Cellubrevin Is a Resident Protein of Insulin-sensitive GLUT4 Glucose Transporter Vesicles in 3T3-L1 Adipocytes

Abstract

Insulin stimulates glucose transport in muscle and fat cells by inducing translocation of GLUT4 glucose transporters from a storage site to the cell surface. The mechanism of this translocation and the identity of the storage site are unknown, but it has been hypothesized that transporters recycle between an insulin-sensitive pool, endosomes, and the cell surface. Upon cell homogenization and fractionation, the storage site migrates with light microsomes (LDM) separate from the plasma membrane fraction (PM). Cellubrevin is a recently identified endosomal protein that may be involved in the reexocytosis of recycling endosomes. Here we describe that cellubrevin is expressed in 3T3-L1 adipocytes and is more abundant in the LDM than in the PM. Cellubrevin was markedly induced during differentiation of 3T3-L1 fibroblasts into adipocytes, in parallel with GLUT4, and the development of insulin regulated traffic. In response to insulin, the cellubrevin content decreased in the LDM and increased in the PM, suggesting translocation akin to that of the GLUT4 glucose transporter. Vesicle-associated membrane protein 2 (VAMP-2)/synaptobrevin-II, a protein associated with regulated exocytosis in secretory cells, also redistributed in response to insulin. Both cellubrevin and VAMP-2 were susceptible to cleavage by tetanus toxin. Immunopurified GLUT4-containing vesicles contained cellubrevin and VAMP-2, and immunopurified cellubrevin-containing vesicles contained GLUT4 protein, but undiscernible amounts of VAMP-2. These observations suggest that cellubrevin and VAMP-2 are constituents of the insulin-regulated pathway of membrane traffic. These results are the first demonstration that cellubrevin is present in a regulated intracellular compartment. We hypothesize that cellubrevin and VAMP-2 may be present in different subsets of GLUT4-containing vesicles.

Sedimentation and immunological analyses of GLUT4 and α2‐Na,K‐ATPase subunit‐containing vesicles from rat skeletal muscle: evidence for segregation

“…Identification of proteins resident in GLUT4-containing vesicles has been taken as prima facie evidence that such proteins may play some functional role in the sorting and delivery of the GLUT4 transporter to the cell surface in response to insulin binding. Several candidate proteins have indeed been identified by this approach and these include, for example, secretory carrier associated membrane proteins (SCAMPS) [26], vesicle-associated membrane proteins (VAMPS) [27], cellubrevin [28], gp160/vp165, a glycoprotein with aminopeptidase activity [25,29] and rab4, a low molecular weight GTP-binding protein implicated in vesicular trafficking [30,31]. The GLUT4-containing vesicles investigated in this study are, we believe, of non-endosomal nature given that we have recently shown that they lack immunoreactive transferrin receptors, generally accepted as a good marker of the endosomal/PM recyclying pathway [31].…”

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

“…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].…”

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

“…Polyclonal anti-green fluorescent protein (GFP) antibody, a transferrin-tetramethylrhodamine conjugate, ProLong Antifade coverslip mounting solution, and Oregon green-conjugated phalloidin were purchased from Molecular Probes (Eugene, OR). Rabbit polyclonal antibodies, one raised to the N-terminal 20 amino acids of VAMP3 and the other raised to the cytosolic amino acids of a GST-VAMP2 fusion protein, were prepared as described (Volchuk et al, 1995). Sheep anti-mouse and nonimmune IgGs were purchased from ICN Biomedicals (Aurora, OH).…”

“…Specific SNARE isoforms are expressed in muscle and fat cells, i.e., VAMP2 and VAMP3/cellubrevin (hereafter called VAMP3) (Cain et al, 1992;Volchuk et al, 1994Volchuk et al, , 1995, syntaxin4 Volchuk et al, 1996), and SNAP-23 (SNAP-25-like protein of 23 kDa) (Wang et al, 1997;Wong et al, 1997;Rea et al, 1998). The participation of the target membrane (t)-SNAREs syntaxin4 (Cheatham et al, 1996;Olson et al, 1997) and SNAP-23 (Rea et al, 1998;Foran et al, 1999;Foster et al, 1999b) in GLUT4 translocation has been implicated with the use of various molecular and biochemical approaches, such as the introduction of botulinum toxins and neutralizing antibodies into 3T3-L1 adipocytes.…”