Karvar Serhan, Yao Xuebiao, Crothers James M, Liu Yuechueng, Forte John G
Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720, USA.
J Biol Chem. 2002 Dec 20;277(51):50030-5. doi: 10.1074/jbc.M207694200. Epub 2002 Oct 16.
The soluble N-ethylmaleimide-sensitive factor attachment protein of 25 kDa (SNAP-25) plays an important role in vesicle trafficking. Together with vesicle-associated membrane protein-2 (VAMP-2) and syntaxin, SNAP-25 forms a ternary complex implicated in docking and fusion of secretory vesicles with the plasma membrane during exocytosis. These so-called SNARE proteins are believed to regulate tubulovesicle trafficking and fusion during the secretory cycle of the gastric parietal cell. Here we examined the cellular localization and functional importance of SNAP-25 in parietal cell cultures. Adenoviral constructs were used to express SNAP-25 tagged with cyan fluorescent protein, VAMP-2 tagged with yellow fluorescent protein, and SNAP-25 in which the C-terminal 25 amino acids were deleted (SNAP-25 Delta181-206). Membrane fractionation experiments and fluorescent imaging showed that SNAP-25 is localized to the apical plasma membrane. The expression of the mutant SNAP-25 Delta181-226 inhibited the acid secretory response of parietal cells. Also, SNAP Delta181-226 bound poorly in vitro with recombinant syntaxin-1 compared with wild type SNAP-25, indicating that pairing between syntaxin-1 and SNAP-25 is required for parietal cell activation. Dual expression of SNAP-25 tagged with cyan fluorescent protein and VAMP-2 tagged with yellow fluorescent protein revealed a dynamic change in distribution associated with acid secretion. In resting cells, SNAP-25 is at the apical plasma membrane and VAMP-2 is associated with cytoplasmic H,K-ATPase-rich tubulovesicles. After stimulation, the two proteins co-localize on the apical plasma membrane. These data demonstrate the functional significance of SNAP-25 as a SNARE protein in the parietal cell and show the dynamic stimulation-associated redistribution of VAMP-2 from H,K-ATPase-rich tubulovesicles to co-localize with SNAP-25 on the apical plasma membrane.
25 kDa的可溶性N - 乙基马来酰亚胺敏感因子附着蛋白（SNAP - 25）在囊泡运输中起重要作用。SNAP - 25与囊泡相关膜蛋白2（VAMP - 2）和 syntaxin一起形成三元复合物，参与胞吐过程中分泌性囊泡与质膜的对接和融合。这些所谓的SNARE蛋白被认为在胃壁细胞的分泌周期中调节微管泡的运输和融合。在此，我们研究了SNAP - 25在壁细胞培养物中的细胞定位和功能重要性。使用腺病毒构建体表达用青色荧光蛋白标记的SNAP - 25、用黄色荧光蛋白标记的VAMP - 2以及C末端25个氨基酸缺失的SNAP - 25（SNAP - 25 Delta181 - 206）。膜分级分离实验和荧光成像表明，SNAP - 25定位于顶端质膜。突变体SNAP - 25 Delta181 - 226的表达抑制了壁细胞的酸分泌反应。此外，与野生型SNAP - 25相比，SNAP Delta181 - 226在体外与重组syntaxin - 1的结合较差，表明syntaxin - 1和SNAP - 25之间的配对是壁细胞激活所必需的。用青色荧光蛋白标记的SNAP - 25和用黄色荧光蛋白标记的VAMP - 2的双重表达揭示了与酸分泌相关的分布动态变化。在静息细胞中，SNAP - 25位于顶端质膜，而VAMP - 2与富含细胞质H，K - ATP酶的微管泡相关。刺激后，这两种蛋白在顶端质膜上共定位。这些数据证明了SNAP - 25作为壁细胞中SNARE蛋白的功能重要性，并显示了VAMP - 2从富含H，K - ATP酶的微管泡到与SNAP - 25在顶端质膜上共定位的动态刺激相关再分布。
