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 G(o), 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 G(o), In addition, raft association may aid in sorting of GAP-43 into axonally directed vesicles in the trans-Golgi network.