Journal
MOLECULAR BIOLOGY OF THE CELL
Volume 20, Issue 6, Pages 1845-1854Publisher
AMER SOC CELL BIOLOGY
DOI: 10.1091/mbc.E08-09-0944
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Funding
- Wellcome Trust Research Career Development fellowship [070835]
- Medical Research Council Senior Research fellowship [G0601597]
- Tenovus Scotland research [S06/1]
- Diabetes UK [BDA:RD06/0003167]
- MRC [G0601597] Funding Source: UKRI
- Medical Research Council [G0601597] Funding Source: researchfish
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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.
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