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Abstract: PO1669

Topology Mapping of Membrane-Inserted ApoL1

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

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

ApoL1 inserts into membranes at pH 6 where it has anion permease activity. Titration of the cis compartment to pH 7.5 suppresses the anion permease and activates a cation channel. How ApoL1 is arrayed in the membrane-inserted form at various pH values is unclear.

Methods

Cys was substituted for Ser or Thr at positions throughout ApoL1 and resulting mutants expressed in E. coli. Purified mutants were allowed to insert into phospholipid vesicles at pH 6.0, held at pH 6.0 or titrated to pH 7.5, and reacted with extravesicular membrane-impermeant fluorescent Cys modifying reagent, Alexafluor-568-maleimide. Unreacted reagent was quenched. Non-membrane-inserted protein was removed by chaotropic extraction and Sepharose 4B chromatography. Membrane-associated protein was separated on SDS-PAGE along with ApoL1 standards for quantification. Ratio of fluorescence intensity to mass of ApoL1 protein was normalized to that of protein modified after detergent denaturation to determine relative accessibility of each Cys to the modifying reagent.

Results

Cys substitutions were generated at amino acid positions 40, 80, 149, 173, 186, 200, 204, 226, 247, and 365. We found three patterns of reactivity after membrane insertion. Cys at positions 40, 149, and 365 showed reactivity that was roughly comparable to that in detergent solution with little difference between pH 6.0 and 7.5, consistent with exposure to the aqueous solution on the cis face of the membrane under all conditions. In contrast, Cys at positions 186, 226 and 247 showed decreased reactivity after membrane insertion that was similar at both pH 6.0 and pH 7.5; these positions are not fully accessible from the external solution. Finally, Cys at positions 80, 173, 200, and 204 had decreased reactivity at pH 6.0 with increase in reactivity at pH 7.5, suggesting these positions may be initially buried in the membrane upon insertion at low pH, but titration to neutral pH induces a structural transition that exposes them to the external solution.

Conclusion

Mapping accessibilty of individual amino acid positions in ApoL1 support a model in which a substantial structural transition accompanies the pH shift-induced activation of the cation channel.

Relative reactivity of membrane-inserted cysteine substitution mutants at pH 6.0 or 7.5
 S40CS80CS149CT173CS186CS200CT204CT226CS247CS365C
pH 6.00.880.320.880.560.600.190.420.230.420.85
pH 7.50.882.341.182.490.670.741.250.240.370.63

Funding

  • NIDDK Support