Tetanus toxin-mediated cleavage of cellubrevin impairs exocytosis of transferrin receptor-containing vesicles in CHO cells.

Abstract

Abstract. Cellubrevin is a member of the synaptobrevin/VAMP family of SNAREs, which has a broad tissue distribution. In fibroblastic cells it is concentrated in the vesicles which recycle transferrin receptors but its role in membrane trafficking and fusion remains to be demonstrated. Cellubrevin, like the synaptic vesicle proteins synaptobrevins I and 1I, can be cleaved by tetanus toxin, a metallo-endoprotease which blocks neurotransmitter release. However, nonneuronal cells are unaffected by the toxin due to lack of cell surface receptors for its heavy chain. To determine whether cellubrevin cleavage impairs exocytosis of recycling vesicles, we tested the effect of tetanus toxin light chain on the release of preinternalized transferrin from streptolysin-O-perforated CHO cells.The release was found to be temperature and ATP dependent as well as NEM sensitive. Addition of tetanus toxin light chain, but not of a proteolytically inactive form of the toxin, resulted in a partial inhibition of transferrin release which correlated with the toxinmediated cleavage of cellubrevin. The residual release of transferrin occurring after complete ceUubrevin degradation was still ATP dependent. Our results indicate that cellubrevin plays an important role in the constitutive exocytosis of vesicles which recycle plasmalemma receptors. The incomplete inhibition of transferrin release produced by the toxin suggests the existence of a cellubrevin-independent exocytotic mechanism, which may involve tetanus toxininsensitive proteins of the synaptobrevin/VAMP family.

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

“…It has been implicated in recycling of transferrin receptors to the plasma membrane [19], secretion of α-granules in platelets [21,22], recycling of T-cell receptors to the immunological synapses [23], and membrane trafficking during cell migration [24,25]. Using a cell fusion assay, we functionally examined the membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs, syntaxin1, syntaxin4, SNAP-23 and SNAP-25.…”

“…Like VAMP2, it can be cleaved by tetanus toxin, a metallo-endoprotease which blocks synaptic exocytosis [18,19]. VAMP3 is present in recycling endosomes and endosome-derived vesicles [18].…”

“…It colocalizes with endocytosed transferrin receptors [18] and the glucose transporter GLUT4 in adipocytes [20], and is also present in α-granules in platelets [21]. VAMP3 has been implicated in recycling of transferrin receptors to the plasma membrane [19], secretion of α-granules in platelets [21,22], recycling of T-cell receptors to the immunological synapses [23], and membrane trafficking during cell migration [24,25]. Surprisingly, VAMP3 knockout mice have no obvious phenotype [26,27], which likely results from compensation by VAMP2 [28] and/or other v-SNARE proteins.…”

A phosphoinositide conversion mechanism for exit from endosomes

“…PI4K2and its lipid product PI(4)P regulate endosomal sorting of the vesicular (v) -SNARE VAMP3, required for TfR recycling (Galli, Chilcote et al 1994;Jovic, Kean et al 2014). In yeast, secretory vesicles are formed at PI(4)P-and Ypt32p (homologues to mammalian Rab11)-enriched subdomains at the TGN, followed by a Ypt32p-to-Sec4 (homologues to mammalian Rab8), switch as additional exocyst subunits are assembled (Mizuno-Yamasaki, Medkova et al 2010).…”

VAMP2, but Not VAMP3/Cellubrevin, Mediates Insulin-dependent Incorporation of GLUT4 into the Plasma Membrane of L6 Myoblasts

“…Randhawa et al 1996;Martin et al, 1996Martin et al, , 1998Malide et al, 1997), whereas VAMP3 has been detected in the recycling endosome and was in part responsible for TfR recycling (Galli et al, 1994;Martin et al, 1996).…”