Decoding P4-ATPase substrate interactions

Cellular membranes display a diversity of functions that are conferred by the unique composition and organization of their proteins and lipids. One important aspect of lipid organization is the asymmetric distribution of phospholipids (PLs) across the plasma membrane. The unequal distribution of key PLs between the cytofacial and exofacial leaflets of the bilayer creates physical surface tension that can be used to bend the membrane; and like Ca²⁺, a chemical gradient that can be used to transduce biochemical signals. PL flippases in the type IV P-type ATPase (P4-ATPase) family are the principle transporters used to set and repair this PL gradient and the asymmetric organization of these membranes are encoded by the substrate specificity of these enzymes. Thus, understanding the mechanisms of P4-ATPase substrate specificity will help reveal their role in membrane organization and cell biology. Further, decoding the structural determinants of substrate specificity provides investigators the opportunity to mutationally tune this specificity to explore the role of particular PL substrates in P4-ATPase cellular functions. This work reviews the role of P4-ATPases in membrane biology, presents our current understanding of P4-ATPase substrate specificity, and discusses how these fundamental aspects of P4-ATPase enzymology may be used to enhance our knowledge of cellular membrane biology.     

Phospholipid‐flipping activity of P4‐ATPase drives membrane curvature

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

Partitioning of the plasma membrane Ca2+-ATPase into lipid rafts in primary neurons: effects of cholesterol depletion

Spatial and temporal alterations in intracellular calcium [Ca(2+)](i) play a pivotal role in a wide array of neuronal functions. Disruption in Ca(2+) homeostasis has been implicated in the decline in neuronal function in brain aging and in neurodegenerative disorders. The plasma membrane Ca(2+)-ATPase (PMCA) is a high affinity Ca(2+) transporter that plays a crucial role in the termination of [Ca(2+)](i) signals and in the maintenance of low [Ca(2+)](i) essential for signaling. Recent evidence indicates that PMCA is uniquely sensitive to its lipid environment and is stimulated by lipids with ordered acyl chains. Here we show that both PMCA and its activator calmodulin (CaM) are partitioned into liquid-ordered, cholesterol-rich plasma membrane microdomains or 'lipid rafts' in primary cultured neurons. Association of PMCA with rafts was demonstrated in preparations isolated by sucrose density gradient centrifugation and in intact neurons by confocal microscopy. Total raft-associated PMCA activity was much higher than the PMCA activity excluded from these microdomains. Depletion of cellular cholesterol dramatically inhibited the activity of the raft-associated PMCA with no effect on the activity of the non-raft pool. We propose that association of PMCA with rafts represents a novel mechanism for its regulation and, consequently, of Ca(2+) signaling in the central nervous system.     

Characterization of native and reconstituted plasma membrane H+-ATPase from the plasma membrane ofBeta vulgaris

Summary Characteristics of the native and reconstituted H⁺-ATPase from the plasma membrane of red beet (Beta vulgaris L.) were examined. The partially purified, reconstituted H⁺-ATPase retained characteristics similar to those of the native plasma membrane H⁺-ATPase following reconstitution into proteoliposomes. ATPase activity and H⁺ transport of both enzymes were inhibited by vanadate, DCCD, DES and mersalyl. Slight inhibition of ATPase activity associated with native plasma membranes by oligomycin, azide, molybdate or NO ₃ ^– was eliminated during solubilization and reconstitution, indicating the loss of contaminating ATPase activities. Both native and reconstituted ATPase activities and H⁺ transport showed a pH optimum of 6.5, required a divalent cation (Co²⁺>Mg²⁺>Mn²⁺>Zn²⁺>Ca²⁺), and preferred ATP as substrate. The Mg:ATP kinetics of the two ATPase activities were similar, showing simple Michaelis-Menten kinetics. Saturation occurred between 3 and 5mM Mg: ATP, with aK _m of 0.33 and 0.46mM Mg: ATP for the native and reconstituted enzymes, respectively. The temperature optimum for the ATPase was shifted from 45 to 35°C following reconstitution. Both native and reconstituted H⁺-ATPases were stimulated by monovalent ions. Native plasma membrane H⁺-ATPase showed an order of cation preference of K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Cs⁺>Li⁺>choline⁺. This basic order was unchanged following reconstitution, with K⁺, NH ₄ ⁺ , Rb⁺ and Cs⁺ being the preferred cations. Both enzymes were also stimulated by anions although to a lesser degree. The order of anion preference differed between the two enzymes. Salt stimulation of ATPase activity was enhanced greatly following reconstitution. Stimulation by KCl was 26% for native ATPase activity, increasing to 228% for reconstituted ATPase activity. In terms of H⁺ transport, both enzymes required a cation such as K⁺ for maximal transport activity, but were stimulated preferentially by Cl^– even in the presence of valinomycin. This suggests that the stimulatory effect of anions on enzyme activity is not simply as a permeant anion, dissipating a positive interior membrane potential, but may involve a direct anion activation of the plasma membrane H⁺-ATPase.     

胡杨愈伤组织质膜的两相分离法及其H~+-ATPase的特性

以胡杨愈伤组织为材料,用PEG3350/DextranT500构成的两相系统提取质膜微囊,研究质膜H+-AT-Pase的特性。结果显示:由6.3%PEG3350、6.3%DextranT500、KCl、磷酸缓冲液(pH7.8)和蔗糖构成的两相系统提取膜微囊的H+-ATPase活性分别被Na3VO4、KNO3、NaN3抑制了约75%、2.6%和1.3%。方向性检测显示原位膜微囊占提取质膜微囊的90%,翻转膜微囊仅占10%。去垢剂对质膜H+-ATPase活性的影响说明0.015%的TritonX-100和0.01%～0.1%的Brij58适用于测定质膜H+-ATPase活性。Lineweaver-Burk动力学分析该酶的Km值为0.65mmol·L-1,Vmax为37.59μmolPi·mg-1protein·h-1。研究结果表明:两相法提取的质膜微囊主要是正向密闭的膜微囊;胡杨愈伤组织质膜H+-ATPase的最适pH为6.5,最适温度为37℃左右。     

A protein activator of the plasma membrane Ca++-ATPase of heart sarcolemma

A detergent extract of dog or beef heart sarcolemmal vesicles was prepared and found to have a stimulatory effect on the Ca⁺⁺-ATPase of plasma membranes from human erythrocyte and cardiac sarcolemma. A procedure is described which enriches the activating fraction. The protein nature of the preparation is illustrated by its sensitivity to boiling and to the proteolytic enzyme(s) trypsin and chymotrypsin. SDS polyacrylamide gels indicate that the protein(s) involved have a molecular weight of 56 and 60 kDa. The sarcolemmal activator can stimulate the Ca⁺⁺-ATPase activity of the isolated enzyme more than 100% in the presence of saturating amounts of calmodulin. The activation is calcium dependent, being greatest at approximately 10µm Ca⁺⁺, free, but does not change theK _m for Ca⁺⁺. A possible physiological role for the activator is discussed.     

Influence of growth conditions on the composition of the plasma membrane from yeast and on kinetic properties of two membrane functions

The influence of different growth conditions on the phospholipid composition and on two membrane functions, the Mg-ATPase and the purine transport system, was investigated. Addition of cholinechloride to the growth medium led to a certain rise in the amount of phosphatidylcholine, whereas supplementation with ethanolamine resulted in a considerably higher portion of phosphatidylethanolamine. When yeast cells were cultured at lower temperatures we found more short-chain fatty acids with a higher content of monounsaturated chains as compared to higher growth temperatures. Addition of paraquat, a herbicide which enhances lipid peroxidation by free radicals, reduced the amount of unsaturated fatty acids without influencing their chain length. The altered membrane composition had no influence on the basic mechanism of interaction between ATPase, MgATP, and free Mg2+ ions. However, several kinetic constants such as Km, Vmax, Ka, and especially Ki were influenced to some extent. Whereas the affinity of the purine transport system to its substrate was not significantly changed by the growth conditions, an effect on Vmax could be seen. Lower growth temperatures clearly led to higher maximal uptake velocities. The presence of paraquat during growth resulted in a considerable decrease of Vmax.     

Effects of air pollutants on proton and sucrose transport at the plasma membrane of Ricinus communis

The effects of the air pollutants O₃, SO₂ and NO₂ on aspects of sucrose/proton cotransport across the plasma membrane of Ricinus communis plants have been investigated. The H⁺-ATPase hydrolytic activity in cotyledon plasma membrane vesicles purified by phase partitioning showed small stimulations by Na₂SO₃ or NaNO₃ added separately or together to the assay medium. ATPase activity from plants pretreated by fumigation with SO₂ or O₃ also showed an increase, the effect of O₃ being quite marked. Plasma membrane H⁺-pumping in KI-treated microsomal fractions and medium acidification by intact cotyledons both showed small decreases in the presence of Na₂SO₃ or NaNO₂. Both Na₂SO₃ and NaNO₂ at high concentrations (2 mol m^–3) had significant effects on sucrose uptake by intact cotyledons, although sucrose efflux was unaffected. No significant effects on sucrose uptake or efflux by intact cotyledons were observed in plants pretreated by fumigation with SO₂ or O₃. Proton-coupled sucrose transport in isolated plasma membrane vesicles was inhibited in the presence of Na₂SO₃ or NaNO₂. However, both pollutants also significantly inhibited the uptake of acetate by the vesicles, indicating a dissipation of the pH gradient across the membrane. It was concluded that no specific aspect of the sucrose/proton cotransport mechanism was damaged by these air pollutants, and that the effects of these pollutants on carbohydrate partitioning are more likely to be due to general effects on membrane integrity or on other aspects such as leaf carbohydrate metabolism.     

Sterol transfer,PI4P consumption,and control of membrane lipid order by endogenous OSBP

The network of proteins that orchestrate the distribution of cholesterol among cellular organelles is not fully characterized. We previously proposed that oxysterol‐binding protein (OSBP) drives cholesterol/PI4P exchange at contact sites between the endoplasmic reticulum (ER) and the trans‐Golgi network (TGN). Using the inhibitor OSW‐1, we report here that the sole activity of endogenous OSBP makes a major contribution to cholesterol distribution, lipid order, and PI4P turnover in living cells. Blocking OSBP causes accumulation of sterols at ER/lipid droplets at the expense of TGN, thereby reducing the gradient of lipid order along the secretory pathway. OSBP consumes about half of the total cellular pool of PI4P, a consumption that depends on the amount of cholesterol to be transported. Inhibiting the spatially restricted PI4‐kinase PI4KIIIβ triggers large periodic traveling waves of PI4P across the TGN. These waves are cadenced by long‐range PI4P production by PI4KIIα and PI4P consumption by OSBP. Collectively, these data indicate a massive spatiotemporal coupling between cholesterol transport and PI4P turnover via OSBP and PI4‐kinases to control the lipid composition of subcellular membranes.     

Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris

Summary A vanadate-sensitive H⁺-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H⁺-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H⁺ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H⁺ transport activity was inhibited half-maximally between 1 and 5 M vanadate (Na₃VO₄) and nearly abolished by 100 M vanadate. ATPase activity of native plasma membrane showed aK _i for vanadate inhibition of 9.5 M, and was inhibited up to 80% by 15 to 20 M vanadate (Na₃VO₄). ATPase activity of the reconstituted vesicles showed aK _i of 2.6 M for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme.     

铝处理下小麦幼苗根系膜脂过氧化作用 和质膜微囊ATP酶活性的变化

采用水培的方法研究了Al对小麦（Triticum aestivum L.cv Yangmai No.5）幼苗的生长、根尖组织膜脂过氧化作用、保护酶的活性和质膜结合酶H     

The kinetics of P515 in relation to the lipid composition of the thylakoid membrane

Flash-induced P515 absorbance changes have been studied in dark-adapted chloroplasts isolated from spinach plants grown under two different light intensities. The slow component (reaction 2), normally present in the P515 response of chloroplasts isolated from plants grown at an intensity of 60 W · m^–2, was largely reduced in chloroplasts isolated from plants grown at an intensity of 6 W · m^–2. This reduction of the slow component in the P515 response appeared to be coincident with an alteration in the lipid composition of the thylakoid membrane. Mainly the ratio monogalactosyldiacylglycerol to digalactosyldiacylglycerol appeared to be altered. In thylakoids from plants grown at 6 W · m^–2, the ratio was approximately 35% lower than that of plants grown at 60 W · m^–2. The amount of both cytochromeb ₅₆₃ and cytochromef was largely reduced in chloroplasts isolated from plants grown at low light intensity. These results may indicate a possible correlation between structural organization of the thylakoid membrane and the kinetics of the flash-induced P515 response.     

RNA--induced silencing of the plasma membrane Ca2+-ATPase 2 in neuronal cells: effects on Ca2+ homeostasis and cell viability

Intraneuronal calcium ([Ca(2+)](i)) regulation is altered in aging brain, possibly because of the changes in critical Ca(2+) transporters. We previously reported that the levels of the plasma membrane Ca(2+)-ATPase (PMCA) and the V(max) for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNA(i) techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro--transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca(2+)](i) revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca(2+)](i), slower recovery from KCl-induced Ca(2+) transients, and incomplete return to pre-stimulation Ca(2+) levels. Primary neurons and SH-SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca(2+) signaling and enhanced susceptibility to stresses that can alter the regulation of Ca(2+) homeostasis.     

Novel GFP-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane

Phosphatidylinositol-4-phosphate (PI4P) plays a crucial role in cellular functions, including protein trafficking, and is mainly located in the cytoplasmic surface of intracellular membranes, which include the trans-Golgi network (TGN) and the plasma membrane. However, many PI4P-binding domains of membrane-associated proteins are localized only to the TGN because of the requirement of a second binding protein such as ADP-ribosylation factor 1 (ARF1) in order to be stably localized to the specific membrane. In this study, we developed new probes that were capable of detecting PI4P at the plasma membrane using the known TGN-targeting PI4P-binding domains. The PI4P-specific binding pleckstrin homology (PH) domain of various proteins including CERT, OSBP, OSH1, and FAPP1 was combined with the N-terminal moderately hydrophobic domain of the short-form of Aplysia phosphodiesterase 4 (S(N30)), which aids in plasma membrane association but cannot alone facilitate this association. As a result, we found that the addition of S(N30) to the N-terminus of the GFP-fused PH domain of OSBP (S(N30)-GFP-OSBP-PH), OSH1 (S(N30)-GFP-OSH1-PH), or FAPP1 (S(N30)-GFP-FAPP1-PH) could induce plasma membrane localization, as well as retain TGN localization. The plasma membrane localization of S(N30)-GFP-FAPP1-PH is mediated by PI4P binding only, whereas those of S(N30)-GFP-OSBP-PH and S(N30)-GFP-OSH1-PH are mediated by either PI4P or PI(4,5)P₂ binding. Taken together, we developed new probes that detect PI4P at the plasma membrane using a combination of a moderately hydrophobic domain with the known TGN-targeting PI4P-specific binding PH domain.     

Cercospora beticola toxins. IX. Relationship between structure of beticolins, inhibition of plasma membrane H+-ATPase and partition in lipid membranes

Beticolins are yellow toxins produced by the fungus Cercospora beticola . The effect of one of them, beticolin-1. has been investigated on corn root plasma membrane H⁺-ATPase (EC 3.6.1.35) at different purification levels (plasma membrane fraction, partially, or highly purified enzyme). The results obtained demonstrated that (1) the purified proton pump was inhibited directly by low amounts of the toxin (I₅₀= 1.62 ± 0.18 μM), (2) the biological effects of beticolin-1 were similar to those of CBT ( Cercospora beticola toxin). Furthermore, it was established that the efficiency of the different beticolins was clearly related to their ability to interact with the lipid bilayers, determined by fluorometric studies: the toxins that exhibited the lower I₅₀ (50% inhibitory concentrations) values were the molecules that had the lowest partition coefficient to liposomes.