A disease-causing mutation illuminates the protein membrane topology of the kidney-expressed prohibitin homology (PHB) domain protein podocin

J Biol Chem. 2014 Apr 18;289(16):11262-11271. doi: 10.1074/jbc.M113.521773. Epub 2014 Mar 4.


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.

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

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Caenorhabditis elegans
  • Caenorhabditis elegans Proteins / genetics
  • Caenorhabditis elegans Proteins / metabolism*
  • Cell Line
  • Cell Membrane / genetics
  • Cell Membrane / metabolism*
  • Cell Membrane / pathology
  • Cholesterol / genetics
  • Cholesterol / metabolism
  • Glomerular Filtration Barrier / metabolism*
  • Glomerular Filtration Barrier / pathology
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Mechanotransduction, Cellular*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • Mutation, Missense*
  • Nephrotic Syndrome / congenital
  • Nephrotic Syndrome / genetics
  • Nephrotic Syndrome / metabolism
  • Nephrotic Syndrome / pathology
  • Protein Structure, Tertiary


  • Caenorhabditis elegans Proteins
  • Intracellular Signaling Peptides and Proteins
  • MEC-2 protein, C elegans
  • Membrane Proteins
  • NPHS2 protein
  • Cholesterol

Supplementary concepts

  • Nephrotic syndrome, idiopathic, steroid-resistant