Pierre P, Pepperkok R, Kreis T E
Département de Biologie Cellulaire, Sciences III, Université, Genève, Switzerland.
J Cell Sci. 1994 Jul;107 ( Pt 7):1909-20. doi: 10.1242/jcs.107.7.1909.
CLIP-170 is a microtubule-binding protein isolated from HeLa cells that is involved in the interaction of endosomes with microtubules. The basic N-terminal domain of CLIP-170 binds to microtubules in vitro. To characterize further the functional domains of this cytoplasmic linker protein, we have transiently expressed intact and mutant forms of CLIP-170 in mammalian cells (HeLa and Vero cells) and show that the tandem repeat present in the N-terminal domain is essential for its binding to microtubules in vivo as previously found in vitro. With increasing levels of expression of CLIP-170, the sites with which the peripheral ends of microtubules interact enlarge, eventually forming large patches, which finally lead to the apparent bundling of microtubules. These patches do not form when the C-terminal domain is absent from the transfected protein. Modification of the microtubule-binding region, particularly of the tandem repeat motif, modulates the binding of CLIP-170 to microtubules. Overexpressed CLIP-170 appears neither to interact with nor to influence the organization of the intermediate filaments, and collapsing the network of intermediate filaments with microinjected antibodies against vimentin has no effect on the distribution of CLIP-170. These data suggest that CLIP-170 has at least two functional domains in vivo, an N-terminal microtubule-binding domain, and a C-terminal domain that is involved in the anchoring of microtubules to peripheral cytoplasmic structures.
CLIP-170是一种从人宫颈癌HeLa细胞中分离出的微管结合蛋白，参与内体与微管的相互作用。CLIP-170的基本N端结构域在体外可与微管结合。为了进一步表征这种细胞质连接蛋白的功能结构域，我们在哺乳动物细胞（HeLa和Vero细胞）中瞬时表达了完整和突变形式的CLIP-170，结果表明，N端结构域中存在的串联重复序列对于其在体内与微管结合至关重要，这与之前在体外的发现一致。随着CLIP-170表达水平的增加，微管外周末端相互作用的位点扩大，最终形成大的斑块，最终导致微管明显成束。当转染蛋白缺少C端结构域时，这些斑块不会形成。微管结合区域的修饰，特别是串联重复基序的修饰，会调节CLIP-170与微管的结合。过表达的CLIP-170似乎既不与中间丝相互作用，也不影响中间丝的组织，用抗波形蛋白的微注射抗体破坏中间丝网络对CLIP-170的分布没有影响。这些数据表明，CLIP-170在体内至少有两个功能结构域，一个是N端微管结合结构域，另一个是C端结构域，参与微管与外周细胞质结构的锚定。
