A Disease-causing Mutation Illuminates the Protein Membrane Topology of the Kidney-expressed Prohibitin Homology (PHB) Domain Protein Podocin

Abstract

Background: Mutations in the stomatin family protein podocin are the most common genetic cause of proteinuria. Results: A conserved proline residue of podocin is essential for its membrane topology. Conclusion: This study confirms a hairpin-like structure of the membrane-attached PHB domain protein and its significance for cholesterol recruitment. Significance: Podocin P118L elucidates the pathogenic implication in kidney disease and identifies a novel family of PHB domain proteins.

Endoplasmic reticulum–retained podocin mutants are massively degraded by the proteasome

“…However, a small part of the Pod wt pool was also glycosylated, consistent with the observations of WT podocin and stomatin by other authors (11,12). We tested by immunoblot the protein expression pattern of a series of podocin mutants that present different subcellular localizations, confirming that only ERretained mutants, such as Pod R168C , were enriched in N-glycosylated forms ( Figure 1E).…”

“…To test whether the difference in protein levels of Pod wt and Pod R138Q was due to these differential glycosylation patterns, we also created podocyte cell lines stably expressing HA-tagged Pod wt and Pod R138Q bearing mutations in the amino acid residues required for Nglycosylation, N199 and N355 (Pod wt(N 199Q, N 355S) and Pod R138Q(N 199Q, N 355S) ). Immunoblots of protein extracts from these cells revealed that although the podocin triplet disappears with mutation of residues N199 and N355 ( Figure S1B), glycosylation alone does not account for the differences in protein levels of Pod wt and Pod R138Q , as there remains increased amounts of Pod wt(N 199Q, N 355S) compared to Pod R138Q(N 199Q, N 355S) .Our results showed that the mutant Pod R138Q was predominantly N-glycosylated, similarly to Pod P118L (12), another ER-retained podocin 2 by guest on May 10, 2018 http://www.jbc.org/ Downloaded from Podocin R138Q quality control and ERAD mutant, suggesting that the majority of intracellular Pod R138Q has a transmembrane topology (11,12). However, a small part of the Pod wt pool was also glycosylated, consistent with the observations of WT podocin and stomatin by other authors (11,12).…”

“…Our results showed that the mutant Pod R138Q was predominantly N-glycosylated, similarly to Pod P118L (12), another ER-retained podocin 2 by guest on May 10, 2018 http://www.jbc.org/ Downloaded from Podocin R138Q quality control and ERAD mutant, suggesting that the majority of intracellular Pod R138Q has a transmembrane topology (11,12). However, a small part of the Pod wt pool was also glycosylated, consistent with the observations of WT podocin and stomatin by other authors (11,12).…”

“…The upper bands of these triplets correspond to N-glycosylated forms, which is particularly intriguing since it implies that the C-terminus of most Pod R138Q is inside the ER lumen. N-glycosylation, and evidence of transmembrane topology, has been already described for stomatin, for the short isoform of podocin and for podocin P118L , another ER-retained podocin mutant (11,12,23). Whereas Pro118, an amino acid which is highly conserved throughout the stomatin family, is located within the hydrophobic intramembrane region and is responsible for the kink of the hairpin topology (12), Arg138 and Arg168, which are also very well conserved, are located within the PHB domain, far away from this region.…”

“…Podocin has a predicted hairpin-loop topology, with both the amino (N)-and C-termini facing the cytoplasm, and its hydrophobic domain anchored either to the inner leaflet of the plasma membrane (PM) or to the outer leaflet of the ER-membrane (7). However, an alternative transmembrane topology has been described for stomatin and podocin, particularly when a conserved proline residue, critical for the kink of the hairpin topology, found at position 118 in podocin, is mutated, and has been linked to N-glycosylated forms of these proteins (11,12). In addition, although the stability and degradation of podocin has been associated with a short internalization motif located in its C-terminus and to an interaction with the ubiquitin ligase Ubr4 (13,14), the mechanisms of Pod R138Q degradation are still unexplored.…”