Bubeck P, Pistor S, Wehland J, Jockusch B M
Cell Biology, Zoological Institute, Technical University of Braunschweig, Germany.
J Cell Sci. 1997 Jun;110 ( Pt 12):1361-71. doi: 10.1242/jcs.110.12.1361.
Vinculin, a prominent protein component of microfilament-membrane attachment sites, consists of three major domains: an N-terminal, compact head and a C-terminal rod-like tail that are connected by a flexible, proline-rich hinge. In vitro, the protein has been shown to interact with numerous ligands, including other components of the microfilament system. To characterize the ligand recruitment ability of the different vinculin domains in a cellular environment, we used a novel approach of comprising chimeric proteins of either the vinculin head, hinge or tail regions, fused to the membrane anchor sequence of ActA, a surface protein of the intracellular bacterial pathogen Listeria monocytogenes. When PtK2 cells were transfected with the corresponding constructs, the ActA membrane anchor directed the chimeric polypeptides to mitochondrial membranes. In this position, they accumulated microfilament proteins, as seen by immunofluorescence analysis. A chimera comprising the full length vinculin clone recruited a substantial amount of the cellular F-actin, the vasodilator stimulated phosphoprotein (VASP) and paxillin, but little alpha-actinin and talin. The presence of only the vinculin head directed some of the fusion protein to focal contacts, and alpha-actinin recruitment was still ineffective. Prominent recruitment of F-actin and of VASP required the presence of the tail and proline-rich hinge, respectively. Reducing the vinculin tail to short pieces harboring only one of the two F-actin binding sequences, which were defined by in vitro experiments, resulted in loss of activity, possibly by incorrect polypeptide folding. The proline-rich hinge domain could be exchanged for the analogous region of the ActA protein, and the number of such proline-clusters, containing an FPPPP motif, correlated with the extent of VASP recruitment. The results show that this system can be used to analyze in vivo the activity of vinculin domains responsible for the assembly of various cytoskeletal ligands.
纽蛋白是微丝-膜附着位点的一种重要蛋白质成分，由三个主要结构域组成：一个N端紧密头部和一个C端杆状尾部，它们通过一个富含脯氨酸的柔性铰链相连。在体外，该蛋白已被证明能与多种配体相互作用，包括微丝系统的其他成分。为了在细胞环境中表征纽蛋白不同结构域的配体募集能力，我们采用了一种新方法，即将纽蛋白头部、铰链区或尾部区域的嵌合蛋白与细胞内细菌病原体单核细胞增生李斯特菌的表面蛋白ActA的膜锚定序列融合。当用相应构建体转染PtK2细胞时，ActA膜锚定序列将嵌合多肽导向线粒体膜。在这个位置，通过免疫荧光分析可以看到它们积累了微丝蛋白。包含全长纽蛋白克隆的嵌合体募集了大量细胞内的丝状肌动蛋白、血管舒张刺激磷蛋白（VASP）和桩蛋白，但很少募集α-辅肌动蛋白和踝蛋白。仅存在纽蛋白头部会使部分融合蛋白定位于粘着斑，而α-辅肌动蛋白的募集仍然无效。丝状肌动蛋白和VASP的显著募集分别需要尾部和富含脯氨酸的铰链区的存在。将纽蛋白尾部缩短为仅包含体外实验确定的两个丝状肌动蛋白结合序列之一的短片段，可能会因多肽折叠不正确而导致活性丧失。富含脯氨酸的铰链区可以被ActA蛋白的类似区域替换，并且含有FPPPP基序的此类脯氨酸簇的数量与VASP募集的程度相关。结果表明，该系统可用于体内分析负责各种细胞骨架配体组装的纽蛋白结构域的活性。
