Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex

Katz, Luba; Hanson, Phyllis I.; Heuser, John E.; Brennwald, Patrick

doi:10.1093/emboj/17.21.6200

Cited by 97 publications

(75 citation statements)

References 26 publications

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“…t-SNAREs, also termed syntaxins, consist of three domains: the regulatory N terminus, the coiled-coil domain required for SNARE complex formation (7)(8)(9)(10)(11), and the transmembrane domain (TMD), by which most SNAREs are anchored to membranes (12). Transmembrane domains of SNAREs are important for SNARE function.…”

Section: It Is Presently Not Clear How the Function Of Snare Proteinsmentioning

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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Section: It Is Presently Not Clear How the Function Of Snare Proteinsmentioning

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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“…This core machinery for vesicle fusion is comprised of soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins that are associated with transport vesicles (v-SNAREs) and the target membrane (t-SNAREs), respectively (Sollner et al, 1993). The complex of SNARE proteins is comprised of four parallel helical bundles that are thought to position both membranes and provide the energy for the formation of a fusion pore (Katz et al, 1998;Sutton et al, 1998;Weber et al, 1998). In yeast, the l(2)gl homologs Sro7/Sro77 interact directly with Sec9, a t-SNARE for vesicle fusion at the plasma membrane, and loss of both gene products with homology to l(2)gl results in a cold-sensitive growth defect with an accumulation of post-Golgi transport vesicles (Lehman et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Mammalian Homolog of Drosophila Tumor Suppressor Lethal (2) Giant Larvae Interacts with Basolateral Exocytic Machinery in Madin-Darby Canine Kidney Cells

Müsch

Cohen

Yeaman

et al. 2002

MBoC

172

118

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The Drosophila tumor suppressor protein lethal (2) giant larvae [l(2)gl] is involved in the establishment of epithelial cell polarity during development. Recently, a yeast homolog of the protein has been shown to interact with components of the post-Golgi exocytic machinery and to regulate a late step in protein secretion. Herein, we characterize a mammalian homolog of l(2)gl, called Mlgl, in the epithelial cell line Madin-Darby canine kidney (MDCK). Consistent with a role in cell polarity, Mlgl redistributes from a cytoplasmic localization to the lateral membrane after contact-naive MDCK cells make cell-cell contacts and establish a polarized phenotype. Phosphorylation within a highly conserved region of Mlgl is required to restrict the protein to the lateral domain, because a recombinant phospho-mutant is distributed in a nonpolar manner. Membrane-bound Mlgl from MDCK cell lysates was coimmunoprecipitated with syntaxin 4, a component of the exocytic machinery at the basolateral membrane, but not with other plasma membrane solubleN-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins that are either absent from or not restricted to the basolateral membrane domain. These data suggest that Mlgl contributes to apico-basolateral polarity by regulating basolateral exocytosis.

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“…On encroachment to the target compartment, SNAREs from the donor or vesicular membranes (v-SNAREs) form complexes in trans with cognate SNARE partners from the opposing target membrane (t-SNAREs). The SNARE complex usually involves three or four SNARE molecules, each contributing one or two ␣-helical cores to the formation of a four-helix coiled-coil bundle (Sutton et al, 1998;Katz et al, 1998). Formation of this complex is necessary and sufficient for membrane fusion in vitro but necessitates interactions between specific and precisely arrayed SNARE partners (Weber et al, 1998;McNew et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

t-SNARE Phosphorylation Regulates Endocytosis in Yeast

Gurunathan

Marash

Weinberger

et al. 2002

MBoC

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Earlier we demonstrated that activation of a ceramide-activated protein phosphatase (CAPP) conferred normal growth and secretion to yeast lacking their complement of exocytic v-SNAREs (Snc1,2) or bearing a temperature-sensitive mutation in an exocytic t-SNARE (Sso2). CAPP activation led to Sso dephosphorylation and enhanced the assembly of t-SNAREs into functional complexes. Thus, exocytosis in yeast is modulated by t-SNARE phosphorylation. Here, we show that endocytic defects in cells lacking the v-and t-SNAREs involved in endocytosis are also rescued by CAPP activation. Yeast lacking the Tlg1 or Tlg2 t-SNAREs, the Snc v-SNAREs, or both, undergo endocytosis after phosphatase activation. CAPP activation correlated with restored uptake of FM4-64 to the vacuole, the uptake and degradation of the Ste2 receptor after mating factor treatment, and the dephosphorylation and assembly of Tlg1,2 into SNARE complexes. Activation of the phosphatase by treatment with C 2 -ceramide, VBM/ELO gene inactivation, or by the overexpression of SIT4 was sufficient to confer rescue. Finally, we found that mutation of single PKA sites in Tlg1 (Ser31 to Ala31) or Tlg2 (Ser90 to Ala90) was sufficient to restore endocytosis, but not exocytosis, to snc cells. These results suggest that endocytosis is also modulated by t-SNARE phosphorylation in vivo.

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Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex

Cited by 97 publications

References 26 publications

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

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

Mammalian Homolog of Drosophila Tumor Suppressor Lethal (2) Giant Larvae Interacts with Basolateral Exocytic Machinery in Madin-Darby Canine Kidney Cells

t-SNARE Phosphorylation Regulates Endocytosis in Yeast

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