Molecular characterization of two functional domains of CLIP-170 in vivo.
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.
Pierre
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Département de Biologie Cellulaire, Sciences III, Université, Genève, Switzerland.
Pepperkok
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Kreis
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009363:Neoplasm Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014709:Vero Cells
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C076744:cytoplasmic linker protein 170