The transport of protein and lipid between intracellular compartments is believed to occur by vesicular carriers. Motor proteins have been proposed to transport these vesicles along microtubules to their destinations. The best characterized of these motor proteins is kinesin. Recently, a family of kinesin-related proteins (KRPs) has been identified and proposed to function in transporting different intracellular vesicles. However, for none of these motors is the membrane receptor in the vesicle known. We will utilize a combination of affinity purification methods to identify receptors in native KRP/receptor complexes present in rat brain. One purification will involve binding of KRPs to microtubules in the presence of AMP-PNP whereas the second will utilize immunopurification. Functional KRP/receptor complexes will be reconstituted into proteoliposomes and analyzed for microtubule binding and for motility in the presence of ATP. Membrane receptor proteins will be further identified by crosslinking techniques and Triton X-114 extraction. Candidate receptor proteins will be eluted from SDS-PAGE gels and partial peptide sequences will be obtained for cloning the cDNAs encoding the receptors from a rat brain library. In addition, anti- peptide antibodies to the receptors will be generated. These tools will allow us to further investigate the roles of KRPs and their receptors in intracellular transport.
