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 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 Double-transmembrane domain reduces fusion rate by increasing lipid-protein mismatch

2020 ◽  
Author(s):  
B. Bu ◽  
Z. Tian ◽  
D. Li ◽  
K. Zhang ◽  
B. Ji ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Fusion Rate ◽  
Protein Receptor ◽  
In Vitro Reconstitution ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Pass Energy ◽  
Autophagosome Maturation

ABSTRACTMembrane fusion mediated by Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins is an important cellular process. For neuronal SNAREs, the single transmembrane domain has been proposed to pass zippering energy to membranes for inducing fast fusion. In contrast, the SNARE protein, syntaxin 17, for membrane fusion involved in autophagosome maturation contains an unusual V-shape double-transmembrane domain that may influence its capability to pass energy. Here, we showed that this double-transmembrane domain significantly reduces fusion with an in vitro reconstitution system. Through theoretic modelling, we found that this V-shape double-transmembrane domain increases lipid-protein mismatch, which reduces the energy transduction for fusion. Moreover, our model also revealed the involvement of 2-3 SNAREs in a general fusion process.SIGNIFICANT STATEMENTSoluble N-ethylmaleimide-sensitive factor activating protein receptors (SNAREs) serve as the molecular machine to mediate membrane fusion. The zipper formation of core structure extending to membranes by two single transmembrena domains (TMDs) is the main driving force of membrane fusion. The role of TMD in fusion is unclear. By adding an extra TMD, we found that the hydrophobic mismatch effect between the thickness of the membrane and the length of TMDs plays an important role in regulating fusion.

scholarly journals Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway

2021 ◽  
Author(s):  
Luther J. Davis ◽  
Nicholas A. Bright ◽  
James R. Edgar ◽  
Michael D.J. Parkinson ◽  
Lena Wartosch ◽  
...  
Keyword(s):  
Pore Formation ◽  
Endocytic Pathway ◽  
Multivesicular Bodies ◽  
Lysosomal Hydrolases ◽  
Attachment Protein ◽  
Body Protein ◽  
Protein Carrier ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Pore Expansion

To provide insights into the kiss-and-run and full fusion events resulting in endocytic delivery to lysosomes, we investigated conditions causing increased tethering and pore formation between late endocytic organelles in HeLa cells. Knockout of the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) VAMP7 and VAMP8 showed, by electron microscopy, the accumulation of tethered LAMP (lysosome associated membrane protein)-carrier vesicles around multivesicular bodies, as well as the appearance of ‘hourglass’ profiles of late endocytic organelles attached by filamentous tethers, but did not prevent endocytic delivery to lysosomal hydrolases. Subsequent depletion of the SNARE YKT6 reduced this delivery, consistent with it compensating for the absence of VAMP7 and VAMP8. We also investigated filamentous tethering between multivesicular bodies and enlarged endolysosomes following depletion of CHMP6 (charged multi-vesicular body protein 6) and provide the first evidence that pore formation commences at the edge of tether arrays, with pore expansion required for full membrane fusion.

Roles of the cytoplasmic and transmembrane domains of syntaxins in intracellular localization and trafficking

2001 ◽  
Vol 114 (17) ◽  
pp. 3115-3124 ◽  
Author(s):  
Kazuo Kasai ◽  
Kimio Akagawa
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Transmembrane Domains ◽  
Cytoplasmic Domains ◽  
Immunofluorescence Analysis ◽  
Attachment Protein ◽  
Transport Pathways ◽  
Sensitive Factor ◽  
Protein Receptors

Syntaxins are target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) involved in docking and fusion of vesicles in exocytosis and endocytosis. Many syntaxin isoforms have been isolated, and each one displays a distinct intracellular localization pattern. However, the signals that drive the specific intracellular localization of syntaxins are poorly understood. In this study, we used indirect immunofluorescence analysis to examine the localization of syntaxin chimeras, each containing a syntaxin transmembrane domain fused to a cytoplasmic domain derived from a different syntaxin. We show that the cytoplasmic domains of syntaxins 5, 6, 7 and 8 have important effects on intracellular localization. We also demonstrate that the transmembrane domain of syntaxin 5 is sufficient to localize the chimera to the compartment expected for wild-type syntaxin 5. Additionally, we find that syntaxins 6, 7 and 8, but not syntaxin 5, are present at the plasma membrane, and that these syntaxins cycle through the plasma membrane by virtue of their cytoplasmic domains. Finally, we find that di-leucine-based motifs in the cytoplasmic domains of syntaxins 7 and 8 are necessary for their intracellular localization and trafficking via distinct transport pathways. Combined, these results suggest that both the cytoplasmic and the transmembrane domains play important roles in intracellular localization and trafficking of syntaxins.

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.

scholarly journals Interaction of SNAREs with ArfGAPs Precedes Recruitment of Sec18p/NSF

2007 ◽  
Vol 18 (8) ◽  
pp. 2852-2863 ◽  
Author(s):  
Christina Schindler ◽  
Anne Spang
Keyword(s):  
Conformational Change ◽  
Atp Hydrolysis ◽  
Small Gtpase ◽  
Snare Complex ◽  
Coat Proteins ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Target Membrane

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are key components of the fusion machinery in vesicular transport and in homotypic membrane fusion. We previously found that ADP-ribosylation factor GTPase activating proteins (ArfGAPs) promoted a conformational change on SNAREs that allowed recruitment of the small GTPase Arf1p in stoichiometric amounts. Here, we show that the ArfGAP Gcs1p accelerates vesicle (v)-target membrane (t)-SNARE complex formation in vitro, indicating that ArfGAPs may act as folding chaperones. These SNARE complexes were resolved in the presence of ATP by the yeast homologues of α-soluble N-ethylmaleimide-sensitive factor attachment protein and N-ethylmaleimide-sensitive factor, Sec17p and Sec18p, respectively. In addition, Sec18p and Sec17p also recognized the “activated” SNAREs even when they were not engaged in v-t-SNARE complexes. Here again, the induction of a conformational change by ArfGAPs was essential. Surprisingly, recruitment of Sec18p to SNAREs did not require Sec17p or ATP hydrolysis. Moreover, Sec18p displaced prebound Arf1p from SNAREs, indicating that Sec18p may have more than one function: first, to ensure that all vesicle coat proteins are removed from the SNAREs before the engagement in a trans-SNARE complex; and second, to resolve cis-SNARE complexes after fusion has occurred.

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 SNARE Function Is Not Involved in Early Endosome Docking

2008 ◽  
Vol 19 (12) ◽  
pp. 5327-5337 ◽  
Author(s):  
Ulf Geumann ◽  
Sina Victoria Barysch ◽  
Peer Hoopmann ◽  
Reinhard Jahn ◽  
Silvio O. Rizzoli
Keyword(s):  
Phosphatidyl Inositol ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Vitro Assay ◽  
Receptor Proteins ◽  
Transport Vesicles ◽  
Early Endosomes ◽  
Sensitive Factor ◽  
Rab Effector

Docking and fusion of transport vesicles constitute elementary steps in intracellular membrane traffic. While docking is thought to be initiated by Rab-effector complexes, fusion is mediated by SNARE (N-ethylmaleimide-sensitive factor [NSF] attachment receptor) proteins. However, it has been recently debated whether SNAREs also play a role in the establishment or maintenance of a stably docked state. To address this question, we have investigated the SNARE dependence of docking and fusion of early endosomes, one of the central sorting compartments in the endocytic pathway. A new, fluorescence-based in vitro assay was developed, which allowed us to investigate fusion and docking in parallel. Similar to homotypic fusion, docking of early endosomes is dependent on the presence of ATP and requires physiological temperatures. Unlike fusion, docking is insensitive to the perturbation of SNARE function by means of soluble SNARE motifs, SNARE-specific Fab fragments, or by a block of NSF activity. In contrast, as expected, docking is strongly reduced by interfering with the synthesis of phosphatidyl inositol (PI)-3 phosphate, with the function of Rab-GTPases, as well as with early endosomal autoantigen 1 (EEA1), an essential tethering factor. We conclude that docking of early endosomes is independent of SNARE function.

scholarly journals Reduced O-GlcNAcylation of SNAP-23 promotes cisplatin resistance by inducing exosome secretion in ovarian cancer

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Luomeng Qian ◽  
Xiaoshan Yang ◽  
Shaohui Li ◽  
Hang Zhao ◽  
Yunge Gao ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Protein Modification ◽  
Snare Complex ◽  
Ovarian Cancer Cells ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor

AbstractExosomes have been associated with chemoresistance in various cancers, but such a role in ovarian cancer is not yet clear. Here, using in vitro cell-based and in vivo mouse model experiments, we show that downregulation of O-GlcNAcylation, a key post-translational protein modification, promotes exosome secretion. This increases exosome-mediated efflux of cisplatin from cancer cells resulting in chemoresistance. Mechanistically, our data indicate that downregulation of O-GlcNAclation transferase (OGT) reduces O-GlcNAclation of SNAP-23. Notably, O-GlcNAcylation of SNAP-23 is vital for regulating exosome release in ovarian cancer cells. Reduced O-GlcNAclation of SNAP-23 subsequently promotes the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of SNAP-23, VAMP8, and Stx4 proteins. This enhances exosome release causing chemoresistance by increasing the efflux of intracellular cisplatin.

scholarly journals Sequential actions of phosphatidylinositol phosphates regulate phagosome-lysosome fusion

2018 ◽  
Vol 29 (4) ◽  
pp. 452-465 ◽  
Author(s):  
Andreas Jeschke ◽  
Albert Haas
Keyword(s):  
Lysosomal Hydrolases ◽  
Attachment Protein ◽  
Free System ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
A Cell ◽  
Phosphatidylinositol Phosphates ◽  
Phosphatidylinositol 4

Phagosomes mature into phagolysosomes by sequential fusion with early endosomes, late endosomes, and lysosomes. Phagosome-with-lysosome fusion (PLF) results in the delivery of lysosomal hydrolases into phagosomes and in digestion of the cargo. The machinery that drives PLF has been little investigated. Using a cell-free system, we recently identified the phosphoinositide lipids (PIPs) phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 4-phosphate (PI(4)P) as regulators of PLF. We now report the identification and the PIP requirements of four distinct subreactions of PLF. Our data show that (i) PI(3)P and PI(4)P are dispensable for the disassembly and activation of (phago)lysosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptors, that (ii) PI(3)P is required only after the tethering step, and that (iii) PI(4)P is required during and after tethering. Moreover, our data indicate that PI(4)P is needed to anchor Arl8 (Arf-like GTPase 8) and its effector homotypic fusion/vacuole protein sorting complex (HOPS) to (phago)lysosome membranes, whereas PI(3)P is required for membrane association of HOPS only. Our study provides a first link between PIPs and established regulators of membrane fusion in late endocytic trafficking.

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