Kinesin-related proteins represent a large class of microtubule motor proteins with diverse roles in a variety of essential cellular processes. One important component of the motor complex is the light chain, whose function s poorly understood. We have identified a protein called yeast Cik1p which interacts with the non-motor domain of the Kar3 kinesin heavy chain-related protein, and possesses several properties expected of a kinesin light chain. The Cik1p-Kar3p complex has been hypothesized to crosslink and slide microtubules. Cik1p is important for Kar3p localization and function. We plan to use a series of biochemical, genetic and molecular approaches to characterize the structure and function of Cik1p. Biochemical approaches will be used to characterize the subunit composition, size and stability of the Cik1p-Kar3p complex. We will determine if Cik1p is stably associated with Kar3p as expected for a Kar3p light chain. We will attempt to reconstitute the Cik1p-Kar3p complex in vitro and analyze its structure using electron microscopy. To help elucidate the function of the Cik1p-Kar3p complex, we will determine if Cik1p in conjunction with the non-motor domain of Kar3p is capable of binding microtubules. The possibility that the Cik1p-Kar3p complex can bundle microtubules and slide them past one another will also be tested. To dissect how Cik1p functions, different functional domains of Cik1p will be analyzed. Sequences potentially involved in dimerization, and those important for Kar3p interaction, nuclear localization and spindle pole body/microtubule localization will be identified using a variety of biochemical and genetic approaches. Importantly, Cik 1p and Kar3p are thought to be differentially compartmentalized within the cell during the yeast life cycle, and Cik1p is appears to be the critical subunit for mediating the differential localization. We will attempt to determine if different isoforms of Cik1p exist and are responsible for regulating the nuclear/cytoplasmic compartmentalization. Finally, in order to identify other components that either function similarly to Cik1p, or that operate within the same or redundant pathways to those of Cik1p, we will characterize five genes, which when present in high copy, suppress the temperature-sensitive growth defects of Cik1 cells. The suppressor genes will be sequenced and the predicted protein sequences examined for similarities to Cik1p and other components of microtubule motor complexes. Disruption phenotypes of the suppressor genes and the subcellular localization of the encoded products will be determined to examine the potential role of these genes in microtubule-associated processes. We expect these studies to enhance our understanding of how microtubule motor complexes are assembled, targeted to the proper subcellular location, and function.
