Endosomal lipid flippases and their related diseases

 In eukaryotic cell membranes, phospholipids are asymmetrically distributed between the two leaflets of the lipid bilayer. For example, the extracellular leaflet of the plasma membrane (PM) is enriched with phosphatidylcholine and sphingomyelin, while the cytosolic leaflet of the PM is enriched with phosphatidylserine (PS) and phosphatidylethanolamine. The asymmetric distribution of PS in the PM is crucial for cell life, since PS in the extracellular leaflet of the PM is recognized as an “eat-me” signal by phagocytes. Inside the cells, a high PS concentration in the cytosolic leaflet of the PM is essential to facilitate various cellular events through the recruitment of signaling molecules such as protein kinase C and Akt.The asymmetric distribution of phospholipids is believed to be generated in part by phospholipid translocases, or “flippases.” The proteins responsible for flippase activity are type IV P-type ATPase (P₄-ATPases). P-type ATPases are multispan transmembrane pumps that use ATP hydrolysis as an energy source. P-type ATPases undergo autophosphorylation of a conserved aspartate residue during the catalytic cycle, hence the designation of “P”-type. P₄-ATPases are unique in that they are phospholipid transporters whereas other types of P-type ATPases are ion transporters.The human genome contains 14 P₄-ATPases, and mutations in some P₄-ATPases cause inherited genetic diseases. For example, mutations in ATP8B1 are associated with intrahepatic cholestasis and also cause hearing loss. Mutations in ATP8A2 are associated with a severe neurological disorder characterized by cerebellar ataxia, mental retardation, and dysequilibrium syndrome (CAMRQ).¹ Despite the accumulating evidence highlighting the physiological importance of P₄-ATPases, how dysfunction of P₄-ATPases causes diseases is poorly understood.In a recent study, we revealed the cellular function of the P₄-ATPase, ATP8A1.² ATP8A1 localizes at recycling endosomes (REs), an organelle that functions in recycling transport of internalized molecules back to the PM, thus defining the amount of proteins at the PM. PS is most concentrated in REs among intracellular organelles and we roughly estimated that 70 and 30% of PS are localized in the cytosolic and the luminal leaflets of RE membranes, respectively.² ATP8A1 generates the asymmetric transbilayer distribution of PS at REs. The knockdown of ATP8A1 halted recycling traffic from REs to the PM. At the mechanistic level, we found that EHD1, a dynamin-like membrane fission protein, lost its RE localization upon ATP8A1 knockdown and EHD1 knockdown also blocked recycling traffic. EHD1 bound PS in vitro and lost its membrane localization in cells that are defective in PS synthesis. Thus, we propose that PS flipping by ATP8A1 recruits EHD1 to RE membranes, thereby regulating the recycling traffic from REs to the PM (Fig. 1). Open in a separate windowFigure 1.Model of flippase-related diseases. Under normal conditions, flippases (e.g., ATP8A1 and ATP8A2) translocate PS to the cytosolic leaflet of RE membranes. PS recruits EHD1 to REs, and then EHD1 participates in the fission of RE membranes to generate transport vesicles that contain cell surface receptors. In flippase-dysfunctional situations, PS levels in the cytosolic leaflet of REs would be low. This impairs the PS/EHD1/membrane traffic axis, leading to a lower abundance of cell surface receptors that are critical for responses to extracellular ligands.ATP8A2 is a tissue-specific ATP8A1 paralogue. We found that a CAMRQ-causative mutation of ATP8A2 (I376M) lost its ATPase and flippase activity toward PS. ATP8A2 is not endogenously expressed in COS-1 cells. Interestingly, the phenotype that was caused by the loss of ATP8A1 in COS-1 cells, was restored by the exogenous expression of wild-type ATP8A2, but not I376M mutant ATP8A2. Moreover, cortical neurons prepared from ATP8A2 knockout mice showed lower abundance of transferrin receptors at the PM. Together, these results indicate that ATP8A2 functions in the recycling traffic in neurons, and that CAMRQ may result from the defect in recycling of important neurological receptor proteins from REs to the PM. One possible candidate protein is very low-density lipoprotein receptor (VLDLR). VLDLR is a receptor for reelin, an extracellular protein that guides neuronal migration in the cerebral cortex and cerebellum. VLDLR circulates between the PM and endosomes (possibly REs) by recycling traffic.³ Significantly, mutations in VLDLR gene are also linked to CAMRQ.^4,5 Therefore, impaired recycling traffic of VLDLR to the PM in neurons with dysfunctional ATP8A2 (I376M) may cause lower expression of VLDLR at the PM, leading to reduced reelin signaling, abnormal neuronal development, and neurological disorder.dATP8B, a P₄-ATPase in Drosophila melanogaster was recently reported to cause an impaired response to cVA pheromone (a sex-specific social cue) and mislocalization of the pheromone receptor in cVA-sensing neurons.⁶ The impaired response to the pheromone in dATP8B mutant was rescued by expressing bovine ATP8A2. Therefore, from insects to mammals, phospholipid flippases may define the localization of neuronal receptors to the PM.Lastly, our findings may explain the phenotype of ATP8A1 knockout mice.⁷ ATP8A1 knockout mice are vital but show deficiencies in hippocampus-dependent learning. Hippocampus-dependent learning involves modification of synaptic strength, and one cellular mechanism for tuning synaptic strength is long-term potentiation (LTP). During LTP, REs supply glutamate receptors to the post-synaptic membrane.⁸ Therefore, we speculate that impaired glutamate receptor traffic from REs to the post-synaptic membranes during LTP may underlie the deficiency in learning in ATP8A1 knockout mice. In agreement with this hypothesis, the dominant-negative form of EHD1 inhibits glutamate receptor traffic during LTP.⁸Many P₄-ATPases are expressed in the Golgi/endosomes and the PM. We expect that they contribute redundantly to the phospholipid asymmetry and membrane traffic through organelles. Simultaneous ablations of P₄-ATPases may dissect their roles and will give more insight into flippase-mediated cellular processes and -related diseases.     

Characterization of the membrane bound Mg2+-ATPase of rat skeletal muscle

A procedure was developed to isolate a membrane fraction of rat skeletal muscle which contains a highly active Mg²⁺-ATPase (5–25 μmol P_i/mg min). The rate of ATP hydrolysis by the Mg²⁺-ATPase was nonlinear but decayed exponentially (first-order rate constant ≥0.2 s^?1 at 37°C). The rapid decline in the ATPase activity depended on the presence of ATP or its nonhydrolyzable analog 5′-adenylyl imidodiphosphate (AdoPP[NH]P). Once inactivated, removal of ATP from the medium did not immediately restore the original activity. ATP- or AdoPP[NH]P-dependent inactivation could be blocked by concanavalin A, wheat germ agglutinin or rabbit antiserum against the membrane. Additions of these proteins after ATP addition prevented further inactivation but did not restore the original activity. Low concentrations of ionic and nonionic detergents increased the rate of ATP-dependent inactivation. Higher concentrations of detergents, which solubilize the membrane completely, inactivated the Mg²⁺-ATPase. Cross-linking the membrane components with glutaraldehyde prevented ATP-dependent inactivation and decreased the sensitivity of the Mg²⁺-ATPase to detergents. It is proposed that the regulation of the Mg²⁺-ATPase by ATP requires the mobility of proteins within the membrane. Cross-linking the membrane proteins with lectins, antiserum or glutaraldehyde prevents inactivation; increasing the mobility with detergents accelerates ATP-dependent inactivation.     

Genetic effects of ATP1A2 in familial hemiplegic migraine type II and animal models

Na⁺/K⁺-ATPase alpha 2 (Atp1a2) is an integral plasma membrane protein belonging to the P-type ATPase family that is responsible for maintaining the sodium (Na⁺) and potassium (K⁺) gradients across cellular membranes with hydrolysis of ATP. Atp1a2 contains two subunits, alpha and beta, with each having various isoforms and differential tissue distribution. In humans, mutations in ATP1A2 are associated with a rare form of hereditary migraines with aura known as familial hemiplegic migraine type II. Genetic studies in mice have revealed other neurological effects of Atp1a2 in mice including anxiety, fear, and learning and motor function disorders. This paper reviews the recent findings in the literature concerning Atp1a2.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products

Two distinct membrane fractions containing H⁺-ATPase activity were prepared from red beet. One fraction contained a H⁺-ATPase activity that was inhibited by NO₃⁻ while the other contained a H⁺-ATPase inhibited by vanadate. We have previously proposed that these H⁺-ATPases are associated with tonoplast (NO₃⁻-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP²⁻, and Mg²⁺ concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg²⁺) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.

Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP²⁻ in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.

Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP²⁻ complex.

The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H⁺-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg²⁺ could modulate ATPase activity at both the tonoplast and plasma membrane.

     

Colocalization of the (Pro)renin Receptor/Atp6ap2 with H+-ATPases in Mouse Kidney but Prorenin Does Not Acutely Regulate Intercalated Cell H+-ATPase Activity

The (Pro)renin receptor (P)RR/Atp6ap2 is a cell surface protein capable of binding and non-proteolytically activate prorenin. Additionally, (P)RR is associated with H⁺-ATPases and alternative functions in H⁺-ATPase regulation as well as in Wnt signalling have been reported. Kidneys express very high levels of H⁺-ATPases which are involved in multiple functions such as endocytosis, membrane protein recycling as well as urinary acidification, bicarbonate reabsorption, and salt absorption. Here, we wanted to localize the (P)RR/Atp6ap2 along the murine nephron, exmaine whether the (P)RR/Atp6ap2 is coregulated with other H⁺-ATPase subunits, and whether acute stimulation of the (P)RR/Atp6ap2 with prorenin regulates H⁺-ATPase activity in intercalated cells in freshly isolated collecting ducts. We localized (P)PR/Atp6ap2 along the murine nephron by qPCR and immunohistochemistry. (P)RR/Atp6ap2 mRNA was detected in all nephron segments with highest levels in the collecting system coinciding with H⁺-ATPases. Further experiments demonstrated expression at the brush border membrane of proximal tubules and in all types of intercalated cells colocalizing with H⁺-ATPases. In mice treated with NH₄Cl, NaHCO₃, KHCO₃, NaCl, or the mineralocorticoid DOCA for 7 days, (P)RR/Atp6ap2 and H⁺-ATPase subunits were regulated but not co-regulated at protein and mRNA levels. Immunolocalization in kidneys from control, NH₄Cl or NaHCO₃ treated mice demonstrated always colocalization of PRR/Atp6ap2 with H⁺-ATPase subunits at the brush border membrane of proximal tubules, the apical pole of type A intercalated cells, and at basolateral and/or apical membranes of non-type A intercalated cells. Microperfusion of isolated cortical collecting ducts and luminal application of prorenin did not acutely stimulate H⁺-ATPase activity. However, incubation of isolated collecting ducts with prorenin non-significantly increased ERK1/2 phosphorylation. Our results suggest that the PRR/Atp6ap2 may form a complex with H⁺-ATPases in proximal tubule and intercalated cells but that prorenin has no acute effect on H⁺-ATPase activity in intercalated cells.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: II. H/ATP Stoichiometry of an Anion-Sensitive H-ATPase

The H⁺/ATP stoichiometry was determined for an anion-sensitive H⁺-ATPase in membrane vesicles believed to be derived from tonoplast. Initial rates of proton influx were measured by monitoring the alkalinization of a weakly buffered medium (pH 6.13) following the addition of ATP to a suspension of membrane vesicles of Beta vulgaris L. Initial rates of ATP hydrolysis were measured in an assay where ATP hydrolysis is coupled to NADH oxidation and monitored spectrophotometrically (A₃₄₀) or by monitoring the release of ³²P from [γ-³²P]ATP. Inasmuch as this anion-sensitive H⁺-ATPase is strongly inhibited by NO₃⁻, initial rates of H⁺ influx and ATP hydrolysis were measured in the absence and presence of NO₃⁻ to account for ATPase activity not involved in H⁺ transport. The NO₃⁻-sensitive activities were calculated and used to estimate the ratio of H⁺ transported to ATP hydrolyzed. These measurements resulted in an estimate of the H⁺/ATP stoichiometry of 1.96 ± 0.14 suggesting that the actual stoichiometry is 2 H⁺ transported per ATP hydrolyzed. When compared with the reported values of the electrochemical potential gradient for H⁺ across the tonoplast measured in vivo, our result suggests that the H⁺-ATPase does not operate near equilibrium but is regulated by cellular factors other than energy supply.     

Regulation of PI(3)K/Akt signalling and cellular transformation by inositol polyphosphate 4‐phosphatase‐1

Akt is a crucial phosphoinositide 3-kinase (PI(3)K) effector that regulates cell proliferation and survival. PI(3)K-generated signals, PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂, direct Akt plasma membrane engagement. Pathological Akt plasma membrane association promotes oncogenesis. PtdIns(3,4)P₂ is degraded by inositol polyphosphate 4-phosphatase-1 (4-ptase-1) forming PtdIns(3)P; however, the role of 4-ptase-1 in regulating the activation and function of Akt is unclear. In mouse embryonic fibroblasts lacking 4-ptase-1 (^−/−MEFs), the Akt-pleckstrin homology (PH) domain was constitutively membrane-associated both in serum-starved and agonist-stimulated cells, in contrast to ^+/+MEFs, in which it was detected only at the plasma membrane following serum stimulation. Epidermal growth factor (EGF) stimulation resulted in increased Ser⁴⁷³ and Thr³⁰⁸-Akt phosphorylation and activation of Akt-dependent signalling in ^−/−MEFs, relative to ^+/+MEFs. Significantly, loss of 4-ptase-1 resulted in increased cell proliferation and decreased apoptosis. SV40-transformed ^−/−MEFs showed increased anchorage-independent cell growth and formed tumours in nude mice. This study provides the first evidence, to our knowledge, that 4-ptase-1 controls the activation of Akt and thereby cell proliferation, survival and tumorigenesis.     

Thermodynamic analysis of F1‐ATPase rotary catalysis using high‐speed imaging

F₁-ATPase (F₁) is a rotary motor protein fueled by ATP hydrolysis. Although the mechanism for coupling rotation and catalysis has been well studied, the molecular details of individual reaction steps remain elusive. In this study, we performed high-speed imaging of F₁ rotation at various temperatures using the total internal reflection dark-field (TIRDF) illumination system, which allows resolution of the F₁ catalytic reaction into elementary reaction steps with a high temporal resolution of 72 µs. At a high concentration of ATP, F₁ rotation comprised distinct 80° and 40° substeps. The 80° substep, which exhibited significant temperature dependence, is triggered by the temperature-sensitive reaction, whereas the 40° substep is triggered by ATP hydrolysis and the release of inorganic phosphate (P_i). Then, we conducted Arrhenius analysis of the reaction rates to obtain the thermodynamic parameters for individual reaction steps, that is, ATP binding, ATP hydrolysis, P_i release, and TS reaction. Although all reaction steps exhibited similar activation free energy values, ΔG^‡ = 53–56 kJ mol⁻¹, the contributions of the enthalpy (ΔH^‡), and entropy (ΔS^‡) terms were significantly different; the reaction steps that induce tight subunit packing, for example, ATP binding and TS reaction, showed high positive values of both ΔH^‡ and ΔS^‡. The results may reflect modulation of the excluded volume as a function of subunit packing tightness at individual reaction steps, leading to a gain or loss in water entropy.     

Conversion of the 2 Cl−/1 H+ antiporter ClC‐5 in a NO3−/H+ antiporter by a single point mutation

Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent''s disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl⁻/1 H⁺. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO₃⁻/H⁺ antiporter atClCa, led to coupled NO₃⁻/H⁺ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO₃⁻/H⁺ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of ∼7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.     

Effect of ε subunit on the rotation of thermophilic Bacillus F1-ATPase

F₁-ATPase is an ATP-driven motor in which γε rotates in the α₃β₃-cylinder. It is attenuated by MgADP inhibition and by the ε subunit in an inhibitory form. The non-inhibitory form of ε subunit of thermophilic Bacillus PS3 F₁-ATPase is stabilized by ATP-binding with micromolar K_d at 25 °C. Here, we show that at [ATP] > 2 μM, ε does not affect rotation of PS3 F₁-ATPase but, at 200 nM ATP, ε prolongs the pause of rotation caused by MgADP inhibition while the frequency of the pause is unchanged. It appears that ε undergoes reversible transition to the inhibitory form at [ATP] below K_d.     

The Parkinson-associated human P5B-ATPase ATP13A2 protects against the iron-induced cytotoxicity

P-type ion pumps are membrane transporters that have been classified into five subfamilies termed P₁–P₅. The ion transported by the P₅-ATPases is not known. Five genes named ATP13A1–ATP13A5 that belong to the P₅-ATPase group are present in humans. Loss-of-function mutations in the ATP13A2 gene (PARK9, OMIM 610513) underlay a form of Parkinson's disease (PD) known as the Kufor–Rakeb syndrome (KRS), which belongs to the group of syndromes of neurodegeneration with brain iron accumulation (NBIA).Here we report that the cytotoxicity induced by iron exposure was two-fold reduced in CHO cells stably expressing the ATP13A2 recombinant protein (ATP13A2). Moreover, the iron content in ATP13A2 cells was lower than control cells stably expressing an inactive mutant of ATP13A2. ATP13A2 expression caused an enlargement of lysosomes and late endosomes. ATP13A2 cells exhibited a reduced iron-induced lysosome membrane permeabilization (LMP). These results suggest that ATP13A2 overexpression improves the lysosome membrane integrity and protects against the iron-induced cell damage.     

Lipid specific activation of the murine P4-ATPase Atp8a1 (ATPase II)

The asymmetric transbilayer distribution of phosphatidylserine (PS) in the mammalian plasma membrane and secretory vesicles is maintained, in part, by an ATP-dependent transporter. This aminophospholipid "flippase" selectively transports PS to the cytosolic leaflet of the bilayer and is sensitive to vanadate, Ca(2+), and modification by sulfhydryl reagents. Although the flippase has not been positively identified, a subfamily of P-type ATPases has been proposed to function as transporters of amphipaths, including PS and other phospholipids. A candidate PS flippase ATP8A1 (ATPase II), originally isolated from bovine secretory vesicles, is a member of this subfamily based on sequence homology to the founding member of the subfamily, the yeast protein Drs2, which has been linked to ribosomal assembly, the formation of Golgi-coated vesicles, and the maintenance of PS asymmetry. To determine if ATP8A1 has biochemical characteristics consistent with a PS flippase, a murine homologue of this enzyme was expressed in insect cells and purified. The purified Atp8a1 is inactive in detergent micelles or in micelles containing phosphatidylcholine, phosphatidic acid, or phosphatidylinositol, is minimally activated by phosphatidylglycerol or phosphatidylethanolamine (PE), and is maximally activated by PS. The selectivity for PS is dependent upon multiple elements of the lipid structure. Similar to the plasma membrane PS transporter, Atp8a1 is activated only by the naturally occurring sn-1,2-glycerol isomer of PS and not the sn-2,3-glycerol stereoisomer. Both flippase and Atp8a1 activities are insensitive to the stereochemistry of the serine headgroup. Most modifications of the PS headgroup structure decrease recognition by the plasma membrane PS flippase. Activation of Atp8a1 is also reduced by these modifications; phosphatidylserine-O-methyl ester, lysophosphatidylserine, glycerophosphoserine, and phosphoserine, which are not transported by the plasma membrane flippase, do not activate Atp8a1. Weakly translocated lipids (PE, phosphatidylhydroxypropionate, and phosphatidylhomoserine) are also weak Atp8a1 activators. However, N-methyl-phosphatidylserine, which is transported by the plasma membrane flippase at a rate equivalent to PS, is incapable of activating Atp8a1 activity. These results indicate that the ATPase activity of the secretory granule Atp8a1 is activated by phospholipids binding to a specific site whose properties (PS selectivity, dependence upon glycerol but not serine, stereochemistry, and vanadate sensitivity) are similar to, but distinct from, the properties of the substrate binding site of the plasma membrane flippase.     

The Binding Mode of ATP Revealed by the Solution Structure of the N-domain of Human ATP7A

We report the solution NMR structures of the N-domain of the Menkes protein (ATP7A) in the ATP-free and ATP-bound forms. The structures consist of a twisted antiparallel six-stranded β-sheet flanked by two pairs of α-helices. A protein loop of 50 amino acids located between β3 and β4 is disordered and mobile on the subnanosecond time scale. ATP binds with an affinity constant of (1.2 ± 0.1) × 10⁴ m⁻¹ and exchanges with a rate of the order of 1 × 10³ s⁻¹. The ATP-binding cavity is considerably affected by the presence of the ligand, resulting in a more compact conformation in the ATP-bound than in the ATP-free form. This structural variation is due to the movement of the α1-α2 and β2-β3 loops, both of which are highly conserved in copper(I)-transporting P_IB-type ATPases. The present structure reveals a characteristic binding mode of ATP within the protein scaffold of the copper(I)-transporting P_IB-type ATPases with respect to the other P-type ATPases. In particular, the binding cavity contains mainly hydrophobic aliphatic residues, which are involved in van der Waal''s interactions with the adenine ring of ATP, and a Glu side chain, which forms a crucial hydrogen bond to the amino group of ATP.     

Roles of Interaction between Actuator and Nucleotide Binding Domains of Sarco(endo)plasmic Reticulum Ca2+-ATPase as Revealed by Single and Swap Mutational Analyses of Serine 186 and Glutamate 439

Roles of hydrogen bonding interaction between Ser¹⁸⁶ of the actuator (A) domain and Glu⁴³⁹ of nucleotide binding (N) domain seen in the structures of ADP-insensitive phosphorylated intermediate (E2P) of sarco(endo)plasmic reticulum Ca²⁺-ATPase were explored by their double alanine substitution S186A/E439A, swap substitution S186E/E439S, and each of these single substitutions. All the mutants except the swap mutant S186E/E439S showed markedly reduced Ca²⁺-ATPase activity, and S186E/E439S restored completely the wild-type activity. In all the mutants except S186E/E439S, the isomerization of ADP-sensitive phosphorylated intermediate (E1P) to E2P was markedly retarded, and the E2P hydrolysis was largely accelerated, whereas S186E/E439S restored almost the wild-type rates. Results showed that the Ser¹⁸⁶-Glu⁴³⁹ hydrogen bond stabilizes the E2P ground state structure. The modulatory ATP binding at sub-mm∼mm range largely accelerated the EP isomerization in all the alanine mutants and E439S. In S186E, this acceleration as well as the acceleration of the ATPase activity was almost completely abolished, whereas the swap mutation S186E/E439S restored the modulatory ATP acceleration with a much higher ATP affinity than the wild type. Results indicated that Ser¹⁸⁶ and Glu⁴³⁹ are closely located to the modulatory ATP binding site for the EP isomerization, and that their hydrogen bond fixes their side chain configurations thereby adjusts properly the modulatory ATP affinity to respond to the cellular ATP level.Sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a)² is a representative member of P-type ion-transporting ATPases and catalyzes Ca²⁺ transport coupled with ATP hydrolysis (Fig. 1) (1–9). In the catalytic cycle, the enzyme is activated by binding of two Ca²⁺ ions at the transport sites (E2 to E1Ca₂, steps 1–2) and then autophosphorylated at Asp³⁵¹ with MgATP to form ADP-sensitive phosphoenzyme (E1P, step 3), which can react with ADP to regenerate ATP. Upon formation of this EP, the bound Ca²⁺ ions are occluded in the transport sites (E1PCa₂). The subsequent isomeric transition to ADP-insensitive form (E2P) results in a change in the orientation of the Ca²⁺ binding sites and reduction of their affinity, and thus Ca²⁺ release into lumen (steps 4 and 5). Finally, the hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca²⁺-unbound form (E2, step 6). E2P can also be formed from P_i in the presence of Mg²⁺ and the absence of Ca²⁺ by reversal of its hydrolysis.Open in a separate windowFIGURE 1.Reaction cycle of sarco(endo)plasmic reticulum Ca²⁺-ATPase.The cytoplasmic three domains N, A, and P largely move and change their organization states during the Ca²⁺ transport cycle (10–22). These changes are linked with the rearrangements in the transmembrane helices. In the EP isomerization (loss of ADP sensitivity) and Ca²⁺ release, the A domain largely rotates (by ∼110° parallel to membrane plane), intrudes into the space between the N and P domains, and the P domain largely inclines toward the A domain. Thus in E2P, these domains produce the most compactly organized state (see Fig. 2 for the change E1Ca₂·AlF₄⁻·ADP →E2·MgF₄²⁻ as the model for the overall process E1PCa₂·ADP^‡ → E2·P_i).Open in a separate windowFIGURE 2.Structure of SERCA1a and formation of Ser¹⁸⁶-Glu⁴³⁹ hydrogen bond between the A and N domains. The coordinates for the structures E1Ca₂·AlF₄⁻·ADP, (the analog for the transition state of the phosphoryl transfer E1PCa₂·ADP^‡, left panel) and E2·MgF₄²⁻ (E2·P_i analog (21), right panel) of Ca²⁺-ATPase were obtained from the Protein Data Bank (PDB accession code 1T5T and 1WPG, respectively (12, 14)). The arrows indicate approximate movements of the A and P domains in the change from E1Ca₂·AlF₄⁻ ·ADP to E2·MgF₄²⁻. Ser¹⁸⁶ and Glu⁴³⁹ are depicted as van der Waals spheres. These two residues form a hydrogen bond in E2·MgF₄²⁻ (see inset). The phosphorylation site Asp³⁵¹, two Ca²⁺ at the transport sites and ADP with AlF₄⁻ at the catalytic site in E1Ca₂·AlF₄⁻·ADP, MgF₄²⁻ bound at the catalytic site in E2·MgF₄²⁻ are depicted. The TGES¹⁸⁴ loop and Val²⁰⁰ loop of the A domain and Tyr¹²² on the top part of M2 are shown. These elements produce three interaction networks between A and P domains and M2 (Tyr¹²²) in E2·MgF₄²⁻ (23–26). M1′ and M1-M10 are also indicated.We have found that the interactions between the A and P domains at the Val²⁰⁰-loop (Asp¹⁹⁶-Asp²⁰³) with the residues of the P domain (Arg⁶⁷⁸/Glu⁶⁸⁰/Arg⁶⁵⁶/Asp⁶⁶⁰) (23) and at the Tyr¹²² hydrophobic cluster (24–26) (see Fig. 2) play critical roles for Ca²⁺ deocclusion/release in E2PCa₂ → E2P + 2Ca²⁺ after the loss of ADP sensitivity (E1PCa₂ to E2PCa₂ isomerization). The proper length of the A/M1′ linker is critical for inducing the inclining motion of the A and P domains for the Ca²⁺ deocclusion and release from E2PCa₂ (27, 28). The importance of the interdomain interaction between Arg⁶⁷⁸ (P) and Asp²⁰³ (A) in stabilizing the E2P and E2 intermediates and its influence on modulatory ATP activation were pointed out by the mutation R678A (29). Regarding the N domain, the importance of Glu⁴³⁹ in the EP isomerization and E2P hydrolysis was previously noted by its alanine substitution, and possible importance of its interaction with Ser¹⁸⁶ on the A domain has been suggested since Glu⁴³⁹ forms a hydrogen bond with Ser¹⁸⁶ in the E2P analog structures (29) (see Fig. 2). The Darier disease-causing mutations of Ser¹⁸⁶ of SERCA2b, S186P and S186F also alter the kinetics of the EP processing and its importance as the residue in the immediate vicinity of TGES¹⁸⁴ has been pointed out (30, 31). Notably also, Glu⁴³⁹ is situated near the adenine binding pocket and its importance in the ATP binding and ATP-induced structural change have been shown (32, 33). In the structure E2(TG)AMPPCP (E2·ATP), Glu⁴³⁹ interacts with the modulatory ATP binding via Mg²⁺, and is involved in the acceleration of the Ca²⁺-ATPase cycle (16).Considering these critical findings on each of Glu⁴³⁹ and Ser¹⁸⁶, it is crucial to reveal the role of the Ser¹⁸⁶-Glu⁴³⁹ hydrogen-bonding interaction between the A and N domains in the EP processing and its ATP modulation (i.e. regulatory ATP-induced acceleration). We therefore made a series of mutants on both Ser¹⁸⁶ and Glu⁴³⁹ including the swap substitution mutant, S186A, E439A, S186A/E439A, S186E, E439S, S186E/E439S, and explored their kinetic properties. Results showed that the Ser¹⁸⁶-Glu⁴³⁹ hydrogen bond is critical for the stabilization of the E2P ground state structure, and possibly functioning as to make the E2P resident time long enough for Ca²⁺ release (E2PCa₂ → E2P + 2Ca²⁺) thus to avoid its hydrolysis without Ca²⁺ release. Results also revealed that the side-chain configurations of Ser¹⁸⁶ and Glu⁴³⁹ are fixed by their hydrogen bond, thereby conferring the proper modulatory ATP binding to occur at the cellular ATP level to accelerate the rate-limiting EP isomerization.     

CGL160-mediated recruitment of the coupling factor CF1 is required for efficient thylakoid ATP synthase assembly,photosynthesis, and chloroplast development in Arabidopsis

Chloroplast ATP synthases consist of a membrane-spanning coupling factor (CF_O) and a soluble coupling factor (CF₁). It was previously demonstrated that CONSERVED ONLY IN THE GREEN LINEAGE160 (CGL160) promotes the formation of plant CF_O and performs a similar function in the assembly of its c-ring to that of the distantly related bacterial Atp1/UncI protein. Here, we show that in Arabidopsis (Arabidopsis thaliana) the N-terminal portion of CGL160 (AtCGL160N) is required for late steps in CF₁-CF_O assembly. In plants that lacked AtCGL160N, CF₁-CF_O content, photosynthesis, and chloroplast development were impaired. Loss of AtCGL160N did not perturb c-ring formation, but led to a 10-fold increase in the numbers of stromal CF₁ subcomplexes relative to that in the wild type. Co-immunoprecipitation and protein crosslinking assays revealed an association of AtCGL160 with CF₁ subunits. Yeast two-hybrid assays localized the interaction to a stretch of AtCGL160N that binds to the DELSEED-containing CF₁-β subdomain. Since Atp1 of Synechocystis (Synechocystis sp. PCC 6803) could functionally replace the membrane domain of AtCGL160 in Arabidopsis, we propose that CGL160 evolved from a cyanobacterial ancestor and acquired an additional function in the recruitment of a soluble CF₁ subcomplex, which is critical for the modulation of CF₁-CF_O activity and photosynthesis.

The green-lineage specific N-terminal domain of CGL160 recruits coupling factor 1 and its lack affects chloroplast development, photosynthesis and thylakoid ATP synthase assembly in Arabidopsis.

IN A NUTSHELL Background: Thylakoid ATP synthases are impressive molecular engines that harness the light-driven proton gradient to generate ATP during photosynthesis. Their molecular mode of operation and atomic structure have been elucidated, but their assembly process is still under investigation. Specific auxiliary factors assist in ATP synthase assembly and prevent the accumulation of dead-end products or deleterious intermediates. CGL160 is one such factor and consists of a membrane and an N-terminal domain. The membrane domain of CGL160 is distantly related to bacterial Atp1 proteins, which are also present in cyanobacteria. Previous studies demonstrated that CGL160 promotes efficient formation of the membranous c-ring of thylakoid ATP synthases in Arabidopsis thaliana. Question: What is the function of the green lineage-specific N-terminal domain of CGL160 in thylakoid ATP synthase assembly, and what is the evolutionary relationship between CGL160 and Atp1? Findings: Here, we showed that the N-terminal domain of CGL160 is required for the late steps in thylakoid ATP synthase assembly and recruits the stromal ATP synthase intermediate coupling factor CF₁. The assembly step is critical for chloroplast development in the dark, ATP synthase activity, and photosynthesis in A. thaliana. We also revealed that Atp1 from the cyanobacterium Synechocystis spec PCC 6803 could functionally replace the membrane domain of CGL160 in A. thaliana. These results indicated that Atp1 operates in c-ring assembly in cyanobacteria and that CGL160 evolved from its cyanobacterial ancestor Atp1. However, CGL160 acquired an additional function in linking a soluble ATP synthase intermediate to a membranous subcomplex. Next steps: The next steps are to identify all auxiliary factors required for the assembly of thylakoid ATP synthases and to understand their precise function in ATP synthase formation. Detailed knowledge of the factors and the assembly process could provide elegant strategies for adjusting proton circuits and altering the ATP budget in crops or other photosynthetic organisms.