We have identified a novel truncated isoform of βI-spectrin that associates with the Golgi complex and participates in sorting Na,K-ATPase to the plasma membrane. Examination of cultured Madin-Darby canine kidney (MDCK) cells with a panel of monoclonal antibodies to red cell βI-spectrin revealed a subset that immunolocalized to the Golgi apparatus and recognized a novel 220 kDa peptide (in contrast to the 240 kDa plasma membrane-associatedβII-spectrin). Antibodies that recognized the C-terminus of red cellβI-spectrin were not Golgi-reactive, suggesting that Golgi spectrin is an alternatively-spliced isoform of βI-spectrin with a C-terminal truncation. Golgi βI-spectrin was found to exist as a functional complex with a recently-described truncated Golgi-specific ankyrin, and theα-subunit of Na,K-ATPase, in immunoprecipitation assays. Stable transfection of full-length βI-spectrin cDNA constructs in MDCK cells resulted in Golgi localization of the transfected product. Transfection of an array of deletion constructs showed that the extreme N-terminal sequences ofβI-spectrin are both necessary and sufficient for Golgi localization. Overexpression of the minimal Golgi-localizing βI-spectrin construct in MDCK cells resulted in growth retardation, altered cell morphology, a disruption of endogenous Golgi spectrin localization, and a pronounced failure of Na,K-ATPase assembly at the basolateral plasma membrane by structural and functional criteria. Our results have identified a novel spectrin-ankyrin skeleton associated with the Golgi apparatus and involved in vesicular trafficking in cultured renal epithelial cells. We suggest that an intact Golgi-associated spectrin cytoskeleton is necessary for the appropriate targeting and sorting of Na,K-ATPase to the basolateral plasma membrane.
