CENTRACTIN (ARP1) ASSOCIATES WITH SPECTRIN REVEALING A POTENTIAL MECHANISM TO LINK DYNACTIN TO INTRACELLULAR ORGANELLES

Centractin (Arp1), an actin-related protein, is a component of the dyn actin complex. To investigate potential functions of the protein, we u sed transient transfections to overexpress centractin in mammalian cel ls. We observed that the overexpressed polypeptide formed filamentous structures that were significantly longer and more variable in length than those observed in the native dynactin complex. The centractin fil aments were distinct from conventional actin in subunit composition an d pharmacology as demonstrated by the absence of immunoreactivity of t hese filaments with an actin-specific antibody, by resistance to treat ment with the drug cytochalasin D, and by the inability to bind phallo idin. We examined the transfected cells for evidence of specific assoc iations of the novel centractin filaments with cellular organelles or cytoskeletal proteins. Using immunocytochemistry we observed the coloc alization of Golgi marker proteins with the centractin polymers. Addit ional immunocytochemical analysis using antibodies to non-erythroid sp ectrin (fodrin) and Golgi-spectrin (beta I Sigma) revealed that spect rin colocalized with the centractin filaments in transfected cells. Bi ochemical assays demonstrated that spectrin was present in dynactin-en riched cellular fractions, was coimmunoprecipitated from rat brain cyt osol using antibodies to dynactin subunits, and was coeluted with dyna ctin using affinity chromatography. Immunoprecipitations and affinity chromatography also revealed that actin is not a bona fide component o f dynactin. Our results indicate that spectrin is associated with the dynactin complex. We suggest a model in which dynactin associates with the Golgi through an interaction between the centractin filament of t he dynactin complex and a spectrin-linked cytoskeletal network.