Arni S, Keilbaugh S A, Ostermeyer A G, Brown D A
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794-5215, USA.
J Biol Chem. 1998 Oct 23;273(43):28478-85. doi: 10.1074/jbc.273.43.28478.
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.
GAP - 43是发育中和再生神经元轴突生长锥中一种丰富的蛋白质。我们发现，GAP - 43在通过Triton X - 100从PC12嗜铬细胞瘤细胞中提取分离的耐去污剂膜（DRM）中富集，并且在原位提取细胞后可在耐去污剂的质膜残余物中检测到。GAP - 43在半胱氨酸-3和半胱氨酸-4处被棕榈酰化。任一半胱氨酸残基的突变都会阻止其与DRM的结合。一种包含GAP - 43前20个氨基酸残基并与β-半乳糖苷酶融合的杂交蛋白比GAP - 43本身更有效地靶向DRM。我们得出结论，串联棕榈酰化的半胱氨酸残基可将GAP - 43靶向DRM，定义了一种新的DRM靶向信号。我们提出，串联或紧密间隔的饱和脂肪酰链会在细胞膜的液态有序相或具有相似性质的相中分配到结构域或“筏”中。去污剂提取后，这些筏被分离为DRM。在体外可能受GAP - 43调节的脑特异性异源三聚体G蛋白Go，也在PC12细胞的DRM中富集。将GAP - 43靶向筏可能有助于通过Go促进信号传导。此外，筏的结合可能有助于将GAP - 43分选到反式高尔基体网络中向轴突定向的囊泡中。
