Biochem. J. (2008) 409
(407–416) (Printed in Great Britain)
A gain-of-function mutant of Munc18-1 stimulates secretory granule recruitment and exocytosis and reveals a direct interaction of Munc18-1 with Rab3
Margaret E. GRAHAM1, Mark T. W. HANDLEY1, Jeff W. BARCLAY, Leo F. CIUFO, Stephanie L. BARROW, Alan MORGAN and Robert D. BURGOYNE2
The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool L69 3BX, U.K.
Munc18-1 plays a crucial role in regulated exocytosis in neurons and neuroendocrine cells through modulation of vesicle docking and membrane fusion. The molecular basis for Munc18 function is still unclear, as are the links with Rabs and SNARE [SNAP (soluble N-ethylmaleimide-sensitive factor-attachment protein) receptor] proteins that are also required. Munc18-1 can bind to SNAREs through at least three modes of interaction, including binding to the closed conformation of syntaxin 1. Using a gain-of-function mutant of Munc18-1 (E466K), which is based on a mutation in the related yeast protein Sly1p, we have identified a direct interaction of Munc18-1 with Rab3A, which is increased by the mutation. Expression of Munc18-1 with the E466K mutation increased exocytosis in adrenal chromaffin cells and PC12 cells (pheochromocytoma cells) and was found to increase the density of secretory granules at the periphery of PC12 cells, suggesting a stimulatory effect on granule recruitment through docking or tethering. Both the increase in exocytosis and changes in granule distribution appear to require Munc18-1 E466K binding to the closed form of syntaxin 1, suggesting a role for this interaction in bridging Rab- and SNARE-mediated events in exocytosis.
Key words: exocytosis, Sec8, secretory granule, soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) protein.
Abbreviations used: EGFP, enhanced green fluorescent protein; GDP[b-S], guanosine 5´-[b-thio]diphosphate; GH, growth hormone; GST, glutathione transferase; GTP[S], guanosine 5´-[g-thio]triphosphate; Hsc70, heat-shock cognate 70 stress protein; PBT, PBS containing 0.3% (w/v) BSA and 0.1% (v/v) Triton X-100; PC12 cells, pheochromocytoma cells; SM, Sec1/Munc18-like; SNAP, soluble N-ethylmaleimide-sensitive factor-attachment protein; SNARE, SNAP receptor; VAMP, vesicle-associated membrane protein; Vps, vacuolar protein sorting.
1These authors contributed equally to this work.
2To whom correspondence should be addressed (email burgoyne@liv.ac.uk).
Received 10 August 2007/27 September 2007; accepted 8 October 2007
Published as BJ Immediate Publication 8 October 2007, doi:10.1042/BJ20071094
© The Authors Journal compilation © 2008 Biochemical Society
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