The cysteine-string domain of the secretory vesicle cysteine-string protein is required for membrane targeting.
The post-translational addition of palmitic acid residues to cysteine-string protein (Csp) was originally thought to form the basis for membrane association of this secretory-vesicle protein. However, subsequent work showed that chemical depalmitoylation of Csp does not result in its release from membranes. We have confirmed these findings and employed [3H]palmitate labelling of PC12 cells to demonstrate that Csp1 remains associated with membranes following the complete removal of palmitic acid residues. Although palmitoylation is not essential for the stable membrane association of Csp, its role in membrane targeting has not been assessed. To examine this, we constructed a Csp mutant protein with seven cysteines replaced by serines in the cysteine-string domain. In contrast to wild-type Csps, this mutant protein was not targeted to membranes when expressed in PC12 or HeLa cells. We conclude that although a palmitoylated cysteine-string domain is not required for stable membrane association of Csp, it is essential for initial membrane targeting.
The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK.
L H
LH
Chamberlain
R D
RD
Burgoyne
D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D019811:Hydroxylamine / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D016716:PC12 Cells / Q000378:metabolism; D010169:Palmitic Acids / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine
D050956:HSP40 Heat-Shock Proteins; D008565:Membrane Proteins; D010169:Palmitic Acids; D011994:Recombinant Proteins; C088460:cysteine string protein; D019811:Hydroxylamine; D012694:Serine