Apically-located P4-ATPase1-Lem1 complex internalizes phosphatidylserine and regulates motility-dependent invasion and egress in <i>Toxoplasma gondii</i>
Kai Chen, Xiyu Huang, Ute Distler, Stefan Tenzer, Özlem Günay-Esiyok, Nishith Gupta
Abstract
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 (TgP4-ATPase1-5) and three non-catalytic partner proteins (TgLem1-3) in the intracellular protozoan pathogen Toxoplasma gondii.However, their flipping activity, physiological relevance and functional coupling remain unknown.Herein, we demonstrate that TgP4-ATPase1 and TgLem1 work together to translocate phosphatidylserine (PtdSer) during the lytic cycle of T. gondii.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 proximitydependent 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 T. gondii.