Mutations in the retromer complex, which is involved in sorting integral membrane proteins from endosomes to cellular compartments, are associated with atypical parkinsonism, but how these mutations affect retromer function remains unclear. Through a quantitative proteomic analysis of the retromer interactome, McMillan et al. reveal a new mechanism for perturbed endosomal sorting in parkinsonism.

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 (ER) 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 podocin wt or podocin R138Q . Although it has been proposed that podocin has a hairpin topology, we present evidence for podocin R138Q N-glycosylation, suggesting that most of the protein has a transmembrane topology. We find that N-glycosylated podocin R138Q has a longer half-life than non-glycosylated podocin R138Q , and that the latter is far more rapidly degraded than podocin wt . Consistent with its rapid degradation, podocin R138Q is exclusively degraded by the proteasome, whereas podocin wt is degraded by both the proteasomal and the lysosomal proteolytic machineries. In addition, we demonstrate an enhanced interaction of podocin R138Q with calnexin as the mechanism of ER retention. Calnexin knock-down enriches the podocin R138Q non-glycosylated fraction, whereas preventing exit from the calnexin cycle increases the glycosylated fraction. Altogether, we propose a model in which hairpin podocin R138Q is rapidly degraded by the proteasome, whereas transmembrane podocin R138Q degradation is delayed due to entry into the calnexin cycle.Nephrotic syndrome (NS) is clinically characterized by proteinuria, edema, hypoalbuminemia and hyperlipidemia, and is a consequence of glomerular filtration barrier (GFB) dysfunction. The prognosis of steroid-resistant nephrotic syndrome (SRNS) is poor, with a high proportion of patients rapidly developing end-stage renal disease, requiring dialysis or transplantation (1,2). The GFB is comprised principally of podocytes, specialised epithelial cells which interdigitate at junctions known as slit diaphragms (SDs). Mutations in the NPHS2 gene, encoding the slit diaphragm (SD) protein podocin, are the most frequent monogenic cause of SRNS in childhood (3,4 Podocin R138Q quality control and ERAD North America (4), and is associated with an earlyonset and rapidly progressing form of the disease (5). In accordance with the severe clinical phenotype, podocin p.R138Q (Pod R138Q ) is retained in the ER of podocytes and does not reach the SD, thus impairing correct functioning of the GFB (6,7).Podocin belongs to the stomatin and prohibitin homology domain (PHB) protein family and is specifically expressed in podocytes. It has been proposed that podocin acts as a molecular scaffold for other SD proteins in lipid raft membrane subdomains. For example, podocin interacts with both nephrin and CD2AP through its carboxy (C)-terminus, and participates in various signaling...