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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >A single amino acid near the C terminus of the synaptosome- associated protein of 25 kDa (SNAP-25) is essential for exocytosis in chromaffin cells
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A single amino acid near the C terminus of the synaptosome- associated protein of 25 kDa (SNAP-25) is essential for exocytosis in chromaffin cells

机译：突触小体相关蛋白C末端25 kDa（SNAP-25）的单个氨基酸对于嗜铬细胞的胞吐作用至关重要

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摘要

Amperometry in chromaffin cells expressing green fluorescent protein (GFP) fused to synaptosome- associated protein of 25 kDa (SNAP-25) have been used to test the involvement of single amino acids in exocytotic function, overcoming some of the limitations of studies based on Botulinum neurotoxin cleavage, as this occurs at defined sites of the protein. Constructs containing either the whole SNAP-25 polypeptide or several deleted forms lacking its C-terminal domain were heavily overexpressed in transfected cells. All GFP-fusions were located in both the cytoplasm and the plasma membrane. Although a coustruct containing com- plete SNAP-25 sustained normal secretion, removal of four or more amino acids of its C terminus greatly altered the overall rate and extent of exocytosis. Further mutational analysis proved that Leu_203, the fourth residue from the C terminus, is critical for secretion. Kinetics of single granule fusions from cells expressing truncated forms showed slow onset and decay times when compared with control cells expressing full SNAP- 25. Thus, these data provide direct evidence for the involve- ment of a specific residue of SNAP-25 in exocytosis and show that overexpression of GFP-SNAP contructs combined with single vesicle fusion measurements constitutes a powerful approach to dissect the structural elements playing a role in individual steps of the exocytotic cascade.
机译：嗜铬细胞表达绿色荧光蛋白（GFP）与25 kDa突触体相关蛋白（SNAP-25）融合的安培法已用于测试单个氨基酸在胞吐功能中的参与，克服了基于肉毒杆菌的研究的某些局限性神经毒素裂解，因为它发生在蛋白质的定义位点。含有完整SNAP-25多肽或缺少C末端结构域的几种缺失形式的构建体在转染细胞中大量过量表达。所有GFP融合体均位于细胞质和质膜中。尽管含有完整SNAP-25的Coucou可以维持正常分泌，但是去除其C末端的四个或更多氨基酸会大大改变胞吐作用的整体速率和程度。进一步的突变分析证明，C末端的第四个残基Leu_203对分泌至关重要。与表达完整SNAP-25的对照细胞相比，来自表达截短形式的细胞的单颗粒融合动力学显示出缓慢的起效和衰变时间。因此，这些数据提供了SNAP-25特定残基参与胞吐作用的直接证据。结果表明，GFP-SNAP结构的过表达与单囊泡融合测量相结合构成了一种强大的方法，可用于解析在胞吐级联反应的各个步骤中发挥作用的结构元件。

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