The overall goal of this project is to identify the biochemical and cellular mechanisms that determine how the kinesin microtubule motor protein assembles onto the correct vesicle cargos in the cell and to dissect the minimum components required for plus-end directed microtubule based motility. In this proposal I have designed experiments using an in vitro motility assay from Dictyostelium extracts and the Dictyostelium conventional kinesin homologue, K3 to study mechanisms of vesicle transport and cargo binding. Conventional chromatography and affinity approaches will be used to purify proteins and the in vitro assay will be used to test the minimum amount of factors required for motility. In vivo roles of kinesin and associated proteins will be elucidated by gene replacement and mutagenesis as well as by immunofluorescence and tagging of the protein with Green-Fluorescent protein for live images of cells.
The specific aims of this project are: 1) Characterization of the in vivo function of the conventional kinesin K3 Dictyostelium homologue 2) Functional analysis and mapping of the conventional kinesin tail domain 3) Identification and characterization of K3 kinesin-protein and kinesin-membrane associations.
