scholarly journals Capture and release of partially zipped trans-SNARE complexes on intact organelles

2009 ◽  
Vol 185 (3) ◽  
pp. 535-549 ◽  
Author(s):  
Matthew L. Schwartz ◽  
Alexey J. Merz
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Attachment Protein ◽  
Binding Partner ◽  
Straightforward Method ◽  
Protein Receptors ◽  
Trans Complex ◽  
In Trans ◽  
Capture And Release ◽  
Protein Attachment

Soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptors (SNAREs) are hypothesized to trigger membrane fusion by complexing in trans through their membrane-distal N termini and zippering toward their membrane-embedded C termini, which in turn drives the two membranes together. In this study, we use a set of truncated SNAREs to trap kinetically stable, partially zipped trans-SNARE complexes on intact organelles in the absence of hemifusion and content mixing. We show that the C-terminal zippering of SNARE cytoplasmic domains controls the onset of lipid mixing but not the subsequent transition from hemifusion to full fusion. Moreover, we find that a partially zipped nonfusogenic trans-complex is rescued by Sec17, a universal SNARE cochaperone. Rescue occurs independently of the Sec17-binding partner Sec18, and it exhibits steep cooperativity, indicating that Sec17 engages multiple stalled trans-complexes to drive fusion. These experiments delineate distinct functions within the trans-complex, provide a straightforward method to trap and study prefusion complexes on native membranes, and reveal that Sec17 can rescue a stalled, partially zipped trans-complex.

scholarly journals Munc13-4 is a limiting factor in the pathway required for platelet granule release and hemostasis

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 869-877 ◽  
Author(s):  
Qiansheng Ren ◽  
Christian Wimmer ◽  
Michael C. Chicka ◽  
Shaojing Ye ◽  
Yi Ren ◽  
...  
Keyword(s):  
Wild Type Mouse ◽  
Limiting Factor ◽  
Attachment Protein ◽  
Altered Expression ◽  
Dense Granules ◽  
Protein Receptors ◽  
Snare Regulators ◽  
Protein Attachment ◽  
Granule Release ◽  
Platelet Secretion

Abstract Activation-dependent platelet granule release is mediated by integral membrane proteins called soluble N-ethylmaleimide–sensitive fusion protein attachment protein receptors (SNAREs) and their regulators; however, the mechanisms for this process are ill-defined. To further characterize platelet secretion, we analyzed the function of platelets from Unc13dJinx mice. Platelets from these animals lack the putative vesicle priming factor, Munc13-4, and have a severe secretion defect. Release from dense granules was completely ablated and that from α-granules and lysosomes was severely compromised. Unc13dJinx platelets showed attenuated aggregation and, consequently, Unc13dJinx mice had prolonged tail-bleeding times. The secretion defect was not due to altered expression of SNAREs or SNARE regulators, defective granule biogenesis, or faulty platelet activation. The defective release could be rescued by adding recombinant Munc13-4 to permeabilized Unc13dJinx platelets. In wild-type mouse platelets, Munc13-4 levels were lower than those of SNAREs suggesting that Munc13-4 could be a limiting component of the platelets' secretory machinery. Consistently, Munc13-4 levels directly correlated with the extent of granule release from permeabilized platelets and from intact, heterozygous Unc13dJinx platelets. These data highlight the importance of Munc13-4 in platelets and indicate that it is a limiting factor required for platelet secretion and hemostasis.

scholarly journals A Histidine Switch in Hemagglutinin-Neuraminidase Triggers Paramyxovirus-Cell Membrane Fusion

2008 ◽  
Vol 83 (4) ◽  
pp. 1727-1741 ◽  
Author(s):  
Anuja Krishnan ◽  
Santosh K. Verma ◽  
Prashant Mani ◽  
Rahul Gupta ◽  
Suman Kundu ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Sendai Virus ◽  
Biological Activities ◽  
Viral Envelope ◽  
Attachment Protein ◽  
Histidine Residues ◽  
Human Parainfluenza Virus ◽  
Mimicking Peptides

ABSTRACT Most paramyxovirus fusion proteins require coexpression of and activation by a homotypic attachment protein, hemagglutinin-neuraminidase (HN), to promote membrane fusion. However, the molecular mechanism of the activation remains unknown. We previously showed that the incorporation of a monohistidylated lipid into F-virosome (Sendai viral envelope containing only fusion protein) enhanced its fusion to hepatocytes, suggesting that the histidine residue in the lipid accelerated membrane fusion. Therefore, we explored whether a histidine moiety in HN could similarly direct activation of the fusion protein. In membrane fusion assays, the histidine substitution mutants of HN (H247A of Sendai virus and H245A of human parainfluenza virus 3) had impaired membrane fusion promotion activity without significant changes in other biological activities. Synthetic 30-mer peptides corresponding to regions of the two HN proteins containing these histidine residues rescued the fusion promoting activity of the mutants, whereas peptides with histidine residues substituted by alanine did not. These histidine-containing peptides also activated F-virosome fusion with hepatocytes both in the presence and in the absence of mutant HN in the virosome. We provide evidence that the HN-mimicking peptides promote membrane fusion, revealing a specific histidine “switch” in HN that triggers fusion.

scholarly journals The Vesicle- and Target-SNARE Proteins That Mediate Glut4 Vesicle Fusion Are Localized in Detergent-insoluble Lipid Rafts Present on Distinct Intracellular Membranes

2002 ◽  
Vol 277 (51) ◽  
pp. 49750-49754 ◽  
Author(s):  
Luke H. Chamberlain ◽  
Gwyn W. Gould
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Lipid Rafts ◽  
Significant Proportion ◽  
Vesicle Fusion ◽  
Spatial Integration ◽  
Attachment Protein ◽  
Intracellular Membranes ◽  
Syntaxin 4 ◽  
Protein Attachment

Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle solubleN-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target solubleN-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of syntaxin 4 (∼35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-β-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.

scholarly journals Disruption of the Dimer-Dimer Interaction of the Mumps Virus Attachment Protein Head Domain, Aided by an Anion Located at the Interface, Compromises Membrane Fusion Triggering

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Marie Kubota ◽  
Iori Okabe ◽  
Shin-ichi Nakakita ◽  
Ayako Ueo ◽  
Yuta Shirogane ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Host Cell ◽  
Receptor Binding ◽  
Mumps Virus ◽  
Viral Envelope ◽  
Attachment Protein ◽  
F Protein ◽  
Virus Attachment ◽  
Head Domain

ABSTRACT Mumps virus (MuV), an enveloped negative-strand RNA virus belonging to the family Paramyxoviridae, enters the host cell through membrane fusion mediated by two viral envelope proteins, an attachment protein hemagglutinin-neuraminidase (MuV-HN) and a fusion (F) protein. However, how the binding of MuV-HN to glycan receptors triggers membrane fusion is not well understood. The crystal structure of the MuV-HN head domain forms a tetramer (dimer of dimers) like other paramyxovirus attachment proteins. In the structure, a sulfate ion (SO42−) was found at the interface between two dimers, which may be replaced by a hydrogen phosphate ion (HPO42−) under physiological conditions. The anion is captured by the side chain of a positively charged arginine residue at position 139 of one monomer each from both dimers. Substitution of alanine or lysine for arginine at this position compromised the fusion support activity of MuV-HN without affecting its cell surface expression, glycan-receptor binding, and interaction with the F protein. Furthermore, the substitution appeared to affect the tetramer formation of the head domain as revealed by blue native-PAGE analysis. These results, together with our previous similar findings with the measles virus attachment protein head domain, suggest that the dimer-dimer interaction within the tetramer may play an important role in triggering membrane fusion during paramyxovirus entry. IMPORTANCE Despite the use of effective live vaccines, mumps outbreaks still occur worldwide. Mumps virus (MuV) infection typically causes flu-like symptoms and parotid gland swelling but sometimes leads to orchitis, oophoritis, and neurological complications, such as meningitis, encephalitis, and deafness. MuV enters the host cell through membrane fusion mediated by two viral proteins, a receptor-binding attachment protein, and a fusion protein, but its detailed mechanism is not fully understood. In this study, we show that the tetramer (dimer of dimers) formation of the MuV attachment protein head domain is supported by an anion located at the interface between two dimers and that the dimer-dimer interaction plays an important role in triggering the activation of the fusion protein and causing membrane fusion. These results not only further our understanding of MuV entry but provide useful information about a possible target for antiviral drugs.

scholarly journals The human SNARE protein Ykt6 mediates its own palmitoylation at C-terminal cysteine residues

2004 ◽  
Vol 384 (2) ◽  
pp. 233-237 ◽  
Author(s):  
Michael VEIT
Keyword(s):  
Fusion Protein ◽  
Binding Site ◽  
Covalent Attachment ◽  
Receptor Protein ◽  
Cysteine Residues ◽  
Snare Protein ◽  
Attachment Protein ◽  
Present Evidence ◽  
Protein Receptor ◽  
Protein Attachment

The yeast SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) protein Ykt6 was shown to mediate palmitoylation of the fusion factor Vac8 in a reaction essential for the fusion of vacuoles. Here I present evidence that hYkt6 (human Ykt6) has self-palmitoylating activity. Incubation of recombinant hYkt6 with [3H]Pal-CoA ([3H]palmitoyl-CoA) leads to covalent attachment of palmitate to C-terminal cysteine residues. The N-terminal domain of human Ykt6 contains a Pal-CoA binding site and is required for the reaction.

scholarly journals Close Is Not Enough

2000 ◽  
Vol 150 (1) ◽  
pp. 105-118 ◽  
Author(s):  
James A. McNew ◽  
Thomas Weber ◽  
Francesco Parlati ◽  
Robert J. Johnston ◽  
Thomas J. Melia ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Active Role ◽  
Snare Complex ◽  
Active Process ◽  
Attachment Protein ◽  
Helical Bundle ◽  
Active Mechanism ◽  
Target Membrane

Is membrane fusion an essentially passive or an active process? It could be that fusion proteins simply need to pin two bilayers together long enough, and the bilayers could do the rest spontaneously. Or, it could be that the fusion proteins play an active role after pinning two bilayers, exerting force in the bilayer in one or another way to direct the fusion process. To distinguish these alternatives, we replaced one or both of the peptidic membrane anchors of exocytic vesicle (v)- and target membrane (t)-SNAREs (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor) with covalently attached lipids. Replacing either anchor with a phospholipid prevented fusion of liposomes by the isolated SNAREs, but still allowed assembly of trans-SNARE complexes docking vesicles. This result implies an active mechanism; if fusion occurred passively, simply holding the bilayers together long enough would have been sufficient. Studies using polyisoprenoid anchors ranging from 15–55 carbons and multiple phospholipid-containing anchors reveal distinct requirements for anchors of v- and t-SNAREs to function: v-SNAREs require anchors capable of spanning both leaflets, whereas t-SNAREs do not, so long as the anchor is sufficiently hydrophobic. These data, together with previous results showing fusion is inhibited as the length of the linker connecting the helical bundle-containing rod of the SNARE complex to the anchors is increased (McNew, J.A., T. Weber, D.M. Engelman, T.H. Sollner, and J.E. Rothman, 1999. Mol. Cell. 4:415–421), suggests a model in which one activity of the SNARE complex promoting fusion is to exert force on the anchors by pulling on the linkers. This motion would lead to the simultaneous inward movement of lipids from both bilayers, and in the case of the v-SNARE, from both leaflets.

scholarly journals Structural Changes Are Associated with SolubleN-Ethylmaleimide-sensitive Fusion Protein Attachment Protein Receptor Complex Formation

1997 ◽  
Vol 272 (44) ◽  
pp. 28036-28041 ◽  
Author(s):  
Dirk Fasshauer ◽  
Henning Otto ◽  
William K. Eliason ◽  
Reinhard Jahn ◽  
Axel T. Brünger
Keyword(s):  
Complex Formation ◽  
Fusion Protein ◽  
Structural Changes ◽  
Receptor Complex ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Protein Attachment

Regulation of SNAP-25 trafficking and function by palmitoylation

2010 ◽  
Vol 38 (1) ◽  
pp. 163-166 ◽  
Author(s):  
Jennifer Greaves ◽  
Gerald R. Prescott ◽  
Oforiwa A. Gorleku ◽  
Luke H. Chamberlain
Keyword(s):  
Fusion Protein ◽  
Intracellular Trafficking ◽  
Neuroendocrine Cells ◽  
Snare Proteins ◽  
Receptor Protein ◽  
Attachment Protein ◽  
Rich Region ◽  
Protein Receptor ◽  
And Function ◽  
Protein Attachment

The SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) protein SNAP-25 (25 kDa synaptosome-associated protein) is essential for regulated exocytosis in neuronal and neuroendocrine cells. Whereas the majority of SNARE proteins contain transmembrane domains, SNAP-25 is instead anchored to membranes by the palmitoylation of a central cysteine-rich region. In this review, we discuss the mechanisms of SNAP-25 palmitoylation and how this modification regulates the intracellular trafficking and exocytotic function of this essential protein.

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