The p-type ATPase superfamily

P-type ATPases function to provide homeostasis in higher eukaryotes, but they are essentially ubiquitous, being found in all domains of life. Thever and Saier [J Memb Biol 2009;229:115-130] recently reported analyses of eukaryotic P-type ATPases, dividing them into nine functionally characterized and 13 functionally uncharacterized (FUPA) families. In this report, we analyze P-type ATPases in all major prokaryotic phyla for which complete genome sequence data are available, and we compare the results with those for eukaryotic P-type ATPases. Topological type I (heavy metal) P-type ATPases predominate in prokaryotes (approx. tenfold) while type II ATPases (specific for Na(+),K(+), H(+) Ca(2+), Mg(2+) and phospholipids) predominate in eukaryotes (approx. twofold). Many P-type ATPase families are found exclusively in prokaryotes (e.g. Kdp-type K(+) uptake ATPases (type III) and all ten prokaryotic FUPA familes), while others are restricted to eukaryotes (e.g. phospholipid flippases and all 13 eukaryotic FUPA families). Horizontal gene transfer has occurred frequently among bacteria and archaea, which have similar distributions of these enzymes, but rarely between most eukaryotic kingdoms, and even more rarely between eukaryotes and prokaryotes. In some bacterial phyla (e.g. Bacteroidetes, Flavobacteria and Fusobacteria), ATPase gene gain and loss as well as horizontal transfer occurred seldom in contrast to most other bacterial phyla. Some families (i.e. Kdp-type ATPases) underwent far less horizontal gene transfer than other prokaryotic families, possibly due to their multisubunit characteristics. Functional motifs are better conserved across family lines than across organismal lines, and these motifs can be family specific, facilitating functional predictions. In some cases, gene fusion events created P-type ATPases covalently linked to regulatory catalytic enzymes. In one family (FUPA Family 24), a type I ATPase gene (N-terminal) is fused to a type II ATPase gene (C-terminal) with retention of function only for the latter. Several pseudogene-encoded nonfunctional ATPases were identified. Genome minimalization led to preferential loss of P-type ATPase genes. We suggest that in prokaryotes and some unicellular eukaryotes, the primary function of P-type ATPases is protection from extreme environmental stress conditions. The classification of P-type ATPases of unknown function into phylogenetic families provides guides for future molecular biological studies.     

Phylogenetic analysis of P5 P-type ATPases, a eukaryotic lineage of secretory pathway pumps

Eukaryotes encompass a remarkable variety of organisms and unresolved lineages. Different phylogenetic analyses have lead to conflicting conclusions as to the origin and associations between lineages and species. In this work, we investigated evolutionary relationship of a family of cation pumps exclusive for the secretory pathway of eukaryotes by combining the identification of lineage-specific genes with phylogenetic evolution of common genes. Sequences of P5 ATPases, which are regarded to be cation pumps in the endoplasmic reticulum (ER), were identified in all eukaryotic lineages but not in any prokaryotic genome. Based on a protein alignment we could group the P5 ATPases into two subfamilies, P5A and P5B that, based on the number of negative charges in conserved trans-membrane segment 4, are likely to have different ion specificities. P5A ATPases are present in all eukaryotic genomes sequenced so far, while P5B ATPases appear to be lost in three eukaryotic lineages; excavates, entamoebas and land plants. A lineage-specific gene expansion of up to four different P5B ATPases is seen in animals.     

Inventory of the superfamily of P-type ion pumps in Arabidopsis

A total of 45 genes encoding for P-type ATPases have been identified in the complete genome sequence of Arabidopsis. Thus, this plant harbors a primary transport capability not seen in any other eukaryotic organism sequenced so far. The sequences group in all five subfamilies of P-type ATPases. The most prominent subfamilies are P(1B) ATPases (heavy metal pumps; seven members), P(2A) and P(2B) ATPases (Ca(2+) pumps; 14 in total), P(3A) ATPases (plasma membrane H(+) pumps; 12 members including a truncated pump, which might represent a pseudogene or an ATPase-like protein with an alternative function), and P(4) ATPases (12 members). P(4) ATPases have been implicated in aminophosholipid flipping but it is not known whether this is a direct or an indirect effect of pump activity. Despite this apparent plethora of pumps, Arabidopsis appears to be lacking Na(+) pumps and secretory pathway (PMR1-like) Ca(2+)-ATPases. A cluster of Arabidopsis heavy metal pumps resembles bacterial Zn(2+)/Co(2+)/Cd(2+)/Pb(2+) transporters. Two members of the cluster have extended C termini containing putative heavy metal binding motifs. The complete inventory of P-type ATPases in Arabidopsis is an important starting point for reverse genetic and physiological approaches aiming at elucidating the biological significance of these pumps.     

Phospholipid‐flipping activity of P4‐ATPase drives membrane curvature

P4‐ATPases are phospholipid flippases that translocate phospholipids from the exoplasmic/luminal to the cytoplasmic leaflet of biological membranes. All P4‐ATPases in yeast and some in other organisms are required for membrane trafficking; therefore, changes in the transbilayer lipid composition induced by flippases are thought to be crucial for membrane deformation. However, it is poorly understood whether the phospholipid‐flipping activity of P4‐ATPases can promote membrane deformation. In this study, we assessed membrane deformation induced by flippase activity via monitoring the extent of membrane tubulation using a system that allows inducible recruitment of Bin/amphiphysin/Rvs (BAR) domains to the plasma membrane (PM). Enhanced phosphatidylcholine‐flippase activity at the PM due to expression of ATP10A, a member of the P4‐ATPase family, promoted membrane tubulation upon recruitment of BAR domains to the PM. This is the important evidence that changes in the transbilayer lipid composition induced by P4‐ATPases can deform biological membranes.     

P4 ATPases - Lipid flippases and their role in disease

P4 ATPases (type 4 P-type ATPases) are multispan transmembrane proteins that have been implicated in phospholipid translocation from the exoplasmic to the cytoplasmic leaflet of biological membranes. Studies in Saccharomyces cerevisiae have indicated that P4 ATPases are important in vesicle biogenesis and are required for vesicular trafficking along several intracellular vesicular transport routes. Although little is known about mammalian P4 ATPases, some members of this subfamily appear to be associated with human disease or mouse pathophysiology. ATP8B1, a phosphatidylserine translocase, is the most extensively studied mammalian P4 ATPase. This protein is important for maintaining the detergent resistant properties of the apical membrane of the hepatocyte. Mutations in ATP8B1 give rise to severe liver disease. Furthermore, a role for Atp8b3 in mouse sperm cell capacitation has been suggested, whereas deficiency of Atp10a and Atp10d leads to insulin resistance and obesity in mice. Here we review the present status on the pathophysiological consequences of P4 ATPase deficiency.     

DNA-dependent adenosinetriphosphatase A is the eukaryotic analogue of the bacteriophage T4 gene 44 protein: immunological identity of DNA replication-associated ATPases.

We report the construction of three stable murine hybridomas that secrete monoclonal antibodies which recognize calf thymus DNA-dependent adenosinetriphosphatase A. All three of the antibodies react specifically with calf thymus ATPase A and the gene 44 protein from the bacteriophage T4 DNA-dependent ATPase. Each of the three anti-ATPase A antibodies appears to recognize a different epitope and none of the antibodies inhibit DNA-dependent ATP hydrolysis by ATPase A. Furthermore, one of the antibodies has been shown to react with two different preparations of HeLa cell DNA-dependent ATPases and a yeast DNA-dependent ATPase, all of which have been implicated in the enzymology of DNA replication. These findings provide strong evidence for the role of ATPase A in DNA replication. These observations lead us to conclude that, apart from the nucleotide binding sites, there are at least three epitopes common to both the bacteriophage and eukaryotic DNA-dependent ATPases that we have examined and that the different preparations of the eukaryotic ATPases contain the same DNA-dependent ATPase.     

Ca2+ Induces Spontaneous Dephosphorylation of a Novel P5A-type ATPase

P5 ATPases constitute the least studied group of P-type ATPases, an essential family of ion pumps in all kingdoms of life. Although P5 ATPases are present in every eukaryotic genome analyzed so far, they have remained orphan pumps, and their biochemical function is obscure. We show that a P5A ATPase from barley, HvP5A1, locates to the endoplasmic reticulum and is able to rescue knock-out mutants of P5A genes in both Arabidopsis thaliana and Saccharomyces cerevisiae. HvP5A1 spontaneously forms a phosphorylated reaction cycle intermediate at the catalytic residue Asp-488, whereas, among all plant nutrients tested, only Ca(2+) triggers dephosphorylation. Remarkably, Ca(2+)-induced dephosphorylation occurs at high apparent [Ca(2+)] (K(i) = 0.25 mm) and is independent of the phosphatase motif of the pump and the putative binding site for transported ligands located in M4. Taken together, our results rule out that Ca(2+) is a transported substrate but indicate the presence of a cytosolic low affinity Ca(2+)-binding site, which is conserved among P-type pumps and could be involved in pump regulation. Our work constitutes the first characterization of a P5 ATPase phosphoenzyme and points to Ca(2+) as a modifier of its function.     

Evolution of proton pumping ATPases: Rooting the tree of life

Proton pumping ATPases are found in all groups of present day organisms. The F-ATPases of eubacteria, mitochondria and chloroplasts also function as ATP synthases, i.e., they catalyze the final step that transforms the energy available from reduction/oxidation reactions (e.g., in photosynthesis) into ATP, the usual energy currency of modern cells. The primary structure of these ATPases/ATP synthases was found to be much more conserved between different groups of bacteria than other parts of the photosynthetic machinery, e.g., reaction center proteins and redox carrier complexes.These F-ATPases and the vacuolar type ATPase, which is found on many of the endomembranes of eukaryotic cells, were shown to be homologous to each other; i.e., these two groups of ATPases evolved from the same enzyme present in the common ancestor. (The term eubacteria is used here to denote the phylogenetic group containing all bacteria except the archaebacteria.) Sequences obtained for the plasmamembrane ATPase of various archaebacteria revealed that this ATPase is much more similar to the eukaryotic than to the eubacterial counterpart. The eukaryotic cell of higher organisms evolved from a symbiosis between eubacteria (that evolved into mitochondria and chloroplasts) and a host organism. Using the vacuolar type ATPase as a molecular marker for the cytoplasmic component of the eukaryotic cell reveals that this host organism was a close relative of the archaebacteria.A unique feature of the evolution of the ATPases is the presence of a non-catalytic subunit that is paralogous to the catalytic subunit, i.e., the two types of subunits evolved from a common ancestral gene. Since the gene duplication that gave rise to these two types of subunits had already occurred in the last common ancestor of all living organisms, this non-catalytic subunit can be used to root the tree of life by means of an outgroup; that is, the location of the last common ancestor of the major domains of living organisms (archaebacteria, eubacteria and eukaryotes) can be located in the tree of life without assuming constant or equal rates of change in the different branches.A correlation between structure and function of ATPases has been established for present day organisms. Implications resulting from this correlation for biochemical pathways, especially photosynthesis, that were operative in the last common ancestor and preceding life forms are discussed.     

Linking phospholipid flippases to vesicle-mediated protein transport

Type IV P-type ATPases (P4-ATPases) are a large family of putative phospholipid translocases (flippases) implicated in the generation of phospholipid asymmetry in biological membranes. P4-ATPases are typically the largest P-type ATPase subgroup found in eukaryotic cells, with five members in Saccharomyces cerevisiae, six members in Caenorhabditis elegans, 12 members in Arabidopsis thaliana and 14 members in humans. In addition, many of the P4-ATPases require interaction with a noncatalytic subunit from the CDC50 gene family for their transport out of the endoplasmic reticulum (ER). Deficiency of a P4-ATPase (Atp8b1) causes liver disease in humans, and studies in a variety of model systems indicate that P4-ATPases play diverse and essential roles in membrane biogenesis. In addition to their proposed role in establishing and maintaining plasma membrane asymmetry, P4-ATPases are linked to vesicle-mediated protein transport in the exocytic and endocytic pathways. Recent studies have also suggested a role for P4-ATPases in the nonvesicular intracellular trafficking of sterols. Here, we discuss the physiological requirements for yeast P4-ATPases in phospholipid translocase activity, transport vesicle budding and ergosterol metabolism, with an emphasis on Drs2p and its noncatalytic subunit, Cdc50p.     

Characterization of a P-type Ca(2+)-ATPase from Flavobacterium odoratum.

In most bacterial cell types studied, low intracellular free calcium is maintained by a variety of secondary exchangers which utilize transmembrane ion gradients. Prokaryotic calcium ATPases appear to be extremely uncommon, and none have been reported in Gram-negative organisms. We demonstrate ATP-dependent calcium uptake in everted membrane vesicles of Flavobacterium odoratum, a common Gram-negative soil and water bacterium. Calcium is transported with an apparent initial rate of 10 nmol/min mg of protein. It is inhibited by 20 microM orthovanadate, a specific P-type ATPase inhibitor, but significantly, it is unaffected by the addition of N-ethylmaleimide, N,N-dicyclohexylcarbodiimide, valinomycin, or nigericin. Because the Ca(2+)-ATPase makes up a high proportion of the total ATPase activity it is easily detected by a soluble ATP hydrolysis assay, with an initial rate for calcium-dependent ATPase activity in vesicles of 25-40 nmol/min.mg at pH 7.8 and 25 degrees C. The calcium-dependent activity is preferentially solubilized by the detergent C12E8 and can be precipitated at 55-80% ammonium sulfate in a fraction free of other contaminating ATPase activities. This partially purified fraction is enriched 15-fold and demonstrates an apparent Km for calcium of 2 microM, and for ATP of 130 microM. The IC50 for vanadate is 1.6 microM. These values are similar to those obtained for the eukaryotic sarcoplasmic reticulum calcium ATPase. The enzyme is rapidly phosphorylated by [gamma-32P]ATP in a calcium-dependent, vanadate-inhibitable manner. The phosphorylated species migrates with an apparent molecular mass of 60 kDa by NaDodSO4-polyacrylamide gel electrophoresis, and the phosphoryl group is sensitive to alkaline conditions, a characteristic of the acylphosphate linkage found in ATPases. These data demonstrate that the majority of calcium transport in F. odoratum is facilitated by a P-type ATPase.     

Functions of phospholipid flippases

Asymmetrical distribution of phospholipids is generally observed in the eukaryotic plasma membrane. Maintenance and changes of this phospholipid asymmetry are regulated by ATP-driven phospholipid translocases. Accumulating evidence indicates that type 4 P-type ATPases (P4-ATPases, also called flippases) translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the plasma membrane and internal membranes. Among P-type ATPases, P4-ATPases are unique in that they are associated with a conserved membrane protein of the Cdc50 family as a non-catalytic subunit. Recent studies indicate that flippases are involved in various cellular functions, including transport vesicle formation and cell polarity. In this review, we will focus on the functional aspect of phospholipid flippases.     

Structure of the actuator domain from the Archaeoglobus fulgidus Cu(+)-ATPase

Copper homeostasis is maintained in part by membrane-bound P(1B)-type ATPases that are found in all organisms and drive the transport of this essential, yet toxic, metal ion across cellular membranes. CopA from Archaeoglobus fulgidus is a hyperthermophilic member of this ATPase subfamily and is homologous to the human Wilson and Menkes disease ATPases. To gain insight into Cu(+)-ATPase function, the structure of the CopA actuator domain (A-domain) was determined to 1.65 A resolution. The CopA A-domain functions to couple ATP hydrolysis in the ATP binding domain (ATPBD) with structural rearrangements of critical transmembrane segments. Its fold is quite similar to that of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) A-domain, with the exception of an external loop region. On the basis of sequence and structural comparisons, specific residues that probably interact with the CopA ATPBD have been identified. Comparisons to the Wilson and Menkes disease A-domains reveal the presence of an additional loop that may be associated with regulatory functions in eukaryotic Cu(+)-ATPases. Finally, several mutations in the Wilson and Menkes disease ATPases occur in the A-domain, and their likely effects on function can be inferred from the CopA A-domain structure.     

A putative plant aminophospholipid flippase, the Arabidopsis P4 ATPase ALA1, localizes to the plasma membrane following association with a β-subunit

Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner leaflet and sphingolipids in the outer leaflet. This unequal distribution of lipids between leaflets is, amongst several proposed functions, hypothesized to be a prerequisite for endocytosis. P4 ATPases, belonging to the P-type ATPase superfamily of pumps, are involved in establishing lipid asymmetry across plasma membranes, but P4 ATPases have not been identified in plant plasma membranes. Here we report that the plant P4 ATPase ALA1, which previously has been connected with cold tolerance of Arabidopsis thaliana, is targeted to the plasma membrane and does so following association in the endoplasmic reticulum with an ALIS protein β-subunit.     

Genomic comparison of P-type ATPase ion pumps in Arabidopsis and rice

Members of the P-type ATPase ion pump superfamily are found in all three branches of life. Forty-six P-type ATPase genes were identified in Arabidopsis, the largest number yet identified in any organism. The recent completion of two draft sequences of the rice (Oryza sativa) genome allows for comparison of the full complement of P-type ATPases in two different plant species. Here, we identify a similar number (43) in rice, despite the rice genome being more than three times the size of Arabidopsis. The similarly large families suggest that both dicots and monocots have evolved with a large preexisting repertoire of P-type ATPases. Both Arabidopsis and rice have representative members in all five major subfamilies of P-type ATPases: heavy-metal ATPases (P1B), Ca2+-ATPases (endoplasmic reticulum-type Ca2+-ATPase and autoinhibited Ca2+-ATPase, P2A and P2B), H+-ATPases (autoinhibited H+-ATPase, P3A), putative aminophospholipid ATPases (ALA, P4), and a branch with unknown specificity (P5). The close pairing of similar isoforms in rice and Arabidopsis suggests potential orthologous relationships for all 43 rice P-type ATPases. A phylogenetic comparison of protein sequences and intron positions indicates that the common angiosperm ancestor had at least 23 P-type ATPases. Although little is known about unique and common features of related pumps, clear differences between some members of the calcium pumps indicate that evolutionarily conserved clusters may distinguish pumps with either different subcellular locations or biochemical functions.     

Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase

P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na⁺/K⁺-ATPase or Ca²⁺-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.     

The evolution of ATPase activity in SMC proteins

The SMC (structural maintenance of chromosomes) proteins are a highly conserved and ubiquitous family of ATPases, found in nearly all living organisms examined, where they play crucial roles in transmission of the hereditary material. However, the extent to which efficient ATP hydrolysis is required for SMC function has been a matter of some debate. Here we investigate the potential functional significance of ATP binding and hydrolysis in different eukaryotic SMC proteins, both by comparing the conservation of conserved ATPase motifs and by exploring potential coevolution between associated domains. In this way, we have been able to account for the reduced requirement for ATPase activity in cohesin's SMC3 and demonstrate the greater apparent conservation requirements for such activity in condensin SMC proteins. Finally, we explore possible interactions between the SMC and non-SMC components of the condensin complex that are required for full condensin activity and may modulate ATPase activity in the holocomplex.     

Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism

Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N-terminal membrane-binding amphipathic helix attached to the main ATPase domain. In infected cells, 2C is involved in the formation of membrane vesicles, where it co-localizes with viral RNA replication complexes, but its precise role in virus replication has not been elucidated. We show here that deletion of the predicted N-terminal amphipathic helix enables overexpression in Escherichia coli of a highly soluble truncated protein, 2C(34–318), that has ATPase and RNA binding activity. ATPase activity was abrogated by point mutations in the Walker A (K116A) and B (D160A) motifs and Motif C (N207A) in the active site. Unliganded 2C(34–318) exhibits concentration-dependent self-association to yield oligomeric forms, the largest of which is tetrameric. Strikingly, in the presence of ATP and RNA, FMDV 2C(34–318) containing the N207A mutation, which binds but does not hydrolyze ATP, was found to oligomerize specifically into hexamers. Visualization of FMDV 2C-ATP-RNA complexes by negative stain electron microscopy revealed hexameric ring structures with 6-fold symmetry that are characteristic of AAA+ ATPases. ATPase assays performed by mixing purified active and inactive 2C(34–318) subunits revealed a coordinated mechanism of ATP hydrolysis. Our results provide new insights into the structure and mechanism of picornavirus 2C proteins that will facilitate new investigations of their roles in infection.     

The type 4 subfamily of P-type ATPases, putative aminophospholipid translocases with a role in human disease

The maintenance of phospholipid asymmetry in membrane bilayers is a paradigm in cell biology. However, the mechanisms and proteins involved in phospholipid translocation are still poorly understood. Members of the type 4 subfamily of P-type ATPases have been implicated in the translocation of phospholipids from the outer to the inner leaflet of membrane bilayers. In humans, several inherited disorders have been identified which are associated with loci harboring type 4 P-type ATPase genes. Up to now, one inherited disorder, Byler disease or progressive familial intrahepatic cholestasis type 1 (PFIC1), has been directly linked to mutations in a type 4 P-type ATPase gene. How the absence of an aminophospholipid translocase activity relates to this severe disease is, however, still unclear. Studies in the yeast Saccharomyces cerevisiae have recently identified important roles for type 4 P-type ATPases in intracellular membrane- and protein-trafficking events. These processes require an (amino)phospholipid translocase activity to initiate budding or fusion of membrane vesicles from or with other membranes. The studies in yeast have greatly contributed to our cell biological insight in membrane dynamics and intracellular-trafficking events; if this knowledge can be translated to mammalian cells and organs, it will help to elucidate the molecular mechanisms which underlie severe inherited human diseases such as Byler disease.     

Phospholipid Flippase Activities and Substrate Specificities of Human Type IV P-type ATPases Localized to the Plasma Membrane

Type IV P-type ATPases (P4-ATPases) are believed to translocate aminophospholipids from the exoplasmic to the cytoplasmic leaflets of cellular membranes. The yeast P4-ATPases, Drs2p and Dnf1p/Dnf2p, flip nitrobenzoxadiazole-labeled phosphatidylserine at the Golgi complex and nitrobenzoxadiazole-labeled phosphatidylcholine (PC) at the plasma membrane, respectively. However, the flippase activities and substrate specificities of mammalian P4-ATPases remain incompletely characterized. In this study, we established an assay for phospholipid flippase activities of plasma membrane-localized P4-ATPases using human cell lines stably expressing ATP8B1, ATP8B2, ATP11A, and ATP11C. We found that ATP11A and ATP11C have flippase activities toward phosphatidylserine and phosphatidylethanolamine but not PC or sphingomyelin. By contrast, ATPase-deficient mutants of ATP11A and ATP11C did not exhibit any flippase activity, indicating that these enzymes catalyze flipping in an ATPase-dependent manner. Furthermore, ATP8B1 and ATP8B2 exhibited preferential flippase activities toward PC. Some ATP8B1 mutants found in patients of progressive familial intrahepatic cholestasis type 1 (PFIC1), a severe liver disease caused by impaired bile flow, failed to translocate PC despite their delivery to the plasma membrane. Moreover, incorporation of PC mediated by ATP8B1 can be reversed by simultaneous expression of ABCB4, a PC floppase mutated in PFIC3 patients. Our findings elucidate the flippase activities and substrate specificities of plasma membrane-localized human P4-ATPases and suggest that phenotypes of some PFIC1 patients result from impairment of the PC flippase activity of ATP8B1.