Endoplasmic reticulum-retained podocin mutants are massively degraded by the proteasome.

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.