Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin

NPHS2 was recently identified as a gene whose mutations cause autosomal rec essive steroid-resistant nephrotic syndrome. Its product, podocin, is a new member of the stomatin family, which consists of hairpin-like integral mem brane proteins with intracellular NH2- and COOH-termini. Podocin is express ed in glomerular podocytes, but its subcellular distribution and interactio n with other proteins are unknown. Here we show, by immunoelectron microsco py, that podocin localizes to the podocyte foot process membrane, at the in sertion site of the slit diaphragm. Podocin accumulates in an oligomeric fo rm in lipid rafts of the slit diaphragm. Moreover, GST pull-down experiment s reveal that podocin associates via its COOH-terminal domain with CD2AP, a cytoplasmic binding partner of nephrin, and with nephrin itself That podoc in interacts with CD2AP and nephrin in vivo is shown by coimmunoprecipitati on of these proteins from glomerular extracts. Furthermore, in vitro studie s reveal direct interaction of podocin and CD2AP. Hence, as with the erythr ocyte lipid raft protein stomatin, podocin is present in high-order oligome rs and may serve a scaffolding function. We postulate that podocin serves i n the structural organization of the slit diaphragm and the regulation of i ts filtration function.