Pathogenic podocin variants exhibit distinct defects in trafficking, membrane organization, and degradation pathways.

Nephrotic syndrome is frequently associated with pathogenic variants in NPHS2 (podocin), including the common and severe R138Q substitution. Using conditionally immortalized human podocytes expressing Myc-tagged podocin variants (G92C, V180M, R138Q, R238S, and R291W), we systematically compared variant-specific defects in plasma-membrane trafficking, detergent-resistant microdomain (DRM) localization, and protein stability. All variants displayed reduced plasma membrane abundance and altered DRM distribution. Among them, R138Q-podocin showed uniquely reduced protein stability. Consistent with previous reports, quantitative proteomics revealed a strong enrichment of endoplasmic reticulum (ER) quality-control and ubiquitin-proteasome components in the R138Q interactome, confirming its identity as an ER-associated degradation substrate. Proteasome inhibition with MG132 stabilized R138Q-podocin and restored its trafficking to both the plasma membrane and DRMs, indicating that impaired stability-rather than an intrinsic trafficking defect-restricts its surface localization. Proteomic profiling further identified caveolin-1, CDCP1, and myosin VI as previously unrecognized podocin interactors. These findings expand the podocin interaction network and suggest potential roles in adhesion-associated membrane organization. Collectively, these results demonstrate that pathogenic podocin variants disrupt podocyte function through distinct mechanisms involving degradation, trafficking, and membrane microdomain association, providing insight into variant-specific disease pathways in nephrotic syndrome. SYNOPSIS: This study examined the trafficking, membrane localization, and stability of disease-associated podocin variants. All variants showed reduced plasma-membrane abundance and altered detergent-resistant microdomain distribution, whereas only R138Q-podocin exhibited marked proteasomal degradation. Quantitative proteomics confirmed the endoplasmic reticulum-associated degradation signature of R138Q and identified caveolin-1, CDCP1, and myosin VI as previously unrecognized podocin interactors. These findings reveal variant-specific mechanisms governing podocin stability and membrane organization.