Plant proton pumps

Chemiosmotic circuits of plant cells are driven by proton (H(+)) gradients that mediate secondary active transport of compounds across plasma and endosomal membranes. Furthermore, regulation of endosomal acidification is critical for endocytic and secretory pathways. For plants to react to their constantly changing environments and at the same time maintain optimal metabolic conditions, the expression, activity and interplay of the pumps generating these H(+) gradients have to be tightly regulated. In this review, we will highlight results on the regulation, localization and physiological roles of these H(+)- pumps, namely the plasma membrane H(+)-ATPase, the vacuolar H(+)-ATPase and the vacuolar H(+)-PPase.     

Phytohormone effects on hydrolytic activity of phosphohydrolases in red beet (Beta vulgaris L.) tonoplasts

The effects of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellic acid (GA₃) and kinetin on the hydrolytic activity of proton pumps (adenosine triphosphatase, H⁺-ATPase, pyrophosphatase, H⁺-PPase) of tonoplasts isolated from stored red beet (Beta vulgaris L. cv. Bordo) roots were studied. Results suggest that the phytohormones can regulate the hydrolytic activities of H⁺-ATPase and H⁺-PPase of the vacuolar membrane. Each of the proton pumps of the tonoplast has its own regulators in spite of similar localization and functions. IAA and kinetin seem to be regulators of the hydrolytic activity for H⁺-PPase whereas for H⁺-ATPase it may be GA₃. Stimulation of enzyme activity by all hormones occurred at concentrations of 10^–6 to 10^–7 M.Abbreviations IAA indole-3-acetic acid - ABA abscisic acid - GA₃ gibberellic acid - H⁺-ATPase adenosine triphosphatase - H⁺-PPase pyrophosphatase - ATP adenosine triphosphate - Tris Tris (hydroxymethyl)-aminomethane - MES (2[N-Morpholino]) ethane sulfonic acid - EDTA ethylene diamine tetraacetic acid - P_i inorganic phosphate     

Response of the plant root to aluminum stress: Analysis of the inhibition of the root elongation and changes in membrane function

Pea root elongation was strongly inhibited in the presence of a low concentration of Al (5 μM). In Al-treated root, the epidermis was markedly injured and characterized by an irregular layer of cells of the root surface. Approximately 30% of total absorbed Al accumulated in the root tip and Al therein was found to cause the inhibition of whole root elongation. Increasing concentrations of Ca²⁺ effectively ameliorated the inhibition of root elongation by Al and 1 mM of CaCl₂ completely repressed the inhibition of root elongation by 50 μM Al. The ameliorating effect of Ca²⁺ was due to the reduction of Al uptake. H⁺-ATPase and H⁺-PPase activity as well as ATP and PPidependent H⁺ transport activity of vacuolar membrane vesicles prepared from barley roots increased to a similar extent by the treatment with 50 μM AlCl₃. The rate of increase of the amount of H⁺-ATPase and H⁺-PPase was proportional to that of protein content measured by immunoblot analysis with antibodies against the catalytic subunit of the vacuolar H⁺-ATPase and H⁺-PPase of mung bean. The increase of both activities was discussed in relation to the physiological tolerance mechanism of barley root against Al stress.     

Activation and significance of vacuolar H+-ATPase in Saccharomyces cerevisiae adaptation and resistance to the herbicide 2,4-dichlorophenoxyacetic acid

The stimulation of the activity of the H(+)-ATPase present in the vacuolar membrane (V-ATPase) of Saccharomyces cerevisiae is here described in response to a moderate stress induced by 2,4-dichlorophenoxyacetic acid (2,4-D). This in vivo activation (up to 5-fold) took place essentially during the adaptation period, preceding cell division under herbicide stress, in coordination with a marked activation of plasma membrane H(+)-ATPase (PM-ATPase) (up to 30-fold) and the decrease of intracellular and vacuolar pH values, suggesting that activation may be triggered by acidification. Single deletion of VMA1 and genes encoding other V-ATPase subunits led to a more extended period of adaptation and to slower growth under 2,4-D stress. Results suggest that a functional V-ATPase is required to counteract, more rapidly and efficiently, the dissipation of the physiological H(+)-gradient across vacuolar membrane registered during 2,4-D adaptation.     

Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast

Nitric oxide (NO), an endogenous signaling molecule in animals and plants, mediates responses to abiotic and biotic stresses. Our previous work demonstrated that 100 μM sodium nitroprusside (SNP, an NO donor) treatment of maize seedlings increased K⁺ accumulation in roots, leaves and sheathes, while decreasing Na⁺ accumulation (Zhang et al. in J Plant Physiol Mol Biol 30:455–459, 2004b). Here we investigate how NO regulates Na⁺, K⁺ ion homeostasis in maize. Pre-treatment with 100 μM SNP for 2 days improved later growth of maize plants under 100 mM NaCl stress, as indicated by increased dry matter accumulation, increased chlorophyll content, and decreased membrane leakage from leaf cells. An NO scavenger, methylene blue (MB-1), blocked the effect of SNP. These results indicated that SNP-derived NO enhanced maize tolerance to salt stress. Further analysis showed that NaCl induced a transient increase in the NO level in maize leaves. Both NO and NaCl treatment stimulated vacuolar H⁺-ATPase and H⁺-PPase activities, resulting in increased H⁺-translocation and Na⁺/H⁺ exchange. NaCl-induced H⁺-ATPase and H⁺-PPase activities were diminished by MB-1. 1-Butanol, an inhibitor of phosphatidic acid (PA) production by phospholipase D (PLD), reduced NaCl- and NO-induced H⁺-ATPase activation. In contrast, applied PA stimulated H⁺-ATPase activity. These results suggest that NO acts as a signal molecule in the NaCl response by increasing the activities of vacuolar H⁺-ATPase and H⁺-PPase, which provide the driving force for Na⁺/H⁺ exchange. PLD and PA play an important role in this process.     

Protective effect of exogenous polyamines on root tonoplast function against salt stress in barley seedlings

Salinity stress is one of the most serious factors limiting the productivity of agricultural crops. A possible survival strategy of plants under saline conditions is to sequester excess Na⁺ in the vacuole by vacuolar Na⁺/H⁺ antiport using a pH gradient generated by H⁺-ATPasc (EC 3.6.1.35) and H⁺-Pyrophosphatase (H⁺-PPase; EC 3.6.1.1) to maintain a higher K⁺/Na⁺ ratio in cytoplasm. The effect of exogenously applied polyamines (PAs) in stabilizing root tonoplast integrity and function against salt stress in the barley (Hordeum vulgare L.) seedlings was investigated. The NaCl-induced reductions in the contents of phospholipids and PAs in tonoplast vesicles isolated from barely seedling roots, as well as the activities of H⁺-ATPase, H⁺-PPase and vacuolar Na⁺/H⁺ antiport were all partially restored by the application of 0.5 mM putrescine and 0.5 mM spermidine, especially the former. The above results indicated that one of the mechanisms involved in attenuating salt injury in barley seedlings by exogenous PAs application was to maintain tonoplast integrity and function under saline conditions. Moreover, the possible mechanism involved in counteracting detrimental effects of salt on the barley seedlings by the application of exogenous PAs was discussed.     

Alterations in the plasma membrane polypeptide pattern of tomato roots (Lycopersicon esculentum) during the development of arbuscular mycorrhiza

Changes induced by arbuscular mycorrhizal (AM) formation in the plasma membrane polypeptide pattern of tomato roots have been assessed by 2D-PAGE analysis. Plasma membrane fractions were isolated by aqueous two-phase partitioning from control and mycorrhizal tomato root microsomes. Analysis of 2D-PAGE gels revealed that AM colonization induces at the plasma membrane level two major changes in protein synthesis: down-regulation of some constitutive polypeptides and synthesis of new polypeptides or endomycorrhizins. A comparison of changes induced by two different levels of AM colonization showed that 16 polypeptides were differentially displayed at both AM colonization stages, while some others were transiently regulated. Five of the differentially displayed plasma membrane polypeptides at both AM colonization stages were selected for N-terminal amino acid sequencing. Reliable sequences were obtained for two of the selected spots. Sequence alignment search indicated that one of the sequenced polypeptides showed 75% identity to the N-terminal sequence of the 69 kDa catalytic subunit of the vacuolar type H(+)-ATPase of several plants. The possible significance of these findings is discussed in relation to the functioning of the AM symbiosis.     

丛枝菌根真菌对植物根尖分生区和根冠细胞的侵染*

一般说来,从枝菌根（ＡＭ）真菌大多数是从植物根系根毛区（成熟区）侵入和扩展的,在显微镜下往往看不到根尖分生区和根冠表皮细胞被ＡＭ真菌侵染的特征。这就很容易给人们造成一种假象,似乎ＡＭ真菌不能侵染根尖分生区和根冠表皮细胞,即它们对ＡＭ真菌是免疫的。然而笔者多次于显微镜下看到ＡＭ真菌侵染根尖分生区和根冠表皮细胞,并形成典型的泡囊、丛枝、菌丝等结构。这一现象导致作者在温室盆栽和大田条件下研究了玫瑰红巨孢囊霉（ Ｇｉｇａｓｐｏｒａ　ｒｏｓｅａ Ｎｉｃｏｌ　＆ Ｓｃｈｅｎｃｋ）、珠状巨孢囊霉（Ｇｉｇａｓｐｏｒａ　ｍａｒｇａｒｉｔａ　Ｂｅｃｋｅｒ　＆　Ｈａｌｌ）、根内球囊霉（Ｇｌｏｍｕｓ　ｏｍｔｒａｒａｄｉｃｅｓ　ｓｃｈｅｎｃｋ　＆　Ｓｍｉｔｈ、摩西球囊霉（Ｇｌｏｍｕｓ ｍｏｓｓｅａｅ （Ｎｉｃｏｌ　＆ Ｇｅｒｄ．） Ｇｅｒｄｅｍａｎｎ　＆ Ｔｒａｐｐｅ）、地表球囊霉（ Ｇｌｏｍｕｓ ｖｅｒｓｉｆｏｒｍｅ（ Ｋａｒｓｔｅｎ）Ｂｅｒｃｈ）和弯丝硬囊霉（ Ｓｃｌｅｒｏｃｙｓｔｉｓ　ｓｉｎｕｏｓａ　Ｇｅｒｄｅｍａｎｎ　＆ Ｂａｋｈｉ）对棉花（Ｇｏｓｓｙｐｉｕｍ　ｈｉｒｓｕｔｕｍ　Ｌ．）、烟草（Ｎｉｃｏｔｉａｎａ　 ｔａｂａｃｕｍ Ｌ．）和白     

Increased Polyamines Conjugated to Tonoplast Vesicles Correlate with Maintenance of the H<Superscript>+</Superscript>-ATPase and H<Superscript>+</Superscript>-PPase Activities and Enhanced Osmotic Stress Tolerance in Wheat

The effects of osmotic stress on H⁺-ATPase and H⁺-PPase activities and the levels of covalently conjugated polyamines (CC-PAs) and noncovalently conjugated polyamines (NCC-PAs) were investigated using tonoplast vesicles isolated from the roots of wheat (Triticum aestivum L.) seedlings differing in drought-tolerance. The results showed that after polyethylene glycol (PEG) 6,000 (–0.55MPa) treatment for 7 days, seedling leaf relative water content (LRWC), relative dry weight increase rate (RDWIR) and root H⁺-ATPase and H⁺-PPase activities from the drought-sensitive cultivar Yangmai No. 9 decreased more markedly than those from the drought-tolerant cultivar Yumai No. 18. At the same time, the increase of the NCC-spermidine (NCC-Spd) and CC-putrescine (CC-Put) levels in root tonoplast vesicles from Yumai No. 18 was more obvious than that from Yangmai No. 9. Exogenous Spd treatment alleviated osmotic stress injury to Yangmai No. 9 seedlings, coupled with marked increases of tonoplast NCC-Spd levels and H⁺-ATPase and H⁺-PPase activities. Treatments with methylglyoxyl bis (guanyl hydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), and phenanthrolin, an inhibitor of transglutaminase (TGase), significantly inhibited the osmotically induced increases of NCC-Spd and CC-Put levels, respectively, in root tonoplast vesicles from Yumai No. 18 seedlings. Both MGBG and phenanthrolin treatments markedly promoted osmotically induced decreases of tonoplast H⁺-ATPase and H⁺-PPase activities and osmotic stress tolerance of seedlings of this cultivar. These results suggest that the NCC-Spd and CC-Put present in tonoplast vesicles isolated from wheat seedling roots might enhance the adaptation of seedlings to osmotic stress via maintenance of tonoplast H⁺-ATPase and H⁺-PPase activities.     

Arbuscular mycorrhizal colonization of tomato by Gigaspora and Glomus species in the presence of root flavonoids

The effect of flavonoids isolated from arbuscular mycorrhizal (AM) colonized and noncolonized clover roots on the number of entry points and percentage of root colonization of tomato (Lycopersicum esculentum L.) by Gigaspora rosea, Gi margarita, Glomus mosseae and G. intrarradices symbionts was determined. With fungi of both genera, a correlation between the number of entry points and the percentage of root colonization was found in the presence of some of the tested flavonoids. The flavonoids acacetin and rhamnetin, present in AM clover roots, inhibited the formation of AM penetration structures and the AM colonization of tomato roots, whereas the flavonoid 5,6,7,8,9-hydroxy chalcone, which could not be detected in AM clover root, inhibited both parameters. The flavonoid quercetin, which was present in AM clover roots, stimulated the penetration and root colonization of tomato by Gigaspora. However, the flavonoids 5,6,7,8-hydroxy-4'-methoxy flavone and 3,5,6,7,4'-hydroxy flavone, which was not found in AM clover root, increased the number of entry points and the AM colonization of tomato roots by Gigaspora. These results indicated that flavonoids could be imnplicated in the process of regulation of AM colonization in plant root, but its role is highly complex and depend not only on flavonoids, but also on AM fungal genus or even species.     

Indolacetic and humic acids induce lateral root development through a concerted plasmalemma and tonoplast H<Superscript>+</Superscript> pumps activation

Increasing evidences have indicated that humic substances can induce plant growth and productivity by functioning as an environmental source of auxinic activity. Here we comparatively evaluate the effects of indole-3-acetic acid (IAA) and humic acids (HA) isolated from two different soils (Inseptsol and Ultisol) and two different organic residues (vermicompost and sewage sludge) on root development and on activities of plasmalemma and tonoplast H⁺ pumps from maize roots. The data show that HA isolated from these different sources as well as low IAA concentrations (10⁻¹⁰ and 10⁻¹⁵ M) improve root growth through a markedly proliferation of lateral roots along with a differential activation not only of the plasmalemma but also of vacuolar H⁺-ATPases and H⁺-pyrophosphatase. Further, the vacuolar H⁺-ATPase had a peak of stimulation in a range from 10⁻⁸ to 10⁻¹⁰ M IAA, whereas the H⁺-pyrophosphatase was sensitive to a much broader range of IAA concentrations from 10⁻³ to 10⁻¹⁵ M. It is proposed a complementary view of the acid growth mechanism in which a concerted activation of the plasmalemma and tonoplast H⁺ pumps plays a key role in the root cell expansion process driven by environment-derived molecules endowed with auxinic activity, such as that of humic substances.     

V H-ATPase along the yeast secretory pathway: Energization of the ER and Golgi membranes

H⁺ transport driven by V H⁺-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca²⁺-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H⁺ transport activity of P H⁺-ATPase by more than 75% while that of V H⁺-ATPase remained unchanged. ER H⁺ ATPase exhibits higher resistance to bafilomycin (I₅₀ = 38.4 nM) than Golgi and vacuole pumps (I₅₀ = 0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H⁺ transport mediated by V H⁺-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H⁺-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.     

Effects of colchicine on the accumulation of vacuolar H+-pyrophosphatase and H+-ATPase in germinating Acacia mangium seeds and the recovery effects by sucrose, indole butyric acid and 6-benzyladenine

Plant vacuoles were isolated from cotyledons of germinatingAcacia mangium seeds, which had been treated with or withoutcolchicine, to measure vacuolar membrane pyrophosphate (PPi)- andATP-dependent H⁺ transport activities, and enzymaticactivities of H⁺-pyrophosphatase(H⁺-PPase) and H⁺-ATPase. Innon-colchicine-treated seeds, activities of the two enzymes increasedrapidly after seed germination to almost a maximal level on the seventhday. A linear function relationship exists in magnitude between PPi- orATP-dependent H⁺transport activity and its correspondingenzymatic activity. The former regression equation is: PPi-dependentH⁺ transport activity(%A.min^–1.g^–1) =–0.039 + H⁺-PPase activity(units.mg^–1) × 1.574, the latter is:ATP-dependent H⁺ transport activity(%A.min^–1.g^–1) =–0.003 + H⁺-ATPase activity(units.mg^–1) × 0.549. In colchicine-treatedseeds, activities of the two enzymes increased very slowly during 8 daysof germination and the relationship to their respectiveH⁺ transport activities was not in agreement with theabove-mentioned regression equations. PPi- and ATP-dependentH⁺ transport activities were lower than thecorresponding values calculated from H⁺-PPase activityand H⁺-ATPase activity according to the two regressionequations, respectively. However, when sucrose, indole butyric acid(IBA), or 6-benzyladenine (6-BA) were applied exogenously to the seedsfollowing colchicine treatment for 3 days, activities ofH⁺-PPase, H⁺-ATPase, PPi- andATP-dependent H⁺ transport in the 6-day-old seedlingsall increased. By statistical analysis, it was concluded that colchicineinhibits cotyledon vacuolar membrane H⁺-PPase,H⁺-ATPase activities, PPi- and ATP-dependentH⁺ transport activities during seed germination andearly seedling growth of Acacia mangium. The inhibitory effectsof colchicine could be overcome by IBA, 6-BA and sucrose to varyingdegrees.     

Overexpression of <Emphasis Type="Italic">Thellungiella halophila</Emphasis> H<Superscript>+</Superscript>-PPase (<Emphasis Type="Italic">TsVP</Emphasis>) in cotton enhances drought stress resistance of plants

An H⁺-PPase gene, TsVP from Thellungiella halophila, was transferred into two cotton (Gossypium hirsutum) varieties (Lumianyan19 and Lumianyan 21) and southern and northern blotting analysis showed the foreign gene was integrated into the cotton genome and expressed. The measurement of isolated vacuolar membrane vesicles demonstrated that the transgenic plants had higher V–H⁺-PPase activity compared with wild-type plants (WT). Overexpressing TsVP in cotton improved shoot and root growth, and transgenic plants were much more resistant to osmotic/drought stress than the WT. Under drought stress conditions, transgenic plants had higher chlorophyll content, improved photosynthesis, higher relative water content of leaves and less cell membrane damage than WT. We ascribe these properties to improved root development and the lower solute potential resulting from higher solute content such as soluble sugars and free amino acids in the transgenic plants. In this study, the average seed cotton yields of transgenic plants from Lumianyan 19 and Lumianyan 21 were significantly increased compared with those of WT after exposing to drought stress for 21 days at flowering stage. The average seed cotton yields were 51 and 40% higher than in their WT counterparts, respectively. This study benefits efforts to improve cotton yields in arid and semiarid regions.     

Hydrolytic enzymes and ability of arbuscular mycorrhizal fungi to colonize roots

The production of hydrolytic enzymes from external mycelia associated with roots and colonized soybean roots (Glycine max L.) inoculated with different arbuscular-mycorrhizal (AM) fungi of the genus GLOMUS:, and the possible relationship between these activities and the capacity of the AM fungi to colonize plant roots was studied. There were differences in root colonization and plant growth between the GLOMUS: strains, and also between two isolates of G. mosseae. Hydrolytic activities in the root and external mycelia associated with roots differed in the AM fungi tested. Correlations were only found between the endoxyloglucanase activity of the external mycelia associated with roots of the AM fungi tested and the percentage root colonization or plant growth. However, hydrolytic activities of roots colonized by the different endophytes correlated with those of external mycelia. The hydrolytic activities were not qualitatively different because the endoxyloglucanase from AM colonized roots and the external mycelia did not show a high degree of polymorphism in the different species of fungus tested. The possible role of the hydrolytic activity of external hyphae of AM fungi was discussed as a factor affecting fungal ability to colonize the root and influence plant growth.     

Physiological characteristics and regulation mechanisms of the H+ pumps in the plasma membrane and tonoplast of characean cells

The relationship between the physiological characteristics and changes in the activities of H⁺ pumps of the plasma membrane and tonoplast of characean cells is discussed. The large size of the characean internodal cells allows us to perform various experimental operations. The intracellular perfusion technique developed by Tazawaet al. (1976) is a powerful tool for analyzing the characteristics and control mechanisms of the H⁺ pumps (Tazawa and Shimmen 1987, Tazawaet al. 1987, Shimmenet al. 1994) Respiration-dependent changes in the activity of the plasma membrane H⁺ pump are explained by changes in the supply of energy substrate. Photosynthesis-dependent changes in activities of both the plasma membrane and the tonoplast H⁺ pumps are explained in terms of changes in the level of inorganic phosphate in the cytoplasm. Cytoplasmic and vacuolar pHs are suggested to be controlling factors forin vivo activities of the H⁺ pumps. Furthermore, the membrane potential and various ions are considered to bein vivo factors that regulate the activities of the H⁺ pumps. Recipient of the Botanical Society Award of Young Scientists, 1993.     

Regulation of arbuscular mycorrhizal development by plant host and fungus species in alfalfa

Two cvs of alfalfa ( Medicago sativa L.), Gilboa and Moapa 69, were inoculated in glasshouse pots with three arbuscular mycorrhizal (AM) fungi to investigate the efficacy of mycorrhizas with respect to the extent of colonization and sporulation. Paspalum notatum Flugge also was inoculated to describe fungal parameters on a routine pot culture host. Percentage root length of P. notatum colonized by Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe, Glomus intraradices Schenck & Smith, and Gigaspora margarita Becker & Hall increased from 10 to 21 wk, and all fungi sporulated during that period. In alfalfa, only colonization by G. intraradices increased over that time period, and it was the only fungus to sporulate in association with alfalfa at 10 wk. Glomus mosseae did not sporulate after 16–21 wk despite having colonized 30–35% of the root length of both alfalfa cvs. In vitro experiments in which Ri T-DNA-transformed roots of alfalfa were inoculated with AM fungi showed normal mycorrhizal formation by G. intraradices and a hypersensitivity-like response to Gi. margarita . Colonized cells became necrotic, and HPLC analysis indicated increased concentrations of phenolics and isoflavonoids in these root segments. These data strongly support the existence of a degree of specificity between AM fungi and host that might rely on specific biochemical regulatory processes initiated in the host as a result of the attempts at colonization by the fungus.     

Inorganic phosphate uptake in intact vacuoles isolated from suspension-cultured cells of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don under varying Pi status

Inorganic phosphate (Pi) uptake across the vacuolar membrane of intact vacuoles isolated from Catharanthus roseus suspension-cultured cells was measured. Under low Pi status, Pi uptake into the vacuole was strongly activated compared to high Pi status. Since Pi uptake across the vacuolar membrane is correlated with H⁺ pumping, we examined the dependency of H⁺ pumping on plant Pi status. Both H⁺ pumping and the activities of the vacuolar H⁺-pumps, the V-type H⁺-ATPase and the H⁺-PPase were enhanced under low Pi status. Despite this increase in H⁺ pumping, Western blot analysis showed no distinct increase in the amount of proton pump proteins. Possible mechanisms for the activation of Pi uptake into the vacuole under low Pi status are discussed. Miwa Ohnishi and Tetsuro Mimura contributed equally to this work.     

Expression of functional Streptomyces coelicolor H+-pyrophosphatase and characterization of its molecular properties

H(+)-translocating pyrophosphatases (H(+)-PPases) are proton pumps that are found in many organisms, including plants, bacteria and protozoa. Streptomyces coelicolor is a soil bacterium that produces several useful antibiotics. Here we investigated the properties of the H(+)-PPase of S. coelicolor by expressing a synthetic DNA encoding the amino-acid sequence of the H(+)-PPase in Escherichia coli. The H(+)-PPase from E. coli membranes was active at a relatively high pH, stable up to 50 degrees C, and sensitive to N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide and acylspermidine. Enzyme activity increased by 60% in the presence of 120 mM K(+), which was less than the stimulation observed with plant vacuolar H(+)-PPases (type I). Substitutions of Lys-507 in the Gly-Gln-x-x-(Ala/Lys)-Ala motif, which is thought to determine the K(+) requirement of H(+)-PPases, did not alter its K(+) dependence, suggesting that other residues control this feature of the S. coelicolor enzyme. The H(+)-PPase was detected during early growth and was present mainly on the plasma membrane and to a lesser extent on intracellular membranous structures.