Podocin Oligomerization Revealed by FRET Analysis: Sites of Interallelic Interactions

Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its most frequent non-silent variant, R229Q, is only pathogenic when trans-associated to specific, dominant negative 3’ mutations giving the first example of clinically important interallelic interactions in human genetics. We suggested the causal role of an abnormal oligomerization. Here we aimed to determine the oligomerization sites of podocin and the effect of dominant negative substitutions. We show in a series of FRET spectroscopy experiments that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283-382): through the first C-terminal helical region (H1, 283-313), which forms a coiled coil, and through the 332-348 region. We found amino acid substitutions with a dominant negative effect on R229Q to cause a significant change in FRET efficiency, suggesting an altered conformation of pathogenic heterooligomers. Coexpressed podocin variants with a dimerization capacity strongly influenced each other's localization, indicating the primary role of oligomerization in the mediation of interallelic interactions. Interestingly, we found oligomerization to also mediate interallelic complementation: oligomerization with membranous podocin variants with an intact C-terminal tail rescues the endocytosis of podocin mutants lacking the region distal the second oligomerization site. Thus, C-terminal oligomerization of podocin mediates interallelic interactions, potentially modifying the pathogenicity of trans-associated NPHS2 alleles.