Association of GAP-43 with detergent-resistant membranes requires two palmitoylated cysteine residues
Association of GAP-43 with detergent-resistant membranes requires two palmitoylated cysteine residues
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DOI：
10.1074/jbc.273.43.28478
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发表时间：
1998-10-23
影响因子：
4.8
通讯作者：
Brown, DA
中科院分区：
文献类型：
--
作者：
Arni, S;Keilbaugh, SA;Brown, DA
GAP-43是发育和再生神经元的轴突生长锥中丰富的蛋白质。我们发现GAP-43富集在通过Triton X-100提取从PC 12嗜铬细胞瘤细胞分离的耐洗涤剂膜（DRM）中，并且可以在原位提取细胞后的洗涤剂不溶性质膜残余物中检测到，GAP-43在Cys-3和Cys-4处被棕榈酰化。Cys残基的突变阻止与DRM的关联。含有融合到β-半乳糖苷酶的GAP-43的前20个氨基酸残基的杂合蛋白甚至比GAP-43本身更有效地靶向DRM。我们的结论是，串联棕榈酰化半胱氨酸残基可以靶向GAP-43 DRM，定义了一个新的信号DRM靶向。我们建议，串联或紧密间隔的饱和脂肪酰基链分区到域或“筏”在液体有序相，或具有类似性质的相，在细胞膜。这些筏在洗涤剂提取后分离为DRM。脑特异性异源三聚体G蛋白G（o）在体外可被GAP-43调节，也富集在来自PC 12细胞的DRM中。GAP-43靶向筏可能起到促进通过G（o）的信号传导的作用。此外，筏缔合可能有助于GAP-43分选成trans-Golgi网络中的轴突定向囊泡。
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.