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.     

Proton and calcium pumping P-type ATPases and their regulation of plant responses to the environment

Plant plasma membrane H⁺-ATPases and Ca²⁺-ATPases maintain low cytoplasmic concentrations of H⁺ and Ca²⁺, respectively, and are essential for plant growth and development. These low concentrations allow plasma membrane H⁺-ATPases to function as electrogenic voltage stats, and Ca²⁺-ATPases as “off” mechanisms in Ca²⁺-based signal transduction. Although these pumps are autoregulated by cytoplasmic concentrations of H⁺ and Ca²⁺, respectively, they are also subject to exquisite regulation in response to biotic and abiotic events in the environment. A common paradigm for both types of pumps is the presence of terminal regulatory (R) domains that function as autoinhibitors that can be neutralized by multiple means, including phosphorylation. A picture is emerging in which some of the phosphosites in these R domains appear to be highly, nearly constantly phosphorylated, whereas others seem to be subject to dynamic phosphorylation. Thus, some sites might function as major switches, whereas others might simply reduce activity. Here, we provide an overview of the relevant transport systems and discuss recent advances that address their relation to external stimuli and physiological adaptations.

The regulation of plasma membrane H^+-ATPases and autoinhibited Ca2^+-ATPases exhibits a complex and dynamic network of posttranslational regulation. The regulation of plasma membrane H⁺-ATPases and autoinhibited Ca²⁺-ATPases exhibits a complex and dynamic network of posttranslational regulation.

P-type ATPases are found in all domains of life and constitute a large superfamily of membrane-bound pumps that share a common machinery, including a reaction cycle that involves catalytic phosphorylation of an Asp, resulting in a phosphorylated intermediate (reviewed in Palmgren and Nissen, 2011; (hence the name P-type; Box 1). The catalytic phosphoryl-aspartate intermediate is not to be confused with regulatory phosphorylation, which occurs on Ser, Thr, and Tyr residues. Five major families of P-type ATPases have been characterized (P1–5), each of which is divided into a number of subfamilies (named with letters). Plasma membrane H⁺-ATPases are classified as P3A ATPases, whereas Ca²⁺ pumps constitute P2A and P2B ATPases. In plants, these pumps are best characterized in the model plant Arabidopsis thaliana (Arabidopsis).Box 1Enzymology of P-type ATPases.P-type ATPases (reviewed in Palmgren and Nissen, 2011) alternate between two extreme conformations during their catalytic cycle: a high-affinity (with respect to ATP and the ion to be exported) Enzyme1 (E1) state, and a low-affinity Enzyme2 (E2) state. Many P-type ATPases are autoinhibited by built-in molecular constraints, namely their C- and N-terminal (for plasma membrane H⁺-ATPases; Palmgren et al., 1999) or N-terminal (for P2B Ca²⁺-ATPases; Malmström et al., 1997) regulatory (R) domains of approximately 100 amino acid residues, which act as brakes by stabilizing the pumps in a low-affinity conformation (Palmgren and Nissen, 2011), most likely E2. Neutralizing the R domain results in a shift in conformational equilibrium towards a high-affinity state, likely E1. In this way, the R domains of plasma membrane H⁺-ATPases and Ca²⁺-ATPases allow posttranslational modification events to control the turnover numbers of these pumps. A structure of a plasma membrane H⁺-ATPase (from the distantly related yeast S. cerevisiae) in its autoinhibited state has been solved (Heit et al., 2021). Its R domain is situated adjacent to the P domain, which would suggest that the R domain functions to restrict the conformational flexibility of the pump. Normally, the hydrolysis of ATP and transport are tightly coupled in P-type ATPases. Therefore, P-type ATPases hydrolyze bound ATP as soon as their ligand-binding site(s) in the membrane region are occupied, but not before. Thus, increasing the ligand affinity of an ATPase simultaneously increases its turnover number, provided that the concentration of ATP is not limiting, which is rarely the case in cells. A specific feature of plasma membrane H⁺-ATPases is that in the autoinhibited state, ATP hydrolysis is only loosely coupled to H⁺ pumping, whereas pump activation results in tight coupling, with one H⁺ pumped per ATP split (Pedersen et al., 2018).In response to internal and/or external cues, plasma membrane H⁺-ATPase and Ca²⁺-ATPase activities are controlled by intracellular concentrations of H⁺ and Ca²⁺, respectively, via interacting proteins, through posttranslational modification by phosphorylation, and by regulated trafficking of the pump to and from the plasma membrane. Their regulation sometimes involves changes in gene expression and turnover, although this is rare, perhaps because both processes are time- and energy-consuming (Haruta et al., 2018).     

Abolishment of proton pumping and accumulation in the E1P conformational state of a plant plasma membrane H+-ATPase by substitution of a conserved aspartyl residue in transmembrane segment 6

The plasma membrane H(+)-ATPase AHA2 of Arabidopsis thaliana, which belongs to the P-type ATPase superfamily of cation-transporting ATPases, pumps protons out of the cell. To investigate the mechanism of ion transport by P-type ATPases we have mutagenized Asp(684), a residue in transmembrane segment M6 of AHA2 that is conserved in Ca(2+)-, Na(+)/K(+)-, H(+)/K(+)-, and H(+)-ATPases and which coordinates Ca(2+) ions in the SERCA1 Ca(2+)-ATPase. We describe the expression, purification, and biochemical analysis of the Asp(684) --> Asn mutant, and provide evidence that Asp(684) in the plasma membrane H(+)-ATPase is required for any coupling between ATP hydrolysis, enzyme conformational changes, and H(+)-transport. Proton pumping by the reconstituted mutant enzyme was completely abolished, whereas ATP was still hydrolyzed. The mutant was insensitive to the inhibitor vanadate, which preferentially binds to P-type ATPases in the E(2) conformation. During catalysis the Asp(684) --> Asn enzyme accumulated a phosphorylated intermediate whose stability was sensitive to addition of ADP. We conclude that the mutant enzyme is locked in the E(1) conformation and is unable to proceed through the E(1)P-E(2)P transition.     

Structures of P-type transporting ATPases and chromosomal locations of their genes

P-type ATPases (E1E2-ATPases) are primary active transporters which form phospho-intermediates during their catalytic cycle. They are classified into P1 to P4 based on the primary structure and potential transmembrane segments. Although the classic P-type ATPases are cation transporters, two new members have recently been found; one is a flippase catalyzing the flip-flop movement of aminophospholipids, but the substrate and function of the other one remain unknown. It would be interesting to determine whether the cations and aminophospholipids are transported by similar or different mechanisms. P-type ATPases are believed to have been derived from a common ancestor, and their genes are found to be distributed in various chromosomal loci. However, gene duplication events can be traced from the tandem arrangement of genes and their linkage map. Na+/K+- and H+/K+-ATPases have not only closely related a subunits but also similar beta subunits. Renal Na+/K+-ATPase has an additional subunit gamma. Similar small polypeptides (phospholemman, Mat-8 and CHIF), which induce Cl- and K+ currents, have been found. The idea of their functional and structural coupling with P-type ATPases, especially with H+/K+-ATPase, is intriguing. Each P-type ATPase must have specific domains or sequences for its intracellular trafficking (sorting, retention and recycling). Identification of such regions and studies on the molecules playing role in their recognition may facilitate the unveiling of various cellular processes regulated by P-type ATPases.     

Molecular aspects of higher plant P-type Ca(2+)-ATPases

Recent genomic data in the model plant Arabidopsis thaliana reveal the existence of at least 11 Ca(2+)-ATPase genes, and an analysis of expressed sequence tags suggests that the number of calcium pumps in this organism might be even higher. A phylogenetic analysis shows that 11 Ca(2+)-ATPases clearly form distinct groups, type IIA (or ECA for ER-type Ca(2+)-ATPase) and type IIB (ACA for autoinhibited Ca(2+)-ATPase). While plant IIB calcium pumps characterized so far are localized to internal membranes, their animal homologues are exclusively found in the plasma membrane. However, Arabidopsis type IIB calcium pump isoforms ACA8, ACA9 and ACA10 form a separate outgroup and, based on the high molecular masses of the encoded proteins, are good candidates for plasma membrane bound Ca(2+)-ATPases. All known plant type IIB calcium ATPases seem to employ an N-terminal calmodulin-binding autoinhibitor. Therefore it appears that the activity of type IIB Ca(2+)-ATPases in plants and animals is controlled by N-terminal and C-terminal autoinhibitory domains, respectively. Possible functions of plant calcium pumps are described and - beside second messenger functions directly linked to calcium homeostasis - new data on a putative involvement in secretory and salt stress functions 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.     

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.     

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.     

Metal Fluoride Inhibition of a P-type H+ Pump: STABILIZATION OF THE PHOSPHOENZYME INTERMEDIATE CONTRIBUTES TO POST-TRANSLATIONAL PUMP ACTIVATION*

The plasma membrane H⁺-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H⁺/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H⁺-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H⁺-ATPases is labile in the basal state, which may provide an explanation for the low H⁺/ATP coupling ratio of these pumps in the basal state.     

低温、高pH胁迫对水稻幼苗根系质膜、液泡膜ATP酶活性的影响

以耐冷性不同的两个水稻品种为材料,比较研究了幼苗根系质膜、液泡膜ATP酶对低温(8℃)及高pH(8.0)胁迫的反应。结果表明水稻根细胞质膜和液泡膜上均存在Ca3+-ATP酶,但活性远低于H+-ATP酶。耐冷品种武育粳3号经低温(8℃)处理2d,根系质膜和液泡膜H+-ATP酶、Ca2+-ATP酶活性均明显升高,至冷处理12d,H+-ATP酶、Ca2+-ATP酶活性有所下降,但仍与对照相近;而冷敏感品种汕优63经低温(8℃)处理2d,根系质膜H+-ATP酶活性略有升高,而质膜Ca2+-ATP酶以及液泡膜H+-ATP酶、Ca2+-ATP酶活性已明显下降;至冷处理12d,4种酶活性均明显低于对照。高pH胁迫使质膜和液泡膜H+-ATP酶活性下降,而使Ca2+-ATP酶活性上升。高pH胁迫会加剧低温冷害。结果表明,耐冷品种质膜、液泡膜ATP酶比冷敏感品种对低温胁迫有更强的适应能力。     

The plant Ca2+ -ATPase repertoire: biochemical features and physiological functions

Ca(2+)-ATPases are P-type ATPases that use the energy of ATP hydrolysis to pump Ca(2+) from the cytoplasm into intracellular compartments or into the apoplast. Plant cells possess two types of Ca(2+) -pumping ATPase, named ECAs (for ER-type Ca(2+)-ATPase) and ACAs (for auto-inhibited Ca(2+)-ATPase). Each type comprises different isoforms, localised on different membranes. Here, we summarise available knowledge of the biochemical characteristics and the physiological role of plant Ca(2+)-ATPases, greatly improved after gene identification, which allows both biochemical analysis of single isoforms through heterologous expression in yeast and expression profiling and phenotypic analysis of single isoform knock-out mutants.     

Structure of Na+,K+-ATPase at 11-A resolution: comparison with Ca2+-ATPase in E1 and E2 states

Na+,K+-ATPase is a heterodimer of alpha and beta subunits and a member of the P-type ATPase family of ion pumps. Here we present an 11-A structure of the heterodimer determined from electron micrographs of unstained frozen-hydrated tubular crystals. For this reconstruction, the enzyme was isolated from supraorbital glands of salt-adapted ducks and was crystallized within the native membranes. Crystallization conditions fixed Na+,K+-ATPase in the vanadate-inhibited E2 conformation, and the crystals had p1 symmetry. A large number of helical symmetries were observed, so a three-dimensional structure was calculated by averaging both Fourier-Bessel coefficients and real-space structures of data from the different symmetries. The resulting structure clearly reveals cytoplasmic, transmembrane, and extracellular regions of the molecule with densities separately attributable to alpha and beta subunits. The overall shape bears a remarkable resemblance to the E2 structure of rabbit sarcoplasmic reticulum Ca2+-ATPase. After aligning these two structures, atomic coordinates for Ca2+-ATPase were fit to Na+,K+-ATPase, and several flexible surface loops, which fit the map poorly, were associated with sequences that differ in the two pumps. Nevertheless, cytoplasmic domains were very similarly arranged, suggesting that the E2-to-E1 conformational change postulated for Ca2+-ATPase probably applies to Na+,K+-ATPase as well as other P-type ATPases.     

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 mgtB Mg2+ transport locus of Salmonella typhimurium encodes a P-type ATPase.

The mgtB locus codes for one of three distinct Mg2+ transport systems of Salmonella typhimurium. The system encoded by the mgtB locus mediates Mg2+ influx only. The nucleotide sequence of a 4.6-kilobase fragment of DNA carrying mgtB has been determined. Two open reading frames were apparent. The most 5' (mgtC) could encode a hydrophobic protein of up to 25 kDa depending on which translation starts are used. A plasmid carrying this region downstream from a phage T7 promoter expresses a 22.5-kDa protein. The second open reading frame encoded a 101-kDa polypeptide (MgtB) consistent with our previous observation that a plasmid carrying the mgtB locus expresses a 102-kDa protein in maxicells. Insertions into either open reading frame abolished the ability of the plasmid to relieve the requirement for added Mg2+ and to restore Mg2+ uptake to a Mg2+ transport-deficient strain of S. typhimurium. The predicted amino acid sequence of MgtC showed no similarity to any other known protein. In contrast, the predicted sequence of MgtB indicated that it is a member of the family of cation transport P-type ATPases. Strikingly, however, MgtB was significantly more similar to eukaryotic Ca2(+)-ATPases than to prokaryotic P-type ATPases or other classes of eukaryotic P-type ATPases such as the Na+,K(+)-ATPase. MgtB is most closely related to Ca2(+)-ATPases of mammalian sarcoplasmic reticulum and yeast. A number of features of the Ca2(+)-ATPases thought to be important for cation transduction across the membrane are present in MgtB but not in other prokaryotic members of this enzyme family. Unlike the Ca2(+)-ATPases, however, which mediate efflux of cation from the cytosol, MgtB mediates influx of cation into the cytosol.     

Gene knockout studies of Ca2+-transporting ATPases.

The biochemical functions of intracellular and plasma membrane Ca2+-transporting ATPases in the control of cytosolic and organellar Ca2+ levels are well established, but the physiological roles of specific isoforms are less well understood. There appear to be three different types of Ca2+ pumps in mammalian tissues: the sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs), which sequester Ca2+ within the endoplasmic or sarcoplasmic reticulum, the plasma membrane Ca2+-ATPases (PMCAs), which extrude Ca2+ from the cell, and the putative secretory pathway Ca2+-ATPase (SPCA), the function of which is poorly understood. This review describes the results of recent analyses of mouse models with null mutations in the genes encoding SERCA and PMCA isoforms and genetic studies of SERCA and SPCA dysfunction in both humans and model organisms. These studies are yielding important insights regarding the physiological functions of individual Ca2+-transporting ATPases in vivo.     

Molecular dissection of functional domains of the E1E2-ATPase using sodium and calcium pump chimeric molecules.

Proposed models for the catalytic subunit of the E1E2-ATPases (ion pumps) predict that the first four transmembrane domains (M1 - M4) reside in the NH2 terminal one-third of the molecule, and the remainder (M5 - M10) in the COOH terminal one-third. The amino-acid sequences for the 5'-(p-fluorosulfonyl)-benzoyl-adenosine (FSBA) binding region residing just before M5 segment are very well conserved among distinct ion pumps. Taking advantage of these models, we have constructed a set of chicken chimeric ion pumps between the (Na++ K+)-ATPase alpha-subunit and the Ca(2+)-ATPase using the FSBA-binding site as an exchange junction, thereby preserving overall topological structure as E1E2 ATPases. From various functional assays on these chimeric ion pumps, including ouabain-inhibitable ATPase activity, Ca2+ binding, Ca2+ uptake, and subunit assembly based on immuno-coprecipitation, the following conclusions were obtained: (a) A (Na++ K+)-ATPase inhibitor, ouabain, binds to the regions before M4 in the alpha-subunit and exerts its inhibitory effect. (b) The regions after M5 of the (Na++ K+)-ATPase alpha-subunit bind the beta-subunit, even when these regions are incorporated into the corresponding domains in the Ca(2+)-ATPase. (c) The corresponding domains of the Ca(2+)-ATPase, the regions after M5, bind 45Ca even when it is incorporated into the corresponding position of the (Na++ K+)-ATPase alpha-subunit.     

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.     

Anticipating antiport in P-type ATPases

Cation-transporting P-type ATPases show a high degree of structural and functional homology. Nevertheless, for many members of this large family, the molecular mechanism of transport is unclear; namely, whether transport is electrogenic or not and if countertransport is involved remains to be established. In a few well-studied cases such as the Na(+)-K(+)-ATPase, plasma membrane Ca(2+) ATPase (PMCA) and sarcoplasmic reticulum Ca(2+) ATPase (SERCA) countertransport has been clearly demonstrated. New data based on the crystal structure of SERCA now strongly indicate that countertransport could be mandatory for all P-type ATPases. This concept should be verified for other known and for all newly characterized P-type ATPases.