IDENTIFICATION OF SNAP RECEPTORS IN RAT ADIPOSE CELL-MEMBRANE FRACTIONS AND IN SNARE COMPLEXES CO-IMMUNOPRECIPITATED WITH EPITOPE-TAGGED N-ETHYLMALEIMIDE-SENSITIVE FUSION PROTEIN

The vesicle-associated membrane proteins [VAMPs; vesicle SNAP receptor s (v-SNAREs)] present on GLUT4-enriched vesicles prepared from rat adi pose cells [Cain, Trimble and Lienhard (1992) J. Biol. Chem. 267, 1168 1-11684] have been identified as synaptobrevin 2 (VAMP 2) and cellubre vin (VAMP 3) by using isoform-specific antisera. Additional antisera i dentify syntaxins 2 and 4 as the predominant target membrane SNAP rece ptors (t-SNAREs) in the plasma membranes (PM), with syntaxin 3 at one- twentieth the level. Syntaxins 2 and 4 are enriched 5-10-fold in PM co mpared with low-density microsomes (LDM). Insulin treatment results in an 11-fold increase in immunodetectable GLUT4 in PM and smaller (appr ox, 2-fold) increases in VAMP 2 and VAMP 3, whereas the subcellular di stributions of the syntaxins are not altered by insulin treatment. To determine which of the SNAP receptors (SNAREs) in PM might participate in SNARE complexes with proteins from GLUT4 vesicles, complexes were immunoprecipitated with anti-myc antibody from solubilized membranes a fter the addition of myc-epitope-tagged N-ethylmaleimide-sensitive fus ion protein (NSF) and recombinant alpha-soluble NSF attachment protein (alpha-SNAP). These complexes contain VAMPs 2 and 3 and syntaxin 4, b ut not syntaxins 2 or 3. Complex formation requires ATP and is disrupt ed by ATP hydrolysis. When all membrane fractions are prepared from ba sal cells, few or no VAMPs and no syntaxin 4 are immunoprecipitated in SNARE complexes obtained from LDM alone (or from immunoisolated GLUT4 vesicles). The content of syntaxin 4 depends on the presence of PM, a nd participation of VAMPs 2 and 3 is enhanced 4-6-fold by the addition of solubilized GLUT4 vesicles to PM. The latter increase is greater t han can be explained by the 2-fold higher levels of VAMPs added to the reaction mixture. When all membrane fractions are prepared from insul in-stimulated cells, SNARE complexes formed from PM alone contain simi lar levels of syntaxin 4 but 5-6-fold higher levels of VAMPs 2 and 3 c ompared with PM alone from basal cells. Addition of GLUT4 Vesicle prot eins to PM from insulin-treated cells results in a further 2-fold incr ease in VAMP 2 recovered in SNARE complexes, Therefore the VAMPs in PM of insulin-treated but not basal cells, and in GLUT4-vesicles from ce lls in either condition, are in a form that readily forms a SNARE comp lex with PM t-SNAREs and NSF. Insulin seems to activate PM and/or GLUT 4 vesicles so as to increase the efficiency of SNARE complex formation .