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 Pci 2 cells, with a half-life of -~3h, 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 Cys9092 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. Key Words: Synaptic proteins-SNAP-25-Palmitoylation-Membrane association-Mutagenesis-Protein turnover. J. Neurochem. 69, 1864Neurochem. 69, -1869Neurochem. 69, (1997.The synaptosomal associated protein of 25 kDa, SNAP-25, is a member ofthe synaptic vesicle docking! fusion complex that includes syntaxin, synaptobrevin (VAMP), and synaptotagmin (for reviews, see Bennett and Scheller, 1994;Ferro-Novick and Jahn, 1994;Rothman and Warren, 1994; Sudhof, 1995). It has been shown to interact with both syntaxin and synaptobrevin by in vitro binding assays (Chapman et al., 1994; Pevsner et al., 1994; McMahon and Sudhof, 1995). Analysis of cDNA clones of SNAP-25 from mouse (Oyler et al., 1989), chicken (Catsicas et al., 1991), and Drosophila and Torpedo showed that the protein is highly conserved through evolution and is expressed specifically in the nervous systems. SNAP-25 plays an important role in synaptic vesicle docking/fusion. Specific cleavage of the protein by botulinum neurotoxins A and E blocks neurotransmitter release at neuromuscular junctions (Biasi et al., 1993;Schiavo et a!., 1993;Binz et al., 1994). More recently, SNAP-25 homologues from yeast and nonneuronal tissues were cloned, which showed high degrees of homology in the important functional domains (Brennwald et al., 1994;Ravichandran et al., 1996). These results strongly suggest that SNAP-25 and its homologues play fundamental roles in vesicular transport.SNAP-25 is localized primarily to the membrane fractions in both developing and adult brains (Oyier et a!., 1989). It is one of the major palmitoylated proteins found in neurons (Hess et a!., 1992), and this posttranslational modification may play an important role in modulating the physiological functions of the protein. Deletion of a 12-amino acid sequence containing the four Cys residues of SNAP-25 abolished palmitoylation and membrane association of the protein, suggesting that paimitoylation may be required for membrane association (Veit et al., 1996). Two SNAP-25 isoform...
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