SNAP-23 participates in SNARE complex assembly in rat adipose cells

SNARE proteins are required for vesicle docking and fusion in eukaryotic ce lls in processes as diverse as homotypic membrane fusion and synaptic vesic le exocytosis [SNARE stands for SNAP receptor, where SNAP is soluble NSF at tachment protein]. The SNARE proteins syntaxin 4 and vesicle-associated mem brane protein (VAMP) 2/3 also participate in the insulin-stimulated translo cation of GLUT4 from intracellular vesicles to the plasma membrane in adipo se cells. We now report the molecular cloning and characterization of rat S NAP-23, a ubiquitously expressed homologue of the essential neuronal SNARE protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Rat SNAP-23 is 86% and 98%, identical respectively to human and mouse SNAP-23. Southern b lot analysis reveals that the rat, mouse and human SNAP-23 genes encode spe cies-specific isoforms of the same protein. Co-immunoprecipitation of synta xin 4 and SNAP23 shows association of these two proteins in rat adipose cel l plasma membranes, and insulin stimulation does not alter the SNAP-23/synt axin 4 complex. In addition, we demonstrate for the first time the particip ation of SNAP-23, along with syntaxin 4 and VAMP2/3, in the formation of 20 S SNARE complexes prepared using rat adipose cell membranes and recombinan t cr-SNAP and NSF proteins. The stoichiometry of the SNARE complexes formed is essentially identical using membranes from either unstimulated or insul in-stimulated adipose cells. These data demonstrate that rat SNAP-23 associ ates with syntaxin 4 before insulin stimulation and is present in the SNARE complexes known to mediate the translocation of GLUT4 from intracellular v esicles to the plasma membrane of rat adipose cells.