A disease-causing mutation illuminates protein membrane topology of the kidney-expressed PHB domain protein podocin [Cell Biology]

March 4th, 2014 by Schurek, E.-M., Voelker, L. A., Tax, J., Lamkemeyer, T., Rinschen, M. M., Ungrue, D., Kratz, J. E., Sirianant, L., Kunzelmann, K., Chalfie, M., Schermer, B., Benzing, T., Hoehne, M.

Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in C. elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocinP118L) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2P134S). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated due to the fact that the mutant C-termini project extracellularly. PodocinP118L and MEC-2P134S did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6 which is part of the multiprotein-lipid supercomplex indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C-termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.