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 Yeast homotypic vacuole fusion requires the Ccz1–Mon1 complex during the tethering/docking stage

2003 ◽  
Vol 163 (5) ◽  
pp. 973-985 ◽  
Author(s):  
Chao-Wen Wang ◽  
Per E. Stromhaug ◽  
Emily J. Kauffman ◽  
Lois S. Weisman ◽  
Daniel J. Klionsky
Keyword(s):  
Protein Complex ◽  
Complex Interaction ◽  
Snare Complex ◽  
Morphological Evidence ◽  
Vacuole Membrane ◽  
Vacuole Fusion ◽  
Class C ◽  
Hops Complex

The function of the yeast lysosome/vacuole is critically linked with the morphology of the organelle. Accordingly, highly regulated processes control vacuolar fission and fusion events. Analysis of homotypic vacuole fusion demonstrated that vacuoles from strains defective in the CCZ1 and MON1 genes could not fuse. Morphological evidence suggested that these mutant vacuoles could not proceed to the tethering/docking stage. Ccz1 and Mon1 form a stable protein complex that binds the vacuole membrane. In the absence of the Ccz1–Mon1 complex, the integrity of vacuole SNARE pairing and the unpaired SNARE class C Vps/HOPS complex interaction were both impaired. The Ccz1–Mon1 complex colocalized with other fusion components on the vacuole as part of the cis-SNARE complex, and the association of the Ccz1–Mon1 complex with the vacuole appeared to be regulated by the class C Vps/HOPS complex proteins. Accordingly, we propose that the Ccz1–Mon1 complex is critical for the Ypt7-dependent tethering/docking stage leading to the formation of a trans-SNARE complex and subsequent vacuole fusion.

scholarly journals The HOPS/class C Vps complex tethers membranes by binding to one Rab GTPase in each apposed membrane

2015 ◽  
Vol 26 (14) ◽  
pp. 2655-2663 ◽  
Author(s):  
Ruoya Ho ◽  
Christopher Stroupe
Keyword(s):  
Protein Sorting ◽  
Rab Gtpase ◽  
Nucleotide Exchange ◽  
Casein Kinase I ◽  
Late Endosomes ◽  
Membrane Tethering ◽  
Class C ◽  
Intracellular Membrane Fusion ◽  
Rab Effector ◽  
Tethered Membranes

Many Rab GTPase effectors are membrane-tethering factors, that is, they physically link two apposed membranes before intracellular membrane fusion. In this study, we investigate the distinct binding factors needed on apposed membranes for Rab effector–dependent tethering. We show that the homotypic fusion and protein-sorting/class C vacuole protein-sorting (HOPS/class C Vps) complex can tether low-curvature membranes, that is, liposomes with a diameter of ∼100 nm, only when the yeast vacuolar Rab GTPase Ypt7p is present in both tethered membranes. When HOPS is phosphorylated by the vacuolar casein kinase I, Yck3p, tethering only takes place when GTP-bound Ypt7p is present in both tethered membranes. When HOPS is not phosphorylated, however, its tethering activity shows little specificity for the nucleotide-binding state of Ypt7p. These results suggest a model for HOPS-mediated tethering in which HOPS tethers membranes by binding to Ypt7p in each of the two tethered membranes. Moreover, because vacuole-associated HOPS is presumably phosphorylated by Yck3p, our results suggest that nucleotide exchange of Ypt7p on multivesicular bodies (MVBs)/late endosomes must take place before HOPS can mediate tethering at vacuoles.

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 An evolutionary switch in the specificity of an endosomal CORVET tether underlies formation of regulated secretory vesicles in the ciliate Tetrahymena thermophila

2017 ◽  
Author(s):  
Daniela Sparvoli ◽  
Elisabeth Richardson ◽  
Hiroko Osakada ◽  
Xun Lan ◽  
Masaaki Iwamoto ◽  
...  
Keyword(s):  
Tetrahymena Thermophila ◽  
Secretory Granules ◽  
Protein Sorting ◽  
Endocytic Pathway ◽  
Forward Genetics ◽  
Vacuolar Protein Sorting ◽  
Protein Receptor ◽  
Early Endosomes ◽  
Class C ◽  
Vacuolar Protein

SummaryIn the endocytic pathway of animals, two related complexes, called CORVET (Class C Core Vacuole/Endosome Transport) and HOPS (Homotypic fusion and protein sorting), act as both tethers and fusion factors for early and late endosomes, respectively. Mutations in CORVET or HOPS lead to trafficking defects and contribute to human disease including immune dysfunction. HOPS and CORVET are conserved throughout eukaryotes but remarkably, in the ciliate Tetrahymena thermophila, the HOPS-specific subunits are absent while CORVET-specific subunits have proliferated. VPS8 (Vacuolar Protein Sorting), a CORVET subunit, expanded to 6 paralogs in Tetrahymena. This expansion correlated with loss of HOPS within a ciliate subgroup including the Oligohymenophorea, which contains Tetrahymena. As uncovered via forward genetics, a single VPS8 paralog in Tetrahymena (VPS8A) is required to synthesize prominent secretory granules called mucocysts. More specifically, ∆vps8a cells fail to deliver a subset of cargo proteins to developing mucocysts, instead accumulating that cargo in vesicles also bearing the mucocyst sorting receptor, Sor4p. Surprisingly, although this transport step relies on CORVET, it does not appear to involve early endosomes. Instead, Vps8a associates with the late endosomal/lysosomal marker Rab7, indicating target specificity switching occurred in CORVET subunits during the evolution of ciliates. Mucocysts belong to a markedly diverse and understudied class of protist secretory organelles called extrusomes. Our results underscore that biogenesis of mucocysts depends on endolysosomal trafficking, revealing parallels with invasive organelles in apicomplexan parasites and suggesting that a wide array of secretory adaptations in protists, like in animals, depend on mechanisms related to lysosome biogenesis.AbbreviationsLRO(Lysosome-related organelle)HOPS(homotypic fusion and protein sorting complex)CORVET(Class C core Vacuole/Endosome Transport)VPS(vacuolar protein sorting)GRL(granule lattice)GRT(granule tip)Igr(Induced upon granule regeneration)SNARE(Soluble NSF attachment protein receptor)LECA(last eukaryotic common ancestor)

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 The Hsp90 Chaperone Complex Regulates GDI-dependent Rab Recycling

2006 ◽  
Vol 17 (8) ◽  
pp. 3494-3507 ◽  
Author(s):  
Christine Y. Chen ◽  
William E. Balch
Keyword(s):  
Rab Gtpase ◽  
Rab Gtpases ◽  
Coding System ◽  
Golgi Transport ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Hsp90 Activity ◽  
Hsp90 Chaperone ◽  
Chaperone Complex ◽  
Hsp 90 ◽  
Synaptic Vesicle Fusion

Rab GTPase regulated hubs provide a framework for an integrated coding system, the membrome network, that controls the dynamics of the specialized exocytic and endocytic membrane architectures found in eukaryotic cells. Herein, we report that Rab recycling in the early exocytic pathways involves the heat-shock protein (Hsp)90 chaperone system. We find that Hsp90 forms a complex with guanine nucleotide dissociation inhibitor (GDI) to direct recycling of the client substrate Rab1 required for endoplasmic reticulum (ER)-to-Golgi transport. ER-to-Golgi traffic is inhibited by the Hsp90-specific inhibitors geldanamycin (GA), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), and radicicol. Hsp90 activity is required to form a functional GDI complex to retrieve Rab1 from the membrane. Moreover, we find that Hsp90 is essential for Rab1-dependent Golgi assembly. The observation that the highly divergent Rab GTPases Rab1 involved in ER-to-Golgi transport and Rab3A involved in synaptic vesicle fusion require Hsp90 for retrieval from membranes lead us to now propose that the Hsp90 chaperone system may function as a general regulator for Rab GTPase recycling in exocytic and endocytic trafficking pathways involved in cell signaling and proliferation.

scholarly journals TBC-2 Regulates RAB-5/RAB-7-mediated Endosomal Trafficking inCaenorhabditis elegans

2010 ◽  
Vol 21 (13) ◽  
pp. 2285-2296 ◽  
Author(s):  
Laëtitia Chotard ◽  
Ashwini K. Mishra ◽  
Marc-André Sylvain ◽  
Simon Tuck ◽  
David G. Lambright ◽  
...  
Keyword(s):  
Rab Gtpase ◽  
Endosomal Trafficking ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Late Endosomes ◽  
Arginine Finger ◽  
Endosome Maturation ◽  
Hops Complex

During endosome maturation the early endosomal Rab5 GTPase is replaced with the late endosomal Rab7 GTPase. It has been proposed that active Rab5 can recruit and activate Rab7, which in turn could inactivate and remove Rab5. However, many of the Rab5 and Rab7 regulators that mediate endosome maturation are not known. Here, we identify Caenorhabditis elegans TBC-2, a conserved putative Rab GTPase-activating protein (GAP), as a regulator of endosome to lysosome trafficking in several tissues. We show that tbc-2 mutant animals accumulate enormous RAB-7–positive late endosomes in the intestine containing refractile material. RAB-5, RAB-7, and components of the homotypic fusion and vacuole protein sorting (HOPS) complex, a RAB-7 effector/putative guanine nucleotide exchange factor (GEF), are required for the tbc-2(−) intestinal phenotype. Expression of activated RAB-5 Q78L in the intestine phenocopies the tbc-2(−) large late endosome phenotype in a RAB-7 and HOPS complex-dependent manner. TBC-2 requires the catalytic arginine-finger for function in vivo and displays the strongest GAP activity on RAB-5 in vitro. However, TBC-2 colocalizes primarily with RAB-7 on late endosomes and requires RAB-7 for membrane localization. Our data suggest that TBC-2 functions on late endosomes to inactivate RAB-5 during endosome maturation.

scholarly journals Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana

2013 ◽  
Vol 24 (4) ◽  
pp. 510-520 ◽  
Author(s):  
Matyáš Fendrych ◽  
Lukáš Synek ◽  
Tamara Pečenková ◽  
Edita Janková Drdová ◽  
Juraj Sekereš ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Complex Dynamics ◽  
Active Regions ◽  
Snare Complex ◽  
Epifluorescence Microscopy ◽  
Rab Gtpases ◽  
Exocyst Complex ◽  
Protein Receptor ◽  
Outer Domain

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6–green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.

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.

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