High‐affinity binding of the yeast<i>cis</i>‐Golgi t‐SNARE, Sed5p, to wild‐type and mutant Sly1p, a modulator of transport vesicle docking

Stimulus-induced tyrosine phosphorylation of Munc18c was investigated as a potential regulatory mechanism by which the Munc18c-Syntaxin 4 complex can be dissociated in response to divergent stimuli in multiple cell types. Use of [ 32 P]orthophosphate incorporation, pervanadate treatment, and phosphotyrosine-specific antibodies demonstrated that Munc18c underwent tyrosine phosphorylation. Phosphorylation was apparent under basal conditions, but levels were significantly increased within 5 min of glucose stimulation in MIN6 beta cells. Tyrosine phosphorylation of Munc18c was also detected in 3T3L1 adipocytes and increased with insulin stimulation, suggesting that this may be a conserved mechanism. Syntaxin 4 binding to Munc18c decreased as Munc18c phosphorylation levels increased in pervanadate-treated cells, suggesting that phosphorylation dissociates the Munc18c-Syntaxin 4 complex. Munc18c phosphorylation was localized to the N-terminal 255 residues. Mutagenesis of one residue in this region, Y219F, significantly increased the affinity of Munc18c for Syntaxin 4, whereas mutation of three other candidate sites was without effect. Moreover, Munc18c-Y219F expression in MIN6 cells functionally inhibited glucose-stimulated SNARE complex formation and insulin granule exocytosis. These data support a novel and conserved mechanism for the dissociation of Munc18c-Syntaxin 4 complexes in a stimulus-dependent manner to facilitate the increase in Syntaxin 4-VAMP2 association and to promote vesicle/granule fusion.In the early 1990s, the discovery of soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) 2 proteins elucidated the molecular basis for synaptic vesicle exocytosis (1, 2). Vesicle exocytosis entails the pairing of a vesicle-associated membrane protein (VAMP) SNARE with a binary cognate receptor complex at the target membrane composed of SNAP-25/23 and syntaxin proteins (target SNAREs) to form the SNARE core complex (3-7). Shortly thereafter, SNARE protein complexes were identified and found to regulate secretory processes in many diverse cell types ranging from yeast and Caenorhabditis elegans to humans, which has led to the general concept that most types of regulated vesicle fusion occur by a common mechanism.Concurrent with the discovery of SNARE proteins was the discovery of the yeast Sec1 secretory protein, which was found to interact directly with the target SNARE syntaxin (8). Homologs in C. elegans (UNC-18), Drosophila melanogaster (ROP), and mammalian cells (Munc18a-c) were also identified (9 -13). Collectively, the Sec1 and Munc18 protein families are now referred to as "SM" proteins for Sec1 and Munc18. Munc18 proteins are ϳ66 -68 kDa in size and are soluble factors with no transmembrane domain (14). They are found localized to the cytosol and also to the plasma membrane through high affinity binding to their cognate syntaxin (15, 16). Munc18a (also known as Munc18-1/neural Sec1/rat brain Sec1) was demonstrated to interact with Syntaxin 1 in a manner mutually exclusive of the other...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The Stimulus-induced Tyrosine Phosphorylation of Munc18c Facilitates Vesicle Exocytosis

Thurmond

2006

“…The E467K mutant tentatively corresponding to yeast SLY1-20 (53) displayed mildly increased binding to syntaxin 3. The implications of this finding are, however, unclear since Grabowski and Gallwitz (54) (TI-VAMP) (47,55). We have previously shown that overexpression of Munc18-2 modifies the quantity of the other SNAREs complexed with syntaxin 3 (9).…”

Section: Discussionmentioning

confidence: 99%

Munc18-2, a Functional Partner of Syntaxin 3, Controls Apical Membrane Trafficking in Epithelial Cells

Riento¹,

Kauppi²,

Keränen³

et al. 2000

The Sec1-related proteins bind to syntaxin family tSNAREs with high affinity, thus controlling the interaction of syntaxins with their cognate SNARE partners. Munc18-2 is a Sec1 homologue enriched in epithelial cells and forms a complex with syntaxin 3, a t-SNARE localized to the apical plasma membrane. We generated here a set of Munc18-2 point mutants with substitutions in conserved amino acid residues. The mutants displayed a spectrum of different syntaxin binding efficiencies. The in vitro and in vivo binding patterns were highly similar, and the association of the Munc18-2 variants with syntaxin 3 correlated well with their ability to displace SNAP-23 from syntaxin 3 complexes when overexpressed in Caco-2 cells. Even the Munc18-2 mutants that do not detectably bind syntaxin 3 were membrane associated in Caco-2 cells, suggesting that the syntaxin interaction is not the sole determinant of Sec1 protein membrane attachment. Overexpression of the wild-type Munc18-2 was shown to inhibit the apical delivery of influenza virus hemagglutinin (HA). Interestingly, mutants unable to bind syntaxin 3 behaved differently in the HA transport assay. While one of the mutants tested had no effect, one inhibited and one enhanced the apical transport of HA. This implies that Munc18-2 function in apical membrane trafficking involves aspects independent of the syntaxin 3 interaction.It is generally accepted that intracellular membrane trafficking requires compartment-specific membrane-anchored proteins denoted collectively as soluble NSF attachment protein receptors (SNAREs), 1 as well as the general factors N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs), or their homologues (1, 2). The SNARE proteins present on the transport vesicles (v-SNAREs; related to the neuronal synaptobrevin/VAMP proteins) and the target membranes (t-SNAREs; homologues of the neuronal syntaxin and SNAP-25 proteins) assemble into stable core complexes through formation of coiled coil helix bundles, a process which is closely connected to the fusion of membrane bilayers (3-5).Proteins of the Sec1 family (reviewed in Ref. 6) are suggested to play a crucial role in the control of SNARE complex assembly. These proteins bind with high affinity to specific syntaxins, thus modulating the capability of these t-SNAREs to interact with their cognate SNARE partners. In vitro binding assays or in vivo overexpression of Sec1 homologues have provided evidence for an inhibitory role of Sec1 proteins in SNARE complex formation and membrane trafficking (7-12). On the other hand, a wealth of evidence shows that Sec1 action is required for normal physiological function of the intracellular trafficking pathways: loss-of-function mutations in the Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster Sec1-related proteins have been demonstrated to lead to specific blocks in vesicle transport, the phenotypic effects often implying disturbance in consumption of transport vesicles (13)(14)(15)(16)(17)(18)(19)(20)...

“…Anti-Sed5p antibodies were produced and purified as described previously (15). Anti-MYC (9E10) and anti-HA (12CA5) monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc., and Roche Molecular Biochemicals, respectively.…”

Section: Methodsmentioning

confidence: 99%

Evidence for Overlapping and Distinct Functions in Protein Transport of Coat Protein Sec24p Family Members

Peng¹,

A²,

Gallwitz

2000

Self Cite

Budding of transport vesicles from the endoplasmic reticulum in yeast requires the formation, at the budding site, of a coat protein complex (COPII) that consists of two heterodimeric subcomplexes (Sec23p/Sec24p and Sec13p/Sec31p) and the Sar1 GTPase. Sec24p is an essential protein and involved in cargo selection. In addition to Sec24p, the yeast Saccharomyces cerevisiae expresses two non-essential Sec24p-related proteins, termed Sfb2p (product of YNL049c) and Sfb3p/Lst1p (product of YHR098c). We here show that Sfb2p and, less efficiently, Sfb3p/Lst1p are able to bind, like Sec24p, the integral membrane cargo protein Sed5p. We also demonstrate that Sfb2p, like Sec24p and Sfb3p/Lst1p, forms a complex with Sec23p in vivo. Whereas the deletion of SFB2 did not affect transport kinetics of various proteins, the maturation of the glycolipid-anchored plasma membrane protein Gas1p was differentially impaired in sfb3 knock-out cells. We generated several conditional-lethal sec24 mutants that, combined with null alleles of SFB2 and SFB3/LST1, led to a complete block of transport between the endoplasmic reticulum and the Golgi (sec24 -11/⌬sfb2) or to cell death (sec24 -11/⌬sfb3). Of the Sec24p family members, Sfb2p is the least abundant at steady state, but high intracellular concentrations of Sfb2p can rescue sec24 mutants under restrictive conditions. The data presented strongly suggest that the Sec24p-related proteins function as COPII components.