Association of GAP-43 with detergent-resistant membranes requires two palmitoylated cysteine residues.
GAP-43 is an abundant protein in axonal growth cones of developing and regenerating neurons. We found that GAP-43 was enriched in detergent-resistant membranes (DRMs) isolated by Triton X-100 extraction from PC12 pheochromocytoma cells and could be detected in detergent-insoluble plasma membrane remnants after extraction of cells in situ. GAP-43 is palmitoylated at Cys-3 and Cys-4. Mutation of either Cys residue prevented association with DRMs. A hybrid protein containing the first 20 amino acid residues of GAP-43 fused to beta-galactosidase was targeted to DRMs even more efficiently than GAP-43 itself. We conclude that tandem palmitoylated Cys residues can target GAP-43 to DRMs, defining a new signal for DRM targeting. We propose that tandem or closely spaced saturated fatty acyl chains partition into domains or "rafts" in the liquid-ordered phase, or a phase with similar properties, in cell membranes. These rafts are isolated as DRMs after detergent extraction. The brain-specific heterotrimeric G protein Go, which may be regulated by GAP-43 in vitro, was also enriched in DRMs from PC12 cells. Targeting of GAP-43 to rafts may function to facilitate signaling through Go. In addition, raft association may aid in sorting of GAP-43 into axonally directed vesicles in the trans-Golgi network.
Arni
S
S
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794-5215, USA.
Keilbaugh
S A
SA
Ostermeyer
A G
AG
Brown
D A
DA
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003545:Cysteine; D003902:Detergents / Q000494:pharmacology; D019922:GAP-43 Protein / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000032:analysis; D005960:Glucosides / Q000494:pharmacology; D008074:Lipoproteins / Q000378:metabolism*; D009154:Mutation; D017830:Octoxynol / Q000494:pharmacology; D016716:PC12 Cells; D010168:Palmitates; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D051381:Rats
D003902:Detergents; D019922:GAP-43 Protein; D005960:Glucosides; D008074:Lipoproteins; D010168:Palmitates; C018619:octyl-beta-D-glucoside; D017830:Octoxynol; D019204:GTP-Binding Proteins; D003545:Cysteine