Pantophysin Is a Phosphoprotein Component of Adipocyte Transport Vesicles and Associates with GLUT4-containing Vesicles

Abstract

Pantophysin, a protein related to the neuroendocrinespecific synaptophysin, recently has been identified in non-neuronal tissues. In the present study, Northern blots showed that pantophysin mRNA was abundant in adipose tissue and increased during adipogenesis of 3T3-L1 cells. Immunoblot analysis of subcellular fractions showed pantophysin present exclusively in membrane fractions and relatively evenly distributed in the plasma membrane and internal membrane fractions. Sucrose gradient ultracentrifugation demonstrated that pantophysin and GLUT4 exhibited overlapping distribution profiles. Furthermore, immunopurified GLUT4 vesicles contained pantophysin, and both GLUT4 and pantophysin were depleted from this vesicle population following treatment with insulin. Additionally, a subpopulation of immunopurified pantophysin vesicles contained insulin-responsive GLUT4. Consistent with the interaction of synaptophysin with vesicle-associated membrane protein 2 in neuroendocrine tissues, pantophysin associated with vesicle-associated membrane protein 2 in adipocytes. Furthermore, in [ Adipocytes undergo agonist-stimulated exocytosis that is best characterized by the insulin-stimulated translocation of the GLUT4 glucose transporter from a unique intracellular vesicle pool to the plasma membrane (PM) 1 (1, 2). Interestingly, however, adipocytes secrete a variety of other proteins such as adipsin, complement factors C3 and B, the complement-related protein Acrp30, tumor necrosis factor-␣, leptin, and others (3-6). Despite the relatively large volume of vesicle traffic in adipocytes, the pathways and molecular components governing exocytosis of these factors remain undescribed. The transport and recycling of vesicles have been studied in depth using yeast, neuronal and neuroendocrine systems to identify molecular components involved in protein trafficking pathways, and molecular mechanisms of vesicle docking and fusion (7-9). With respect to the insulin-stimulated translocation of GLUT4 in adipocytes, several SNARE complex proteins recently have been shown to be functionally involved in the docking of GLUT4 vesicles. These include the v-SNARE VAMP2 and the t-SNAREs syntaxin-4 and synaptosome-associated protein-23 (10 -15). Potential modulators of SNARE complex formation also have been identified in adipocytes and these include several members of the Rab family of small GTP-binding proteins, members of the Munc18 family of proteins, and synaptotagmin-5 (16 -22). Adipocytes also express the N-ethylmaleimide-sensitive fusion protein and ␣-synaptosome-associated proteins involved in catalyzing vesicle membrane fusion events (23).Another potential modulator of SNARE complex formation is the integral membrane protein synaptophysin. Synaptophysin is a neuroendocrine-specific protein and is characteristically associated with synaptic vesicles as well as small vesicles in these cell types (24,25). The in vivo function of synaptophysin is unclear; however, in vitro studies suggest that synaptophysin may play a role in regulating inter...

Proteomic Analysis of GLUT4 Storage Vesicles Reveals LRP1 to Be an Important Vesicle Component and Target of Insulin Signaling

“…3 and Table 1), Snares and Rabs (supplemental Tables S1 and S2), and components of the retromer complex (supplemental Table S1) that mediates endosome to Golgi retrograde traffic (63). We also identified in reasonable abundance the tetraspan vesicle membrane proteins of the secretory component-associated membrane proteins, physin, and gyrin families (24), all of which had been previously identified as participating in GLUT4 trafficking by immunological means (22,64,65). These were also not seen by Larance et al (13) possibly for the same reasons noted above.…”

Essential Role of Insulin Receptor Substrate-2 in Insulin Stimulation of Glut4 Translocation and Glucose Uptake in Brown Adipocytes

“…The homogenized cells were then subjected to subcellular fractionation to isolate plasma membrane (PM), cytosolic, high density microsomal (HDM), and low density microsomal (LDM) fraction as described previously (25).…”

An adipocentric view of signaling and intracellular trafficking

“…Synaptophysin is a synaptic vesicle protein and the homologue pantophysin is an abundant adipocyte protein [171,172]. Although pantophysin can interact with VAMP2, sucrose gradient fractionation and GLUT4 vesicle immunoabsorption demonstrate that only a small fraction of pantophysin was colocalized with GLUT4 [172].…”