Fig. 9: TgVMP1 and PfVMP1 are lipid scramblases, and functionally conserved along with HsVMP1. | Nature Communications

Fig. 9: TgVMP1 and PfVMP1 are lipid scramblases, and functionally conserved along with HsVMP1.

From: An ER-resident lipid scramblase is crucial for biogenesis and function of apicomplexan parasite secretory organelles

Fig. 9

A The schematic shows empty-liposomes and scramblase-liposomes with the indicated constituents. Addition of dithionite, a membrane impermeable quencher, would decrease the fluorescence of NBD-PS in the outer liposome leaflet, causing a ~50% decrease in the fluorescence signal intensity of the empty-liposomes. On the other hand, continuous translocation of NBD-PS by scramblase between outer and inner leaflets of the scramblase-liposomes would cause a »50% decrease in fluorescence signal intensity. The addition of Triton X-100 would make the liposome membrane permeable to dithionite, causing a decrease in fluorescence signal intensity to almost zero. B The plot shows relative fluorescence intensities (Ft/F0) of empty-liposomes, TgVMP1AIDHA-containing liposomes (TgVMP1-liposomes), and PfVMP1AIDHA-containing liposomes (PfVMP1-liposomes) before, after addition of dithionite, and after addition of Triton X-100. Arrows indicate the time points at which dithionite and Triton X-100 were added. Data are the average of three independent scramblase assays. C +IAA and –IAA intracellular RHTIR1-VMP1AID (TgVMP1KD), RHTIR1-VMP1AID/PfVMP1 (PfVMP1COM), and RHTIR1-VMP1AID/HsVMP1 (HsVMP1COM) parasites were stained for the PV marker GRA1. The number of PVs containing different numbers of parasites is shown as a percentage of the total number of PVs (% Vacuoles) on the y-axis for the indicated parasites on the x-axis. Data are mean ± SD error bar based on ≥100 PVs in each of three independent experiments. The p-values are for the PVs containing a single parasite only. D Purified +IAA and -IAA tachyzoites were evaluated for dense granule biogenesis defect using antibodies to GRA2. The Airyscan micrographs show signal for GRA2, nuclear stain (DAPI), and overlap of the two panels (Merged) for the indicated parasites. Scale bar = 5 µm. The arrowheads indicate perinuclear accumulation of GRA2 in +IAA TgVMP1KD, but not in +IAA PfVMP1COM and +IAA HsVMP1COM parasites, indicating restoration of dense granule biogenesis by PfVMP1 and HsVMP1, respectively. E The percentage of parasites showing perinuclear accumulation of GRA2 in D (% Perinuclear accumulation) is shown on the y-axis for the indicated parasites on the x-axis. Data are mean ± SD error bar based on ≥100 parasites in each of the two independent experiments. Source data are provided as a Source Data file.

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