Figure 4: ALA10 complements the phenotypes of the P4-ATPase-deficient yeast strain ZHY709 and translocates NBD-PS, -PE, -PC and lysoPC.

(a) Functional complementation of a P4-ATPase-deficient yeast strain (ZHY709) expressing wild-type ALA10 (RGSH₁₀-ALA10) and mutant ala10 (ala10D430N) in combination with the β-subunit ALIS1 (RGSH₆-ALIS1). Yeast strain BY4741 (wild type) and ZHY709 transformed with empty plasmids were used as a positive and negative control, respectively. Yeast strains were grown at 30 °C on glucose- (control) or galactose-containing (induced gene expression) plates supplemented with papuamide B (Pap B), duramycin (Dura), and miltefosine (Milte) or on plates incubated at 20 °C, as indicated. The experiment was repeated at least three times with identical results. (b) Immunoblot illustrating the expression of P4-ATPases and co-expressed β-subunits in yeast microsomal membranes (Anti-RGSHis). (c) Subcellular localization of GFP-ALA10 in the presence and absence of RGSH₆-ALIS1. BF, bright field. Scale bar, 5 μm. (d) ZHY709 yeast cells expressing ALA10+ALIS1 were labelled with the indicated NBD-lipids and analysed by flow cytometry. Accumulation of NBD-lipids is shown as a percentage of fluorescence intensity relative to ZHY709 (dotted line), arbitrary values corresponding to 100% are given for all lipids in the Methods section. Data represent means±s.d. (n=3-17). ***P≤0.001, **P≤0.01 and *P≤0.05, significantly different with respect to ZHY709 according to Student’s t-test. (e) Chemical structure of NBD-PC and -lysoPC; black box, choline head group; grey box, NBD. (f) NBD-lysoPC accumulation in ZHY709 cells transformed with different plant P4-ATPases and their respective β-subunits. Data represent means±s.d. (n=3–4). **P≤0.01 and *P≤0.05, significantly different with respect to ZHY709 according to Student’s t-test. (g) ALA10+ALIS1-dependent NBD-PC and -lysoPC uptake and analysis of metabolized lipids. Cells expressing ALA10+ALIS1 or containing empty vector (EMPTY) were incubated with NBD-lipids for 30 min before imaging by fluorescence (NBD) or bright field (BF) microscopy (left). Scale bar, 5 μm. For analysis by thin-layer chromatography (right), lipids were extracted from labelled cells (Cells) and their medium (M). Lipid structures are given to the right in each figure (circle, choline; square, phosphate group; star, NBD group).