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.     

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.     

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.     

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.     

The yeast plasma membrane P4-ATPases are major transporters for lysophospholipids

The transbilayer movement of phospholipids plays an essential role in establishing and maintaining the asymmetric distribution of lipids in biological membranes. The P4-ATPase family has been implicated as the major transporters of the aminoglycerophospholipids in both surface and endomembrane systems. Historically, fluorescent lipid analogs have been used to monitor the lipid transport activity of the P4-ATPases. Recent evidence now demonstrates that lyso-phosphatidylethanolamine (lyso-PtdEtn) and lyso-phosphatidylcholine (lyso-PtdCho) are bona fide biological substrates transported by the yeast plasma membrane ATPases, Dnf1p and Dnf2p, in consort with a second protein Lem3p. Subsequent to transport, the lysophospholipids are acylated by the enzyme Ale1p to produce PtdEtn and PtdCho. The transport of the lysophospholipids occurs at rates sufficient to support all the PtdEtn and PtdCho synthesis required for rapid cell growth. The lysophospholipid transporters also utilize the anti-neoplastic and anti-parasitic ether lipid substrates related to edelfosine. The identification of biological substrates for the plasma membrane ATPases coupled with the power of yeast genetics now provides new tools to dissect the structure and function of the aminoglycerophospholipid transporters.     

Control of Protein and Sterol Trafficking by Antagonistic Activities of a Type IV P-type ATPase and Oxysterol Binding Protein Homologue

The oxysterol binding protein homologue Kes1p has been implicated in nonvesicular sterol transport in Saccharomyces cerevisiae. Kes1p also represses formation of protein transport vesicles from the trans-Golgi network (TGN) through an unknown mechanism. Here, we show that potential phospholipid translocases in the Drs2/Dnf family (type IV P-type ATPases [P4-ATPases]) are downstream targets of Kes1p repression. Disruption of KES1 suppresses the cold-sensitive (cs) growth defect of drs2Δ, which correlates with an enhanced ability of Dnf P4-ATPases to functionally substitute for Drs2p. Loss of Kes1p also suppresses a drs2-ts allele in a strain deficient for Dnf P4-ATPases, suggesting that Kes1p antagonizes Drs2p activity in vivo. Indeed, Drs2-dependent phosphatidylserine translocase (flippase) activity is hyperactive in TGN membranes from kes1Δ cells and is potently attenuated by addition of recombinant Kes1p. Surprisingly, Drs2p also antagonizes Kes1p activity in vivo. Drs2p deficiency causes a markedly increased rate of cholesterol transport from the plasma membrane to the endoplasmic reticulum (ER) and redistribution of endogenous ergosterol to intracellular membranes, phenotypes that are Kes1p dependent. These data suggest a homeostatic feedback mechanism in which appropriately regulated flippase activity in the Golgi complex helps establish a plasma membrane phospholipid organization that resists sterol extraction by a sterol binding protein.     

On the origin of lipid asymmetry: the flip side of ion transport

Membrane lipid asymmetry influences a multitude of cellular functions, including membrane vesiculation, cell division, and lifespan. Most cells retain the bulk of aminophospholipids to the cytosolic membrane leaflet by means of ATP-fuelled flippases or translocases. Converging lines of evidence indicate that members of the P(4) subfamily of P-type ATPases catalyze aminophospholipid transport and create lipid asymmetry in late secretory and endocytic compartments. Yet P-type ATPases usually pump small cations or metal ions. Atomic structures revealed important aspects of the transport mechanism, and sequence homology indicates that this mechanism is conserved throughout the family. Consequently, understanding how P(4) ATPases acquired the ability to translocate phospholipids instead of simple ions has become a major focus of interest.     

Mechanism and significance of P4 ATPase-catalyzed lipid transport: Lessons from a Na/K-pump

Members of the P₄ subfamily of P-type ATPases are believed to catalyze phospholipid transport across membrane bilayers, a process influencing a host of cellular functions. Atomic structures and functional analysis of P-type ATPases that pump small cations and metal ions revealed a transport mechanism that appears to be conserved throughout the family. A challenging problem is to understand how this mechanism is adapted in P₄ ATPases to flip phospholipids. P₄ ATPases form oligomeric complexes with members of the CDC50 protein family. While formation of these complexes is required for P₄ ATPase export from the endoplasmic reticulum, little is known about the functional role of the CDC50 subunits. The Na⁺/K⁺-ATPase and closely-related H⁺/K⁺-ATPase are the only other P-type pumps that are oligomeric, comprising mandatory β-subunits that are strikingly reminiscent of CDC50 proteins. Besides serving a role in the functional maturation of the catalytic α-subunit, the β-subunit also contributes specifically to intrinsic transport properties of the Na⁺/K⁺ pump. As β-subunits and CDC50 proteins likely adopted similar structures to accomplish analogous tasks, current knowledge of the Na⁺/K⁺-ATPase provides a useful guide for understanding the inner workings of the P₄ ATPase class of lipid pumps.     

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.     

Evolution and a revised nomenclature of P4 ATPases,a eukaryotic family of lipid flippases

In all eukaryotic cells, P4 ATPases, also named phospholipid flippases, generate phospholipid asymmetry across biological membranes. This process is essential for cell survival, as it is required for vesicle budding and fusion in the secretory pathway. Several P4 ATPase isoforms can be identified in all sequenced eukaryotic genomes, but their evolution and interrelationships are poorly described. In this study, we conducted a thorough phylogenetic analysis of P4 ATPases in all major eukaryotic super-groups and found that they can be divided into three distinct families, P4A, P4B and P4C ATPases, all of which have an ancient origin. While P4B ATPases have been lost in plants, P4A ATPases are present in all eukaryotic super-groups. P4C ATPases form an intermediate group between the other two but appear to share a common origin with P4A ATPases. Sequence motifs unique to P4 ATPases are situated in the basal ATP hydrolyzing machinery. In addition, no clear signature motifs within P4 ATPase subgroups were found that could be related to lipid specificity, likely pointing to an elaborate transport mechanism in which different amino acid residue combinations in these pumps can result in recognition of the same substrate.     

Pumping lipids with P4-ATPases

While accumulating evidence indicates that P4-ATPases catalyze phospholipid transport across cellular bilayers, their kinship to cation-pumping ATPases has raised fundamental questions concerning the underlying flippase mechanism. Loss of P4-ATPase function perturbs vesicle formation in late secretory and endocytic compartments. An intriguing concept is that P4-ATPases help drive vesicle budding by generating imbalances in transbilayer lipid numbers. Moreover, activation of P4-ATPases by phosphoinositides and other effectors of coat recruitment provide a potential mechanism to confine flippase activity to sites of vesicle biogenesis. These developments have raised considerable interest in understanding the mechanism, regulation and biological implications of P4-ATPase-catalyzed phospholipid transport.     

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.     

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.     

Transport ATPases in biological systems and relationship to human disease: a brief overview

Interest in the field of transport ATPases has grown dramatically during the past 20 years and gained considerable visibility for several reasons. First, it was shown that most transport ATPases can be lumped into only a few categories designated simply as P, V, F, and ABC types, the latter consisting of a large superfamily. Second, it has been shown that many transport ATPases have a clear relevance to human disease. Third, the field of transport ATPases has become rather advanced in the study of the reaction mechanisms and structure–function relationships associated with several of these enzymes. Finally, the Nobel committee recently recognized major accomplishments in this field of research. Here, the author provides a brief discussion of transport ATPases that are present in biological systems and their relevance or possible relevance to human disease.     

A 200-amino acid ATPase module in search of a basic function

A fast growing family of ATPases has recently been highlighted. It was named the AAA family, for ATPases Associated to a variety of cellular Activities. The key feature of the family is a highly conserved module of 230 amino acids present in one or two copies in each protein. Despite extensive sequence conservation, the members of the family fulfil a large diversity of cellular functions: cell cycle regulation, gene expression in yeast and HIV, vesicle-mediated transport, peroxisome assembly, 26S protease function etc. In addition, several members of this family can be found in the same organism (up to 17 in S. cerevisiae). The contrast between functional diversity and structural conservation of the module, from archaebacteria to mammals, suggests that it plays an essential, but as yet unknown, role at key points of the cellular machinery. Two (non-exclusive) such possibilities are: (1) ATP-dependent proteasome function and (2) ATP-dependent anchorage of proteins. Finally, the basic biochemical activity of the AAA module is still a matter of speculation, and we propose that it acts as an ATP-dependent protein clamp.     

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.     

Structure of the ATP binding domain from the Archaeoglobus fulgidus Cu+-ATPase

The P-type ATPases translocate cations across membranes using the energy provided by ATP hydrolysis. CopA from Archaeoglobus fulgidus is a hyperthermophilic ATPase responsible for the cellular export of Cu+ and is a member of the heavy metal P1B-type ATPase subfamily, which includes the related Wilson and Menkes diseases proteins. The Cu+-ATPases are distinct from their P-type counter-parts in ion binding sequences, membrane topology, and the presence of cytoplasmic metal binding domains, suggesting that they employ alternate forms of regulation and novel mechanisms of ion transport. To gain insight into Cu+-ATPase function, the structure of the CopA ATP binding domain (ATPBD) was determined to 2.3 A resolution. Similar to other P-type ATPases, the ATPBD includes nucleotide binding (N-domain) and phosphorylation (P-domain) domains. The ATPBD adopts a closed conformation similar to the nucleotide-bound forms of the Ca2+-ATPase. The CopA ATPBD is much smaller and more compact, however, revealing the minimal elements required for ATP binding, hydrolysis, and enzyme phosphorylation. Structural comparisons to the AMP-PMP-bound form of the Escherichia coli K+-transporting Kdp-ATPase and to the Wilson disease protein N-domain indicate that the five conserved N-domain residues found in P1B-type ATPases, but not in the other families, most likely participate in ATP binding. By contrast, the P-domain includes several residues conserved among all P-type ATPases. Finally, the CopA ATPBD structure provides a basis for understanding the likely structural and functional effects of various mutations that lead to Wilson and Menkes diseases.     

DNA sequence analysis of bacterial toxic heavy metal resistances

Bacterial plasmids have genes that confer highly specific resistances to As, Bi, Cd, Cu, Cr, Hg, Pb, Te, Zn, and other toxic heavy metals. For each toxic cation or anion, generally a different resistance system exists, and these systems may be “linked” together on multiple resistance plasmids. For Cd²⁺, AsO₂ ^?, AsO₄ ^3?, Hg²⁺, and organomercurials, DNA sequence analysis has supplemented direct physiological and biochemical experiments to produce sophisticated understanding. ThecadA ATPase ofS.aureus plasmids is a 727 amino acid membrane ATPase that pumps Cd²⁺ from the cells as rapidly as it is accumulated. This polypeptide is related by sequence to other cation translocating ATPases, including the membrane K⁺ ATPases ofEscherichia coli andStreptococcus faecalis, the H⁺ ATPases of yeast andNeurospora, the Na⁺/K⁺ ATPases of vertebrate animals, and the Ca²⁺ ATPases of rabbit muscle. The conserved residues include the aspartyl residue that is phosphorylated, the lysine involved in ATP binding, and the proline within a membrane translocating region. The arsenate and arsenite translocating ATPase consists of 3 polypeptides (from DNA sequence analysis), including a recognizable ATP binding protein (arsA), an integral membrane protein (arsB gene), and a substrate specificity subunit (arsC gene). Inorganic mercury and organomercurial degradation is carried out by a series of about 6 polypeptides, including 2 soluble intracellular enzymes (organomercurial lyase and mercuric reductase). The latter is related by sequence and function to glutathione reductase and lipoamide dehydrogenase of prokaryotes and eukaryotes. These enzymes are dimeric, FAD-containing, NAD(P)H-dependent oxidoreductases. Other recognizable polypeptides in themer system include a DNA-binding regulatory protein from themerR gene and a Hg²⁺ transport system consisting of a periplasmic Hg²⁺-binding protein (merP gene) and a membrane protein (merT gene) in gram negative systems.     

Calcium and copper transport ATPases: analogies and diversities in transduction and signaling mechanisms

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms. The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca²⁺ with high affinity on one side of the membrane, and releases the bound Ca²⁺ on the other side of the membrane to yield a high free Ca²⁺ gradient. These features are a basic requirement for cellular Ca²⁺ signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes to stabilization of ATP7A copper ATPase in plasma membranes.