title: A disease-causing mutation illuminates the protein membrane topology of the kidney-expressed prohibitin homology (PHB) domain protein podocin.
abstract: 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.
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Schurek
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Linus A
authors.initials: LA
authors.lastname: Völker
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Judit
authors.initials: J
authors.lastname: Tax
authors.affiliation: Cologne Excellence Cluster on Cellular Stress Responses in Ageing-associated Diseases, University of Cologne, 50931 Cologne, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Lamkemeyer
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Markus M
authors.initials: MM
authors.lastname: Rinschen
authors.affiliation: Cologne Excellence Cluster on Cellular Stress Responses in Ageing-associated Diseases, University of Cologne, 50931 Cologne, Germany.
authors.forename: Denise
authors.initials: D
authors.lastname: Ungrue
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027-6902, and.
authors.forename: John E
authors.initials: JE
authors.lastname: Kratz
authors.affiliation: Department of Physiology, University of Regensburg, 93053 Regensburg, Germany.
authors.forename: Lalida
authors.initials: L
authors.lastname: Sirianant
authors.affiliation: Department of Physiology, University of Regensburg, 93053 Regensburg, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Kunzelmann
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027-6902, and.
authors.forename: Martin
authors.initials: M
authors.lastname: Chalfie
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany,; Cologne Excellence Cluster on Cellular Stress Responses in Ageing-associated Diseases, University of Cologne, 50931 Cologne, Germany,; Systems Biology of Ageing Cologne, University of Cologne, 50931 Cologne, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Schermer
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany,; Cologne Excellence Cluster on Cellular Stress Responses in Ageing-associated Diseases, University of Cologne, 50931 Cologne, Germany,; Systems Biology of Ageing Cologne, University of Cologne, 50931 Cologne, Germany,. Electronic address: thomas.benzing@uk-koeln.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Benzing
authors.affiliation: Systems Biology of Ageing Cologne, University of Cologne, 50931 Cologne, Germany,; Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Höhne
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002784:Cholesterol / Q000235:genetics / Q000378:metabolism; D060730:Glomerular Filtration Barrier / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D040542:Mechanotransduction, Cellular; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020125:Mutation, Missense; D009404:Nephrotic Syndrome / Q000151:congenital / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000091402:Prohibitins; D017434:Protein Structure, Tertiary
chemical_list: D029742:Caenorhabditis elegans Proteins; D047908:Intracellular Signaling Peptides and Proteins; C096705:MEC-2 protein, C elegans; D008565:Membrane Proteins; C407223:NPHS2 protein; C000717328:PHB protein, human; D000091402:Prohibitins; C000717338:prohibitin 2, C elegans; D002784:Cholesterol
keywords: Caenorhabditis elegans; Cholesterol-binding Protein; Glycosylation; Lipid Raft; Membrane Topology; PHB Domain; Podocin; Protein Conformation; Steroid-resistant Nephrotic Syndrome; Stomatin Family Proteins