Peptide Mimics of the Vesicular Stomatitis Virus G-protein Transmembrane Segment Drive Membrane Fusion in Vitro

Langosch, Dieter; Brosig, Bettina; Pipkorn, Rüdiger

doi:10.1074/jbc.m102579200

Cited by 46 publications

(59 citation statements)

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“…In addition to length, TM domains may exert a sequencedependent effect by altering lipid packing or membrane curvature within the donor lipid bilayer as part of the fusion process (8,9,29,34,49). In the case of p10, the small volume of the glycine residues and free mobility around the glycineglycine bonds could facilitate additional mobility of the fatty acyl groups of adjacent lipids.…”

Section: Discussionmentioning

confidence: 99%

Palmitoylation, Membrane-Proximal Basic Residues, and Transmembrane Glycine Residues in the Reovirus p10 Protein Are Essential for Syncytium Formation

Shmulevitz

Salsman

Duncan

2003

J Virol

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Avian reovirus and Nelson Bay reovirus are two unusual nonenveloped viruses that induce extensive cell-cell fusion via expression of a small nonstructural protein, termed p10. We investigated the importance of the transmembrane domain, a conserved membrane-proximal dicysteine motif, and an endodomain basic region in the membrane fusion activity of p10. We now show that the p10 dicysteine motif is palmitoylated and that loss of palmitoylation correlates with a loss of fusion activity. Mutational and functional analyses also revealed that a triglycine motif within the transmembrane domain and the membrane-proximal basic region were essential for p10-mediated membrane fusion. Mutations in any of these three motifs did not influence events upstream of syncytium formation, such as p10 membrane association, protein topology, or surface expression, suggesting that these motifs are more intimately associated with the membrane fusion reaction. These results suggest that the rudimentary p10 fusion protein has evolved a mechanism of inducing membrane merger that is highly dependent on the specific interaction of several different motifs with donor membranes. In addition, cross-linking, coimmunoprecipitation, and complementation assays provided no evidence for p10 homo-or heteromultimer formation, suggesting that p10 may be the first example of a membrane fusion protein that does not form stable, higher-order multimers.Protein-mediated membrane fusion is an essential step in numerous cellular processes and in entry of enveloped viruses into cells (20,22,53). The viral fusion proteins involved in enveloped virus entry and virus-induced syncytium formation have been extensively characterized. Membrane fusion is dependent on extensive conformational changes in these complex, multimeric viral proteins that serve to regulate and drive the fusion reaction (46, 52). Much effort has been focused on defining the roles of specific fusion protein motifs in the membrane fusion reaction. Numerous studies have revealed the essential role that viral fusion peptides play in the membrane fusion reaction, serving to destabilize lateral lipid interactions and alter membrane curvature, leading to fusion pore formation (15,19,31,39,48). Studies on the influence of the transmembrane (TM) domains or endodomains of viral fusion proteins on membrane fusion, however, have yielded variable or contradictory results, and specific roles for these motifs remain unclear (1,9,34,35,38).Avian reovirus (ARV) and Nelson Bay reovirus (NBV) are two of only a limited number of nonenveloped viruses that induce cell-cell fusion and multinucleated-syncytium formation (12). Both viruses encode homologous 10-kDa proteins (p10) that, when expressed by themselves in transfected cells, induce extensive cell-cell fusion (45). These fusion-associated small transmembrane (FAST) proteins are significantly smaller than the enveloped virus fusion proteins, containing only 95 to 98 amino acids. The reovirus FAST proteins are also the only examples of nonstructural viral protei...

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Section: Discussionmentioning

confidence: 99%

Palmitoylation, Membrane-Proximal Basic Residues, and Transmembrane Glycine Residues in the Reovirus p10 Protein Are Essential for Syncytium Formation

Shmulevitz

Salsman

Duncan

2003

J Virol

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“…Effect of the Hydrophobic Core Length of KALR on Fusogenicity-Previous studies showed that peptides corresponding to the TM domain of the vesicular stomatitis virus G protein and of SNARE induce in vitro membrane fusion (67)(68)(69). It was proposed that the fusogenicity of TM domains depends on structural flexibility (67)(68)(69).…”

Section: Effect Of the Hydrophobic Core Length Of Kalr On Insertion Intomentioning

confidence: 99%

“…It was proposed that the fusogenicity of TM domains depends on structural flexibility (67)(68)(69). The relationship between TM peptide fusogenicity and structural flexibility was further shown by using a TM model peptide with variable proportions of ␣-helix/␤-sheet-promoting residues (42).…”

Section: Effect Of the Hydrophobic Core Length Of Kalr On Insertion Intomentioning

confidence: 99%

Mode of Membrane Interaction and Fusogenic Properties of a de Novo Transmembrane Model Peptide Depend on the Length of the Hydrophobic Core

Lorin

Charloteaux

Fridmann-Sirkis

et al. 2007

Journal of Biological Chemistry

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Model peptides composed of alanine and leucine residues are often used to mimic single helical transmembrane domains. Many studies have been carried out to determine how they interact with membranes. However, few studies have investigated their lipid-destabilizing effect. We designed three peptides designated KALRs containing a hydrophobic stretch of 14, 18, or 22 alanines/leucines surrounded by charged amino acids. Molecular modeling simulations in an implicit membrane model as well as attenuated total reflection-Fourier transform infrared analyses show that KALR is a good model of a transmembrane helix. However, tryptophan fluorescence and attenuated total reflection-Fourier transform infrared spectroscopy indicate that the extent of binding and insertion into lipids increases with the length of the peptide hydrophobic core. Although binding can be directly correlated to peptide hydrophobicity, we show that insertion of peptides into a membrane is determined by the length of the peptide hydrophobic core. Functional studies were performed by measuring the ability of peptides to induce lipid mixing and leakage of liposomes. The data reveal that whereas KALR 14 does not destabilize liposomal membranes, KALR 18 and KALR 22 induce 40 and 50% of lipid-mixing, and 65 and 80% of leakage, respectively. These results indicate that a transmembrane model peptide can induce liposome fusion in vitro if it is long enough. The reasons for the link between length and fusogenicity are discussed in relation to studies of transmembrane domains of viral fusion proteins. We propose that fusogenicity depends not only on peptide insertion but also on the ability of peptides to destabilize the two leaflets of the liposome membrane.

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“…Alternatively, conformational flexibility of SNARE TMDs may support fusion. In fact, liposomes fuse efficiently when just TMD peptides corresponding to synaptic SNAREs or viral fusion proteins were incorporated into the bilayer (24,60). Since sequence variants displaying more stable ␣-helical conformations in isotropic solution were less fusogenic, fusogenicity may depend on the conformational flexibility of the ␣-helical SNARE TMDs in the membrane.…”

Section: Vam3 Transmembrane Domain Directs Vacuole Fusionmentioning

confidence: 99%

The Transmembrane Domain of Vam3 Affects the Composition ofcis- and trans-SNARE Complexes to Promote Homotypic Vacuole Fusion

Rohde

Dietrich

Langosch

et al. 2003

Journal of Biological Chemistry

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It is presently not clear how the function of SNARE proteins is affected by their transmembrane domains.Here, we analyzed the role of the transmembrane domain of the vacuolar SNARE Vam3 by replacing it by a lipid anchor. Vacuoles with mutant Vam3 fuse poorly and have increased amounts of cis-SNARE complexes, indicating that they are more stable. As a consequence efficient cis-SNARE complex disassembly that occurs at priming as a prerequisite of fusion requires addition of exogenous Sec18. trans-SNARE complexes in this mutant accumulate up to 4-fold over wild type, suggesting that the transmembrane domain of Vam3 is required to transit through this step. Finally, palmitoylation of Vac8, a reaction that also occurs early during priming is reduced by almost one-half. Since palmitoylated Vac8 is required beyond trans-SNARE complex formation, this may partially explain the fusion deficiency.Membrane fusion along the secretory pathway requires the specific interaction of SNAREs 1 that are localized to vesicles and target organelles (1). Fusion reactions are controlled by Sec18/NSF and ␣-SNAP/yeast Sec17 that disassemble preexisting SNARE complexes or activate t-SNAREs (2, 3). Upon docking of a vesicle with its target membrane, SNAREs from opposite membranes form trans-SNARE complexes, which are a prerequisite for complete fusion (4). In vitro studies with recombinant SNAREs, reconstituted into liposomes, indicated that SNAREs can drive bilayer mixing by pairing in a cognate fashion (5, 6).t-SNAREs, also termed syntaxins, consist of three domains: the regulatory N terminus, the coiled-coil domain required for SNARE complex formation (7-11), and the transmembrane domain (TMD), by which most SNAREs are anchored to membranes (12). Transmembrane domains of SNAREs are important for SNARE function. Alterations in synaptobrevin or syntaxin TMDs in Caenorhabditis elegans cause strong neurotransmission defects (13). C. elegans and vertebrates contain syntaxin isoforms that differ only by their TMDs (14 -16). Furthermore, TMDs of syntaxin 1A and synaptobrevin II drive homo-as well as heterodimerization (17)(18)(19). Moreover, the interaction of syntaxin 1A with synaptobrevin, but not SNAP- (24), suggesting that they constitute autonomous fusogenic domains. However, the evidence suggesting that SNAREs themselves may act as catalysts relies mostly on minimal systems. It does not exclude the possibility that SNAREs regulate other factors that could act as fusogens on cellular membranes or enhance SNARE-mediated fusion (25)(26)(27). 25, depends on the TMD of the t-SNARE (20). In addition, in vitro fusion of liposomes requires anchoring of SNAREs via transmembrane domains (21) and tight coupling between the coiled-coil and the TMD (22). Replacing the TMDs of exocytic SNAREs in yeast arrested secretion (23). Interestingly, TMD peptides alone are sufficient to drive fusion of liposomesYeast vacuole fusion occurs in a cascade of priming, docking, and fusion (28). Five SNAREs, the t-SNARE-like proteins Vam3, Vam7, and Vti1, and the v-SNA...

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Peptide Mimics of the Vesicular Stomatitis Virus G-protein Transmembrane Segment Drive Membrane Fusion in Vitro

Cited by 46 publications

References 36 publications

Palmitoylation, Membrane-Proximal Basic Residues, and Transmembrane Glycine Residues in the Reovirus p10 Protein Are Essential for Syncytium Formation

Palmitoylation, Membrane-Proximal Basic Residues, and Transmembrane Glycine Residues in the Reovirus p10 Protein Are Essential for Syncytium Formation

Mode of Membrane Interaction and Fusogenic Properties of a de Novo Transmembrane Model Peptide Depend on the Length of the Hydrophobic Core

The Transmembrane Domain of Vam3 Affects the Composition ofcis- and trans-SNARE Complexes to Promote Homotypic Vacuole Fusion

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