F-ACTIN BINDING-SITE MASKED BY THE INTRAMOLECULAR ASSOCIATION OF VINCULIN HEAD AND TAIL DOMAINS
F-ACTIN BINDING-SITE MASKED BY THE INTRAMOLECULAR ASSOCIATION OF VINCULIN HEAD AND TAIL DOMAINS
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DOI：
10.1038/373261a0
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发表时间：
1995-01-19
期刊：
影响因子：
64.8
通讯作者：
CRAIG, SW
中科院分区：
文献类型：
--
作者：
JOHNSON, RP;CRAIG, SW
尽管黏着斑蛋白存在于F-肌动蛋白附着于质膜的所有位点（1），但肌原纤维与肌膜的连接需要酸（2），尚不清楚黏着斑蛋白如何促进肌动蛋白附着于膜。由于黏着斑蛋白与F-肌动蛋白直接相互作用的生物化学证据存在争议（3-9），目前的肌动蛋白-膜连接模型仅描述黏着斑蛋白的间接作用，作为α-肌动蛋白的系链（10）。我们在这里证明了黏着斑蛋白（11）的95 K头部和30 K尾部结构域之间的分子内缔合掩盖了羧基末端尾部结构域中存在的F-肌动蛋白结合位点。共沉淀和交联测定，并通过透射电子显微镜直接可视化，揭示了F-肌动蛋白和细菌表达的融合蛋白，包含氨基酸811-1066的黏着斑蛋白，和F-肌动蛋白和蛋白水解片段之间的相互作用，黏着斑蛋白包含氨基酸858-1066。纤维蛋白本身既不与F-肌动蛋白共沉积也不交联。黏着斑蛋白的氨基末端95 K头部片段，但不是完整的黏着斑蛋白，抑制共沉降和交联。我们建议黏着斑蛋白组装成一个adherens连接涉及破坏的头-尾相互作用，揭示了一个网站，介导微丝附着。
ALTHOUGH vinculin is present at all sites of F-actin attachment to plasma membranes(1) acid is required for linkage of myofibrils to sarcolemma(2), it is unclear how it promotes attachment of actin to membranes. Because biochemical evidence for a direct interaction of vinculin with F-actin is controversial(3-9), current models of actin-membrane linkages depict only an indirect role for vinculin, as a tether for alpha-actinin(10). We demonstrate here that an intramolecular association between the 95K head and 30K tail domains of vinculin(11) masks an F-actin binding site present in the carboxyterminal tail domain. Cosedimentation and crosslinking assays, and direct visualization by transmission electron microscopy, reveal an interaction between F-actin and a bacterially expressed fusion protein containing amino acids 811-1066 of vinculin, and between F-actin and a proteolytic fragment of vinculin containing amino acids 858-1066. Vinculin itself neither cosediments with nor crosslinks F-actin. The amino-terminal 95K head fragment of vinculin, but not intact vinculin, inhibits both cosedimentation and crosslinking. We propose that assembly of vinculin into an adherens junction involves disruption of the head-tail interaction, revealing a site that mediates microfilament attachment.