ATP9B, a P4-ATPase (a putative aminophospholipid translocase), localizes to the trans-Golgi network in a CDC50 protein-independent manner

Type IV P-type ATPases (P4-ATPases) are putative phospholipid flippases that translocate phospholipids from the exoplasmic (lumenal) to the cytoplasmic leaflet of lipid bilayers and are believed to function in complex with CDC50 proteins. In Saccharomyces cerevisiae, five P4-ATPases are localized to specific cellular compartments and are required for vesicle-mediated protein transport from these compartments, suggesting a role for phospholipid translocation in vesicular transport. The human genome encodes 14 P4-ATPases and three CDC50 proteins. However, the subcellular localization of human P4-ATPases and their interactions with CDC50 proteins are poorly understood. Here, we show that class 5 (ATP10A, ATP10B, and ATP10D) and class 6 (ATP11A, ATP11B, and ATP11C) P4-ATPases require CDC50 proteins, primarily CDC50A, for their exit from the endoplasmic reticulum (ER) and final subcellular localization. In contrast, class 2 P4-ATPases (ATP9A and ATP9B) are able to exit the ER in the absence of exogenous CDC50 expression: ATP9B, but not ATP11B, was able to exit the ER despite depletion of CDC50 proteins by RNAi. Although ATP9A and ATP9B show a high overall sequence similarity, ATP9A localizes to endosomes and the trans-Golgi network (TGN), whereas ATP9B localizes exclusively to the TGN. A chimeric ATP9 protein in which the N-terminal cytoplasmic region of ATP9A was replaced with the corresponding region of ATP9B was localized exclusively to the Golgi. These results indicate that ATP9B is able to exit the ER and localize to the TGN independently of CDC50 proteins and that this protein contains a Golgi localization signal in its N-terminal cytoplasmic region.     

CDC50 proteins are critical components of the human class-1 P4-ATPase transport machinery

Members of the P(4) subfamily of P-type ATPases catalyze phospholipid transport and create membrane lipid asymmetry in late secretory and endocytic compartments. P-type ATPases usually pump small cations and the transport mechanism involved appears conserved throughout the family. How this mechanism is adapted to flip phospholipids remains to be established. P(4)-ATPases form heteromeric complexes with CDC50 proteins. Dissociation of the yeast P(4)-ATPase Drs2p from its binding partner Cdc50p disrupts catalytic activity (Lenoir, G., Williamson, P., Puts, C. F., and Holthuis, J. C. (2009) J. Biol. Chem. 284, 17956-17967), suggesting that CDC50 subunits play an intimate role in the mechanism of transport by P(4)-ATPases. The human genome encodes 14 P(4)-ATPases while only three human CDC50 homologues have been identified. This implies that each human CDC50 protein interacts with multiple P(4)-ATPases or, alternatively, that some human P(4)-ATPases function without a CDC50 binding partner. Here we show that human CDC50 proteins each bind multiple class-1 P(4)-ATPases, and that in all cases examined, association with a CDC50 subunit is required for P(4)-ATPase export from the ER. Moreover, we find that phosphorylation of the catalytically important Asp residue in human P(4)-ATPases ATP8B1 and ATP8B2 is critically dependent on their CDC50 subunit. These results indicate that CDC50 proteins are integral part of the P(4)-ATPase flippase machinery.     

Critical role of the beta-subunit CDC50A in the stable expression, assembly, subcellular localization, and lipid transport activity of the P4-ATPase ATP8A2

P(4)-ATPases have been implicated in the transport of lipids across cellular membranes. Some P(4)-ATPases are known to associate with members of the CDC50 protein family. Previously, we have shown that the P(4)-ATPase ATP8A2 purified from photoreceptor membranes and reconstituted into liposomes catalyzes the active transport of phosphatidylserine across membranes. However, it was unclear whether ATP8A2 functioned alone or as a complex with a CDC50 protein. Here, we show by mass spectrometry and Western blotting using newly generated anti-CDC50A antibodies that CDC50A is associated with ATP8A2 purified from photoreceptor membranes. ATP8A2 expressed in HEK293T cells assembles with endogenous or expressed CDC50A, but not CDC50B, to generate a heteromeric complex that actively transports phosphatidylserine and to a lesser extent phosphatidylethanolamine across membranes. Chimera CDC50 proteins in which various domains of CDC50B were replaced with the corresponding domains of CDC50A were used to identify domains important in the formation of a functional ATP8A2-CDC50 complex. These studies indicate that both the transmembrane and exocytoplasmic domains of CDC50A are required to generate a functionally active complex. The N-terminal cytoplasmic domain of CDC50A appears to play a direct role in the reaction cycle. Mutagenesis studies further indicate that the N-linked oligosaccharide chains of CDC50A are required for stable expression of an active ATP8A2-CDC50A lipid transport complex. Together, our studies indicate that CDC50A is the β-subunit of ATP8A2 and is crucial for the correct folding, stable expression, export from endoplasmic reticulum, and phosphatidylserine flippase activity of ATP8A2.     

Role for Phospholipid Flippase Complex of ATP8A1 and CDC50A Proteins in Cell Migration

Type IV P-type ATPases (P4-ATPases) and CDC50 family proteins form a putative phospholipid flippase complex that mediates the translocation of aminophospholipids such as phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the outer to inner leaflets of the plasma membrane. In Chinese hamster ovary (CHO) cells, at least eight members of P4-ATPases were identified, but only a single CDC50 family protein, CDC50A, was expressed. We demonstrated that CDC50A associated with and recruited P4-ATPase ATP8A1 to the plasma membrane. Overexpression of CDC50A induced extensive cell spreading and greatly enhanced cell migration. Depletion of either CDC50A or ATP8A1 caused a severe defect in the formation of membrane ruffles, thereby inhibiting cell migration. Analyses of phospholipid translocation at the plasma membrane revealed that the depletion of CDC50A inhibited the inward translocation of both PS and PE, whereas the depletion of ATP8A1 inhibited the translocation of PE but not that of PS, suggesting that the inward translocation of cell-surface PE is involved in cell migration. This hypothesis was further examined by using a PE-binding peptide and a mutant cell line with defective PE synthesis; either cell-surface immobilization of PE by the PE-binding peptide or reduction in the cell-surface content of PE inhibited the formation of membrane ruffles, causing a severe defect in cell migration. These results indicate that the phospholipid flippase complex of ATP8A1 and CDC50A plays a major role in cell migration and suggest that the flippase-mediated translocation of PE at the plasma membrane is involved in the formation of membrane ruffles to promote cell migration.     

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.     

Phospholipid Flippase ATP10A Translocates Phosphatidylcholine and Is Involved in Plasma Membrane Dynamics

We showed previously that ATP11A and ATP11C have flippase activity toward aminophospholipids (phosphatidylserine (PS) and phosphatidylethanolamine (PE)) and ATP8B1 and that ATP8B2 have flippase activity toward phosphatidylcholine (PC) (Takatsu, H., Tanaka, G., Segawa, K., Suzuki, J., Nagata, S., Nakayama, K., and Shin, H. W. (2014) J. Biol. Chem. 289, 33543–33556). Here, we show that the localization of class 5 P4-ATPases to the plasma membrane (ATP10A and ATP10D) and late endosomes (ATP10B) requires an interaction with CDC50A. Moreover, exogenous expression of ATP10A, but not its ATPase-deficient mutant ATP10A(E203Q), dramatically increased PC flipping but not flipping of PS or PE. Depletion of CDC50A caused ATP10A to be retained at the endoplasmic reticulum instead of being delivered to the plasma membrane and abrogated the increased PC flipping activity observed by expression of ATP10A. These results demonstrate that ATP10A is delivered to the plasma membrane via its interaction with CDC50A and, specifically, flips PC at the plasma membrane. Importantly, expression of ATP10A, but not ATP10A(E203Q), dramatically altered the cell shape and decreased cell size. In addition, expression of ATP10A, but not ATP10A(E203Q), delayed cell adhesion and cell spreading onto the extracellular matrix. These results suggest that enhanced PC flipping activity due to exogenous ATP10A expression alters the lipid composition at the plasma membrane, which may in turn cause a delay in cell spreading and a change in cell morphology.     

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 P4 ATPase Phospholipid Translocases (Flippases) in Human and Rat Pancreatic Beta Cells: THEIR GENE SILENCING INHIBITS INSULIN SECRETION*

The negative charge of phosphatidylserine in lipid bilayers of secretory vesicles and plasma membranes couples the domains of positively charged amino acids of secretory vesicle SNARE proteins with similar domains of plasma membrane SNARE proteins enhancing fusion of the two membranes to promote exocytosis of the vesicle contents of secretory cells. Our recent study of insulin secretory granules (ISG) (MacDonald, M. J., Ade, L., Ntambi, J. M., Ansari, I. H., and Stoker, S. W. (2015) Characterization of phospholipids in insulin secretory granules in pancreatic beta cells and their changes with glucose stimulation. J. Biol. Chem. 290, 11075–11092) suggested that phosphatidylserine and other phospholipids, such as phosphatidylethanolamine, in ISG could play important roles in docking and fusion of ISG to the plasma membrane in the pancreatic beta cell during insulin exocytosis. P4 ATPase flippases translocate primarily phosphatidylserine and, to a lesser extent, phosphatidylethanolamine across the lipid bilayers of intracellular vesicles and plasma membranes to the cytosolic leaflets of these membranes. CDC50A is a protein that forms a heterodimer with P4 ATPases to enhance their translocase catalytic activity. We found that the predominant P4 ATPases in pure pancreatic beta cells and human and rat pancreatic islets were ATP8B1, ATP8B2, and ATP9A. ATP8B1 and CDC50A were highly concentrated in ISG. ATP9A was concentrated in plasma membrane. Gene silencing of individual P4 ATPases and CDC50A inhibited glucose-stimulated insulin release in pure beta cells and in human pancreatic islets. This is the first characterization of P4 ATPases in beta cells. The results support roles for P4 ATPases in translocating phosphatidylserine to the cytosolic leaflets of ISG and the plasma membrane to facilitate the docking and fusion of ISG to the plasma membrane during insulin exocytosis.     

ATP11C mutation is responsible for the defect in phosphatidylserine uptake in UPS-1 cells

Type IV P-type ATPases (P4-ATPases) translocate phospholipids from the exoplasmic to the cytoplasmic leaflets of cellular membranes. We and others previously showed that ATP11C, a member of the P4-ATPases, translocates phosphatidylserine (PS) at the plasma membrane. Twenty years ago, the UPS-1 (uptake of fluorescent PS analogs) cell line was isolated from mutagenized Chinese hamster ovary (CHO)-K1 cells with a defect in nonendocytic uptake of nitrobenzoxadiazole PS. Due to its defect in PS uptake, the UPS-1 cell line has been used in an assay for PS-flipping activity; however, the gene(s) responsible for the defect have not been identified to date. Here, we found that the mRNA level of ATP11C was dramatically reduced in UPS-1 cells relative to parental CHO-K1 cells. By contrast, the level of ATP11A, another PS-flipping P4-ATPase at the plasma membrane, or CDC50A, which is essential for delivery of most P4-ATPases to the plasma membrane, was not affected in UPS-1 cells. Importantly, we identified a nonsense mutation in the ATP11C gene in UPS-1 cells, indicating that the intact ATP11C protein is not expressed. Moreover, exogenous expression of ATP11C can restore PS uptake in UPS-1 cells. These results indicate that lack of the functional ATP11C protein is responsible for the defect in PS uptake in UPS-1 cells and ATP11C is crucial for PS flipping in CHO-K1 cells.     

P4-ATPase ATP8A2 acts in synergy with CDC50A to enhance neurite outgrowth

P(4)-ATPases are lipid flippases that transport phospholipids across cellular membranes, playing vital roles in cell function. In humans, the disruption of the P(4)-ATPase ATP8A2 gene causes a severe neurological phenotype. Here, we found that Atp8a2 mRNA was highly expressed in PC12 cells, hippocampal neurons and the brain. Overexpression of ATP8A2 increased the length of neurite outgrowth in NGF-induced PC12 cells and in primary cultures of rat hippocampal neurons. Inducing the loss of function of CDC50A in hippocampal neurons via RNA interference reduced neurite outgrowth, and the co-overexpression of CDC50A and ATP8A2 in PC12 cells enhanced NGF-induced neurite outgrowth. These results indicate that ATP8A2, acting in synergy with CDC50A, performs an important role in neurite outgrowth in neurons.     

Phospholipid flippases: building asymmetric membranes and transport vesicles

Phospholipid flippases in the type IV P-type ATPase family (P4-ATPases) are essential components of the Golgi, plasma membrane and endosomal system that play critical roles in membrane biogenesis. These pumps flip phospholipid across the bilayer to create an asymmetric membrane structure with substrate phospholipids, such as phosphatidylserine and phosphatidylethanolamine, enriched within the cytosolic leaflet. The P4-ATPases also help form transport vesicles that bud from Golgi and endosomal membranes, thereby impacting the sorting and localization of many different proteins in the secretory and endocytic pathways. At the organismal level, P4-ATPase deficiencies are linked to liver disease, obesity, diabetes, hearing loss, neurological deficits, immune deficiency and reduced fertility. Here, we review the biochemical, cellular and physiological functions of P4-ATPases, with an emphasis on their roles in vesicle-mediated protein transport. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Plant and animal type 2B Ca2+-ATPases: evidence for a common auto-inhibitory mechanism

Plant auto-inhibited Ca²⁺-ATPase 8 (ACA8) and animal plasma membrane Ca²⁺-ATPase 4b (PMCA4b) are representatives of plant and animal 2B P-type ATPases with a regulatory auto-inhibitory domain localized at the N- and C-terminus, respectively. To check whether the regulatory domain works independently of its terminal localization and if auto-inhibitory domains of different organisms are interchangeable, a mutant in which the N-terminus of ACA8 is repositioned at the C-terminus and chimeras in which PMCA4b C-terminus is fused to the N- or C-terminus of ACA8 were analysed in the yeast mutant K616 devoid of endogenous Ca²⁺-ATPases. Results show that the regulatory function of the terminal domain is independent from its position in ACA8 and that the regulatory domain belonging to PMCA4b is able to at least partially auto-inhibit ACA8.     

Transport through recycling endosomes requires EHD1 recruitment by a phosphatidylserine translocase

P₄-ATPases translocate aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of membranes. PS is highly enriched in recycling endosomes (REs) and is essential for endosomal membrane traffic. Here, we show that PS flipping by an RE-localized P₄-ATPase is required for the recruitment of the membrane fission protein EHD1. Depletion of ATP8A1 impaired the asymmetric transbilayer distribution of PS in REs, dissociated EHD1 from REs, and generated aberrant endosomal tubules that appear resistant to fission. EHD1 did not show membrane localization in cells defective in PS synthesis. ATP8A2, a tissue-specific ATP8A1 paralogue, is associated with a neurodegenerative disease (CAMRQ). ATP8A2, but not the disease-causative ATP8A2 mutant, rescued the endosomal defects in ATP8A1-depleted cells. Primary neurons from Atp8a2^−/− mice showed a reduced level of transferrin receptors at the cell surface compared to Atp8a2^+/+ mice. These findings demonstrate the role of P₄-ATPase in membrane fission and give insight into the molecular basis of CAMRQ.     

The distinct functional properties of the nucleotide-binding domain of ATP7B, the human copper-transporting ATPase: analysis of the Wilson disease mutations E1064A, H1069Q, R1151H, and C1104F

Copper transport by the P(1)-ATPase ATP7B, or Wilson disease protein (WNDP),1 is essential for human metabolism. Perturbation of WNDP function causes intracellular copper accumulation and severe pathology, known as Wilson disease (WD). Several WD mutations are clustered within the WNDP nucleotide-binding domain (N-domain), where they are predicted to disrupt ATP binding. The mechanism by which the N-domain coordinates ATP is presently unknown, because residues important for nucleotide binding in the better characterized P(2)-ATPases are not conserved within the P(1)-ATPase subfamily. To gain insight into nucleotide binding under normal and disease conditions, we generated the recombinant WNDP N-domain and several WD mutants. Using isothermal titration calorimetry, we demonstrate that the N-domain binds ATP in a Mg(2+)-independent manner with a relatively high affinity of 75 microm, compared with millimolar affinities observed for the P(2)-ATPase N-domains. The WNDP N-domain shows minimal discrimination between ATP, ADP, and AMP, yet discriminates well between ATP and GTP. Similar results were obtained for the N-domain of ATP7A, another P(1)-ATPase. Mutations of the invariant WNDP residues E1064A and H1069Q drastically reduce nucleotide affinities, pointing to the likely role of these residues in nucleotide coordination. In contrast, the R1151H mutant exhibits only a 1.3-fold reduction in affinity for ATP. The C1104F mutation significantly alters protein folding, whereas C1104A does not affect the structure or function of the N-domain. Together, the results directly demonstrate the phenotypic diversity of WD mutations within the N-domain and indicate that the nucleotide-binding properties of the P(1)-ATPases are distinct from those of the P(2)-ATPases.     

小麦根和叶细胞质膜Ca^2＋—ATPase性质的比较

比较了小麦(Triticum aestivum L.cv.Longchun No.13)根和叶细胞质膜Ca~(2 )-ATPase的性质,并结合二者所处的环境和功能方面的差异进行了分析。结果表明:根细胞质膜Ca~(2 )-ATPase在一个较宽的pH范围内有高活性,最适反应温度为45℃;叶细胞质膜Ca~(2 )-ATPase只在一个较窄的pH范围内有高活性,最适反应温度为50℃。根细胞质膜Ca~(2 )-ATPase对ATP的Hill系数为1.6,具有明显的正协同作用;叶细胞质膜Ca~(2 )-ATPase对ATP的Hill系数为1.0,符合米氏动力学类型。两种器官的细胞质膜Ca~(2 )-ATPase受Ca~(2 )激活的Hill系数都小于1,都具有负协同作用。钙调素对酶活性有激活作用,而Mg~(2 )则有抑制作用。     

Phytophthora nicotianae PnPMA1 encodes an atypical plasma membrane H+ -ATPase that is functional in yeast and developmentally regulated

PnPMA1, a gene encoding a putative P-type plasma membrane H(+)-ATPase, has been isolated by differential screening of a Phytophthora nicotianae germinated cyst cDNA library. PnPMA1 is differentially expressed during pathogen asexual development with a more than 10-fold increase in expression in germinated cysts, the stage at which plant infection is initiated, compared to vegetative or sporulating hyphae or motile zoospores. PnPMA1 proteins are encoded by two closely linked genes that have no introns and encode identical proteins having 1,068 amino acid residues and a molecular mass of 116.3kDa. PnPMA1 shows moderate identity (30-50%) to plant and fungal plasma membrane H(+)-ATPases and weak identity to other P-type cation-transporting ATPases. PnPMA1 contains all the catalytic domains characteristic of H(+)-ATPases but also has a distinct domain of approximately 155 amino acids that forms a putative cytoplasmic loop between transmembrane domains 8 and 9, a feature that is not present in PMA1 proteins from other organisms. Polyclonal antibodies raised against the 155 residue domain were shown by immunogold labelling to react with a protein in the plasma membrane of P. nicotianae germinated cysts but not with the plasma membrane of motile zoospores. Genetic complementation experiments demonstrated that the P. nicotianae PnPMA1 is functional in yeast, Saccharomyces cerevisiae.     

The a-subunit of the V-type H+-ATPase interacts with phosphofructokinase-1 in humans

V-type or H+-ATPases are a family of ATP-dependent proton pumps that move protons across the plasma membrane at specialized sites such as kidney epithelial cells and osteoclasts as well as acidifying intracellular compartments. The 100-kDa polytopic a-subunit of this group of ATPases is suggested to play an important role in coupling the two functions of the pump, ATP hydrolysis and proton transport. In man, different a-subunit isoforms are encoded by four genes. ATP6V0A4 encodes a4, which is expressed apically in alpha-intercalated cells in both human and mouse kidney. We sought binding partners for the C terminus of a4 in order to address its potential role in the H+-ATPase complex. Random peptide phage display analysis revealed a consensus motif (WLELRP) with almost complete homology to part of the enzyme phosphofructokinase 1 (PFK-1). Activity of this enzyme is the rate-limiting step in glycolysis. Specificity of a4 binding to this peptide was confirmed by enzyme-linked immunosorbent assay. Protein-protein interaction was further demonstrated by co-immunoprecipitation of a4 with PFK-1 from solubilized human kidney membrane proteins. An in vitro bead-bound PFK-1 pull-down assay showed that this interaction was also true for the ubiquitously expressed a1 subunit. Finally, PFK-1 co-immunolocalized with a4 in alpha-intercalated cells in the collecting ducts of human kidney. These findings indicate a direct link between V-type H+-ATPases and glycolysis via the C-terminal region of the a-subunit of the pump and suggest a novel regulatory mechanism between H+-ATPase function and energy supply. This interaction between the a-subunit and PFK-1 also provides new evidence that the C terminus of this subunit lies cytoplasmically in vivo.     

Comparison of Properties of the Plasma Membrane Ca2+ -ATPase from Wheat Root and Leaf

The properties of plasma membrane Ca2 + -ATPases from wheat ( Triticum aestivum L. cv. Lengchun No. 13) root and leaf were compared, and their different properties were analyzed in association with the differentia of the functions of these two organs and their relevant environments. Root plasma membrane Ca2 + -ATPase showed a high activity in a broad range of pH and an optimum reaction temperature of 45 ℃, while the leaf enzyme activated in a narrow range of pH and an optimum reaction temperature of 50 ℃. Hill coefficient of root plasma membrane Ca2 + -ATPase for ATP was 1.6, revealing an obvious positive cooperativity. In contrast, that of leaf plasma membrane Ca2 +-ATPase was 1.0, being in keeping with Michaelis-Menten dynamics. For Ca2 + activation, Hill coefficient of plasma membrane Ca2 + -ATPases from both organs were less than 1, suggesting that both had negative cooperativity. The enzymes were activated by calmodulin and inhibited by Mg2+.     

Cdc50p Plays a Vital Role in the ATPase Reaction Cycle of the Putative Aminophospholipid Transporter Drs2p

Members of the P₄ subfamily of P-type ATPases are believed to catalyze transport of phospholipids across cellular bilayers. However, most P-type ATPases pump small cations or metal ions, and atomic structures revealed a transport mechanism that is conserved throughout the family. Hence, a challenging problem is to understand how this mechanism is adapted in P₄-ATPases to flip phospholipids. P₄-ATPases form heteromeric complexes with Cdc50 proteins. The primary role of these additional polypeptides is unknown. Here, we show that the affinity of yeast P₄-ATPase Drs2p for its Cdc50-binding partner fluctuates during the transport cycle, with the strongest interaction occurring at a point where the enzyme is loaded with phospholipid ligand. We also find that specific interactions with Cdc50p are required to render the ATPase competent for phosphorylation at the catalytically important aspartate residue. Our data indicate that Cdc50 proteins are integral components of the P₄-ATPase transport machinery. Thus, acquisition of these subunits may have been a crucial step in the evolution of flippases from a family of cation pumps.P-type ATPases form a large family of membrane pumps that are transiently autophosphorylated at a conserved aspartate residue, hence the designation P-type. Prominent examples include the Ca²⁺-ATPase SERCA,⁴ which pumps Ca²⁺ from the cytosol into the lumen of the sarcoplasmic reticulum of skeletal muscle cells (1), and the Na⁺/K⁺-ATPase, which generates the electrochemical gradients for sodium and potassium that are vital to animal cells (2). Transport is accomplished by cyclic changes between two main enzyme conformations, E₁ and E₂, during which the ATPase is phosphorylated by ATP at the aspartate residue and subsequently dephosphorylated. These processes are coupled to vectorial transport and counter-transport by a controlled opening and closing of cytoplasmic and exoplasmic pathways, which give access to the ion-binding sites that are buried inside the membrane-spanning region of the pump (3). A host of crystal structures of the Ca²⁺ pump SERCA in well defined states of the reaction cycle revealed important aspects of the transport mechanism (4, 5). Sequence homology and structures of other ATPases show that this mechanism rests on principles and structural elements that apply to all P-type ATPases (6–8).Although P-type ATPases usually pump small cations or metal ions, members of the P₄ subfamily form a notable exception. A growing body of evidence indicates that P₄-ATPases catalyze phospholipid transport and create membrane lipid asymmetry (9–11). This process contributes to a multitude of cellular functions, including membrane vesiculation, cell division, and life span. The yeast Saccharomyces cerevisiae contains five P₄-ATPases, namely Dnf1p and Dnf2p at the plasma membrane, Drs2p and Dnf3p in the trans-Golgi network, and Neo1p in an endosomal compartment (12–14). Removal of Dnf1p and Dnf2p abolishes inward translocation of 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-labeled analogs of phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) and causes an aberrant exposure of endogenous aminophospholipids at the cell surface (13, 15). Trans-Golgi membranes isolated from a yeast strain that lacks the Dnf proteins and contains a temperature-sensitive drs2 allele display a defect in 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-PS translocation when shifted to the non-permissive temperature (16). The latter finding provides strong evidence that Drs2p is directly coupled to flippase activity, and subsequent studies showed that Drs2p, together with Dnf3p, are required for maintaining PE asymmetry in post-Golgi secretory vesicles (17).Although no P₄-ATPase has been shown to display flippase activity in reconstitution experiments with purified enzyme, the relationship of P₄-ATPases to flippase activity and lipid asymmetry has gained further support from functional studies in various other organisms, including parasites (18), plants (19), worms (20), and mice (21). Besides a common domain organization, P₄-ATPases display a clear sequence homology with cation-transporting P-type pumps. Shared sequence motifs include the canonical phosphorylation site in the P domain, the nucleotide-binding site in the N domain, and a TGES-related sequence in the A domain (22). This implies that P₄-ATPases and cation pumps use the same mechanism to couple ATP hydrolysis to ligand transport. Phospholipid transport by P₄-ATPases would correspond to counter-transport of H⁺ ions by the Ca²⁺ pump and of K⁺ ions by the Na⁺/K⁺-ATPase as the direction of movement is from the exoplasmic to the cytoplasmic leaflet. During the reaction cycle of cation pumps, access to the ion-binding pocket alternates between the two sides of the membrane, with the ions becoming temporarily occluded after each ion binding event (23). How this mechanism is adapted in P₄-ATPases to translocate phospholipids is unclear. Flippases must provide a sizeable hydrophilic pathway for the polar headgroup to pass through the membrane as well as accommodate the hydrophobic nature of the lipid backbone. Whether P₄-ATPases alone are sufficient to accomplish this task is not known.Recent studies revealed that P₄-ATPases form complexes with members of the Cdc50 protein family (24). Cdc50 proteins consist of two membrane spans and a large, N-glycosylated ectodomain with one or more conserved disulfide bonds (25). The yeast family members Cdc50p, Lem3p, and Crf1p can be co-immunoprecipitated with Drs2p, Dnf1p/Dnf2p, and Dnf3p, respectively. Formation of these complexes is required for proper expression and endoplasmic reticulum (ER) export of either partner (24, 26) so that mutation of one member of the complex phenocopies mutations in the other (15, 25). This behavior in yeast is mirrored in other organisms; Ld Ros3, a Lem3p homolog in Leishmania parasites, is needed for proper trafficking of the P₄-ATPase Ld MT (18), whereas the human P₄-ATPase ATP8B1 requires a Cdc50p homolog, CDC50A, for ER exit and delivery to the plasma membrane (27). Moreover, the Arabidopsis P₄-ATPase ALA3 requires its Cdc50-binding partner ALIS1 to complement the lipid transport defect at the plasma membrane in a Δdnf1Δdnf2Δdrs2 yeast mutant (19).Together, the above findings indicate that Cdc50 subunits are indispensable for a proper functioning of P₄-ATPases and that it is the combination of the two that yields a physiologically active transporter. However, these studies have not clarified the primary function of the Cdc50 polypeptide in the complex. Here, we provide the first evidence that Cdc50 subunits play a crucial role in the P₄-ATPase reaction cycle. Using a genetic reporter system, we find that P₄-ATPase-Cdc50 interactions are dynamic and tightly coupled to the ATPase reaction cycle. Moreover, by characterizing the enzymatic properties of a purified P₄-ATPase-Cdc50 complex, we show that catalytic activity relies on direct and specific interactions between the subunit and transporter.