Inward translocation of NBD-PC and NBD-PE across the plasma membrane of Leishmania requires LdMT and LdRos3.

Promastigotes of wild type (wt), ΔLdMT and ΔLdRos3 lines were labelled with NBD-lipids for 30 min at 2°C and than washed and analysed by flow cytometry (A–C) or visualised by fluorescence microscopy (D). ATP depletion was achieved by preincubation with 5 mM 2-deoxyglucose and 20 mM sodium azide. To abolish the proton electrochemical gradient 50 µM of the protonophore CCCP was used. As a control LdMT-GFP and LdRos3-GFP on episomal Leishmania expression vectors were reintroduced in ΔLdMT and ΔLdRos3 mutants, respectively; control, non-labelled cells showing the intrinsic fluorescence of the GFP fusion proteins. Data are normalized to NBD-PC internalization of wild-type parasites; 100% corresponds to 468±96 a.u. NBD-PC. Data represent the means ± SE of at least three independent experiments. For statistical analysis Welch's test was performed. Significant differences in the lipid uptake of the mutants compared to the wild type are denoted by asterisks (** p = 0.05; * p = 0.01).