Association of N-Ethylmaleimide Sensitive Fusion (NSF) Protein and Soluble NSF Attachment Proteins-α and -γ with Glucose Transporter-4-Containing Vesicles in Primary Rat Adipocytes

Abstract

To investigate the role of N-ethylmaleimide sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAP)-containing fusion complexes in glucose transporter-4 (GLUT4) membrane trafficking, the subcellular distributions of NSF, alpha-SNAP, and gamma-SNAP in primary rat adipocytes were determined. A large fraction of the NSF and SNAPs were associated with intracellular membranes, distributed between the low-density microsomes (LDM) and high-density microsomes. Very little of the NSF and SNAPs were associated with the plasma membrane fraction. This distribution did not change after insulin stimulation. Approximately 75% of the NSF and SNAPs in the LDM fraction were coimmunoprecipitated with 85% of the GLUT4 and 60% of the vesicle associated membrane proteins (VAMPs; synaptobrevins) VAMP-2 and cellubrevin in anti-GLUT4 immunoadsorptions. In contrast to NSF and the SNAPs, the beta-coatomer protein (beta-COP) found in the LDM fraction was excluded from GLUT4 vesicles. When LDM fractions were solubilized with Thesit (octaethylene glycol dodecyl ether) or Triton X-100, approximately 40% of the alpha-SNAP was colocalized with NSF on glycerol gradients in large (approximately 20S), ATP-sensitive complexes. VAMP-2 and cellubrevin are concentrated in the LDM fractions and in GLUT4 vesicles; both were excluded from these complexes. These data suggest that the steady state association of NSF and the SNAPs with GLUT4 vesicles and cell membranes is independent of the formation of fusion complexes.

Binary Interactions of the SNARE Proteins Syntaxin-4, SNAP23, and VAMP-2 and Their Regulation by Phosphorylation

“…There are now several lines of evidence to support the hypothesis that these isoforms of v-and t-SNAREs are required for GLUT4 translocation in response to insulin (14,22,29,45,46). The mechanisms by which insulin triggers exocytosis in these cell types are not yet known, but regulation by insulin may occur at the step of GLUT4 vesicle formation or translocation to the membrane (27,47) rather than at the docking and fusion steps.…”

Insulin action on glucose transporters through molecular switches, tracks and tethers

“…NSF recruitment to the cis-SNARE complex requires SNAP [53]. NSF is believed to associate with GLUT4 vesicles and cell membranes, but this interaction itself does not impact on the formation of fusion complexes [54]. ATPase-deficient NSF (that binds SNAREs but cannot disassemble them) predominantly affected intracellular membrane fusion events involved in GLUT4 cycling from the endosomal system to the SC, but not to the PM [55] in rat adipocytes.…”

Insulin Signaling and the Regulation of Glucose Transport

“…Although these SNARE interactions are essential, none of the core proteins appear to be direct targets of insulin action. On the other hand, several important SNARE accessory proteins such as Munc18c, Synip, and NSF (N-ethylmaleimide sensitive factor) may be involved in the control of Glut4 docking and fusion events and might be targets of insulin action (19)(20)(21)(22). In fact, Munc18c heterozygous knockout mice are less insulin sensitive than wild-type mice, with reduced insulinstimulated Glut4 translocation in skeletal muscle (23).…”