Apically-located P4-ATPase1-Lem1 complex internalizes phosphatidylserine and regulates motility-dependent invasion and egress in Toxoplasma gondii .

The membrane asymmetry regulated by P4-ATPases is crucial for the functioning of eukaryotic cells. The underlying spatial translocation or flipping of specific lipids is usually assured by respective P4-ATPases coupled to conforming non-catalytic subunits. Our previous work has identified five P4-ATPases (P4-ATPase1-5) and three non-catalytic partner proteins (Lem1-3) in the intracellular protozoan pathogen, . However, their flipping activity, physiological relevance and functional coupling remain unknown. Herein, we demonstrate that P4-ATPase1 and Lem1 work together to translocate phosphatidylserine (PtdSer) during the lytic cycle of . Both proteins localize in the plasma membrane at the invasive (apical) end of its acutely-infectious tachyzoite stage. The genetic knockout of P4-ATPase1 and conditional depletion of Lem1 in tachyzoites severely disrupt the asexual reproduction and translocation of PtdSer across the plasma membrane. Moreover, the phenotypic analysis of individual mutants revealed a requirement of lipid flipping for the motility, egress and invasion of tachyzoites. Not least, the proximity-dependent biotinylation and reciprocal immunoprecipitation assays demonstrated the physical interaction of P4-ATPase1 and Lem1. Our findings disclose the mechanism and significance of PtdSer flipping during the lytic cycle and identify the P4-ATPase1-Lem1 heterocomplex as a potential drug target in .