scholarly journals Mammalian Late Vacuole Protein Sorting Orthologues Participate in Early Endosomal Fusion and Interact with the Cytoskeleton

2004 ◽  
Vol 15 (3) ◽  
pp. 1197-1210 ◽  
Author(s):  
Simon C. W. Richardson ◽  
Stanley C. Winistorfer ◽  
Viviane Poupon ◽  
J. Paul Luzio ◽  
Robert C. Piper
Keyword(s):  
Protein Sorting ◽  
Early Endosome ◽  
Endocytic Pathway ◽  
Rab Proteins ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Early Endosomes ◽  
Sensitive Factor ◽  
Factor Attachment Protein Receptor

In Saccharomyces cerevisiae, the class C vacuole protein sorting (Vps) proteins, together with Vam2p/Vps41p and Vam6p/Vps39p, form a complex that interacts with soluble N-ethylmaleimide-sensitive factor attachment protein receptor and Rab proteins to “tether” vacuolar membranes before fusion. To determine a role for the corresponding mammalian orthologues, we examined the function, localization, and protein interactions of endogenous mVps11, mVps16, mVps18, mVam2p, and mVam6. We found a significant proportion of these proteins localized to early endosome antigen-1 and transferrin receptor-positive early endosomes in Vero, normal rat kidney, and Chinese hamster ovary cells. Immunoprecipitation experiments showed that mVps18 not only interacted with Syntaxin (Syn)7, vesicle-associated membrane protein 8, and Vti1-b but also with Syn13, Syn6, and the Sec1/Munc18 protein mVps45, which catalyze early endosomal fusion events. Moreover, anti-mVps18 antibodies inhibited early endosome fusion in vitro. Mammalian mVps18 also associated with mVam2 and mVam6 as well as with the microtubule-associated Hook1 protein, an orthologue of the Drosophila Hook protein involved in endosome biogenesis. Using in vitro binding and immunofluorescence experiments, we found that mVam2 and mVam6 also associated with microtubules, whereas mVps18, mVps16, and mVps11 associated with actin filaments. These data indicate that the late Vps proteins function during multiple soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated fusion events throughout the endocytic pathway and that their activity may be coordinated with cytoskeletal function.

scholarly journals Syntaxin 7 and VAMP-7 are SolubleN-Ethylmaleimide–sensitive Factor Attachment Protein Receptors Required for Late Endosome–Lysosome and Homotypic Lysosome Fusion in Alveolar Macrophages

2000 ◽  
Vol 11 (7) ◽  
pp. 2327-2333 ◽  
Author(s):  
Diane McVey Ward ◽  
Jonathan Pevsner ◽  
Matthew A. Scullion ◽  
Michael Vaughn ◽  
Jerry Kaplan
Keyword(s):  
Alveolar Macrophages ◽  
Transmembrane Domain ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Early Endosomes ◽  
Sensitive Factor ◽  
Protein Receptors

Endocytosis in alveolar macrophages can be reversibly inhibited, permitting the isolation of endocytic vesicles at defined stages of maturation. Using an in vitro fusion assay, we determined that each isolated endosome population was capable of homotypic fusion. All vesicle populations were also capable of heterotypic fusion in a temporally specific manner; early endosomes, isolated 4 min after internalization, could fuse with endosomes isolated 8 min after internalization but not with 12-min endosomes or lysosomes. Lysosomes fuse with 12-min endosomes but not with earlier endosomes. Using homogenous populations of endosomes, we have identified Syntaxin 7 as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome–lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late endosome and lysosome fusion as well as heterotypic late endosome–lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was a SNARE required for late endosome–lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both late endosome–lysosome fusion and homotypic lysosome fusion in vitro. These studies indicate that: 1) fusion along the endocytic pathway is a highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and human VAMP-7, are involved in fusion of vesicles in the late endocytic pathway in alveolar macrophages.

scholarly journals Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking

2015 ◽  
Vol 26 (3) ◽  
pp. 530-536 ◽  
Author(s):  
Jessica B. A. Sadler ◽  
Nia J. Bryant ◽  
Gwyn W. Gould
Keyword(s):  
Plasma Membrane ◽  
Snare Complex ◽  
Cell Type ◽  
Attachment Protein ◽  
Systematic Analysis ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Factor Attachment Protein Receptor

The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes.

scholarly journals Synaptotagmin Isoforms Couple Distinct Ranges of Ca2+, Ba2+, and Sr2+ Concentration to SNARE-mediated Membrane Fusion

2005 ◽  
Vol 16 (10) ◽  
pp. 4755-4764 ◽  
Author(s):  
Akhil Bhalla ◽  
Ward C. Tucker ◽  
Edwin R. Chapman
Keyword(s):  
Membrane Fusion ◽  
Synaptic Vesicles ◽  
Binding Protein ◽  
Divalent Cations ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Factor Attachment Protein Receptor ◽  
Stimulation Of

Ca2+-triggered exocytosis of synaptic vesicles is controlled by the Ca2+-binding protein synaptotagmin (syt) I. Fifteen additional isoforms of syt have been identified. Here, we compared the abilities of three syt isoforms (I, VII, and IX) to regulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in vitro in response to divalent cations. We found that different isoforms of syt couple distinct ranges of Ca2+, Ba2+, and Sr2+ to membrane fusion; syt VII was ∼400-fold more sensitive to Ca2+ than was syt I. Omission of phosphatidylserine (PS) from both populations of liposomes completely abrogated the ability of all three isoforms of syt to stimulate fusion. Mutations that selectively inhibit syt·target-SNARE (t-SNARE) interactions reduced syt stimulation of fusion. Using Sr2+ and Ba2+, we found that binding of syt to PS and t-SNAREs can be dissociated from activation of fusion, uncovering posteffector-binding functions for syt. Our data demonstrate that different syt isoforms are specialized to sense different ranges of divalent cations and that PS is an essential effector of Ca2+·syt action.

Gβγ Interferes with Ca2+-Dependent Binding of Synaptotagmin to the Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor (SNARE) Complex

2007 ◽  
Vol 72 (5) ◽  
pp. 1210-1219 ◽  
Author(s):  
Eun-Ja Yoon ◽  
Tatyana Gerachshenko ◽  
Bryan D. Spiegelberg ◽  
Simon Alford ◽  
Heidi E. Hamm
Keyword(s):  
Snare Complex ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Factor Attachment Protein Receptor

scholarly journals Syntaxin 16 is a master recruitment factor for cytokinesis

2013 ◽  
Vol 24 (23) ◽  
pp. 3663-3674 ◽  
Author(s):  
Hélia Neto ◽  
Alexandra Kaupisch ◽  
Louise L. Collins ◽  
Gwyn W. Gould
Keyword(s):  
Membrane Vesicles ◽  
Attachment Protein ◽  
Recycling Endosome ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Late Telophase ◽  
Intracellular Organelles ◽  
Factor Attachment Protein Receptor

Recently it was shown that both recycling endosome and endosomal sorting complex required for transport (ESCRT) components are required for cytokinesis, in which they are believed to act in a sequential manner to bring about secondary ingression and abscission, respectively. However, it is not clear how either of these complexes is targeted to the midbody and whether their delivery is coordinated. The trafficking of membrane vesicles between different intracellular organelles involves the formation of soluble N-ethylmalei­mide–sensitive factor attachment protein receptor (SNARE) complexes. Although membrane traffic is known to play an important role in cytokinesis, the contribution and identity of intracellular SNAREs to cytokinesis remain unclear. Here we demonstrate that syntaxin 16 is a key regulator of cytokinesis, as it is required for recruitment of both recycling endosome–associated Exocyst and ESCRT machinery during late telophase, and therefore that these two distinct facets of cytokinesis are inextricably linked.

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