SNAP-25 palmitoylation and plasma membrane targeting require a functional secretory pathway.
Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated membrane protein essential for neurotransmitter release from synaptic terminals. We used neuronal cell lines to study the biosynthesis and posttranslational processing of SNAP-25 to investigate how palmitoylation contributes to the subcellular localization of the protein. SNAP-25 was synthesized as a soluble protein that underwent palmitoylation approximately 20 min after synthesis. Palmitoylation of the protein coincided with its stable membrane association. Treatment of cells with brefeldin A or other disrupters of transport inhibited palmitoylation of newly synthesized SNAP-25 and abolished membrane association. These results demonstrate that the processing of SNAP-25 and its targeting to the plasma membrane depend on an intact transport mechanism along the exocytic pathway. The kinetics of SNAP-25 palmitoylation and membrane association and the sensitivity of these parameters to brefeldin A suggest a novel trafficking pathway for targeting proteins to the plasma membrane. In vitro, SNAP-25 stably associated with membranes was not released from the membrane after chemical deacylation. We propose that palmitoylation of SNAP-25 is required for initial membrane targeting of the protein but that other interactions can maintain membrane association in the absence of fatty acylation.
Gonzalo
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Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Linder
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D000215:Acylation; D000818:Animals; D001693:Biological Transport, Active; D020126:Brefeldin A; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003517:Cyclopentanes / Q000494:pharmacology; D019922:GAP-43 Protein / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D016716:PC12 Cells; D010169:Palmitic Acids / Q000737:chemistry / Q000378:metabolism; D017729:Presynaptic Terminals / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25
D003517:Cyclopentanes; D019922:GAP-43 Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D010169:Palmitic Acids; D011500:Protein Synthesis Inhibitors; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D020126:Brefeldin A; D019204:GTP-Binding Proteins