Figure 9.

In vitro lipid transfer activities of the predicted Sfh3 sterol-binding mutants. Transfer activities for the indicated Sfh3 variants were measured using DHE (A), [³H]cholesterol (B), CTL (C), [³H]PtdIns (D), and [Pyr]PtdIns (E) as transfer substrates. Assay incubations were for 10 min for the real-time fluorescence-based DHE and CTL assays and 30 min for [³H]cholesterol, [³H]PtdIns and [Pyr]PtdIns transfer assays. All assays were performed at 37 °C, and protein input was clamped at 10 μg for all experiments. For DHE, CTL, and [Pyr]PtdIns assays, values for spontaneous transfer (no protein) were determined, and spontaneous transfer backgrounds were subtracted from each measurement. Transfer activities were normalized to Sfh3 activity. The averages of triplicate determinations from two independent experiments for DHE, CTL, and [Pyr]PtdIns, and triplicate determinations from three independent experiments for [³H]cholesterol and [³H]PtdIns, are shown. Error bars represent standard deviations for the [³H]cholesterol and [³H]PtdIns assays and mean ± range for DHE, CTL, and [Pyr]PtdIns assays.