CHARACTERIZATION OF A MINUS END-DIRECTED KINESIN-LIKE MOTOR PROTEIN FROM CULTURED-MAMMALIAN-CELLS

Using the CHO2 monoclonal antibody raised against CHO spindles (Sellit to, C., M. Kimble, and R. Kuriyama. 1992. Cell Motil. Cytoskeleton. 22 :7-24) we identified a 66-kD protein located at the interphase centros ome and mitotic spindle. Isolated cDNAs for the antigen encode a 622-a mino acid polypeptide. Sequence analysis revealed the presence of 340- amino acid residues in the COOH terminus, which is homologous to the m otor domain conserved among other members of the kinesin superfamily. The protein is composed of a central alpha-helical portion with globul ar domains at both NH2 and COOH termini, and the epitope to the monocl onal antibody resides in the central alpha-helical stalk. A series of deletion constructs were created for in vitro analysis of microtubule interactions. While the microtubule binding and bundling activities re quire both the presence of the COOH terminus and the alpha-helical dom ain, the NH2-terminal half of the antigen lacked the ability to intera ct with microtubules. The full-length as well as deleted proteins cons isting of the COOH-terminal motor and the central alpha-helical stalk supported microtubule gliding, with velocity ranging from 1.0 to 8.4 m u m/minute. The speed of microtubule movement decreased with decreasin g lengths of the central stalk attached to the COOH-terminal motor. Th e microtubules moved with their plus end leading, indicating that the antigen is a minus end-directed motor. The CHO2 sequence shows 86% ide ntify to HSET, a gene located at the centromeric end of the human MHC region in chromosome 6 (Ando, A., Y. Y. Kikuti, H. Kawata, N. Okamoto, T. Imai, T. Eki, K. Yokoyama, E. Soeda, T. Ikemura, K. Abe, and H. In oko. 1994. Immunogenetics. 39:194-200), indicating that HSET might rep resent a human homologue of the CHO2 antigen.