Fukuda Mitsunori, Ogata Yukie, Saegusa Chika, Kanno Eiko, Mikoshiba Katsuhiko
Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Biochem J. 2002 Jul 1;365(Pt 1):173-80. doi: 10.1042/BJ20011877.
Synaptotagmin VII (Syt VII) has been proposed to regulate several different types of Ca2+-dependent exocytosis, but its subcellular localization (lysosome or plasma membrane) and the number of alternative splicing isoforms of Syt VII (single or multiple forms) are matters of controversy. In the present study, we show by reverse transcriptase-PCR analysis that mouse Syt VII has one major isoform (Syt VIIalpha), the original Syt VII, and two minor isoforms (Syt VIIbeta and Syt VIIgamma), which contain unique insertions (of 44 and 116 amino acids respectively) in the spacer domain between the transmembrane and C2 domains of Syt VIIalpha. Similar results were obtained with respect to rat and human Syt VII mRNA expression. An antibody against the N-terminal domain of mouse Syt VII [anti-(Syt VII-N)], which specifically recognized recombinant Syt VII but not other Syt isoforms expressed in COS-7 cells, recognized two major, closely co-migrating bands (p58 and p60) and minor bands of approx. 65 kDa in mouse brain. Immunoaffinity purification of proteins that bind the anti-(Syt VII-N) antibody, and peptide sequence analysis revealed that: (i) the major p58 and p60 bands are identified as adenylate cyclase-associated protein 2; (ii) actin-binding protein is localized at the plasma membrane; and (iii) Syt VIIalpha (65 kDa) is the major Syt VII isoform, but with a much lower expression level than previously thought. It was also shown that FLAG-Syt VII-green-fluorescence-protein fusion protein stably expressed in PC12 cells is localized in the perinuclear region (co-localization with TGN38 protein, even after brefeldin A treatment) and in the tips of neurites (co-localization with Syt I), and not in the plasma membrane.
突触结合蛋白 VII（Syt VII）被认为可调节几种不同类型的钙依赖性胞吐作用，但其亚细胞定位（溶酶体或质膜）以及 Syt VII 的可变剪接异构体数量（单一或多种形式）仍存在争议。在本研究中，我们通过逆转录酶 - PCR 分析表明，小鼠 Syt VII 有一个主要异构体（Syt VIIα），即原始的 Syt VII，以及两个次要异构体（Syt VIIβ 和 Syt VIIγ），它们在 Syt VIIα 的跨膜和 C2 结构域之间的间隔区分别含有独特的插入片段（分别为 44 和 116 个氨基酸）。在大鼠和人类 Syt VII mRNA 表达方面也获得了类似结果。一种针对小鼠 Syt VII N 端结构域的抗体[抗 -（Syt VII - N）]，它能特异性识别重组 Syt VII，但不能识别在 COS - 7 细胞中表达的其他 Syt 异构体，在小鼠脑中识别出两条主要的、迁移率相近的条带（p58 和 p60）以及约 65 kDa 的次要条带。对与抗 -（Syt VII - N）抗体结合的蛋白质进行免疫亲和纯化和肽序列分析表明：（i）主要的 p58 和 p60 条带被鉴定为腺苷酸环化酶相关蛋白 2；（ii）肌动蛋白结合蛋白定位于质膜；（iii）Syt VIIα（65 kDa）是主要的 Syt VII 异构体，但其表达水平比先前认为的要低得多。还表明，在 PC12 细胞中稳定表达的 FLAG - Syt VII - 绿色荧光蛋白融合蛋白定位于核周区域（即使在布雷菲德菌素 A 处理后仍与 TGN38 蛋白共定位）和神经突尖端（与 Syt I 共定位），而不在质膜中。
