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Vol. 20, Issue 6, 1845-1854, March 15, 2009

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The Hydrophobic Cysteine-rich Domain of SNAP25 Couples with Downstream Residues to Mediate Membrane Interactions and Recognition by DHHC Palmitoyl Transferases

Jennifer Greaves^*, Gerald R. Prescott^*, Yuko Fukata^,, Masaki Fukata^,, Christine Salaun, and Luke H. Chamberlain^*

*Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom; Division of Membrane Physiology, National Institute for Physiological Sciences, Okazaki 444-8787, Japan; PRESTO, Japan Science and Technology Agency, Tokyo 102-0075, Japan; and INSERM U845, Faculte de Medecine Paris Descartes–Site Necker, 75730 Paris Cedex 15, France

Submitted September 17, 2008; Revised December 11, 2008; Accepted January 12, 2009
Monitoring Editor: Thomas F.J. Martin

SNAP25 is synthesized as a soluble protein but must associate with the plasma membrane to function in exocytosis; however, this membrane-targeting pathway is poorly defined. SNAP25 contains a palmitoylated cysteine-rich domain with four cysteines, and we show that coexpression of specific DHHC palmitoyl transferases is sufficient to promote SNAP25 membrane association in HEK293 cells. siRNA-mediated knockdown of its SNARE partner, syntaxin 1A, does not affect membrane interaction of SNAP25 in PC12 cells, whereas specific cysteine-to-alanine mutations perturb membrane binding, which is restored by leucine substitutions. These results suggest a role for cysteine hydrophobicity in initial membrane interactions of SNAP25, and indeed other hydrophobic residues in the cysteine-rich domain are also important for membrane binding. In addition to the cysteine-rich domain, proline-117 is also essential for SNAP25 membrane binding, and experiments in HEK293 cells revealed that mutation of this residue inhibits membrane binding induced by coexpression with DHHC17, but not DHHC3 or DHHC7. These results suggest a model whereby SNAP25 interacts autonomously with membranes via its hydrophobic cysteine-rich domain, requiring only sufficient expression of partner DHHC proteins for stable membrane binding. The role of proline-117 in SNAP25 palmitoylation is one of the first descriptions of elements within substrate proteins that modulate DHHC specificity.

Address correspondence to: Luke H. Chamberlain (Luke.Chamberlain{at}ed.ac.uk)

Abbreviations used: CSP, cysteine-string protein; DHHC, aspartic acid-histidine-histidine-cysteine motif; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; HEK, human embryonic kidney; PAT, palmitoyl transferase; PC12, pheochromocytoma-12; SNAP25, synaptosomal-associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide–sensitive factor attachment protein receptor.

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