Endoplasmic Reticulum Membrane Insertion of APOL1 Risk Alleles Disrupts Calcium Homeostasis and Is Mitigated by Autophagic Clearance

Background: Apolipoprotein 1 ( APOL1 ) risk alleles (G1 and G2) are major genetic drivers of APOL1 -associated kidney disease in populations of African ancestry. Prior studies implicated membrane-associated channel activity and ion dysregulation in APOL1 cytotoxicity, but how APOL1 risk alleles engage intracellular membranes, disrupt endoplasmic reticulum calcium, and are handled by quality-control pathways remains incompletely defined. Methods: We used a split-green fluorescent protein topology system to visualize and quantify APOL1 membrane insertion and domain-deletion mutants to define structural requirements for insertion and toxicity. Endoplasmic reticulum calcium was monitored using a genetically encoded fluorescent, endoplasmic reticulum-specific calcium indicator. APOL1 degradation was assessed using autophagy and proteasome modulators, immunoblotting, and LC3 colocalization. In vivo relevance was tested in transgenic Drosophila nephrocytes expressing human APOL1 alleles with or without dietary rapamycin. Results: APOL1 inserted robustly into the endoplasmic reticulum membrane under basal cellular conditions, and its pore-forming and membrane-addressing domains contributed strongly to insertion and cytotoxicity. APOL1-G1 and APOL1-G2 showed greater endoplasmic reticulum membrane insertion than APOL1-G0, accompanied by greater endoplasmic reticulum calcium depletion. These findings support a model in which risk allele-associated misfolding exposes hydrophobic regions that promote aberrant endoplasmic reticulum membrane engagement. APOL1 accumulated after autophagy inhibition, decreased after rapamycin treatment, and localized to LC3-positive autophagosomal structures, whereas proteasomal inhibition did not accumulate APOL1 nor induce its toxicity. Rapamycin reduced APOL1 protein levels, improved cell viability, and mitigated risk allele-induced nephrocyte hypertrophy and filtration dysfunction in vivo . Conclusions: These findings identified enhanced endoplasmic reticulum membrane insertion, endoplasmic reticulum calcium depletion, and autophagy-lysosomal clearance as linked features of APOL1 risk allele toxicity.