Sec22b-dependent assembly of endoplasmic reticulum Q-SNARE proteins

2008 ◽  
Vol 410 (1) ◽  
pp. 93-100 ◽  
Author(s):  
Takehiro Aoki ◽  
Masaki Kojima ◽  
Katsuko Tani ◽  
Mitsuo Tagaya
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Cd Spectroscopy ◽  
Snare Proteins ◽  
Peripheral Membrane Proteins ◽  
Protein Receptor ◽  
Snare Motif ◽  
Α Helix ◽  
Protein Attachment

SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins involved in membrane fusion usually contain a conserved α-helix (SNARE motif) that is flanked by a C-terminal transmembrane domain. They can be classified into Q-SNARE and R-SNARE based on the structural property of their motifs. Assembly of four SNARE motifs (Qa, b, c and R) is supposed to trigger membrane fusion. We have previously shown that ER (endoplasmic reticulum)-localized syntaxin 18 (Qa) forms a complex with BNIP1 (Qb), p31/Use1 (Qc), Sec22b (R) and several peripheral membrane proteins. In the present study, we examined the interaction of syntaxin 18 with other SNAREs using pulldown assays and CD spectroscopy. We found that the association of syntaxin 18 with Sec22b induces an increase in α-helicity of their SNARE motifs, which results in the formation of high-affinity binding sites for BNIP1 and p31. This R-SNARE-dependent Q-SNARE assembly is quite different from the assembly mechanisms of SNAREs localized in organelles other than the ER. The implication of the mechanism of ER SNARE assembly is discussed in the context of the physiological roles of the syntaxin 18 complex.

scholarly journals Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

IUCrJ ◽  
2014 ◽  
Vol 1 (6) ◽  
pp. 505-513 ◽  
Author(s):  
Asma Rehman ◽  
Julia K. Archbold ◽  
Shu-Hong Hu ◽  
Suzanne J. Norwood ◽  
Brett M. Collins ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Blood Glucose Control ◽  
Vital Role ◽  
Snare Complex ◽  
Snare Proteins ◽  
Regulatory Role ◽  
Sm Proteins ◽  
Protein Receptor

Membrane fusion is essential for human health, playing a vital role in processes as diverse as neurotransmission and blood glucose control. Two protein families are key: (1) the Sec1p/Munc18 (SM) and (2) the solubleN-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins. Whilst the essential nature of these proteins is irrefutable, their exact regulatory roles in membrane fusion remain controversial. In particular, whether SM proteins promote and/or inhibit the SNARE-complex formation required for membrane fusion is not resolved. Crystal structures of SM proteins alone and in complex with their cognate SNARE proteins have provided some insight, however, these structures lack the transmembrane spanning regions of the SNARE proteins and may not accurately reflect the native state. Here, we review the literature surrounding the regulatory role of mammalian Munc18 SM proteins required for exocytosis in eukaryotes. Our analysis suggests that the conflicting roles reported for these SM proteins may reflect differences in experimental design. SNARE proteins appear to require C-terminal immobilization or anchoring, for example through a transmembrane domain, to form a functional fusion complex in the presence of Munc18 proteins.

scholarly journals Analysis of Sec22p in Endoplasmic Reticulum/Golgi Transport Reveals Cellular Redundancy in SNARE Protein Function

2002 ◽  
Vol 13 (9) ◽  
pp. 3314-3324 ◽  
Author(s):  
Yiting Liu ◽  
Charles Barlowe
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Protein Function ◽  
Secretory Pathway ◽  
Snare Proteins ◽  
Snare Protein ◽  
Intracellular Membrane ◽  
Protein Receptor ◽  
Intracellular Membrane Fusion

Membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins form heteromeric complexes that are required for intracellular membrane fusion and are proposed to encode compartmental specificity. In yeast, the R-SNARE protein Sec22p acts in transport between the endoplasmic reticulum (ER) and Golgi compartments but is not essential for cell growth. Other SNARE proteins that function in association with Sec22p (i.e., Sed5p, Bos1p, and Bet1p) are essential, leading us to question how transport through the early secretory pathway is sustained in the absence of Sec22p. In wild-type strains, we show that Sec22p is directly required for fusion of ER-derived vesicles with Golgi acceptor membranes. Insec22Δ strains, Ykt6p, a related R-SNARE protein that operates in later stages of the secretory pathway, is up-regulated and functionally substitutes for Sec22p. In vivo combination of thesec22Δ mutation with a conditionalykt6-1 allele results in lethality, consistent with a redundant mechanism. Our data indicate that the requirements for specific SNARE proteins in intracellular membrane fusion are less stringent than appreciated and suggest that combinatorial mechanisms using both upstream-targeting elements and SNARE proteins are required to maintain an essential level of compartmental organization.

scholarly journals SLY1 and Syntaxin 18 specify a distinct pathway for procollagen VII export from the endoplasmic reticulum

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Cristina Nogueira ◽  
Patrik Erlmann ◽  
Julien Villeneuve ◽  
António JM Santos ◽  
Emma Martínez-Alonso ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Protein Secretion ◽  
Family Members ◽  
Retrograde Transport ◽  
Cytoplasmic Domain ◽  
Snare Complex ◽  
Snare Proteins ◽  
General Protein

TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum (ER). In this study, we report a connection between the cytoplasmic domain of TANGO1 and SLY1, a protein that is required for membrane fusion. Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the recruitment of COPII components, general protein secretion, and retrograde transport of the KDEL-containing protein BIP, and ERGIC53. SLY1 is known to interact with the ER-specific SNARE proteins Syntaxin 17 and 18, however only Syntaxin 18 was required for Procollagen VII export. Neither SLY1 nor Syntaxin 18 was required for the export of the equally bulky Procollagen I from the ER. Altogether, these findings reveal the sorting of bulky collagen family members by TANGO1 at the ER and highlight the existence of different export pathways for secretory cargoes one of which is mediated by the specific SNARE complex containing SLY1 and Syntaxin 18.

scholarly journals A common mechanism for the regulation of vesicular SNAREs on phospholipid membranes

2004 ◽  
Vol 377 (3) ◽  
pp. 781-785 ◽  
Author(s):  
Kuang HU ◽  
Colin RICKMAN ◽  
Joe CARROLL ◽  
Bazbek DAVLETOV
Keyword(s):  
Membrane Fusion ◽  
Snare Complex ◽  
Common Mechanism ◽  
Cell Physiology ◽  
Phospholipid Membranes ◽  
Intracellular Traffic ◽  
Protein Receptor ◽  
Liposomal Membranes ◽  
Eukaryotic Organisms ◽  
Protein Attachment

The SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) family of proteins is essential for membrane fusion in intracellular traffic in eukaryotic organisms. v-SNAREs (vesicular SNAREs) must engage target SNAREs in the opposing membrane to form the fusogenic SNARE complex. Temporal and spatial control of membrane fusion is important for many aspects of cell physiology and may involve the regulation of the SNAREs resident on intracellular membranes. Here we show that the v-SNARE synaptobrevin 2, also known as VAMP (vesicle-associated membrane protein) 2, is restricted from forming the SNARE complex in chromaffin granules from adrenal medullae to the same degree as in brain-purified synaptic vesicles. Our analysis indicates that the previously reported synaptophysin–synaptobrevin interaction is not likely to be involved in regulation of the v-SNARE. Indeed, the restriction can be reproduced for two distinct v-SNARE homologues, synaptobrevin 2 and cellubrevin/VAMP3, by reconstituting them in pure liposomal membranes. Overall, our data uncover a common mechanism for the control of SNARE engagement where intact phospholipid membranes rather than proteins down-regulate vesicular SNAREs in different cellular organelles.

scholarly journals The yeast Batten disease orthologue Btn1 controls endosome–Golgi retrograde transport via SNARE assembly

2011 ◽  
Vol 195 (2) ◽  
pp. 203-215 ◽  
Author(s):  
Rachel Kama ◽  
Vydehi Kanneganti ◽  
Christian Ungermann ◽  
Jeffrey E. Gerst
Keyword(s):  
Disease Gene ◽  
Transmembrane Domain ◽  
Batten Disease ◽  
Retrograde Transport ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Late Endosomes ◽  
Target Membrane ◽  
Opposing Effects ◽  
Protein Attachment

The human Batten disease gene CLN3 and yeast orthologue BTN1 encode proteins of unclear function. We show that the loss of BTN1 phenocopies that of BTN2, which encodes a retromer accessory protein involved in the retrieval of specific cargo from late endosomes (LEs) to the Golgi. However, Btn1 localizes to Golgi and regulates soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) function to control retrograde transport. Specifically, BTN1 overexpression and deletion have opposing effects on phosphorylation of the Sed5 target membrane SNARE, on Golgi SNARE assembly, and on Golgi integrity. Although Btn1 does not interact physically with SNAREs, it regulates Sed5 phosphorylation by modulating Yck3, a palmitoylated endosomal kinase. This may involve modification of the Yck3 lipid anchor, as substitution with a transmembrane domain suppresses the deletion of BTN1 and restores trafficking. Correspondingly, deletion of YCK3 mimics that of BTN1 or BTN2 with respect to LE–Golgi retrieval. Thus, Btn1 controls retrograde sorting by regulating SNARE phosphorylation and assembly, a process that may be adversely affected in Batten Disease patients.

scholarly journals Multiple features within Sed5p mediate it’s COPI-independent trafficking and Golgi localization

2019 ◽  
Author(s):  
Guanbin Gao ◽  
David K. Banfield
Keyword(s):  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Yeast Cells ◽  
Multiple Features ◽  
Protein Retention ◽  
Golgi Retention ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Snare Motif ◽  
Sm Protein

ABSTRACTProtein retention and the transport of proteins and lipids into and out of the Golgi is intimately linked to the biogenesis and homeostasis of this sorting hub of eukaryotic cells. Of particular importance are membrane proteins that mediate membrane fusion events with and within the Golgi – the Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). In the Golgi of budding yeast cells a single syntaxin - the SNARE Sed5p - oversees membrane fusion within the Golgi. Determining how Sed5p is localized to and trafficked within the Golgi is critical to informing our understanding of the mechanism(s) of biogenesis and homeostasis of this organelle. Here we establish that the Golgi retention and trafficking of Sed5p between the Golgi and the ER is independent of COPI function, the composition of the transmembrane domain, and binding of the Sec1-Munc18 (SM) protein Sly1p. Rather, the steady state localization of Sed5p to the Golgi appears to be primarily conformation-based relying on intra-molecular associations between the Habc domain and SNARE-motif.

scholarly journals Localization, Dynamics, and Protein Interactions Reveal Distinct Roles for ER and Golgi SNAREs

1998 ◽  
Vol 141 (7) ◽  
pp. 1489-1502 ◽  
Author(s):  
Jesse C. Hay ◽  
Judith Klumperman ◽  
Viola Oorschot ◽  
Martin Steegmaier ◽  
Christin S. Kuo ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Protein Interactions ◽  
The Other ◽  
Snare Proteins ◽  
Attachment Protein ◽  
Golgi Transport ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Coated Membranes ◽  
Factor Attachment Protein Receptor

ER-to-Golgi transport, and perhaps intraGolgi transport involves a set of interacting soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins including syntaxin 5, GOS-28, membrin, rsec22b, and rbet1. By immunoelectron microscopy we find that rsec22b and rbet1 are enriched in COPII-coated vesicles that bud from the ER and presumably fuse with nearby vesicular tubular clusters (VTCs). However, all of the SNAREs were found on both COPII- and COPI-coated membranes, indicating that similar SNARE machinery directs both vesicle pathways. rsec22b and rbet1 do not appear beyond the first Golgi cisterna, whereas syntaxin 5 and membrin penetrate deeply into the Golgi stacks. Temperature shifts reveal that membrin, rsec22b, rbet1, and syntaxin 5 are present together on membranes that rapidly recycle between peripheral and Golgi-centric locations. GOS-28, on the other hand, maintains a fixed localization in the Golgi. By immunoprecipitation analysis, syntaxin 5 exists in at least two major subcomplexes: one containing syntaxin 5 (34-kD isoform) and GOS-28, and another containing syntaxin 5 (41- and 34-kD isoforms), membrin, rsec22b, and rbet1. Both subcomplexes appear to involve direct interactions of each SNARE with syntaxin 5. Our results indicate a central role for complexes among rbet1, rsec22b, membrin, and syntaxin 5 (34 and 41 kD) at two membrane fusion interfaces: the fusion of ER-derived vesicles with VTCs, and the assembly of VTCs to form cis-Golgi elements. The 34-kD syntaxin 5 isoform, membrin, and GOS-28 may function in intraGolgi transport.

scholarly journals A Novel Site of Action for α-SNAP in the SNARE Conformational Cycle Controlling Membrane Fusion

2008 ◽  
Vol 19 (3) ◽  
pp. 776-784 ◽  
Author(s):  
Marcin Barszczewski ◽  
John J. Chua ◽  
Alexander Stein ◽  
Ulrike Winter ◽  
Rainer Heintzmann ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Site Of Action ◽  
Snare Motif ◽  
Liposome Fusion

Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of α-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of α-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of α-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of α-SNAP potently inhibits Ca2+-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an α-SNAP mutant defective in NSF activation is used. We conclude that α-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for α-SNAP in the SNARE cycle that drives exocytotic membrane fusion.

scholarly journals Involvement of Syntaxin 18, an Endoplasmic Reticulum (ER)-localized SNARE Protein, in ER-mediated Phagocytosis

2006 ◽  
Vol 17 (9) ◽  
pp. 3964-3977 ◽  
Author(s):  
Kiyotaka Hatsuzawa ◽  
Taku Tamura ◽  
Hitoshi Hashimoto ◽  
Hiromi Hashimoto ◽  
Sachihiko Yokoya ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Functional Role ◽  
Transmembrane Domain ◽  
Dominant Negative ◽  
Snare Proteins ◽  
Snare Protein ◽  
Fcγ Receptor ◽  
J774 Cells ◽  
Phagosomal Membrane ◽  
Interfering Rna

The endoplasmic reticulum (ER) is thought to play an important structural and functional role in phagocytosis. According to this model, direct membrane fusion between the ER and the plasma or phagosomal membrane must precede further invagination, but the exact mechanisms remain elusive. Here, we investigated whether various ER-localized SNARE proteins are involved in this fusion process. When phagosomes were isolated from murine J774 macrophages, we found that ER-localized SNARE proteins (syntaxin 18, D12, and Sec22b) were significantly enriched in the phagosomes. Fluorescence and immuno-EM analyses confirmed the localization of syntaxin 18 in the phagosomal membranes of J774 cells stably expressing this protein tagged to a GFP variant. To examine whether these SNARE proteins are required for phagocytosis, we generated 293T cells stably expressing the Fcγ receptor, in which phagocytosis occurs in an IgG-mediated manner. Expression in these cells of dominant-negative mutants of syntaxin 18 or D12 lacking the transmembrane domain, but not a Sec22b mutant, impaired phagocytosis. Syntaxin 18 small interfering RNA (siRNA) selectively decreased the efficiency of phagocytosis, and the rate of phagocytosis was markedly enhanced by stable overexpression of syntaxin 18 in J774 cells. Therefore, we conclude that syntaxin 18 is involved in ER-mediated phagocytosis, presumably by regulating the specific and direct fusion of the ER and plasma or phagosomal membranes.

scholarly journals Snarepins Are Functionally Resistant to Disruption by Nsf and αSNAP

2000 ◽  
Vol 149 (5) ◽  
pp. 1063-1072 ◽  
Author(s):  
Thomas Weber ◽  
Francesco Parlati ◽  
James A. McNew ◽  
Robert J. Johnston ◽  
Benedikt Westermann ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Snare Proteins ◽  
Attachment Protein ◽  
Receptor Proteins ◽  
Protein Receptor ◽  
The Face ◽  
Target Membrane ◽  
The Moment ◽  
Protein Attachment ◽  
Lines Of Evidence

SNARE (SNAP [soluble NSF {N-ethylmaleimide–sensitive fusion protein} attachment protein] receptor) proteins are required for many fusion processes, and recent studies of isolated SNARE proteins reveal that they are inherently capable of fusing lipid bilayers. Cis-SNARE complexes (formed when vesicle SNAREs [v-SNAREs] and target membrane SNAREs [t-SNAREs] combine in the same membrane) are disrupted by the action of the abundant cytoplasmic ATPase NSF, which is necessary to maintain a supply of uncombined v- and t-SNAREs for fusion in cells. Fusion is mediated by these same SNARE proteins, forming trans-SNARE complexes between membranes. This raises an important question: why doesn't NSF disrupt these SNARE complexes as well, preventing fusion from occurring at all? Here, we report several lines of evidence that demonstrate that SNAREpins (trans-SNARE complexes) are in fact functionally resistant to NSF, and they become so at the moment they form and commit to fusion. This elegant design allows fusion to proceed locally in the face of an overall environment that massively favors SNARE disruption.

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