Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Abstract

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the Nterminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the Nterminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins.

How Pig Sperm Prepares to Fertilize: Stable Acrosome Docking to the Plasma Membrane

“…The exclusive presence of 80 kDa SNARE complex in the capacitated cavitated membrane fraction supports the possibility of SNARE-mediated docking and priming of PM and OAM. A role of the syntaxin 1B/SNAP 23/VAMP 3 complex in the regulation of exocytosis has been reported for other cell types [25]. Moreover, the conformational switch of a specific syntaxin isoform (i.e.…”

Analysis of SNARE-Mediated Exocytosis Using a Cell Fusion Assay

“…Multiple readout systems have been developed to detect fusion of the cells that express flipped v-SNAREs (v-cells) and the cells that express flipped t-SNARE proteins (t-cells) Hu et al, 2003;Hu et al, 2007). In the microscopic assay shown in Fig.…”

“…There are putative N-glycosylation motifs (Asn-X-Ser/Thr) in VAMPs. Our previous studies (Hu et al, 2003;Hu et al, 2007) showed that N-glycosylation disrupts the function of flipped SNAREs, and that treatment of tunicamycin, an antibiotic that inhibits N-glycosylation (Elbein, 1984), restores the fusion activities of flipped SNAREs. To prevent N-glycosylation of VAMPs 1, 4, 5, 7 and 8, v-cells expressing these VAMP proteins and control cells are incubated in cell culture medium containing 10 g/ml of tunicamycin during transfection.…”

“…To prevent N-glycosylation of VAMPs 1, 4, 5, 7 and 8, v-cells expressing these VAMP proteins and control cells are incubated in cell culture medium containing 10 g/ml of tunicamycin during transfection. Since we have mutated the putative N-glycosylation sites in flipped VAMP2 (Hu et al, 2003) and VAMP3 proteins (Hu et al, 2007), v-cells that express VAMPs 2 or 3 are not treated with tunicamycin. Since flipped syntaxins 1 or 4, SNAP-25 proteins are not N-glycosylated (Hu et al, 2003;Hu et al, 2007), the t-cells are not treated with tunicamycin.…”

Enterohaemorrhagic E. coli modulates an ARF6:Rab35 signaling axis to prevent recycling endosome maturation during infection

“…Amongst the cell surface receptors depleted is the Transferrin Receptor (TfR), the prototypical recycling protein, which cycles between the PM and the early and recycling endosome compartments [5]. We demonstrated that the reduction in TfR levels on the cell surface is not due to degradation, as total cellular levels of TfR are unaltered during infection, and that injection of EspG results in the movement of the TfR to cytosolic vesicles positive for markers of recycling endosomes [4], [6], [7]. Therefore, we hypothesize that EspG may inhibit the recycling of internalized cell surface receptors back to the PM.…”