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 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 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 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 New Component of the Vacuolar Class C-Vps Complex Couples Nucleotide Exchange on the Ypt7 Gtpase to Snare-Dependent Docking and Fusion

2000 ◽  
Vol 151 (3) ◽  
pp. 551-562 ◽  
Author(s):  
Andrew E. Wurmser ◽  
Trey K. Sato ◽  
Scott D. Emr
Keyword(s):  
Transport Process ◽  
Rab Gtpase ◽  
Nucleotide Exchange ◽  
Large Protein ◽  
Downstream Effector ◽  
Large Protein Complex ◽  
Transport Vesicles ◽  
Multiple Reactions ◽  
Class C ◽  
Vacuolar Protein

The class C subset of vacuolar protein sorting (Vps) proteins (Vps11, Vps18, Vps16 and Vps33) assembles into a vacuole/prevacuole-associated complex. Here we demonstrate that the class C-Vps complex contains two additional proteins, Vps39 and Vps41. The COOH-terminal 148 amino acids of Vps39 direct its association with the class C-Vps complex by binding to Vps11. A previous study has shown that a large protein complex containing Vps39 and Vps41 functions as a downstream effector of the active, GTP-bound form of Ypt7, a rab GTPase required for the fusion of vesicular intermediates with the vacuole (Price, A., D. Seals, W. Wickner, and C. Ungermann. 2000. J. Cell Biol. 148:1231–1238). Here we present data that indicate that this complex also functions to stimulate nucleotide exchange on Ypt7. We show that Vps39 directly binds the GDP-bound and nucleotide-free forms of Ypt7 and that purified Vps39 stimulates nucleotide exchange on Ypt7. We propose that the class C-Vps complex both promotes Vps39-dependent nucleotide exchange on Ypt7 and, based on the work of Price et al., acts as a Ypt7 effector that tethers transport vesicles to the vacuole. Thus, the class C-Vps complex directs multiple reactions during the docking and fusion of vesicles with the vacuole, each of which contributes to the overall specificity and efficiency of this transport process.

scholarly journals Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase

2008 ◽  
Vol 182 (6) ◽  
pp. 1141-1151 ◽  
Author(s):  
Christopher L. Brett ◽  
Rachael L. Plemel ◽  
Braden T. Lobingier ◽  
Marissa Vignali ◽  
Stanley Fields ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signal Propagation ◽  
Rab Gtpase ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Guanosine Triphosphate ◽  
Casein Kinase I ◽  
Gtp Hydrolysis ◽  
Nucleotide Exchange Factor

Rab guanosine triphosphatases (GTPases) are pivotal regulators of membrane identity and dynamics, but the in vivo pathways that control Rab signaling are poorly defined. Here, we show that the GTPase-activating protein Gyp7 inactivates the yeast vacuole Rab Ypt7 in vivo. To efficiently terminate Ypt7 signaling, Gyp7 requires downstream assistance from an inhibitory casein kinase I, Yck3. Yck3 mediates phosphorylation of at least two Ypt7 signaling targets: a tether, the Vps-C/homotypic fusion and vacuole protein sorting (HOPS) subunit Vps41, and a SNARE, Vam3. Phosphorylation of both substrates is opposed by Ypt7-guanosine triphosphate (GTP). We further demonstrate that Ypt7 binds not one but two Vps-C/HOPS subunits: Vps39, a putative Ypt7 nucleotide exchange factor, and Vps41. Gyp7-stimulated GTP hydrolysis on Ypt7 therefore appears to trigger both passive termination of Ypt7 signaling and active kinase-mediated inhibition of Ypt7's downstream targets. We propose that signal propagation through the Ypt7 pathway is controlled by integrated feedback and feed-forward loops. In this model, Yck3 enforces a requirement for the activated Rab in docking and fusion.

scholarly journals Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles

2004 ◽  
Vol 167 (6) ◽  
pp. 1087-1098 ◽  
Author(s):  
Rutilio A. Fratti ◽  
Youngsoo Jun ◽  
Alexey J. Merz ◽  
Nathan Margolis ◽  
William Wickner
Keyword(s):  
Membrane Fusion ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Membrane Microdomains ◽  
Rab Gtpase ◽  
Protein Protein Interactions ◽  
Px Domain ◽  
Protein Partitioning ◽  
Membrane Fusion Proteins ◽  
Rab Effector

Membrane microdomains are assembled by lipid partitioning (e.g., rafts) or by protein–protein interactions (e.g., coated vesicles). During docking, yeast vacuoles assemble “vertex” ring-shaped microdomains around the periphery of their apposed membranes. Vertices are selectively enriched in the Rab GTPase Ypt7p, the homotypic fusion and vacuole protein sorting complex (HOPS)–VpsC Rab effector complex, SNAREs, and actin. Membrane fusion initiates at vertex microdomains. We now find that the “regulatory lipids” ergosterol, diacylglycerol and 3- and 4-phosphoinositides accumulate at vertices in a mutually interdependent manner. Regulatory lipids are also required for the vertex enrichment of SNAREs, Ypt7p, and HOPS. Conversely, SNAREs and actin regulate phosphatidylinositol 3-phosphate vertex enrichment. Though the PX domain of the SNARE Vam7p has direct affinity for only 3-phosphoinositides, all the regulatory lipids which are needed for vertex assembly affect Vam7p association with vacuoles. Thus, the assembly of the vacuole vertex ring microdomain arises from interdependent lipid and protein partitioning and binding rather than either lipid partitioning or protein interactions alone.

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 The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER

2003 ◽  
Vol 162 (3) ◽  
pp. 403-412 ◽  
Author(s):  
Pierre Morsomme ◽  
Cristina Prescianotto-Baschong ◽  
Howard Riezman
Keyword(s):  
Golgi Complex ◽  
Protein Sorting ◽  
Secretory Proteins ◽  
Rab Gtpase ◽  
Cargo Protein ◽  
Conserved Oligomeric Golgi Complex ◽  
Sorting Process ◽  
Conserved Oligomeric Golgi

Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1–1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1–1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.

scholarly journals Mapping of Vps21 and HOPS Binding Sites in Vps8 and Effect of Binding Site Mutants on Endocytic Trafficking

2010 ◽  
Vol 9 (4) ◽  
pp. 602-610 ◽  
Author(s):  
Agnes Pawelec ◽  
Janja Arsić ◽  
Ralf Kölling
Keyword(s):  
Binding Sites ◽  
Carboxypeptidase Y ◽  
Rab Gtpase ◽  
Physical Interaction ◽  
Endosomal Trafficking ◽  
Core Complex ◽  
Content Type ◽  
Multiple Contacts ◽  
Late Endosomes ◽  
Cargo Protein

ABSTRACT Vps8 is a subunit of the CORVET tethering complex, which is involved in early-to-late endosome fusion. Here, we examine the role of Vps8 in membrane fusion at late endosomes in Saccharomyces cerevisiae. We demonstrate that Vps8 associates with membranes and that this association is independent of the class C/HOPS core complex and, contrary to a previous report, also independent of the Rab GTPase Vps21. Our data indicate that Vps8 makes multiple contacts with membranes. One of these membrane binding regions could be mapped to the N-terminal part of the protein. By two-hybrid analysis, we obtained evidence for a physical interaction between Vps8 and the Rab5 homologue Vps21. In addition, the interaction with the HOPS core complex was confirmed by immunoprecipitation experiments. By deletion analysis, the Vps21 and HOPS binding sites were mapped in Vps8. Deletions that abrogated HOPS core complex binding had a strong effect on the turnover of the endocytic cargo protein Ste6 and on vacuolar sorting of carboxypeptidase Y. In contrast, deletions that abolished Vps21 binding showed only a modest effect. This suggests that the Vps21 interaction is not essential for endosomal trafficking but may be important for some other aspect of Vps8 function.

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.

Sign in / Sign up

Export Citation Format

Share Document