Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells.
Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells.
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DOI：
10.1152/ajpcell.00041.2005
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发表时间：
2005-05
期刊：
影响因子：
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通讯作者：
Guangmu Li;Qiongqiong Yang;E. Alexander;J. Schwartz
中科院分区：
文献类型：
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作者：
Guangmu Li;Qiongqiong Yang;E. Alexander;J. Schwartz
集合管中的H（+）转运通过将载有H（+）-ATP酶的小泡胞吐插入顶膜来调节。可溶性N-乙基马来酰亚胺敏感性融合蛋白附着蛋白（SNAP）受体（SNARE）蛋白对于胞吐作用至关重要。Syntaxin 1A包含三个主要结构域，SNARE N、H3和羧基末端跨膜结构域。几种突触融合蛋白亚型通过H3结构域形成SNARE融合复合物;只有突触融合蛋白1A通过其H3结构域也结合H（+）-ATP酶。这提出了在突触融合蛋白1A的H3结构域中存在H（+）-ATP酶和SNARE蛋白的单独结合位点的可能性。在syntaxin 1A的H3结构域中进行一系列截短，并表达谷胱甘肽S-转移酶（GST）融合蛋白。我们测定了大鼠肾匀浆中与GST-突触融合蛋白分子复合的H（+）-ATP酶和SNARE蛋白的量。全长突触融合蛋白亚型和突触融合蛋白-1ADeltaC [氨基酸（aa）1-264]与H（+）-ATP酶和SNAP 23以及囊泡相关膜多肽（VAMP）形成复合物。发现H3部分中的一个盒结合H（+）-ATP酶（aa 235-264），另一个盒结合SNAP 23和VAMP（aa 190-234），结合程度与全长突触融合蛋白相当。然而，没有SNARE N（aa 1-160）的单独aa 235-264盒不结合，但需要连接到SNARE N以结合H（+）-ATP酶。当这种嵌合体结构被横切到内髓集合管细胞中时，它抑制细胞内pH恢复，这是H（+）-ATP酶介导的分泌的一个指标。我们的结论是，在突触融合蛋白1A的H3结构域是一个独特的盒，参与结合的H（+）-ATP酶的顶膜，并赋予特异性的突触融合蛋白1A的过程中的H（+）-ATP酶胞吐。
H(+) transport in the collecting duct is regulated by exocytic insertion of H(+)-ATPase-laden vesicles into the apical membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain. Several syntaxin isoforms form SNARE fusion complexes through the H3 domain; only syntaxin 1A, through its H3 domain, also binds H(+)-ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H(+)-ATPase and for SNARE proteins. A series of truncations in the H3 domain of syntaxin 1A were made and expressed as glutathione S-transferase (GST) fusion proteins. We determined the amount of H(+)-ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1ADeltaC [amino acids (aa) 1-264] formed complexes with H(+)-ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound H(+)-ATPase (aa 235-264) and another that bound SNAP23 and VAMP (aa 190-234) to an equivalent degree as full-length syntaxin. However, the aa 235-264 cassette alone without the SNARE N (aa 1-160) does not bind but requires ligation to the SNARE N to bind H(+)-ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of H(+)-ATPase mediated secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding of the H(+)-ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of H(+)-ATPase exocytosis.