Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43.
Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43.
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DOI：
10.1083/jcb.108.2.613
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发表时间：
1989-02
期刊：
影响因子：
--
通讯作者：
Virág I
中科院分区：
文献类型：
--
作者：
Skene JH;Virág I
生长锥是伸长轴突末端的活动装置，是广泛且动态的膜-细胞骨架相互作用以及将新膜插入生长轴突的场所。生长锥膜中最丰富的蛋白质之一是一种名为 GAP-43 的蛋白质，在轴突发育或再生期间，大多数神经元的合成急剧增加。我们已经开始通过探究 GAP-43 蛋白如何与这些膜相互作用来探索 GAP-43 在生长锥膜功能中的作用。膜洗涤实验表明，成熟的 GAP-43 与生长锥膜紧密结合，并且 Triton X-114 溶解的 GAP-43 在富含去污剂的相和缺乏去污剂的相之间的分配表明具有相当大的疏水性。蛋白质的疏水行为由二价阳离子调节，特别是锌和钙。用 [35S] 蛋氨酸对新生大鼠脑中的 GAP-43 进行体内标记表明，GAP-43 最初是作为可溶性蛋白质合成的，在翻译后附着在膜上。在组织培养中，大鼠大脑皮层细胞和神经元样 PC12 细胞均主动将 [3H] 棕榈酸掺入 GAP-43 中。与细胞体分离的分离生长锥也将标记的脂肪酸掺入 GAP-43 中，表明成熟蛋白质上脂肪酸部分的活跃更新。用中性羟胺水解酯样键可去除结合的脂肪酸，并在 GAP-43 上暴露出新的硫醇基团，这表明脂肪酸附着在蛋白质仅有的两个半胱氨酸残基上，位于氨基末端的短疏水结构域中。二价阳离子对蛋白质疏水行为的调节表明，含有大量带负电残基的其他结构域也可能有助于 GAP-43 膜相互作用。我们的观察表明 GAP-43 与生长锥膜存在动态且可逆的相互作用。
Growth cones, the motile apparatus at the ends of elongating axons, are sites of extensive and dynamic membrane-cytoskeletal interaction and insertion of new membrane into the growing axon. One of the most abundant proteins in growth cone membranes is a protein designated GAP- 43, whose synthesis increases dramatically in most neurons during periods of axon development or regeneration. We have begun to explore the role of GAP-43 in growth cone membrane functions by asking how the protein interacts with those membranes. Membrane-washing experiments indicate that mature GAP-43 is tightly bound to growth cone membranes, and partitioning of Triton X-114-solubilized GAP-43 between detergent- enriched and detergent-depleted phases indicates considerable hydrophobicity. The hydrophobic behavior of the protein is modulated by divalent cations, particularly zinc and calcium. In vivo labeling of GAP-43 in neonatal rat brain with [35S]methionine shows that GAP-43 is initially synthesized as a soluble protein that becomes attached to membranes posttranslationally. In tissue culture, both rat cerebral cortex cells and neuron-like PC12 cells actively incorporate [3H]palmitic acid into GAP-43. Isolated growth cones detached from their cell bodies also incorporate labeled fatty acid into GAP-43, suggesting active turnover of the fatty acid moieties on the mature protein. Hydrolysis of ester-like bonds with neutral hydroxylamine removes the bound fatty acid and exposes new thiol groups on GAP-43, suggesting that fatty acid is attached to the protein's only two cysteine residues, located in a short hydrophobic domain at the amino terminus. Modulation of the protein's hydrophobic behavior by divalent cations suggests that other domains, containing large numbers of negatively charged residues, might also contribute to GAP-43-membrane interactions. Our observations suggest a dynamic and reversible interaction of GAP-43 with growth cone membranes.