ATP10A deficiency results in male-specific infertility in mice.

Over 8% of couples worldwide are affected by infertility and nearly half of these cases are due to male-specific issues where the underlying cause is often unknown. Therefore, discovery of new genetic factors contributing to male-specific infertility in model organisms can enhance our understanding of the etiology of this disorder. Here we show that murine ATP10A, a phospholipid flippase, is highly expressed in male reproductive organs, specifically the testes and vas deferens. Therefore, we tested the influence of ATP10A on reproduction by examining fertility of Atp10A knockout mice. Our findings reveal that Atp10A deficiency leads to male-specific infertility, but does not perturb fertility in the females. The Atp10A deficient male mice exhibit smaller testes, reduced sperm count (oligozoospermia) and lower sperm motility (asthenozoospermia). Additionally, Atp10A deficient mice display testes and vas deferens histopathological abnormalities, as well as altered total and relative amounts of hormones associated with the hypothalamic-pituitary-gonadal axis. Surprisingly, circulating testosterone is elevated 2-fold in the Atp10A knockout mice while luteinizing hormone, follicle stimulating hormone, and inhibin B levels were not significantly different from WT littermates. The knockout mice also exhibit elevated levels of gonadotropin receptors and alterations to ERK, p38 MAPK, Akt, and cPLA2-dependent signaling in the testes. Atp10A was knocked out in the C57BL/6J background, which also carries an inactivating nonsense mutation in the closely related lipid flippase, Atp10D. We have corrected the Atp10D nonsense mutation using CRISPR/Cas9 and determined that loss of Atp10A alone is sufficient to cause infertility in male mice. Collectively, these findings highlight the critical role of ATP10A in male fertility in mice and provide valuable insights into the underlying molecular mechanisms.

Glucosylceramide flippases contribute to cellular glucosylceramide homeostasis.

Lipid transport is an essential cellular process with importance to human health, disease development, and therapeutic strategies. Type IV P-type ATPases (P4-ATPases) have been identified as membrane lipid flippases by utilizing nitrobenzoxadiazole (NBD)-labeled lipids as substrates. Among the 14 human type IV P-type ATPases, ATP10D was shown to flip NBD-glucosylceramide (GlcCer) across the plasma membrane. Here, we found that conversion of incorporated GlcCer (d18:1/12:0) to other sphingolipids is accelerated in cells exogenously expressing ATP10D but not its ATPase-deficient mutant. These findings suggest that 1) ATP10D flips unmodified GlcCer as well as NBD-GlcCer at the plasma membrane and 2) ATP10D can translocate extracellular GlcCer, which is subsequently converted to other metabolites. Notably, exogenous expression of ATP10D led to the reduction in cellular hexosylceramide levels. Moreover, the expression of GlcCer flippases, including ATP10D, also reduced cellular hexosylceramide levels in fibroblasts derived from patients with Gaucher disease, which is a lysosomal storage disorder with excess GlcCer accumulation. Our study highlights the contribution of ATP10D to the regulation of cellular GlcCer levels and maintaining lipid homeostasis.

Direct evidence of lipid transport by the Drs2-Cdc50 flippase upon truncation of its terminal regions.

P4-ATPases in complex with Cdc50 subunits are lipid flippases that couple ATP hydrolysis with lipid transport to the cytoplasmic leaflet of membranes to create lipid asymmetry. Such vectorial transport has been shown to contribute to vesicle formation in the late secretory pathway. Some flippases are regulated by autoinhibitory regions that can be destabilized by protein kinase-mediated phosphorylation and possibly by binding of cytosolic proteins. In addition, the binding of lipids to flippases may also induce conformational changes required for the activity of these transporters. Here, we address the role of phosphatidylinositol-4-phosphate (PI4P) and the terminal autoinhibitory tails on the lipid flipping activity of the yeast lipid flippase Drs2-Cdc50. By functionally reconstituting the full-length and truncated forms of Drs2 in a 1:1 complex with the Cdc50 subunit, we provide compelling evidence that lipid flippase activity is exclusively detected for the truncated Drs2 variant and is dependent on the presence of the phosphoinositide PI4P. These findings highlight the critical role of phosphoinositides as lipid co-factors in the regulation of lipid transport by the Drs2-Cdc50 flippase.

Type IV P-Type ATPases: Recent Updates in Cancer Development, Progression, and Treatment.

Adaptations of cancer cells for survival are remarkable. One of the most significant properties of cancer cells to prevent the immune system response and resist chemotherapy is the altered lipid metabolism and resulting irregular cell membrane composition. The phospholipid distribution in the plasma membrane of normal animal cells is distinctly asymmetric. Lipid flippases are a family of enzymes regulating membrane asymmetry, and the main class of flippases are type IV P-type ATPases (P4-ATPases). Alteration in the function of flippases results in changes to membrane organization. For some lipids, such as phosphatidylserine, the changes are so drastic that they are considered cancer biomarkers. This review will analyze and discuss recent publications highlighting the role that P4-ATPases play in the development and progression of various cancer types, as well as prospects of targeting P4-ATPases for anti-cancer treatment.

Cell detoxification of secondary metabolites by P4-ATPase-mediated vesicle transport.

Mechanisms for cellular detoxification of drug compounds are of significant interest in human health. Cyclosporine A (CsA) and tacrolimus (FK506) are widely known antifungal and immunosuppressive microbial natural products. However, both compounds can result in significant side effects when used as immunosuppressants. The insect pathogenic fungus Beauveria bassiana shows resistance to CsA and FK506. However, the mechanisms underlying the resistance have remained unknown. Here, we identify a P4-ATPase gene, BbCRPA, from the fungus, which confers resistance via a unique vesicle mediated transport pathway that targets the compounds into detoxifying vacuoles. Interestingly, the expression of BbCRPA in plants promotes resistance to the phytopathogenic fungus Verticillium dahliae via detoxification of the mycotoxin cinnamyl acetate using a similar pathway. Our data reveal a new function for a subclass of P4-ATPases in cell detoxification. The P4-ATPases conferred cross-species resistance can be exploited for plant disease control and human health protection.

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 (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.

TMEM30A is essential for hair cell polarity maintenance in postnatal mouse cochlea.

BACKGROUND: Phosphatidylserine is translocated to the inner leaflet of the phospholipid bilayer membrane by the flippase function of type IV P-tape ATPase (P4-ATPase), which is critical to maintain cellular stability and homeostasis. Transmembrane protein 30A (TMEM30A) is the ß-subunit of P4-ATPase. Loss of P4-ATPase function causes sensorineural hearing loss and visual dysfunction in human. However, the function of TMEM30A in the auditory system is unclear. METHODS: P4-ATPase subtype expression in the cochlea was detected by immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) at different developmental stages. Hair cell specific TMEM30A knockout mice and wild-type littermates were used for the following functional and morphological analysis. Auditory function was evaluated by auditory brainstem response. We investigated hair cell and stereocilia morphological changes by immunofluorescence staining. Scanning electron microscopy was applied to observe the stereocilia ultrastructure. Differentially expressed transcriptomes were analyzed based on RNA-sequencing data from knockout and wild-type mouse cochleae. Differentially expressed genes were verified by qRT-PCR. RESULTS: TMEM30A and subtypes of P4-ATPase are expressed in the mouse cochlea in a temporal-dependent pattern. Deletion of TMEM30A in hair cells impaired hearing onset due to progressive hair cell loss. The disrupted kinocilia placement and irregular distribution of spectrin-α in cuticular plate indicated the hair cell planar polarity disruption in TMEM30A deletion hair cells. Hair cell degeneration begins at P7 and finishes around P14. Transcriptional analysis indicates that the focal adhesion pathway and stereocilium tip-related genes changed dramatically. Without the TMEM30A chaperone, excessive ATP8A2 accumulated in the cytoplasm, leading to overwhelming endoplasmic reticulum stress, which eventually contributed to hair cell death. CONCLUSIONS: Deletion of TMEM30A led to disrupted planar polarity and stereocilia bundles, and finally led to hair cell loss and auditory dysfunction. TMEM30A is essential for hair cell polarity maintenance and membrane homeostasis. Our study highlights a pivotal role of TMEM30A in the postnatal development of hair cells and reveals the possible mechanisms underlying P4-ATPase-related genetic hearing loss.

Exploring the Phospholipid Transport Mechanism of ATP8A1-CDC50.

P4-ATPase translocates lipids from the exoplasmic to the cytosolic plasma membrane leaflet to maintain lipid asymmetry distribution in eukaryotic cells. P4-ATPase is associated with severe neurodegenerative and metabolic diseases such as neurological and motor disorders. Thus, it is important to understand its transport mechanism. However, even with progress in X-ray diffraction and cryo-electron microscopy techniques, it is difficult to obtain the dynamic information of the phospholipid transport process in detail. There are still some problems required to be resolved: (1) when does the lipid transport happen? (2) How do the key residues on the transmembrane helices contribute to the free energy of important states? In this work, we explore the phospholipid transport mechanism using a coarse-grained model and binding free energy calculations. We obtained the free energy landscape by coupling the protein conformational changes and the phospholipid transport event, taking ATP8A1-CDC50 (the typical subtype of P4-ATPase) as the research object. According to the results, we found that the phospholipid would bind to the ATP8A1-CDC50 at the early stage when ATP8A1-CDC50 changes from E2P to E2Pi-PL state. We also found that the electrostatic effects play crucial roles in the phospholipid transport process. The information obtained from this work could help us in designing novel drugs for P-type flippase disorders.

Lipid Transport by Candida albicans Dnf2 Is Required for Hyphal Growth and Virulence.

Candida albicans is a common cause of human mucosal yeast infections, and invasive candidiasis can be fatal. Antifungal medications are limited, but those targeting the pathogen cell wall or plasma membrane have been effective. Therefore, virulence factors controlling membrane biogenesis are potential targets for drug development. P4-ATPases contribute to membrane biogenesis by selecting and transporting specific lipids from the extracellular leaflet to the cytoplasmic leaflet of the bilayer to generate lipid asymmetry. A subset of heterodimeric P4-ATPases, including Dnf1-Lem3 and Dnf2-Lem3 from Saccharomyces cerevisiae, transport phosphatidylcholine (PC), phosphatidylethanolamine (PE), and the sphingolipid glucosylceramide (GlcCer). GlcCer is a critical lipid for Candida albicans polarized growth and virulence, but the role of GlcCer transporters in virulence has not been explored. Here, we show that the Candida albicans Dnf2 (CaDnf2) requires association with CaLem3 to form a functional transporter and flip fluorescent derivatives of GlcCer, PC, and PE across the plasma membrane. Mutation of conserved substrate-selective residues in the membrane domain strongly abrogates GlcCer transport and partially disrupts PC transport by CaDnf2. Candida strains harboring dnf2-null alleles (dnf2ΔΔ) or point mutations that disrupt substrate recognition exhibit defects in yeast-to-hypha growth transition, filamentous growth, and virulence in systemically infected mice. The influence of CaDNF1 deletion on the morphological phenotypes is negligible, although the dnf1ΔΔ dnf2ΔΔ strain was less virulent than the dnf2ΔΔ strain. These results indicate that the transport of GlcCer and/or PC by plasma membrane P4-ATPases is important for the pathogenicity of Candida albicans.

Two types of type IV P-type ATPases independently re-establish the asymmetrical distribution of phosphatidylserine in plasma membranes.

Phospholipids are asymmetrically distributed between the lipid bilayer of plasma membranes in which phosphatidylserine (PtdSer) is confined to the inner leaflet. ATP11A and ATP11C, type IV P-Type ATPases in plasma membranes, flip PtdSer from the outer to the inner leaflet, but involvement of other P4-ATPases is unclear. We herein demonstrated that once PtdSer was exposed on the cell surface of ATP11A-/-ATP11C-/- mouse T cell line (W3), its internalization to the inner leaflet of plasma membranes was negligible at 15 °C. However, ATP11A-/-ATP11C-/- cells internalized the exposed PtdSer at 37 °C, a temperature at which trafficking of intracellular membranes was active. In addition to ATP11A and 11C, W3 cells expressed ATP8A1, 8B2, 8B4, 9A, 9B, and 11B, with ATP8A1 and ATP11B being present at recycling endosomes. Cells deficient in four P4-ATPases (ATP8A1, 11A, 11B, and 11C) (QKO) did not constitutively expose PtdSer on the cell surface but lost the ability to re-establish PtdSer asymmetry within 1 hour, even at 37 °C. The expression of ATP11A or ATP11C conferred QKO cells with the ability to rapidly re-establish PtdSer asymmetry at 15 °C and 37 °C, while cells expressing ATP8A1 or ATP11B required a temperature of 37 °C to achieve this function, and a dynamin inhibitor blocked this process. These results revealed that mammalian cells are equipped with two independent mechanisms to re-establish its asymmetry: the first is a rapid process involving plasma membrane flippases, ATP11A and ATP11C, while the other is mediated by ATP8A1 and ATP11B, which require an endocytosis process.

Regulatory Roles of N- and C-Terminal Cytoplasmic Regions of P4-ATPases.

P4-ATPases, which are subfamily members of P-type ATPase superfamily, translocate membrane lipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet, thus regulating trans-bilayer lipid asymmetry. Mammalian P4-ATPases localize to the specific subcellular organelles or the plasma membrane where they translocate the specific lipids. Although recent advances in the structural analysis of P4-ATPases have improved our understanding of lipid transporting machinery, the mechanism of substrate specificity and the regulatory mechanism of the enzymes remain largely unknown. Recent studies have uncovered several specific localization and regulatory mechanisms of P4-ATPases. Here, we review the current understanding of the regulatory mechanism of P4-ATPase activity and localization in mammalian cells.

ATP8B2-Mediated Asymmetric Distribution of Plasmalogens Regulates Plasmalogen Homeostasis and Plays a Role in Intracellular Signaling.

Plasmalogens are a subclass of glycerophospholipid containing vinyl-ether bond at the sn-1 position of glycerol backbone. Ethanolamine-containing plasmalogens (plasmalogens) are major constituents of cellular membranes in mammalian cells and de novo synthesis of plasmalogens largely contributes to the homeostasis of plasmalogens. Plasmalogen biosynthesis is regulated by a feedback mechanism that senses the plasmalogen level in the inner leaflet of the plasma membrane and regulates the stability of fatty acyl-CoA reductase 1 (Far1), a rate-limiting enzyme for plasmalogen biosynthesis. However, the molecular mechanism underlying the localization of plasmalogens in cytoplasmic leaflet of plasma membrane remains unknown. To address this issue, we attempted to identify a potential transporter of plasmalogens from the outer to the inner leaflet of plasma membrane by focusing on phospholipid flippases, type-IV P-type adenosine triphosphatases (P4-ATPase), localized in the plasma membranes. We herein show that knockdown of ATP8B2 belonging to the class-1 P4-ATPase enhances localization of plasmalogens but not phosphatidylethanolamine in the extracellular leaflet and impairs plasmalogen-dependent degradation of Far1. Furthermore, phosphorylation of protein kinase B (AKT) is downregulated by lowering the expression of ATP8B2, which leads to suppression of cell growth. Taken together, these results suggest that enrichment of plasmalogens in the cytoplasmic leaflet of plasma membranes is mediated by ATP8B2 and this asymmetric distribution of plasmalogens is required for sensing plasmalogens as well as phosphorylation of AKT.

CDC50 Orthologues in Plasmodium falciparum Have Distinct Roles in Merozoite Egress and Trophozoite Maturation.

In model organisms, type IV ATPases (P4-ATPases) require cell division control protein 50 (CDC50) chaperones for their phospholipid flipping activity. In the malaria parasite Plasmodium falciparum, guanylyl cyclase alpha (GCα) is an integral membrane protein that is essential for release (egress) of merozoites from their host erythrocytes. GCα is unusual in that it contains both a C-terminal cyclase domain and an N-terminal P4-ATPase domain of unknown function. We sought to investigate whether any of the three CDC50 orthologues (termed A, B, and C) encoded by P. falciparum are required for GCα function. Using gene tagging and conditional gene disruption, we demonstrate that CDC50B and CDC50C but not CDC50A are expressed in the clinically important asexual blood stages and that CDC50B is a binding partner of GCα whereas CDC50C is the binding partner of another putative P4-ATPase, phospholipid-transporting ATPase 2 (ATP2). Our findings indicate that CDC50B has no essential role for intraerythrocytic parasite maturation but modulates the rate of parasite egress by interacting with GCα for optimal cGMP synthesis. In contrast, CDC50C is essential for blood stage trophozoite maturation. Additionally, we find that the CDC50C-ATP2 complex may influence parasite endocytosis of host cell hemoglobin and consequently hemozoin formation. IMPORTANCE Malaria morbidity arises due to successive rounds of replication of Plasmodium parasites within red blood cells. Mature daughter merozoites are released from infected erythrocytes to invade new cells in a tightly regulated process termed egress. Previous studies have shown that a unique bifunctional guanylyl cyclase, GCα, initiates egress by synthesis of cGMP. GCα has an N-terminal P4-ATPase domain of unknown function. In model organisms, P4-ATPases function through interaction with a CDC50 partner protein. Here, we investigate the role of CDC50 orthologues in P. falciparum and show that GCα binds CDC50B, an interaction that regulates egress efficiency. We also find that CDC50C is essential and binds a putative P4-ATPase, ATP2, in a complex that influences endocytosis of host hemaglobin. Our results highlight the heterogenous and critical role of CDC50 proteins in P. falciparum.

Structural insights into the activation of autoinhibited human lipid flippase ATP8B1 upon substrate binding.

SignificanceATP8B1 is a P4 ATPase that maintains membrane asymmetry by transporting phospholipids across the cell membrane. Disturbance of lipid asymmetry will lead to the imbalance of the cell membrane and eventually, cell death. Thus, defects in ATP8B1 are usually associated with severe human diseases, such as intrahepatic cholestasis. The present structures of ATP8B1 complexed with its auxiliary noncatalytic partners CDC50A and CDC50B reveal an autoinhibited state of ATP8B1 that could be released upon substrate binding. Moreover, release of this autoinhibition could be facilitated by the bile acids, which are key factors that alter the membrane asymmetry of hepatocytes. This enabled us to figure out a feedback loop of bile acids and lipids across the cell membrane.

CDC50A is required for aminophospholipid transport and cell fusion in mouse C2C12 myoblasts.

Myoblast fusion is essential for the formation of multinucleated muscle fibers and is promoted by transient changes in the plasma membrane lipid distribution. However, little is known about the lipid transporters regulating these dynamic changes. Here, we show that proliferating myoblasts exhibit an aminophospholipid flippase activity that is downregulated during differentiation. Deletion of the P4-ATPase flippase subunit CDC50A (also known as TMEM30A) results in loss of the aminophospholipid flippase activity and compromises actin remodeling, RAC1 GTPase membrane targeting and cell fusion. In contrast, deletion of the P4-ATPase ATP11A affects aminophospholipid uptake without having a strong impact on cell fusion. Our results demonstrate that myoblast fusion depends on CDC50A and may involve multiple CDC50A-dependent P4-ATPases that help to regulate actin remodeling.

P-Type ATPase Apt1 of the Fungal Pathogen Cryptococcus neoformans Is a Lipid Flippase of Broad Substrate Specificity.

Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a ß-subunit. Using heterologous expression in S. cerevisiae, we demonstrate that Apt1p forms a heterodimeric complex with the C. neoformans Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in C. neoformans is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development.

Lipid Transporters Beam Signals from Cell Membranes.

Lipid composition in cellular membranes plays an important role in maintaining the structural integrity of cells and in regulating cellular signaling that controls functions of both membrane-anchored and cytoplasmic proteins. ATP-dependent ABC and P4-ATPase lipid transporters, two integral membrane proteins, are known to contribute to lipid translocation across the lipid bilayers on the cellular membranes. In this review, we will highlight current knowledge about the role of cholesterol and phospholipids of cellular membranes in regulating cell signaling and how lipid transporters participate this process.

Neonatal LPS exposure reduces ATP8A2 level in the prefrontal cortex in mice via increasing IFN-γ level.

Neonatal lipopolysaccharide (LPS) exposure can cause depressive-like behaviors in rodents involving elevated interferon (IFN)-γ. Studies have linked down-regulation of prefrontal cortex (PFC) ATPase phospholipid transporting 8A2(ATP8A2) expression to depressive-like behaviors. In non-neuronal cells, IFN-γ could reduce ATP8A2 expression. Therefore, we hypothesized that neonatal LPS exposure might induce PFC ATP8A2 down-regulation by increasing the IFN-γ level. Here, C57BL6/J mice of both sexes received 3-dose-injections of LPS (50 µg/kg body weight, i.p.) on postnatal day (PND)5, PND7, and PND9. LPS-treated mice showed a transiently decreased PFC ATP8A2 expression indicated by western blot results. Moreover, a significant negative correlation of PFC ATP8A2 expression was found with the IFN-γ level. Using neutralizing mAb, IFN-γ was identified as the key mediator of LPS-induced PFC ATP8A2 decrease indicated by western blot and immunofluorescence results. In sum, neonatal LPS exposure reduced ATP8A2 level in PFC in mice via increasing IFN-γ level. This finding may help further understand the mechanism underlying LPS-induced impairments in brain development and function.

Novel variants in critical domains of ATP8A2 and expansion of clinical spectrum.

ATP8A2 is a P4-ATPase that flips phosphatidylserine across membranes to generate and maintain transmembrane phospholipid asymmetry. Loss-of-function variants cause severe neurodegenerative and developmental disorders. We have identified three ATP8A2 variants in unrelated Iranian families that cause intellectual disability, dystonia, below-average head circumference, mild optic atrophy, and developmental delay. Additionally, all the affected individuals displayed tooth abnormalities associated with defects in teeth development. Two variants (p.Asp825His and p.Met438Val) reside in critical functional domains of ATP8A2. These variants express at very low levels and lack ATPase activity. Inhibitor studies indicate that these variants are misfolded and degraded by the cellular proteasome. We conclude that Asp825, which coordinates with the Mg2+ ion within the ATP binding site, and Met438 are essential for the proper folding of ATP8A2 into a functional flippase. We also provide evidence on the association of tooth abnormalities with defects in ATP8A2, thereby expanding the clinical spectrum of the associated disease.

Aminoglycerophospholipid flipping and P4-ATPases in Toxoplasma gondii.

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.