RECONSTITUTION OF MEMBRANE-FUSION BETWEEN PANCREATIC-ISLET SECRETORY GRANULES AND PLASMA-MEMBRANES - CATALYSIS BY A PROTEIN CONSTITUENT RECOGNIZED BY MONOCLONAL-ANTIBODIES DIRECTED AGAINST GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

An isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isolate d and purified from rabbit brain cytosol has previously been demonstra ted to catalyze membrane fusion (Glaser and Gross, Biochemistry 33 (19 94) 5805-5812; Glaser and Gross, Biochemistry 34 (1995) 12193-12203). Herein, we provide evidence suggesting that this GAPDH isoform can rec onstitute in vitro protein-catalyzed fusion between naturally occurrin g subcellular membrane fractions involved in insulin exocytosis. Utili zing purified rat pancreatic beta-cell plasma membranes and secretory granules, we show that a brain cytosolic factor catalyzed the rapid an d efficient fusion of these two purified membrane fractions which coul d be inhibited by a monoclonal antibody directed against the brain iso form of GAPDH. Moreover, the brain cytosolic factor also catalyzed the fusion of reconstituted vesicles prepared from lipid extracts of isle t plasma membranes and secretory granules. Although the brain cytosoli c factor rapidly catalyzed membrane fusion between islet plasma membra nes and secretory granules, it did not catalyze fusion between one sec retory granule population with another. To identify the potential impo rtance of brain cytosolic factor catalyzed membrane fusion in islet ce lls, we examined extracts of hamster insulinoma tumor cells (HIT cells ) for fusion-catalyzing activity. A protein constituent was present in HIT cell cytosol which was immunologically similar to the rabbit brai n GAPDH isoform. Although native HIT cell cytosol did not catalyze mem brane fusion, removal of an endogenous protein inhibitor unmasked the presence of the protein which catalyzed membrane fusion activity and s uch fusion was ablated by a monoclonal antibody directed against the b rain isoform of GAPDH. Collectively, these results suggest the possibi lity that an isoform of brain GAPDH, also evident in HIT cells, can ca talyze fusion between the two naturally occurring subcellular membrane compartments involved in insulin secretion and suggest a novel paradi gm potentially coupling glycolytic flux with insulin release. (C) 1998 Elsevier Science B.V. All rights reserved.