Endoplasmic Reticulum-Retained Podocin Mutants Are Massively Degraded by the Proteasome

J Biol Chem. 2018 Mar 16;293(11):4122-4133. doi: 10.1074/jbc.RA117.001159. Epub 2018 Jan 30.

Abstract

Podocin is a key component of the slit diaphragm in the glomerular filtration barrier, and mutations in the podocin-encoding gene NPHS2 are a common cause of hereditary steroid-resistant nephrotic syndrome. A mutant allele encoding podocin with a p.R138Q amino acid substitution is the most frequent pathogenic variant in European and North American children, and the corresponding mutant protein is poorly expressed and retained in the endoplasmic reticulum both in vitro and in vivo To better understand the defective trafficking and degradation of this mutant, we generated human podocyte cell lines stably expressing podocinwt or podocinR138Q Although it has been proposed that podocin has a hairpin topology, we present evidence for podocinR138QN-glycosylation, suggesting that most of the protein has a transmembrane topology. We find that N-glycosylated podocinR138Q has a longer half-life than non-glycosylated podocinR138Q and that the latter is far more rapidly degraded than podocinwt Consistent with its rapid degradation, podocinR138Q is exclusively degraded by the proteasome, whereas podocinwt is degraded by both the proteasomal and the lysosomal proteolytic machineries. In addition, we demonstrate an enhanced interaction of podocinR138Q with calnexin as the mechanism of endoplasmic reticulum retention. Calnexin knockdown enriches the podocinR138Q non-glycosylated fraction, whereas preventing exit from the calnexin cycle increases the glycosylated fraction. Altogether, we propose a model in which hairpin podocinR138Q is rapidly degraded by the proteasome, whereas transmembrane podocinR138Q degradation is delayed due to entry into the calnexin cycle.

Keywords: endoplasmic-reticulum-associated protein degradation (ERAD); genetic disease; glycosylation; intracellular trafficking; kidney; podocin; proteasome; steroid-resistant nephrotic syndrome.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calnexin / metabolism*
  • Endoplasmic Reticulum / metabolism*
  • HEK293 Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism*
  • Mutation*
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Transport
  • Proteolysis

Substances

  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Mutant Proteins
  • NPHS2 protein
  • Calnexin
  • Proteasome Endopeptidase Complex