KIFC3, a microtubule minus end-directed motor for the apical transport of annexin XIIIb-associated Triton-insoluble membranes

We have identified and characterized a COOH-terminal motor domain-type kine sin superfamily protein (KIFC), KIFC3, in the kidney. KIFC3 is a minus end- directed microtubule motor protein, therefore it accumulates in regions whe re minus ends of microtubules assemble. In polarized epithelial cells, KIFC 3 is localized on membrane organelles immediately beneath the apical plasma membrane of renal tubular epithelial cells in vivo and polarized MDCK II c ells in vitro. Flotation assay, coupled with detergent extraction, demonstr ated that KIFC3 is associated with Triton X-100-insoluble membrane organell es, and that it overlaps with apically transported TGN-derived vesicles. Th is was confirmed by immunoprecipitation and by GST pulldown experiments sho wing the specific colocalization of KIFC3 and annexin XIIIb, a previously c haracterized membrane protein for apically transported vesicles (Lafont, F. , S. Lecat, P. Verkade, and K. Simons. 1998. J. Cell Biol. 142:1413-1427). Furthermore, we proved that the apical transport of both influenza hemagglu tinin and annexin XIIIb was partially inhibited or accelerated by overexpre ssion of motor-domain less (dominant negative) or full-length KIFC3, respec tively. Absence of cytoplasmic dynein on these annexin XIIIb-associated ves icles and distinct distribution of the two motors on the EM level verified the existence of KIFC3-driven transport in epithelial cells.