A Disease-causing Mutation Illuminates the Protein Membrane Topology of the Kidney-expressed Prohibitin Homology ( PHB) Domain Protein Podocin

作者：Schurek Eva Maria; Voelker Linus A; Tax Judit; Lamkemeyer Tobias; Rinschen Markus M; Ungrue Denise; Kratz John E III; Sirianant Lalida; Kunzelmann Karl; Chalfie Martin; Schermer Bernhard; Benzing Thomas^*; Hoehne Martin

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2014, 289(16): 11262-11271.

DOI：10.1074/jbc.M113.521773

• Caenorhabditis elegans
• Cholesterol-binding Protein
• Glycosylation
• Lipid Raft
• Protein Conformation
• PHB Domain
• Membrane Topology
• Podocin
• Steroid-resistant Nephrotic Syndrome
• Stomatin Family Proteins

Background: Mutations in the stomatin family protein podocin are the most common genetic cause of proteinuria. Results: A conserved proline residue of podocin is essential for its membrane topology. Conclusion: This study confirms a hairpin-like structure of the membrane-attached PHB domain protein and its significance for cholesterol recruitment. Significance: Podocin(P118L) elucidates the pathogenic implication in kidney disease and identifies a novel family of PHB domain proteins.
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 Caenorhabditis 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 (podocin(P118L)) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2(P134S)). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. Podocin(P118L) and MEC-2(P134S) 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.

• 出版日期2014-4-18