The Kar3p kinesin-related protein forms a novel heterodimeric structure with its associated protein Cik1p

Proteins that physically associate with members of the kinesin superfamily are critical for the functional diversity observed for these microtubule mo tor proteins. However, quaternary structures of complexes between kinesins and kinesin-associated proteins are poorly defined. We have analyzed the na ture of the interaction between the Kar3 motor protein, a minus-end-directe d kinesin from yeast, and its associated protein Cik1. Extraction experimen ts demonstrate that Kar3p and Cik1p are tightly associated. Mapping of the interaction domains of the two proteins by two-hybrid analyses indicates th at Kar3p and Cik1p associate in a highly specific manner along the lengths of their respective coiled-coil domains. Sucrose gradient velocity centrifu gation and gel filtration experiments were used to determine the size of th e Kar3-Cik1 complex from both mating pheromone-treated cells and vegetative ly growing cells. These experiments predict a size for this complex that is consistent with that of a heterodimer containing one Kar3p subunit and one Cik1p subunit. Finally, immunoprecipitation of epitope-tagged and untagged proteins confirms that only one subunit of Kar3p and Cik1p are present in the Kar3-Cik1 complex. These findings demonstrate that the Kar3-Cik1 comple x has a novel heterodimeric structure not observed previously for kinesin c omplexes.