Criado M, Gil A, Viniegra S, Gutiérrez L M
Instituto de Neurociencias, Departamento de Neuroquímica, and Facultad de Medicina, Universidad Miguel Hernández, Campus de San Juan, 03550 Alicante, Spain.
Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7256-61. doi: 10.1073/pnas.96.13.7256.
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 construct containing complete 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 Leu203, 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 involvement 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.
在表达与25 kDa突触小体相关蛋白（SNAP - 25）融合的绿色荧光蛋白（GFP）的嗜铬细胞中，安培法已被用于测试单个氨基酸在胞吐功能中的作用，克服了基于肉毒杆菌神经毒素切割的研究的一些局限性，因为这种切割发生在蛋白质的特定位点。含有整个SNAP - 25多肽或几种缺失其C末端结构域的缺失形式的构建体在转染细胞中大量过表达。所有GFP融合蛋白都位于细胞质和质膜中。虽然含有完整SNAP - 25的构建体维持正常分泌，但去除其C末端的四个或更多氨基酸会极大地改变胞吐作用的总体速率和程度。进一步的突变分析证明，C末端的第四个残基Leu203对分泌至关重要。与表达完整SNAP - 25的对照细胞相比，表达截短形式的细胞中单个颗粒融合的动力学显示出缓慢的起始和衰减时间。因此，这些数据为SNAP - 25的特定残基参与胞吐作用提供了直接证据，并表明GFP - SNAP构建体的过表达与单个囊泡融合测量相结合构成了一种强大的方法，用于剖析在胞吐级联反应的各个步骤中起作用的结构元件。
