APOL1 plasma membrane pools resist rapid protein degradation.

Homozygosity of two sequence variants within the human APOL1 gene, called APOL1 G1 and G2, in combination with high Apolipoprotein L1 (APOL1) expression levels are linked to a wide spectrum of renal diseases summarized as APOL1-mediated kidney diseases (AMKDs). Previous studies have shown that inflammatory and immunomodulatory triggers are major contributors to elevated APOL1 protein expression. However, little is known about the stability of APOL1 and the role of protein degradation in regulating its intracellular levels. In this study, we systematically investigated these aspects. To investigate degradation dynamics, we used stable HEK293T cell lines with inducible overexpression of GFP-tagged APOL1 vA/G0, its C-terminal risk variants (G1, G2), N-terminal isoforms (vB1, vB3, vC), as well as the deletion mutant (ΔN59), APOL2, and an APOL1–APOL2 chimeric protein (NTvA-APOL2). Degradation and protein biosynthesis were modified using proteasome inhibitors and cycloheximide, respectively. Treated cells were analyzed using Western blotting, immunofluorescence microscopy and flow cytometry analyses. Moreover, in silico analyses were performed to identify motifs within the APOL1 sequence potentially mediating its degradation. This study shows that APOL1 is subject to remarkably rapid proteasomal degradation, observed for both the APOL1 wildtype (G0) and renal risk variants (RRVs) G1 and G2. Moreover, despite distinct topologies at the intracellular membranes of the APOL1 isoforms, all exhibit rapid protein degradation. In contrast, APOL2 – the closest homolog of APOL1 – demonstrated significantly greater resistance to proteasomal degradation. In silico analyses identified two intrinsically disordered regions (IDRs) present in APOL1 but absent in APOL2, potentially underlying the increased susceptibility to degradation. Notably, APOL1 surface-localized pools were resistant to rapid proteasomal degradation, with no major differences observed between G0 and RRVs. Together our findings suggest that APOL1 stability is highly compartment-specific, with rapid degradation at intracellular pools and pronounced stability at the cell surface. Targeting the stability of APOL1 at the PM represents a promising avenue for the development of novel therapeutic interventions against AMKD.