MOLECULAR CHARACTERIZATION OF 2 FUNCTIONAL DOMAINS OF CLIP-170 IN-VIVO

CLIP-170 is a microtubule-binding protein isolated from HeLa cells tha t is involved in the interaction of endosomes with microtubules. The b asic 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 CLI P-170 in mammalian cells (HeLa and Vero cells) and show that the tande m 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 patch es, which finally lead to the apparent bundling of microtubules. These patches do not form when the C-terminal domain is absent from the tra nsfected protein. Modification of the microtubule-binding region, part icularly of the tandem repeat motif, modulates the binding of CLIP-170 to microtubules. Overexpressed CLIP-170 appears neither to interact w ith nor to influence the organization of the intermediate filaments, a nd 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 dom ains in vivo, an N-terminal microtubule-binding domain, and a C-termin al domain that is involved in the anchoring of microtubules to periphe ral cytoplasmic structures.