Figure 1.

Effect of lipid bilayer composition on AHA2 proton pumping activity. (A) Illustration of the proton pumping assay on proteoliposomes with reconstituted AHA2. The accumulation of protons inside the vesicles was determined by measuring the fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) as a fluorescent ∆pH probe. Reactions were started by the addition of Mg²⁺ to ATP-containing buffer. After reaching saturation conditions, the H⁺ gradient was disrupted by the addition of the protonophore m-chlorophenylhydrazon (CCCP). Valinomycin was always present to mediate K⁺ exchange and prevent the build-up of a transmembrane electrical potential. (B) Lewis structures of the lipids used. (C) Representative ACMA fluorescence trace on proteoliposomes with reconstituted H⁺-ATPase in PC liposomes. The initial slope (dashed line) was taken as a measure for the proton pumping rate. (D) Dot plot showing the initial rates of proton pumping of AHA2 reconstituted in liposomes composed of pure PC and PC in mixture with the indicated phospholipids (30 mol%); the mix contained PC:PE:PS (45:45:10). Colors are the same as in B, except for the mix. Letters above box plots indicate significant differences determined by Tukey’s HSD test (p < 0.05). Box plot center lines show the medians. Box limits indicate the 25th and 75th percentiles. Whiskers are extended to the highest and the lowest values. Data are based on at least three independent reconstitutions measured three times (see number of reconstitutions in Supplementary Table S1).