Johnson R P, Craig S W
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Nature. 1995 Jan 19;373(6511):261-4. doi: 10.1038/373261a0.
Although vinculin is present at all sites of F-actin attachment to plasma membranes and is required for linkage of myofibrils to sarcolemma, 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, current models of actin-membrane linkages depict only an indirect role for vinculin, as a tether for alpha-actinin. We demonstrate here that an intramolecular association between the 95K head and 30K tail domains of vinculin masks an F-actin binding site present in the carboxy-terminal 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.
尽管纽蛋白存在于F-肌动蛋白附着于质膜的所有部位，并且是肌原纤维与肌膜连接所必需的，但尚不清楚它如何促进肌动蛋白与膜的附着。由于纽蛋白与F-肌动蛋白直接相互作用的生化证据存在争议，目前的肌动蛋白-膜连接模型仅将纽蛋白描述为一种间接作用，作为α-辅肌动蛋白的系链。我们在此证明，纽蛋白95K头部和30K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。沉降和交联试验以及透射电子显微镜直接观察显示，F-肌动蛋白与含有纽蛋白811-1066氨基酸的细菌表达融合蛋白之间以及F-肌动蛋白与含有纽蛋白858-1066氨基酸的蛋白水解片段之间存在相互作用。纽蛋白本身既不与F-肌动蛋白共沉降也不与F-肌动蛋白交联。纽蛋白的氨基末端95K头部片段而非完整的纽蛋白会抑制共沉降和交联。我们提出，纽蛋白组装成黏着连接涉及头尾相互作用的破坏，从而暴露出一个介导微丝附着的位点。
