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Abstract: SA-PO984

Topology of Membrane-Inserted ApoL1

Session Information

Category: Glomerular Diseases

  • 1403 Podocyte Biology


  • Edwards, John C., Saint Louis University, Saint Louis, Missouri, United States
  • Bruno, Jonathan M., Saint Louis University, Saint Louis, Missouri, United States

ApoL1 inserts into membranes at low pH where it functions as an anion permease. Titration of the cis compartment to neutral suppresses the anion permeablity and activates a cation channel activity. pH 6.0 is optimal for membrane association that leads to cation channel after titration; pH 5 is optimal for the anion permease activity. As pH is lowered from 6 to 5, the total amount of protein associated with membranes increases along with the anion pemease, but the potential for cation permease after titration is suppressed. We hypothesize that the structure of the membrane inserted ApoL1 is altered by the pH at which it encounters the membrane.


Single cysteine substitution mutant were generated at position 40, 80, 89, 149, 168, 178, 204, 226, 236, 247, 263, 300, 314, 330, and 365, expressed in bacteria and purified. Each protein was mixed with preformed lipid vesicles. Accessibility of the engineered cysteine was probed with AF488-maleimide (AF-Mal) from the cis or trans compartment. Lipid-protein vesicles were separated by chaotropic wash followed by sepharose 4B chromatography. Membrane associated protein was separated by SDS PAGE. AF-Mal modification was detected by in-gel fluorescence, total protein by western blot.


Protein associated at pH 6 was accessible to trans-modification at positions 300, 314, and 330 only, and this was unchanged by subsequent titration to pH 7.5. Protein associated at pH 5 was accessible to trans-modification at the same sites, plus at 365.


ApoL1 associated with vesicles at pH 6 has only one trans-accessible segment detected by these methods, minimally including positions 300 through 330 and we detect no change in topology with transition to conditions that activate the cation channel, consistent with a previously published model (Schaub et al., JBC (2021) 297: 101009). However, association at pH 5 leads to a larger portion of the molecule accessible from the trans comparment, consistent with the entire C-terminal region from 330 to the end translocated across the membrane. This additional translocation would eliminate the transmembrane segment that has been proposed to form the cation selective pore and would explain the decrease in cation channel activity after association at lower pH, additionally predicting different ion permeability activities depending on where along the endocytic pathway ApoL1 inserts into membranes.


  • NIDDK Support