RIGOR-TYPE MUTATION IN THE KINESIN-RELATED PROTEIN HSEG5 CHANGES ITS SUBCELLULAR-LOCALIZATION AND INDUCES MICROTUBULE BUNDLING

HsEg5 is a human kinesin-related motor protein essential for the forma tion of a bipolar mitotic spindle. It interacts with the mitotic centr osomes in a phosphorylation-dependent manner. To investigate further t he mechanisms involved in targetting HsEg5 to the spindle apparatus, w e expressed various mutants of HsEg5 in HeLa cells. All these mutants share a mutation of Thr-112 in the N-terminal motor domain, resulting in the inactivation of the ATP binding domain. In vitro, the HsEg5-T11 2N mutant motor domain showed a nucleotide-independent microtubule ass ociation, typical of a kinesin protein binding to microtubules in a ri gor state. In vivo, overexpression of the HsEg5 rigor mutant in HeLa c ells induced, in interphase, microtubule bundling, and, in mitosis, th e formation of monopolar mitotic spindles similar to those observed af ter microinjection of anti-HsEg5 antibodies. Localization of the HsEg5 rigor mutant on cytoplasmic microtubules did not require the C-termin al tail domain but was lost when the stalk domain was also deleted. Su crose gradient centrifugation experiments showed that microtubule bund ling was most likely caused by the binding of HsEg5 mutants in a dimer ic state. These results demonstrate that the precise subcellular local ization of HsEg5 in vivo is regulated not only by the phosphorylation of the tail domain but also by the oligomeric state of the protein. (C ) 1998 Wiley-Liss, Inc.