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

Kai Chen; Xiyu Huang; Ute Distler; Stefan Tenzer; Özlem Günay-Esiyok; Nishith Gupta

doi:10.1016/j.csbj.2023.02.032

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

Comput Struct Biotechnol J. 2023 Feb 18:21:1893-1906. doi: 10.1016/j.csbj.2023.02.032. eCollection 2023.

Authors

Kai Chen¹, Xiyu Huang¹, Ute Distler², Stefan Tenzer², Özlem Günay-Esiyok¹, Nishith Gupta^{1

3}

Affiliations

¹ Department of Molecular Parasitology, Faculty of Life Sciences, Humboldt University, Berlin, Germany.
² Institute of Immunology, University Medical Center of the Johannes-Gutenberg University, Mainz, Germany.
³ Intracellular Parasite Education and Research Labs (iPEARL), Department of Biological Sciences, Birla Institute of Technology and Science, Pilani (BITS-P), Hyderabad, India.

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 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 T. gondii.

Keywords: BSA, bovine serum albumin; CDC50, Cell Division Control 50; COS, crossover sequence; Cdc50; DAPI, 4′,6-diamidino-2-phenylindole; DHFR-TS, dihydrofolate reductase – thymidylate synthase; HFF, human foreskin fibroblast; HXGPRT, hypoxanthine-xanthine-guanine phosphoribosyltransferase; IAA, indole-3-acetic acid; LEM, Ligand Effector Module; Lem1; NBD, nitrobenzoxadiazole; NBD-lipid; P4-ATPase1; PBS, phosphate-buffered saline; Phosphatidylserine; Phospholipid flipping; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdSer, phosphatidylserine; PtdThr, phosphatidylthreonine; UTR, untranslated region; cGMP, cyclic Guanosine Monophosphate; mAID, (mini) auxin-inducible degron.

Grants and funding

IA/S/19/1/504263/WTDBT_/DBT-Wellcome Trust India Alliance/India