Lipid flipping in the membrane bilayers is a widespread eukaryotic phenomenon that is catalyzed by assorted P4-ATPases. Its occurrence, mechanism, and importance in apicomplexan parasites have remained elusive, however. Here we show that Toxoplasma gondii, an obligate intracellular parasite with high clinical relevance, can salvage
phosphatidylserine (PtdSer) and
phosphatidylethanolamine (PtdEtn) but not
phosphatidylcholine (PtdCho) probes from its milieu. Consistently, the drug analogs of PtdCho are broadly ineffective in the parasite culture. NBD-PtdSer imported to the parasite interior is decarboxylated to NBD-PtdEtn, while the latter is not methylated to yield PtdCho, which confirms the expression of PtdSer
decarboxylase but a lack of PtdEtn
methyltransferase activity and suggests a role of exogenous
lipids in membrane biogenesis of T. gondii. Flow cytometric quantitation of NBD-probes endorsed the selectivity of
phospholipid transport and revealed a dependence of the process on energy and
protein. Accordingly, our further work identified five P4-ATPases (TgP4-ATPase1-5), all of which harbor the signature residues and motifs required for
phospholipid flipping. Of the four
proteins expressed during the lytic cycle, TgP4-ATPase1 is present in the apical plasmalemma; TgP4-ATPase3 resides in the Golgi network along with its noncatalytic partner
Ligand Effector Module 3 (TgLem3), whereas TgP4-ATPase2 and TgP4-ATPase5 localize in the plasmalemma as well as endo/cytomembranes. Last but not least,
auxin-induced degradation of TgP4-ATPase1-3 impaired the parasite growth in human host cells, disclosing their crucial roles during acute
infection. In conclusion, we show selective translocation of PtdEtn and PtdSer at the parasite surface and provide the underlying mechanistic and physiological insights in a model eukaryotic pathogen.