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 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 The yeast vacuolar ABC transporter Ybt1p regulates membrane fusion through Ca2+ transport modulation

2012 ◽  
Vol 448 (3) ◽  
pp. 365-372 ◽  
Author(s):  
Terry L. Sasser ◽  
Mark Padolina ◽  
Rutilio A. Fratti
Keyword(s):  
Abc Transporter ◽  
Fusion Reaction ◽  
Protein Sorting ◽  
Atp Binding Cassette Transporter ◽  
Vacuole Fusion ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Bile Acid Transporter ◽  
Transport Modulation

Ybt1p is a class C ABC transporter (ATP-binding cassette transporter) that is localized to the vacuole of Saccharomyces cerevisiae. Although Ybt1p was originally identified as a bile acid transporter, it has also been found to function in other capacities, including the translocation of phosphatidylcholine to the vacuole lumen, and the regulation of Ca2+ homoeostasis. In the present study we found that deletion of YBT1 enhanced in vitro homotypic vacuole fusion by up to 50% relative to wild-type vacuoles. The increased vacuole fusion was not due to aberrant protein sorting of SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) or recruitment of factors from the cytosol such as Ypt7p and the HOPS (homotypic fusion and vacuole protein sorting) tethering complex. In addition, ybt1Δ vacuoles displayed no observable differences in the formation of SNARE complexes, interactions between SNAREs and HOPS, or formation of vertex microdomains. However, the absence of Ybt1p caused significant changes in Ca2+ transport during fusion. One difference was the prolonged Ca2+ influx exhibited by ybt1Δ vacuoles at the start of the fusion reaction. We also observed a striking delay in SNARE-dependent Ca2+ efflux. As vacuole fusion can be inhibited by high Ca2+ concentrations, we suggest that the delayed efflux in ybt1Δ vacuoles leads to the enhanced SNARE function.

scholarly journals Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor–mediated phagosome formation and maturation in macrophages

2018 ◽  
Vol 29 (14) ◽  
pp. 1753-1762 ◽  
Author(s):  
Chiye Sakurai ◽  
Makoto Itakura ◽  
Daiki Kinoshita ◽  
Seisuke Arai ◽  
Hitoshi Hashimoto ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Interferon Γ ◽  
Fc Receptor ◽  
Attachment Protein ◽  
Iκb Kinase ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Fret Efficiency

SNAP-23 is a plasma membrane-localized soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNARE) involved in Fc receptor (FcR)-mediated phagocytosis. However, the regulatory mechanism underlying its function remains elusive. Using phosphorylation-specific antibodies, SNAP-23 was found to be phosphorylated at Ser95 in macrophages. To understand the role of this phosphorylation, we established macrophage lines overexpressing the nonphosphorylatable S95A or the phosphomimicking S95D mutation. The efficiency of phagosome formation and maturation was severely reduced in SNAP-23-S95D–overexpressing cells. To examine whether phosphorylation at Ser95 affected SNAP-23 structure, we constructed intramolecular Förster resonance energy transfer (FRET) probes of SNAP-23 designed to evaluate the approximation of the N termini of the two SNARE motifs. Interestingly, a high FRET efficiency was detected on the membrane when the S95D probe was used, indicating that phosphorylation at Ser95 caused a dynamic structural shift to the closed form. Coexpression of IκB kinase (IKK) 2 enhanced the FRET efficiency of the wild-type probe on the phagosome membrane. Furthermore, the enhanced phagosomal FRET signal in interferon-γ–activated macrophages was largely dependent on IKK2, and this kinase mediated a delay in phagosome-lysosome fusion. These results suggested that SNAP-23 phosphorylation at Ser95 played an important role in the regulation of SNARE-dependent membrane fusion during FcR-mediated phagocytosis.

Role of tethering factors in secretory membrane traffic

2006 ◽  
Vol 290 (1) ◽  
pp. C11-C26 ◽  
Author(s):  
Elizabeth Sztul ◽  
Vladimir Lupashin
Keyword(s):  
Coiled Coil ◽  
Membrane Traffic ◽  
Cellular Responses ◽  
Regulatory Proteins ◽  
Attachment Protein ◽  
Experimental Systems ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Protein Components

Coiled-coil and multisubunit tethers have emerged as key regulators of membrane traffic and organellar architecture. The restricted subcellular localization of tethers and their ability to interact with Rabs and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) suggests that tethers participate in determining the specificity of membrane fusion. An accepted model of tether function considers them molecular “bridges” that link opposing membranes before SNARE pairing. This model has been extended by findings in various experimental systems, suggesting that tethers may have other functions. Recent reports implicate tethers in the assembly of SNARE complexes, cargo selection and transit, cytoskeletal events, and localized attachment of regulatory proteins. A concept of tethers as scaffolding machines that recruit protein components involved in varied cellular responses is emerging. In this model, tethers function as integration switches that simultaneously transmit information to coordinate distinct processes required for membrane traffic.

scholarly journals Syntaxin 6 Regulates Glut4 Trafficking in 3T3-L1 Adipocytes

2003 ◽  
Vol 14 (7) ◽  
pp. 2946-2958 ◽  
Author(s):  
H. Kumudu I. Perera ◽  
Mairi Clarke ◽  
Nicholas J. Morris ◽  
Wanjin Hong ◽  
Luke H. Chamberlain ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Glucose Transporter ◽  
Attachment Protein ◽  
Insulin Withdrawal ◽  
Sensitive Factor ◽  
Cytosolic Domain ◽  
Protein Receptors ◽  
Adipose Cells

Insulin stimulates the movement of glucose transporter-4 (Glut4)–containing vesicles to the plasma membrane of adipose cells. We investigated the role of post-Golgi t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the trafficking of Glut4 in 3T3-L1 adipocytes. Greater than 85% of syntaxin 6 was found in Glut4-containing vesicles, and this t-SNARE exhibited insulin-stimulated movement to the plasma membrane. In contrast, the colocalization of Glut4 with syntaxin 7, 8, or 12/13 was limited and these molecules did not translocate to the plasma membrane. We used adenovirus to overexpress the cytosolic domain of these syntaxin's and studied their effects on Glut4 traffic. Overexpression of the cytosolic domain of syntaxin 6 did not affect insulin-stimulated glucose transport, but increased basal deGlc transport and cell surface Glut4 levels. Moreover, the syntaxin 6 cytosolic domain significantly reduced the rate of Glut4 reinternalization after insulin withdrawal and perturbed subendosomal Glut4 sorting; the corresponding domains of syntaxins 8 and 12 were without effect. Our data suggest that syntaxin 6 is involved in a membrane-trafficking step that sequesters Glut4 away from traffic destined for the plasma membrane. We speculate that this is at the level of traffic of Glut4 into its unique storage compartment and that syntaxin 16 may be involved.

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.

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.

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