Characterization of the palmitoylation domain of SNAP-25.
SNAP-25 (synaptosomal associated protein of 25 kDa) is a neural specific protein that has been implicated in the synaptic vesicle docking and fusion process. It is tightly associated with membranes, and it is one of the major palmitoylated proteins found in neurons. The functional role of palmitoylation for SNAP-25 is unclear. In this report, we show that the palmitate of SNAP-25 is rapidly turned over in PC12 cells, with a half-life of approximately 3 h, and the half-life for the protein is 8 h. Mutation of Cys to Ser at positions 85, 88, 90, and 92 reduced the palmitoylation to 9, 21, 42, and 35% of the wild-type protein, respectively. Additional mutations of either Cys(85,88) or Cys(90,92) nearly abolished palmitoylation of the protein. A similar effect on membrane binding for the mutant SNAP-25 was observed, which correlated with the degree of palmitoylation. These results suggest that all four Cys residues are involved in palmitoylation and that membrane association of SNAP-25 may be regulated through dynamic palmitoylation.
Lane
S R
SR
Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, U.S.A.
Liu
Y
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine; D017931:DNA Primers; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D009474:Neurons / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D050825:Synaptosomal-Associated Protein 25; D014162:Transfection
D017931:DNA Primers; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D019308:Palmitic Acid; D003545:Cysteine