole fusion relies on the delay between stalk and pore formation (Mattie et al., 2017Mattie S. McNally E.K. Karim M.A. Vali H. Brett C.L. How and why intralumenal membrane fragments form during vacuolar lysosome fusion.Mol. Biol. Cell. 2017; 28: 309-321Crossref PubMed Google Scholar): if pore formation is delayed, the stalk expands across the entire boundary membrane, and, when a pore forms, no membrane is entrapped during fusion. Thus, pore formation must occur rapidly after stalk expansion to form ILFs during vacuole fusion. To better understand how these ILFs form (or not) during fusion, we showed that adding GTPγS, a non-hydrolyzable form of GTP, to cell-free vacuole fusion reactions causes premature stalk formation, preventing ILF formation (Mattie et al., 2017Mattie S. McNally E.K. Karim M.A. Vali H. Brett C.L. How and why intralumenal membrane fragments form during vacuolar lysosome fusion.Mol. Biol. Cell. 2017; 28: 309-321Crossref PubMed Google Scholar), whereas addition of purified, recombinant Vam7 protein (the soluble Qc-SNARE) induces premature pore formation to enhance ILF formation. However, we were unable to demonstrate how the endogenous fusion machinery may perform this function. Because HOPS mediates the interplay between the Rab-GTPase Ypt7 and SNAREs (Wickner, 2010Wickner W. Membrane fusion: Five lipids, four SNAREs, three chaperones, two nucleotides, and a Rab, all dancing in a ring on yeast vacuoles.Annu. Rev. Cell Dev. Biol. 2010; 26: 115-136Crossref PubMed Scopus (215) Google Scholar), we hypothesized that modulating interactions between these proteins may influence ILF formation. Here, we test this hypothesis and confirm that functional interactions between Ypt7, its effector Vps41 within HOPS, and the kinase Yck3 regulate ILF formation during vacuole fusion. To identify a Ypt7 mutation that may alter ILF formation but not vacuole fusion, we first examined vacuole morphology within S. cerevisiae cells harboring different point mutations reported to either lock the Rab-GTPase in a GDP-bound (T22N; Liu et al., 2012Liu T.T. Gomez T.S. Sackey B.K. Billadeau D.D. Burd C.G. Rab GTPase regulation of retromer-mediated cargo export during endosome maturation.Mol. Biol. Cell. 2012; 23: 2505-2515Crossref PubMed Scopus (85) Google Scholar, Lawrence et al., 2014Lawrence G. Brown C.C. Flood B.A. Karunakaran S. Cabrera M. Nordmann M. Ungermann C. Fratti R.A. Dynamic association of the PI3P-interacting Mon1-Ccz1 GEF with vacuoles is controlled through its phosphorylation by the type 1 casein kinase Yck3.Mol. Biol. Cell. 2014; 25: 1608-1619Crossref PubMed Google Scholar) or GTP-bound state (Q68L and N126I; Brett et al., 2008Brett C.L. Plemel R.L. Lobingier B.T. Vignali M. Fields S. Merz A.J. Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase.J. Cell Biol. 2008; 182: 1141-1151Crossref PubMed Scopus (101) Google Scholar, Rana et al., 2015Rana M. Lachmann J. Ungermann C. Identification of a Rab GTPase-activating protein cascade that controls recycling of the Rab5 GTPase Vps21 from the vacuole.Mol. Biol. Cell. 2015; 26: 2535-2549Crossref PubMed Google Scholar), reduce its affinity for nucleotides (D129N; Kucharczyk et al., 2001Kucharczyk R. Kierzek A.M. Slonimski P.P. Rytka J. The Ccz1 protein interacts with Ypt7 GTPase during fusion of multiple transport intermediates with the vacuole in S. cerevisiae.J. Cell Sci. 2001; 114: 3137-3145PubMed Google Scholar), or disrupt GTPase activating protein (GAP)-induced GTP hydrolysis and possibly effector binding (D44N; Vollmer et al., 1999Vollmer P. Will E. Scheglmann D. Strom M. Gallwitz D. Primary structure and biochemical characterization of yeast GTPase-activating proteins with substrate preference for the transport GTPase Ypt7p.Eur. J. Biochem. 1999; 260: 284-290Crossref PubMed Scopus (46) Google Scholar). Mutations of interest should affect the production of ILFs but not vacuole morphology, as fusion itself should not be altered (Mattie et al., 2017Mattie S. McNally E.K. Karim M.A. Vali H. Brett C.L. How and why intralumenal membrane fragments form during vacuolar lysosome fusion.Mol. Biol. Cell. 2017; 28: 309-321Crossref PubMed Google Scholar). We found that cells expressing Ypt7-T22N or Ypt7-D129N contain fragmented vacuoles resembling ypt7Δ cells (Figures 1A and 1B ), confirming that Ypt7 nucleotide binding and activation is required for homotypic vacuole fusion (Wichmann et al., 1992Wichmann H. Hengst L. Gallwitz D. Endocytosis in yeast: evidence for the involvement of a small GTP-binding protein (Ypt7p).Cell. 1992; 71: 1131-1142Abstract Full Text PDF PubMed Scopus (203) Google Scholar, Eitzen et al., 2000Eitzen G. Will E. Gallwitz D. Haas A. Wickner W. Sequential action of two GTPases to promote vacuole docking and fusion.EMBO J. 2000; 19: 6713-6720Crossref PubMed Scopus (73) Google Scholar, Balderhaar et al., 2010Balderhaar H.J. Arlt H. Ostrowicz C. Bröcker C. Sündermann F. Brandt R. Babst M. Ungermann C. The Rab GTPase Ypt7 is linked to retromer-mediated receptor recycling and fusion at the yeast late endosome.J. Cell Sci. 2010; 123: 4085-4094Crossref PubMed Scopus (81) Google Scholar). Nearly all cells expressing Ypt7-Q68L or Ypt7-N126I contained a single large vacuole, confirming that homotypic fusion was enhanced when Ypt7 was constitutively active (Rana et al., 2015Rana M. Lachmann J. Ungermann C. Identification of a Rab GTPase-activating protein cascade that controls recycling of the Rab5 GTPase Vps21 from the vacuole.Mol. Biol. Cell. 2015; 26: 2535-2549Crossref PubMed Google Scholar). However, cells expressing Ypt7-D44N had similar vacuole morphologies as cells expressing wild-type Ypt7 (Figures 1A and 1B). To further characterize Ypt7 mutations that seemed to support fusion, we recorded vacuole fusion events in live cells (Figure 1C; Video S1) and found that vacuoles in Ypt7-D44N cells underwent slightly fewer homotypic fusion events, as compared to wild-type or Ypt7-Q68L cells (Figure 1D). Importantly, fusion events within Ypt7-D44N cells produced significantly fewer visible ILFs, whereas fusion events in Ypt7-Q68L cells produced significantly more ILFs, as compared to wild-type cells (Figure 1E). These preliminary findings suggest that introducing mutations that lock Ypt7 in a GTP-bound state (Q68L) or possibly disrupt Ypt7 effector binding (D44N) may affect the fusion-by-hemifusion reaction to alter ILF formation. We decided to focus our studies on the D44N mutation as it showed the strongest phenotype, and we reasoned that characterizing a mutation that may disrupt Ypt7-HOPS interactions would provide new insights into how the underlying machinery orchestrates vacuole fusion and ILF formation. https://www.cell.com/cms/asset/e241d63e-002c-42e2-88e3-7b6e1b58cea4/mmc2.mp4Loading ... Download .mp4 (2.88 MB) Help with .mp4 files Video S1. Mutations in YPT7 Affect ILF Formation during Vacuole Fusion in Live Cells, Related to Figure 1Videos of vacuole fusion events in live yeast cells expressing wild-type, D44N or Q68L Ypt7 stained with FM4-64 demonstrating the presence or absence of intralumenal fragment formation. To understand how Ypt7-D44N disrupts ILF formation, we first examined its effects on the subreactions of vacuole lipid bilayer fusion in vitro. To start, we isolated vacuoles from cells and measured the kinetics of stalk or pore formation using lipid or content mixing assays, respectively (Haas et al., 1995Haas A. Scheglmann D. Lazar T. Gallwitz D. Wickner W. The GTPase Ypt7p of Saccharomyces cerevisiae is required on both partner vacuoles for the homotypic fusion step of vacuole inheritance.EMBO J. 1995; 14: 5258-5270Crossref PubMed Scopus (233) Google Scholar, Jun and Wickner, 2007Jun Y. Wickner W. Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing.Proc. Natl. Acad. Sci. USA. 2007; 104: 13010-13015Crossref PubMed Scopus (63) Google Scholar). We found that Ypt7-D44N did not affect vacuole lipid mixing (Figure 2A) but severely impaired content mixing (Figure 2B) stimulated by ATP in vitro. To confirm that content mixing was blocked, we imaged isolated vacuoles before and 60 min after fusion was stimulated with ATP (Figure 2C) and calculated vacuole surface area (Figure 2D). As expected, the surface area significantly increased after 60 min when vacuoles isolated from wild-type cells, indicating that full fusion occurred. This increase was blocked by the fusion inhibitor rGyp1-46 (a purified, recombinant protein consisting of the catalytic domain of the Rab-GAP Gyp1; Eitzen et al., 2000Eitzen G. Will E. Gallwitz D. Haas A. Wickner W. Sequential action of two GTPases to promote vacuole docking and fusion.EMBO J. 2000; 19: 6713-6720Crossref PubMed Scopus (73) Google Scholar, Brett et al., 2008Brett C.L. Plemel R.L. Lobingier B.T. Vignali M. Fields S. Merz A.J. Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase.J. Cell Biol. 2008; 182: 1141-1151Crossref PubMed Scopus (101) Google Scholar), confirming that membrane fusion was responsible for the observed increase in vacuole surface area. Importantly, no increase in surface area was observed when vacuoles were isolated from Ypt7-D44N cells, consistent with content mixing results. Thus, we found that the relative amount of lipid mixing to content mixing was greater for the mutant as compared to wild-type (Figure 2E) and the delay between these events was extended in the mutant (Figure 2F). We also noted that fewer ILFs were observed within vacuoles isolated from Ypt7-D44N cells (Figure 2G), consistent with observations made in vivo (Figure 1E). Together, these results indicated that only outer leaflets (not inner leaflets) of lipid bilayers merged and that hemifusion diaphragms may be accumulating between docked vacuoles. To test this hypothesis, we first used a polytopic protein exclusion assay to study hemifusion diaphragm accumulation during vacuole fusion in vitro (Mattie et al., 2017Mattie S. McNally E.K. Karim M.A. Vali H. Brett C.L. How and why intralumenal membrane fragments form during vacuolar lysosome fusion.Mol. Biol. Cell. 2017; 28: 309-321Crossref PubMed Google Scholar). If a hemifusion diaphragm forms at the boundary between apposing organelles, then polytopic proteins with large cytoplasmic domains such as GFP-tagged Vph1, the stalk domain of the V-type H+-ATPase, will be excluded because biophysical constraints prevent it from entering a hybrid bilayer composed of luminal-facing leaflets on both sides (Nikolaus et al., 2010Nikolaus J. Stöckl M. Langosch D. Volkmer R. Herrmann A. Direct visualization of large and protein-free hemifusion diaphragms.Biophys. J. 2010; 98: 1192-1199Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Fusion reactions containing vacuoles isolated from wild-type cells showed uniform distribution of Vph1-GFP on vacuole membranes, i.e., it was present within boundary membranes (Figures 2H and 2I), as previously reported (Wang et al., 2002Wang L. Seeley E.S. Wickner W. Merz A.J. Vacuole fusion at a ring of vertex docking sites leaves membrane fragments within the organelle.Cell. 2002; 108: 357-369Abstract Full Text Full Text" @default.
• W2893281237 created "2018-10-05" @default.
• W2893281237 creator A5057632375 @default.
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• W2893281237 creator A5065510038 @default.
• W2893281237 creator A5088210322 @default.
• W2893281237 creator A5091118370 @default.
• W2893281237 date "2018-10-01" @default.
• W2893281237 modified "2023-10-17" @default.
• W2893281237 title "Rab-Effector-Kinase Interplay Modulates Intralumenal Fragment Formation during Vacuole Fusion" @default.
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