Kinesin and kinesin-related proteins (KRPs) are microtubule (MT)- based motor proteins that transport intracellular particles along MTs, and thus play important roles in organelle placement, vesicle trafficking, meiosis and mitosis.
The aim of the work proposed here is to characterize a novel trimeric kinesin related protein, hereafter referred to as the KRP, from sea urchin (SU) eggs and Drosophila embryos. The purified KRP, which is the first to be isolated from its native cell-type, consists of two polypeptides (Mr 85kd and 95kd) that have a kinesin motor domain complexed with a 115kd accessory subunit. The KRP displays nucleotide sensitive MT binding and bundling activity, and generates force for MT movement in a motility assay. We propose a detailed analysis of the molecular architecture of the trimeric KRP, its """"""""in vitro"""""""" motor activities and its biological functions, focusing mainly on the SU egg KRP, although our analysis of the accessory polypeptide will also involve the Drosophila KRP (whose purification protocol needs to be completed). We will use monoclonal antibodies against the SU motor subunits, and against the SU and fly accessory subunits, to identify cDNAS encoding the corresponding polypeptides. These probes will be used to learn the sequences and structural relationships between the subunits in the native SU KRP. In addition, the results of MT binding, ATPase and motility assays on bacterially-expressed motor polypeptides and the native complex will be compared, to illuminate the functional significance of being components of a trimeric complex. We will probe the biological functions of the complex using mAbs to the 85kd, 95kd and 115kd subunits of the SU KRP for immunolocalization and microinjection experiments on dividing sea urchin eggs and blastomeres. Finally, we will study the function of the fly 115kd homologue using antibodies for cytologicaI studies and by generating mutants that are defective in 115kd function. Thus we will test our hypothesis that, in mitotic cells, the 115kd subunit may regulate or target the motor polypeptides to the site of force- generation, where the KRP drives relative sliding between MTs, for example during assembly or elongation of the mitotic spindle.
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