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 motor proteins. However, quaternary structures of complexes between kinesins and kinesin-associated proteins are poorly defined. We have analyzed the nature of the interaction between the Kar3 motor protein, a minus-end-directed kinesin from yeast, and its associated protein Cik1. Extraction experiments demonstrate that Kar3p and Cik1p are tightly associated. Mapping of the interaction domains of the two proteins by two-hybrid analyses indicates that Kar3p and Cik1p associate in a highly specific manner along the lengths of their respective coiled-coil domains. Sucrose gradient velocity centrifugation and gel filtration experiments were used to determine the size of the Kar3-Cik1 complex from both mating pheromone-treated cells and vegetatively 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 complex has a novel heterodimeric structure not observed previously for kinesin complexes.
Barrett
J G
JG
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.
Manning
B D
BD
Snyder
M
M
D000595:Amino Acid Sequence; D002499:Centrifugation, Density Gradient; D005591:Chemical Fractionation; D019281:Dimerization; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010675:Pheromones / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017421:Sequence Analysis; D013395:Sucrose
C076025:CIK1 protein, S cerevisiae; D005656:Fungal Proteins; C063504:KAR3 protein, S cerevisiae; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D010675:Pheromones; D029701:Saccharomyces cerevisiae Proteins; D013395:Sucrose; D016547:Kinesins