Identification and partial characterization of mitotic centromere- associated kinesin, a kinesin-related protein that associates with centromeres during mitosis

Using antipeptide antibodies to conserved regions of the kinesin motor domain, we cloned a kinesin-related protein that associates with the centromere region of mitotic chromosomes. We call the protein MCAK, for mitotic centromere-associated kinesin. MCAK appears concentrated on centromeres at prophase and persists until telophase, after which time the localization disperses. It is found throughout the centromere region and between the kinetochore plates of isolated mitotic CHO chromosomes, in contrast to two other kinetochore-associated microtubule motors: cytoplasmic dynein and CENP-E (Yen et al., 1992), which are closer to the outer surface of the kinetochore plates. Sequence analysis shows MCAK to be a kinesin-related protein with the motor domain located in the center of the protein. It is 60-70% similar to kif2, a kinesin-related protein originally cloned from mouse brain with a centrally located motor domain (Aizawa et al., 1992). MCAK protein is present in interphase and mitotic CHO cells and is transcribed as a single 3.4-kb message.     

Potential ligands to the [2Fe-2S] Rieske cluster of the cytochrome bc1 complex of Rhodobacter capsulatus probed by site-directed mutagenesis.

The Rieske protein of the ubiquinol-cytochrome c oxidoreductase (bc1 complex or b6f complex) contains a [2Fe-2S] cluster which is thought to be bound to the protein via two nitrogen and two sulfur ligands [Britt, R. D., Sauer, K., Klein, M. P., Knaff, D. B., Kriauciunas, A., Yu, C.-A., Yu, L., & Malkin, R. (1991) Biochemistry 30, 1892-1901; Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. All available Rieske amino acid sequences have carboxyl termini featuring two conserved regions containing four cysteine (Cys) and two or three histidine (His) residues. Site-directed mutagenesis was applied to the Rieske protein of the photosynthetic bacterium Rhodobacter capsulatus, and the mutants obtained were studied biochemically in order to identify which of these conserved residues are the ligands of the [2Fe-2S] cluster. It was found that His159 (in the R. capsulatus numbering) is not a ligand and that the presence of the Rieske protein in the intracytoplasmic membrane is greatly decreased by alteration of any of the remaining six His or Cys residues. Among these mutations, only the substitution Cys155 to Ser resulted in the synthesis of Rieske protein (in a small amount) which contained a [2Fe-2S] cluster with altered biophysical properties. This finding suggested that Cys155 is not a ligand to the cluster. A comparison of the conserved regions of the Rieske proteins with bacterial aromatic dioxygenases (which contain a spectrally and electrochemically similar [2Fe-2S] cluster) indicated that Cys133, His135, Cys153, and His156 are conserved in both groups of enzymes, possibly as ligands to their [2Fe-2S] clusters. These findings led to the proposal that Cys138 and Cys155, which are not conserved in bacterial dioxygenases, may form an internal disulfide bond which is important for the structure of the Rieske protein and the conformation of the quinol oxidation (Qo) site of the bc1 complex.