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.

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 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.

scholarly journals SNARE phosphorylation: a control mechanism for insulin-stimulated glucose transport and other regulated exocytic events

2017 ◽  
Vol 45 (6) ◽  
pp. 1271-1277 ◽  
Author(s):  
Kamilla M.E. Laidlaw ◽  
Rachel Livingstone ◽  
Mohammed Al-Tobi ◽  
Nia J. Bryant ◽  
Gwyn W. Gould
Keyword(s):  
Membrane Fusion ◽  
Molecular Mechanisms ◽  
Membrane Receptors ◽  
Multivesicular Bodies ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Plasma Membrane Receptors ◽  
Regulated Trafficking ◽  
Receptor Family

Trafficking within eukaryotic cells is a complex and highly regulated process; events such as recycling of plasma membrane receptors, formation of multivesicular bodies, regulated release of hormones and delivery of proteins to membranes all require directionality and specificity. The underpinning processes, including cargo selection, membrane fusion, trafficking flow and timing, are controlled by a variety of molecular mechanisms and engage multiple families of lipids and proteins. Here, we will focus on control of trafficking processes via the action of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family of proteins, in particular their regulation by phosphorylation. We will describe how these proteins are controlled in a range of regulated trafficking events, with particular emphasis on the insulin-stimulated delivery of glucose transporters to the surface of adipose and muscle cells. Here, we focus on a few examples of SNARE phosphorylation which exemplify distinct ways in which SNARE machinery phosphorylation may regulate membrane fusion.

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 GLUT4 Recycles via a trans-Golgi Network (TGN) Subdomain Enriched in Syntaxins 6 and 16 But Not TGN38: Involvement of an Acidic Targeting Motif

2003 ◽  
Vol 14 (3) ◽  
pp. 973-986 ◽  
Author(s):  
Annette M. Shewan ◽  
Ellen M. van Dam ◽  
Sally Martin ◽  
Tang Bor Luen ◽  
Wanjin Hong ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Adipocyte Differentiation ◽  
Attachment Protein ◽  
Trans Golgi Network ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Glucose Transporter Glut4 ◽  
Golgi Network ◽  
Endosomal System

Insulin stimulates glucose transport in fat and muscle cells by triggering exocytosis of the glucose transporter GLUT4. To define the intracellular trafficking of GLUT4, we have studied the internalization of an epitope-tagged version of GLUT4 from the cell surface. GLUT4 rapidly traversed the endosomal system en route to a perinuclear location. This perinuclear GLUT4 compartment did not colocalize with endosomal markers (endosomal antigen 1 protein, transferrin) or TGN38, but showed significant overlap with the TGN target (t)-solubleN-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Syntaxins 6 and 16. These results were confirmed by vesicle immunoisolation. Consistent with a role for Syntaxins 6 and 16 in GLUT4 trafficking we found that their expression was up-regulated significantly during adipocyte differentiation and insulin stimulated their movement to the cell surface. GLUT4 trafficking between endosomes and trans-Golgi network was regulated via an acidic targeting motif in the carboxy terminus of GLUT4, because a mutant lacking this motif was retained in endosomes. We conclude that GLUT4 is rapidly transported from the cell surface to a subdomain of thetrans-Golgi network that is enriched in the t-SNAREs Syntaxins 6 and 16 and that an acidic targeting motif in the C-terminal tail of GLUT4 plays an important role in this process.

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 Alterations in platelet secretion differentially affect thrombosis and hemostasis

2018 ◽  
Vol 2 (17) ◽  
pp. 2187-2198 ◽  
Author(s):  
Smita Joshi ◽  
Meenakshi Banerjee ◽  
Jinchao Zhang ◽  
Akhil Kesaraju ◽  
Irina D. Pokrovskaya ◽  
...  
Keyword(s):  
Attachment Protein ◽  
Granule Exocytosis ◽  
Key Points ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Granule Secretion ◽  
Platelet Secretion

Key Points VAMP isoforms regulate the kinetics and extent of platelet granule exocytosis. Manipulating platelet sensitive factor attachment protein receptors alters granule secretion, which affects the hemostatic balance.

scholarly journals The HOPS complex mediates autophagosome–lysosome fusion through interaction with syntaxin 17

2014 ◽  
Vol 25 (8) ◽  
pp. 1327-1337 ◽  
Author(s):  
Peidu Jiang ◽  
Taki Nishimura ◽  
Yuriko Sakamaki ◽  
Eisuke Itakura ◽  
Tomohisa Hatta ◽  
...  
Keyword(s):  
Protein Sorting ◽  
Endocytic Pathway ◽  
Mass Spectrometry Analysis ◽  
Autophagic Flux ◽  
Interacting Protein ◽  
Spectrometry Analysis ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Vacuolar Protein ◽  
Hops Complex

Membrane fusion is generally controlled by Rabs, soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs), and tethering complexes. Syntaxin 17 (STX17) was recently identified as the autophagosomal SNARE required for autophagosome–lysosome fusion in mammals and Drosophila. In this study, to better understand the mechanism of autophagosome–lysosome fusion, we searched for STX17-interacting proteins. Immunoprecipitation and mass spectrometry analysis identified vacuolar protein sorting 33A (VPS33A) and VPS16, which are components of the homotypic fusion and protein sorting (HOPS)–tethering complex. We further confirmed that all HOPS components were coprecipitated with STX17. Knockdown of VPS33A, VPS16, or VPS39 blocked autophagic flux and caused accumulation of STX17- and microtubule-associated protein light chain (LC3)–positive autophagosomes. The endocytic pathway was also affected by knockdown of VPS33A, as previously reported, but not by knockdown of STX17. By contrast, ultraviolet irradiation resistance–associated gene (UVRAG), a known HOPS-interacting protein, did not interact with the STX17–HOPS complex and may not be directly involved in autophagosome–lysosome fusion. Collectively these results suggest that, in addition to its well-established function in the endocytic pathway, HOPS promotes autophagosome–lysosome fusion through interaction with STX17.

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