scholarly journals Phosphorylation of the effector complex HOPS by the vacuolar kinase Yck3p confers Rab nucleotide specificity for vacuole docking and fusion

2012 ◽  
Vol 23 (17) ◽  
pp. 3429-3437 ◽  
Author(s):  
Michael Zick ◽  
William Wickner
Keyword(s):  
Bound State ◽  
Rab Gtpase ◽  
Fusion Reactions ◽  
Gtpase Activating Protein ◽  
Vacuole Fusion ◽  
Protein Receptor ◽  
Membrane Tethering ◽  
Strict Requirement ◽  
Nucleotide Specificity ◽  
Gtpase Cycle

The homotypic fusion of yeast vacuoles requires the Rab-family GTPase Ypt7p and its effector complex, homotypic fusion and vacuole protein sorting complex (HOPS). Although the vacuolar kinase Yck3p is required for the sensitivity of vacuole fusion to proteins that regulate the Rab GTPase cycle—Gdi1p (GDP-dissociation inhibitor [GDI]) or Gyp1p/Gyp7p (GTPase-activating protein)—this kinase phosphorylates HOPS rather than Ypt7p. We addressed this puzzle in reconstituted proteoliposome fusion reactions with all-purified components. In the presence of HOPS and Sec17p/Sec18p, there is comparable fusion of 4-SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) proteoliposomes when they have Ypt7p bearing either GDP or GTP, a striking exception to the rule that only GTP-bound forms of Ras-superfamily GTPases have active conformations. However, the phosphorylation of HOPS by recombinant Yck3p confers a strict requirement for GTP-bound Ypt7p for binding phosphorylated HOPS, for optimal membrane tethering, and for proteoliposome fusion. Added GTPase-activating protein promotes GTP hydrolysis by Ypt7p, and added GDI captures Ypt7p in its GDP-bound state during nucleotide cycling. In either case, the net conversion of Ypt7:GTP to Ypt7:GDP has no effect on HOPS binding or activity but blocks fusion mediated by phosphorylated HOPS. Thus guanine nucleotide specificity of the vacuolar fusion Rab Ypt7p is conferred through downstream posttranslational modification of its effector complex.

scholarly journals The yeast vacuolar Rab GTPase Ypt7p has an activity beyond membrane recruitment of the homotypic fusion and protein sorting–Class C Vps complex

2012 ◽  
Vol 443 (1) ◽  
pp. 205-211 ◽  
Author(s):  
Christopher Stroupe
Keyword(s):  
Protein Sorting ◽  
Rab Gtpase ◽  
Main Function ◽  
Rab Gtpases ◽  
Membrane Recruitment ◽  
Vacuole Fusion ◽  
Protein Receptor ◽  
Head Groups ◽  
Class C ◽  
Hops Complex

A previous report described lipid mixing of reconstituted proteoliposomes made using lipid mixtures that mimic the composition of yeast vacuoles. This lipid mixing required SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor)-attachment protein] receptor} proteins, Sec18p and Sec17p (yeast NSF and α-SNAP) and the HOPS (homotypic fusion and protein sorting)–Class C Vps (vacuole protein sorting) complex, but not the vacuolar Rab GTPase Ypt7p. The present study investigates the activity of Ypt7p in proteoliposome lipid mixing. Ypt7p is required for the lipid mixing of proteoliposomes lacking cardiolipin [1,3-bis-(sn-3′-phosphatidyl)-sn-glycerol]. Omission of other lipids with negatively charged and/or small head groups does not cause Ypt7p dependence for lipid mixing. Yeast vacuoles made from strains disrupted for CRD1 (cardiolipin synthase) fuse to the same extent as vacuoles from strains with functional CRD1. Disruption of CRD1 does not alter dependence on Rab GTPases for vacuole fusion. It has been proposed that the recruitment of the HOPS complex to membranes is the main function of Ypt7p. However, Ypt7p is still required for lipid mixing even when the concentration of HOPS complex in lipid-mixing reactions is adjusted such that cardiolipin-free proteoliposomes with or without Ypt7p bind to equal amounts of HOPS. Ypt7p therefore must stimulate membrane fusion by a mechanism that is in addition to recruitment of HOPS to the membrane. This is the first demonstration of such a stimulatory activity–that is, beyond bulk effector recruitment–for a Rab GTPase.

scholarly journals Identification of a Rab GTPase-activating protein cascade that controls recycling of the Rab5 GTPase Vps21 from the vacuole

2015 ◽  
Vol 26 (13) ◽  
pp. 2535-2549 ◽  
Author(s):  
Meenakshi Rana ◽  
Jens Lachmann ◽  
Christian Ungermann
Keyword(s):  
Endoplasmic Reticulum ◽  
Endocytic Pathway ◽  
Rab Gtpase ◽  
Guanine Nucleotide ◽  
Membrane Recycling ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Guanine Nucleotide Exchange ◽  
Vacuole Fusion ◽  
Nucleotide Exchange Factor

Transport within the endocytic pathway depends on a consecutive function of the endosomal Rab5 and the late endosomal/lysosomal Rab7 GTPases to promote membrane recycling and fusion in the context of endosomal maturation. We previously identified the hexameric BLOC-1 complex as an effector of the yeast Rab5 Vps21, which also recruits the GTPase-activating protein (GAP) Msb3. This raises the question of when Vps21 is inactivated on endosomes. We provide evidence for a Rab cascade in which activation of the Rab7 homologue Ypt7 triggers inactivation of Vps21. We find that the guanine nucleotide exchange factor (GEF) of Ypt7 (the Mon1-Ccz1 complex) and BLOC-1 both localize to the same endosomes. Overexpression of Mon1-Ccz1, which generates additional Ypt7-GTP, or overexpression of activated Ypt7 promotes relocalization of Vps21 from endosomes to the endoplasmic reticulum (ER), which is indicative of Vps21 inactivation. This ER relocalization is prevented by loss of either BLOC-1 or Msb3, but it also occurs in mutants lacking endosome–vacuole fusion machinery such as the HOPS tethering complex, an effector of Ypt7. Importantly, BLOC-1 interacts with the HOPS on vacuoles, suggesting a direct Ypt7-dependent cross-talk. These data indicate that efficient Vps21 recycling requires both Ypt7 and endosome–vacuole fusion, thus suggesting extended control of a GAP cascade beyond Rab interactions.

scholarly journals HOPS drives vacuole fusion by binding the vacuolar SNARE complex and the Vam7 PX domain via two distinct sites

2011 ◽  
Vol 22 (14) ◽  
pp. 2601-2611 ◽  
Author(s):  
Lukas Krämer ◽  
Christian Ungermann
Keyword(s):  
Membrane Fusion ◽  
Snare Complex ◽  
Endomembrane System ◽  
Attachment Protein ◽  
Vacuole Fusion ◽  
Px Domain ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Membrane Tethering

Membrane fusion within the endomembrane system follows a defined order of events: membrane tethering, mediated by Rabs and tethers, assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes, and lipid bilayer mixing. Here we present evidence that the vacuolar HOPS tethering complex controls fusion through specific interactions with the vacuolar SNARE complex (consisting of Vam3, Vam7, Vti1, and Nyv1) and the N-terminal domains of Vam7 and Vam3. We show that homotypic fusion and protein sorting (HOPS) binds Vam7 via its subunits Vps16 and Vps18. In addition, we observed that Vps16, Vps18, and the Sec1/Munc18 protein Vps33, which is also part of the HOPS complex, bind to the Q-SNARE complex. In agreement with this observation, HOPS-stimulated fusion was inhibited if HOPS was preincubated with the minimal Q-SNARE complex. Importantly, artificial targeting of Vam7 without its PX domain to membranes rescued vacuole morphology in vivo, but resulted in a cytokinesis defect if the N-terminal domain of Vam3 was also removed. Our data thus support a model of HOPS-controlled membrane fusion by recognizing different elements of the SNARE complex.

scholarly journals Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion

2005 ◽  
Vol 171 (6) ◽  
pp. 981-990 ◽  
Author(s):  
Christoph Reese ◽  
Andreas Mayer
Keyword(s):  
Positive Curvature ◽  
Fusion Pore ◽  
Viral Fusion ◽  
Fusion Reactions ◽  
Attachment Protein ◽  
Vacuole Fusion ◽  
Protein Receptor ◽  
Pore Opening ◽  
Rate Limiting ◽  
Protein Attachment

Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE)– and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5′-(γ-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems.

scholarly journals The Major Role of the Rab Ypt7p in Vacuole Fusion Is Supporting HOPS Membrane Association

2009 ◽  
Vol 284 (24) ◽  
pp. 16118-16125 ◽  
Author(s):  
Christopher M. Hickey ◽  
Christopher Stroupe ◽  
William Wickner
Keyword(s):  
Fusion Reaction ◽  
Protein Sorting ◽  
Membrane Association ◽  
Rab Gtpase ◽  
Gtpase Activating Protein ◽  
Attachment Protein ◽  
Vacuole Fusion ◽  
Sensitive Factor ◽  
Protein Receptors

Yeast vacuole fusion requires soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), the Rab GTPase Ypt7p, vacuolar lipids, Sec17p and Sec18p, and the homotypic fusion and vacuole protein sorting complex (HOPS). HOPS is a multisubunit protein with direct affinities for SNAREs, vacuolar lipids, and the GTP-bound form of Ypt7p; each of these affinities contributes to HOPS association with the organelle. Using all-purified components, we have reconstituted fusion, but the Rab Ypt7p was not required. We now report that phosphorylation of HOPS by the vacuolar kinase Yck3p blocks HOPS binding to vacuolar lipids, making HOPS membrane association and the ensuing fusion depend on the presence of Ypt7p. In accord with this finding in the reconstituted fusion reaction, the inactivation of Ypt7p by the GTPase-activating protein Gyp1–46p only blocks the fusion of purified vacuoles when Yck3p is present and active. Thus, although Ypt7p may contribute to other fusion functions, its central role is to bind HOPS to the membrane.

scholarly journals Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p

2002 ◽  
Vol 156 (2) ◽  
pp. 315-326 ◽  
Author(s):  
Amy S. Gladfelter ◽  
Indrani Bose ◽  
Trevin R. Zyla ◽  
Elaine S.G. Bardes ◽  
Daniel J. Lew
Keyword(s):  
Transcriptional Activation ◽  
Gtpase Activating Protein ◽  
Gtp Hydrolysis ◽  
Septin Ring ◽  
Turn On ◽  
Ring Assembly ◽  
Bud Emergence ◽  
Assembly Factor ◽  
Gtpase Cycle ◽  
Budding Yeast Cell Cycle

At the beginning of the budding yeast cell cycle, the GTPase Cdc42p promotes the assembly of a ring of septins at the site of future bud emergence. Here, we present an analysis of cdc42 mutants that display specific defects in septin organization, which identifies an important role for GTP hydrolysis by Cdc42p in the assembly of the septin ring. The mutants show defects in basal or stimulated GTP hydrolysis, and the septin misorganization is suppressed by overexpression of a Cdc42p GTPase-activating protein (GAP). Other mutants known to affect GTP hydrolysis by Cdc42p also caused septin misorganization, as did deletion of Cdc42p GAPs. In performing its roles in actin polarization and transcriptional activation, GTP-Cdc42p is thought to function by activating and/or recruiting effectors to the site of polarization. Excess accumulation of GTP-Cdc42p due to a defect in GTP hydrolysis by the septin-specific alleles might cause unphysiological activation of effectors, interfering with septin assembly. However, the recessive and dose-sensitive genetic behavior of the septin-specific cdc42 mutants is inconsistent with the septin defect stemming from a dominant interference of this type. Instead, we suggest that assembly of the septin ring involves repeated cycles of GTP loading and GTP hydrolysis by Cdc42p. These results suggest that a single GTPase, Cdc42p, can act either as a ras-like GTP-dependent “switch” to turn on effectors or as an EF-Tu–like “assembly factor” using the GTPase cycle to assemble a macromolecular structure.

scholarly journals Yeast vacuolar HOPS, regulated by its kinase, exploits affinities for acidic lipids and Rab:GTP for membrane binding and to catalyze tethering and fusion

2015 ◽  
Vol 26 (2) ◽  
pp. 305-315 ◽  
Author(s):  
Amy Orr ◽  
William Wickner ◽  
Scott F. Rusin ◽  
Arminja N. Kettenbach ◽  
Michael Zick
Keyword(s):  
Protein Sorting ◽  
Rab Gtpase ◽  
Attachment Protein ◽  
Functional Relationships ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Membrane Tethering ◽  
Rab Effector ◽  
Supporting Membrane ◽  
Acidic Lipids

Fusion of yeast vacuoles requires the Rab GTPase Ypt7p, four SNAREs (soluble N-ethylmaleimide–sensitive factor attachment protein receptors), the SNARE disassembly chaperones Sec17p/Sec18p, vacuolar lipids, and the Rab-effector complex HOPS (homotypic fusion and vacuole protein sorting). Two HOPS subunits have direct affinity for Ypt7p. Although vacuolar fusion has been reconstituted with purified components, the functional relationships between individual lipids and Ypt7p:GTP have remained unclear. We now report that acidic lipids function with Ypt7p as coreceptors for HOPS, supporting membrane tethering and fusion. After phosphorylation by the vacuolar kinase Yck3p, phospho-HOPS needs both Ypt7p:GTP and acidic lipids to support fusion.

scholarly journals Sec1p and Mso1p C-terminal tails cooperate with the SNAREs and Sec4p in polarized exocytosis

2011 ◽  
Vol 22 (2) ◽  
pp. 230-244 ◽  
Author(s):  
Marion Weber-Boyvat ◽  
Nina Aro ◽  
Konstantin G. Chernov ◽  
Tuula Nyman ◽  
Jussi Jäntti
Keyword(s):  
Close Association ◽  
Adaptor Protein ◽  
Binding Mode ◽  
Snare Complex ◽  
Bimolecular Fluorescence Complementation ◽  
Rab Gtpase ◽  
Temperature Sensitive ◽  
Nucleotide Exchange ◽  
Protein Receptor

The Sec1/Munc18 protein family members perform an essential, albeit poorly understood, function in association with soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes in membrane fusion. The Saccharomyces cerevisiae Sec1p has a C-terminal tail that is missing in its mammalian homologues. Here we show that deletion of the Sec1p tail (amino acids 658–724) renders cells temperature sensitive for growth, reduces sporulation efficiency, causes a secretion defect, and abolishes Sec1p-SNARE component coimmunoprecipitation. The results show that the Sec1p tail binds preferentially ternary Sso1p-Sec9p-Snc2p complexes and it enhances ternary SNARE complex formation in vitro. The bimolecular fluorescence complementation (BiFC) assay results suggest that, in the SNARE-deficient sso2–1 Δsso1 cells, Mso1p, a Sec1p binding protein, helps to target Sec1p(1–657) lacking the C-terminal tail to the sites of secretion. The results suggest that the Mso1p C terminus is important for Sec1p(1–657) targeting. We show that, in addition to Sec1p, Mso1p can bind the Rab-GTPase Sec4p in vitro. The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud. This association depends on the Sec4p guanine nucleotide exchange factor Sec2p. Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p.

scholarly journals Multiple ER–Golgi SNARE transmembrane domains are dispensable for trafficking but required for SNARE recycling

2016 ◽  
Vol 27 (17) ◽  
pp. 2633-2641 ◽  
Author(s):  
Li Chen ◽  
Martin S. Y. Lau ◽  
David K. Banfield
Keyword(s):  
Transmembrane Domain ◽  
Retrograde Transport ◽  
Steady State Distribution ◽  
State Distribution ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Strict Requirement ◽  
Membrane Tether ◽  
Essential Prerequisite

The formation of soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes between opposing membranes is an essential prerequisite for fusion between vesicles and their target compartments. The composition and length of a SNARE’s transmembrane domain (TMD) is also an indicator for their steady-state distribution in cells. The evolutionary conservation of the SNARE TMD, together with the strict requirement of this feature for membrane fusion in biochemical studies, implies that the TMD represents an essential protein module. Paradoxically, we find that for several essential ER- and Golgi-localized SNAREs, a TMD is unnecessary. Moreover, in the absence of a covalent membrane tether, such SNAREs can still support ER–Golgi vesicle transport and recapitulate established genetic interactions. Transport anomalies appear to be restricted to retrograde trafficking, but these defects are overcome by the attachment of a C-terminal lipid anchor to the SNARE. We conclude that the TMD functions principally to support the recycling of Qb-, Qc-, and R-SNAREs and, in so doing, retrograde transport.

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