Secondary Transport Processes Research Articles

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha–beta–PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA–PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha–alpha and alpha–PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha–alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)2.

Transcriptional, metabolic, physiological and developmental responses of switchgrass to phosphorus limitation.

Knowing how switchgrass (Panicum virgatum L.) responds and adapts to phosphorus (P)‐limitation will aid efforts to optimize P acquisition and use in this species for sustainable biomass production. This integrative study investigated the impacts of mild, moderate, and severe P‐stress on genome transcription and whole‐plant metabolism, physiology and development in switchgrass. P‐limitation reduced overall plant growth, increased root/shoot ratio, increased root branching at moderate P‐stress, and decreased root diameter with increased density and length of root hairs at severe P‐stress. RNA‐seq analysis revealed thousands of genes that were differentially expressed under moderate and severe P‐stress in roots and/or shoots compared to P‐replete plants, with many stress‐induced genes involved in transcriptional and other forms of regulation, primary and secondary metabolism, transport, and other processes involved in P‐acquisition and homeostasis. Amongst the latter were multiple miRNA399 genes and putative targets of these. Metabolite profiling showed that levels of most sugars and sugar alcohols decreased with increasing P stress, while organic and amino acids increased under mild and moderate P‐stress in shoots and roots, although this trend reversed under severe P‐stress, especially in shoots.

The Gene Encoding Subunit A of the Vacuolar H+-ATPase From Cotton Plays an Important Role in Conferring Tolerance to Water Deficit.

In plant cells, vacuolar H+-ATPases (V-ATPases) are responsible for deacidification of the cytosol and energisation of the secondary transport processes across the tonoplast. A number of V-ATPase subunit genes have been demonstrated to be involved in the regulation of the plant response to water deficit. However, there are no reports on the role of V-ATPase subunit A (VHA-A) in dehydration tolerance of cotton. In this study, cotton GhVHA-A gene was functionally characterized, especially with regard to its role in dehydration stress tolerance. Expression analysis showed that GhVHA-A was differentially expressed in various cotton organs and was induced by dehydration, low temperature, high salinity, and abscisic acid treatment in leaves. We also report that GhVHA-A improve dehydration tolerance in transgenic tobacco and cotton. Virus-induced gene silencing of GhVHA-A decreased the tolerance of cotton plantlets to dehydration stress. Silencing GhVHA-A decreased chlorophyll content and antioxidant enzyme activities and increased malondialdehyde (MDA) content in cotton under dehydration stress. However, transgenic tobacco expressing GhVHA-A exhibited enhanced dehydration resistance, resulting in reduced leaf water loss, higher average root length, and lower MDA levels under dehydration stress. Meanwhile, overexpression of GhVHA-A in tobacco conferred water deficit tolerance by enhancing osmotic adjustment (proline) and the activities of the antioxidant enzymes superoxide dismutase and peroxidase, thereby enhancing reactive oxygen species detoxification. These results suggest that GhVHA-A plays an important role in conferring resistance to dehydration stress. Our results have identified GhVHA-A as a candidate gene for improving dehydration tolerance in plants.

The interplay between phenotypic and ontogenetic plasticities can be assessed using reaction-diffusion models : The case of Pseudoplatystoma fishes.

Every morphological, behavioral, or even developmental character expression of living beings is coded in its genotype and is expressed in its phenotype. Nevertheless, the interplay between phenotypic and ontogenetic plasticities, that is, the capability to manifest trait variations, is a current field of research that needs morphometric, numerical, or even mathematical modeling investigations. In the present work, we are searching for a phenotypic index able to identify the underlying correlation among phenotypic, ontogenetic, and geographic distribution of the evolutionary development of species of the same genus. By studying the case of Pseudoplatystoma fishes, we use their skin patterns as an auxiliary trait that can be reproduced by means of a reaction diffusion (RD) model. From this model, we infer the phenotypic index in terms of one of the parameters appearing in the mathematical equations. To achieve this objective, we perform extensive numerical simulations and analysis of the model equations and link the parameter variations with different environmental and physicochemical conditions in which the individuals develop, and which may be regulated by the ontogenetic plasticity of the species. Our numerical study indicates that the patterns predicted by a set of reaction diffusion equations are not uniquely determined by the value of the parameters of the equation, but also depend on how the process is initiated and on the spatial distribution of values of these parameters. These factors are therefore significant, since they show that an individual's growth dynamics and apparent secondary transport processes, like advection, can be determinant for the alignment of motifs in a skin pattern. Our results allow us to discern the correlation between phenotypic, ontogenetic, and geographic distribution of the different species of Pseudoplatystoma fishes, thus indicating that RD models represent a useful taxonomic tool able to quantify evolutionary indexes.

Design, fabrication and analysis of stagnation flow microreactors used to study hypergolic reactions.

A novel method to study the condensed phase reactions that occur during the ignition of hypergolic propellants (very fast liquid reactions) using microreactors is presented. Planar counterflow microreactors are used to isolate liquid-phase reactions and diffusion from secondary gas-phase chemical and transport processes that often occur concurrently during the overall ignition process. The counterflow microreactor has made it possible to achieve valuable insight into the preignition mechanisms of hypergolic propellants hitherto not possible using conventional drop or impinging jet tests. In the present paper, the microreactor fabrication, flow field characterization, and reactivity of 2-dimethylaminoethylazide and nitric acid as hypergols are presented. Particle image velocimetry and numerical simulations were conducted to characterize the laminar velocity flow-field from which stagnation point strain rates and contact residence times along the centerline of the microreactor were evaluated. Temperature measurements at the exit of the reactor (as well as at the stagnation point) were used as a measure for the extent of the reaction or the heat released from the reaction. For the hypergols, an increase in reactant flow (or equivalently strain rate at the stagnation point) was found to initially increase reactivity, but eventually resulted in a decrease in temperature, revealing a maxima in temperature and reactivity. The trends indicated a reaction that was initially diffusion or heat loss controlled, which transitioned towards kinetic control at higher strain (flow) rates. This paper details the first comprehensive measurements and analysis of the effects of diffusion based mixing on the interfacial reactions occurring between hypergols.

Coupling between Neurotransmitter Translocation and Protonation State of a Titratable Residue during Na+-Coupled Transport

Coupling between Neurotransmitter Translocation and Protonation State of a Titratable Residue during Na+-Coupled Transport

Morphology and Histochemistry of Juvenile American Alligator (Alligator mississippiensis) Nephrons

Here we present a detailed morphological description of the alligator (Alligator mississippiensis) kidney and nephron. We present a series of histological, histochemical, and immunohistochemical markers that clearly define the seven regions of the alligator nephron. The alligator kidney is composed of many paired (mirrored) lobules on each kidney (lobe). Single nephrons span the width of lobules three times. The fine structure of glomeruli, lying in rows spanning the height of the lobule, is resolved by periodic acid methionine silver (PAMS) and periodic acid Schiff's (PAS) histochemistry. Glomeruli are connected to the proximal tubule (PT) via a neck segment. The PT is alcian blue-negative, making it distinct from the distal tubule (DT), connecting segment (CS), and collecting duct (CD). The PT is clearly identifiable by a PAS-positive brush border membrane. The PT is connected to the DT via an intermediate segment (IS) that makes a 180 degrees turn to connect these tubules. PAMS-positive material is found in the lumens of the PT, IS, and DT. Also, PAMS-positive granules are found in the DT, CS, and CD. Immunolocalization of the Na(+), K(+)-ATPase to the basolateral membrane of the DT, CS, and CD suggests a role of this enzyme in driving primary and secondary transport processes in these segments, including bicarbonate transport into the lumen of the DT (leading to an alkaline urine). Through the techniques described here, we have identified a series of distinct markers to be used by pathologists, veterinarians, and researchers to easily identify alligator nephron segments. Anat Rec, 2009. (c) 2009 Wiley-Liss, Inc.

Activity of tonoplast proton pumps and Na+/H+ exchange in potato cell cultures is modulated by salt

The efficient exclusion of excess Na from the cytoplasm and vacuolar Na(+) accumulation are the main mechanisms for the adaptation of plants to salt stress. This is typically carried out by transmembrane transport proteins that exclude Na(+) from the cytosol in exchange for H(+), a secondary transport process which is energy-dependent and driven by the proton-motive force generated by plasma-membrane and tonoplast proton pumps. Tonoplast enriched-vesicles from control and 150 mM NaCl-tolerant calli lines were used as a model system to study the activity of V-H(+)-PPase and V-H(+)-ATPase and the involvement of Na(+) compartmentalization into the vacuole as a mechanism of salt tolerance in Solanum tuberosum. Both ATP- and pyrophosphate (PP(i))-dependent H(+)-transport were higher in tonoplast vesicles from the salt-tolerant line than in vesicles from control cells. Western blotting of tonoplast proteins confirmed that changes in V-H(+)-PPase activity are correlated with increased protein amount. Conversely, immunodetection of the A-subunit of V-H(+)-ATPase revealed that a mechanism of post-translational regulation is probably involved. Na(+)-dependent dissipation of a pre-established pH gradient was used to measure Na(+)/H(+) exchange in tonoplast vesicles. The initial rates of proton efflux followed Michaelis-Menten kinetics and the V(max) of proton dissipation was 2-fold higher in NaCl-tolerant calli when compared to the control. H(+)-coupled exchange was specific for Na(+) and Li(+) and not for K(+). The increase of both the pH gradient across the tonoplast and the Na(+)/H(+) antiport activity in response to salt strongly suggests that Na(+) sequestration into the vacuole contributes to salt tolerance in potato.

The Emerging Structure of Vacuolar ATPases

Bioenergetics and physiology of primary pumps have been revitalized by new insights into the mechanism of energizing biomembranes. Structural information is becoming available, and the three-dimensional structure of F-ATPase is being resolved. The growing understanding of the fundamental mechanism of energy coupling may revolutionize our view of biological processes. The F- and V-ATPases (vacuolar-type ATPase) exhibit a common mechanical design in which nucleotide-binding on the catalytic sector, through a cycle of conformation changes, drives the transmembrane passage of protons by turning a membrane-embedded rotor. This motor can run in forward or reverse directions, hydrolyzing ATP as it pumps protons uphill or creating ATP as protons flow downhill. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as an ATP-dependent proton pump. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. V- and F-ATPases have similar structure and mechanism of action, and several of their subunits evolved from common ancestors. Electron microscopy studies of V-ATPase revealed its general structure at low resolution. Recently, several structures of V-ATPase subunits, solved by X-ray crystallography with atomic resolution, were published. This, together with electron microscopy low-resolution maps of the whole complex, and biochemistry cross-linking experiments, allows construction of a structural model for a part of the complex that may be used as a working hypothesis for future research.

Remotely sensed mesoscale oceanography of the Central Eastern Pacific and recruitment variability in Dosidicus gigas

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 310:25-32 (2006) - doi:10.3354/meps310025 Remotely sensed mesoscale oceanography of the Central Eastern Pacific and recruitment variability in Dosidicus gigas Claire M. Waluda*, Paul G. Rodhouse British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 0ET, UK *Email: clwa@bas.ac.uk ABSTRACT: The Jumbo flying squid Dosidicus gigas has a short life span and is subject to rapid changes in population size. This species inhabits the Eastern Pacific, one of the most variable oceanic environments in the world, subject to the El Niño Southern Oscillation (ENSO). This study examined the influence of environmental variability on squid abundance off Peru using mesoscale oceanographic indicators derived from remotely sensed satellite imagery. In the fishery area off Peru, squid abundance was positively associated with sea surface temperatures (SST) between 17 and 22°C during July, corresponding to the peak of the fishery season. In the putative hatching area close to the Costa Rica Dome, squid abundance was positively associated with SST of between 24 and 28°C during September, prior to the start of the fishery season. Squid abundance is apparently strongly influenced by mesoscale variability linked to ENSO, with low levels of upwelling during the very strong El Niño of 1997 to 1998 leading to very low catches of squid off Peru. Variability in upwelling off Peru and in the Costa Rica Dome region may drive primary and secondary production and transport processes affecting the planktonic early life stages and also the availability of food and suitable habitat for adult squid. KEY WORDS: Costa Rica Dome · Dosidicus gigas · El Niño Southern Oscillation · Eastern Pacific · Fishery · Flying squid · Mesoscale oceanography · Upwelling Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 310. Online publication date: April 03, 2006 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2006 Inter-Research.

Exogenously Supplied Compatible Solutes Rapidly Ameliorate NaCl-induced Potassium Efflux from Barley Roots

It has been suggested that the role of compatible solutes in plant stress responses is not limited to conventional osmotic adjustment, but also includes some other regulatory or osmoprotective functions. In this study, we hypothesized that one such function is in maintaining cytosolic K+ homeostasis by preventing NaCl-induced K+ leakage from the cell, a feature that may confer salt tolerance in many species, particularly in barley. This hypothesis was investigated using the non-invasive microelectrode ion flux (MIFE) measuring technique. We show that low (0.5-5 mM) concentrations of exogenously supplied proline or betaine significantly reduced NaCl-induced K+ efflux from barley roots in a dose-response manner. This effect was instantaneous, implying that large intracellular concentrations of compatible solutes are not required for an amelioratory role. Exogenously supplied betaine also significantly enhanced NaCl-induced H+ efflux, but only in pre-incubated roots, implying some alternative mechanism of regulation. Sap K+ and Na+ analysis and membrane potential measurements are also consistent with the model that one function of compatible solutes is in maintaining cytosolic K+ homeostasis by preventing NaCl-induced K+ leakage from the cell, possibly through the enhanced activity of H+-ATPase, controlling voltage-dependent outward-rectifying K+ channels and creating the electrochemical gradient necessary for secondary ion transport processes. These data provide the first direct evidence for regulation of ion fluxes across the plasma membrane by physiologically relevant low concentrations of compatible solutes.

Facilitated transport of heavy metals by bacterial colloids in sand columns

The aim of this work is to evaluate the ability of biotic colloids (e.g. bacterial cells) to facilitate the transport of heavy metals in soils, and to identify the main factors influencing colloid transport in order to determine the geochemical conditions where this secondary transport process may become dominant. The model colloids studied in this paper are living cells of Escherichia coli and Ralstonia metallidurans. We studied the transport of mercury zinc, and cadmium in columns of Fontainebleau sand. The properties (i.e. optical and morphological properties, charge (zeta potential, ζ) and hydrophobia (water/hexadecane distribution parameter, K hw )) of the bacterial cells surface were characterised, as well as their potential for heavy metals sorption (kinetic and isotherm). Both surface charge (ζ=-54 and -14 mV) and hydrophobia (K hw =0.25 and 0.05) differ strongly for the two bacteria. Column studies were conducted with bacteria and heavy metals separately or simultaneously. The cell surface differences led to different transport behaviour of the two bacteria, although the retardation factor is close to 1 for both. We observed that colloid mobility increases when increasing bacterial cells concentration and when decreasing the ionic strength. We also observed that bacterial colloids appeared as excellent vectors for Hg, Zn and Cd. Indeed, heavy metals adsorbed on the Fontainebleau sand when injected alone in columns (retardation factors of 1.4; 2.9 and 3.8 for Hg, Zn and Cd, respectively); whereas no retardation (R≃1) is observed when injected in the presence of both bacteria. Moreover, transport of biosorbed metal appears to be 4 to 6 times higher than dissolved heavy-metal.

A journey from mammals to yeast with vacuolar H+-ATPase (V-ATPase).

The vacuolar H+-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. V-ATPase has a structure and mechanism of action similar to F-ATPase and several of their subunits probably evolved from common ancestors. In eukaryotic cells, F-ATPase is confined to the semiautonomous organelles, chloroplasts and mitochondria, which contain their own genes that encode some of the F-ATPase subunits. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the protonmotive force (pmf), V-ATPases function exclusively as ATP-dependent proton pumps. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. It was the survival of the yeast mutant without the active enzyme and yeast genetics that allowed the identification of genuine subunits of the V-ATPase. It also revealed special properties of individual subunits, factors that are involved in the enzyme's biogenesis and assembly, as well as the involvement of V-ATPase in the secretory pathway, endocytosis, and respiration. It may be the insect V-ATPase that unconventionally resides in the plasma membrane of their midgut, that will give the first structure resolution of this complex.

EST Sequencing, Annotation and Macroarray Transcriptome Analysis Identify Preferentially Root‐Expressed Genes in Sugar Beet

Abstract: An integrated approach involving EST sequencing, data mining and multiparallel expression profiling by macroarray analysis was established to classify sugar beet gene products with respect to their expression in three different organs. The DNA sequences of 2996 ESTs derived from young sugar beet plants specified 2048 unique gene products with putative functions in primary and secondary metabolism (651), transport processes (136), signal transduction (78) and cellular organization (39). The cDNA clone collection was the basis for the generation of a macroarray. Sensitivity and reproducibility of our macroarray hybridization procedure were estimated first. The detection limit was found to correspond to 10 ‐ 50 copies of single transcripts per cell. Within an interval of two‐fold variation in signal intensities, reproducibility between spots on the same filter was determined to be 98.9 %, between spots on different filters 89.8 %, and reproducibility after hybridization with two probes synthesized from the same poly(A)+RNA sample was 97.6 %. Expression profiles from roots, leaves and inflorescences of field‐grown plant material were generated. Two different samples of each organ were analysed to reduce sampling effects, which accounted on average for 30.3 % of spots with at least two‐fold deviation. Expression values for each organ were determined by a stringent statistical evaluation of eight hybridizations for each clone. Macroarray expression data were confirmed by Northern blot analysis and quantitative RT‐PCR experiments concerning eleven cDNAs. The analysis was then focused on 76 unique cDNAs, for which the amount of detected transcript in roots was at least twice as high as in the other organs tested. Functions of preferentially root‐expressed candidate genes in taproot morphology and physiology are discussed.

Circadian rhythm of acidification in insect vas deferens regulated by rhythmic expression of vacuolar H(+)-ATPase.

Recent studies have demonstrated that the peripheral tissues of vertebrates and invertebrates contain circadian clocks; however, little is known about their functions and the rhythmic outputs that they generate. To understand clock-controlled rhythms at the cellular level, we investigated a circadian clock located in the reproductive system of a male moth (the cotton leaf worm Spodoptera littoralis) that is essential for the production of fertile spermatozoa. Previous work has demonstrated that spermatozoa are released from the testes in a daily rhythm and are periodically stored in the upper vas deferens (UVD). In this paper, we demonstrate a circadian rhythm in pH in the lumen of the UVD, with acidification occurring during accumulation of spermatozoa in the lumen. The daily rhythm in pH correlates with a rhythmic increase in the expression of a proton pump, the vacuolar H(+)-ATPase (V-ATPase), in the apical portion of the UVD epithelium. Rhythms in pH and V-ATPase persist in light/dark cycles and constant darkness, but are abolished in constant light, a condition that disrupts clock function and renders spermatozoa infertile. Treatment with colchicine impairs the migration of V-ATPase-positive vesicles to the apical cell membrane and abates the acidification of the UVD lumen. Bafilomycin, a selective inhibitor of V-ATPase activity, also prevents the decline in luminal pH. We conclude that the circadian clock generates a rhythm of luminal acidification by regulating the levels and subcellular distribution of V-ATPase in the UVD epithelium. Our data provide the first evidence for circadian control of V-ATPase, the fundamental enzyme that provides the driving force for numerous secondary transport processes. They also demonstrate how circadian rhythms displayed by individual cells contribute to the synchrony of physiological processes at the organ level.

Quick guide: ATP synthase

Also known as… F1Fo ATP synthase, F1Fo ATPase, F-type ATPase. F1 denotes the soluble catalytic headgroup where ATP synthesis and hydrolysis occur, and Fo is the ion-translocating membrane domain (see Fig. 1Fig. 1).Fig. 1An illustration of the E. coli F1Fo. F1 is made up of five different subunits, with a stoichiometry α3β3γδe, and the composition of Fo is abc10–12. Strong evidence supports a mechanism whereby the central stalk of the soluble F1 domain and the oligomeric ring of subunit c of the membrane sector rotate as one (shaded orange), coupling ion movement to ATP synthesis and hydrolysis.View Large Image | View Hi-Res Image | Download PowerPoint SlideWhat is it? An enzyme that uses energy from the transmembrane proton electrochemical gradient, generated by oxidative metabolism or photosynthesis, to produce ATP from ADP and Pi. The enzyme can also act in reverse, hydrolysing ATP and pumping H+ or Na+.Where is it found? In energy-transducing membranes such as the plasma membrane of bacteria and blue–green algae, the inner membrane of mitochondria and the thylakoid membrane of chloroplasts. Hence, they play a fundamental role throughout nature producing the ‘universal energy exchange currency’ ATP.It first came to prominence… in the early 1960s when lollipop-like structures were observed on mitochondrial membranes by electron microscopy. The large soluble domain, F1, can be detached and was shown to hydrolyse ATP. Later, ATP hydrolysis and synthesis were shown to be coupled to H+ translocation through the complex, consistent with Peter Mitchell's chemiosmotic theory.Its first heyday was… probably 1994 when the crystal structure of the F1 headgroup was solved by John E. Walker and colleagues. The structure supported the ‘binding change mechanism’ proposed by Paul D. Boyer — a sequential change in the binding affinities of the alternating catalytic sites in F1 to promote tight substrate binding and product release during catalysis — and provided a structural basis for a rotary mechanism of catalysis. Walker and Boyer were awarded the Nobel Prize for Chemistry in 1997. Its second coming will be when the entire complex of Fo is solved.How does it work? As a molecular motor. There is good evidence that H+ movement occurs at the interface between the fixed subunit a and the mobile subunit c oligomeric ring (see Fig. 1Fig. 1). The oligomeric c ring is attached to the central stalk and forms a spindle within the complex that turns at several thousand revolutions per minute. The asymmetrical central stalk interacts with the subunits of the surrounding catalytic headgroup and couples events at the catalytic sites with H+ movement.Other speculated functions include… maintenance of an electrochemical gradient necessary for secondary transport and, in some bacteria, cytoplasmic pH, by working in reverse, hydrolysing ATP and pumping H+.Does it have any relatives? Yes, the vacuolar ATPase (V1Vo) and archaeal ATPase (A1Ao). The V1Vo ATPase is a H+ pump important for acidifying organelle interiors, and generating electrochemical gradients for secondary transport processes. F1 is also related to DNA and RNA helicases.Does it have commercial potential? It is implicated in many physiological roles and is likely to be a valuable target for therapeutic drugs. An inhibitor protein of human F1Fo, IF1, may be useful for preventing tissue damage of the heart during ischemia by inhibiting ATP hydrolysis. V1Vo in Plasmodium falciparum appears to be a target of antimicrobial agents which interact at the ion-binding site.Where can I find out more?Boyer PD: The ATP Synthase — a splendid molecular machine. Annu Rev Biochem 1997, 66:717-749Walker JE: ATP synthesis by rotary catalysis (Nobel Lecture). Angew Chem Int Ed Engl 1998 , 37:2309-2319Proton motive ATPases. J Exp Biol 2000, 203:1-170.The Mechanism of F1Fo-ATPase. Biochim Biophys Acta 2000, 1458:221-510.

Carrier-mediated transport of riboflavin in Ashbya gossypii.

The filamentous hemiascomycete Ashbya gossypii is used for industrial riboflavin production. We examined riboflavin uptake and excretion at the plasma membrane using riboflavin auxotrophic and overproducing mutants. The riboflavin uptake system had low activity [Vmax = 20 +/- 4 nmol min(-1) g(-1) mycelial dry weight (dw)] and high affinity (KM = 40 +/- 12 microM). Inhibitor studies with the analogs FMN and FAD revealed high specificity of the uptake system. Excretion of riboflavin was not the consequence of non-specific permeability of the plasma membrane. Excretion rates in the mid-production phase were determined to be 2.5 nmol min(-1) g(-1) dw for wild-type cells and 66.7 nmol min(-1) g(-1) dw for an overproducing mutant, respectively. Inhibition of the reverse reaction, riboflavin uptake, led to an increase in apparent riboflavin efflux in the early production phase, indicating the presence of a separate excretion carrier. Riboflavin accumulation in A. gossypii vacuoles leading to product retention was found to be a secondary transport process. To address the question of whether a flux from the vacuoles back into the cytoplasm is present, we characterized efflux in hyphae in which the plasma membrane was permeabilized with digitonin. Efflux kinetics across the vacuolar membrane were unaffected by the lack of vacuolar H+ATPase activity and ATP, suggesting a passive mechanism. Based on the characterization of riboflavin transport processes in this study, the design of new production strains with improved riboflavin excretion may be possible.

The Cellular Biology of Proton-Motive Force Generation by V-ATPases

The vacuolar H(+)-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force, V-ATPases function exclusively as ATP-dependent proton pumps. The proton-motive force generated by V-ATPases in organelles and across plasma membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. The enzyme is also vital for the proper functioning of endosomes and the Golgi apparatus. In contrast to yeast vacuoles, which maintain an internal pH of approximately 5. 5, it is believed that the vacuoles of lemon fruit may have a pH as low as 2. Similarly, some brown and red algae maintain an internal pH as low as 1 in their vacuoles. It was yeast genetics that allowed the identification of the special properties of individual subunits and the discovery of the factors that are involved in V-ATPase biogenesis and assembly. Null mutations in genes encoding V-ATPase subunits of Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations. Treatment of these null mutants with ethyl methanesulphonate causes mutations that suppress the V-ATPase null phenotype, and these cells are able to grow at pH 7.5. The suppressor mutants were denoted as svf (Suppressor of V-ATPase Function). The svf mutations are recessive: crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that were not able to grow at pH 7.5. A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or on the sensitivity and distribution of membrane proteins in different targets was discovered. We termed this gene family VTC (Vacuolar Transporter Chaperon) and discovered four genes in S. cerevisiae that belong to the family. Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in an svf phenotype that was able to grow at pH 7.5. Apparently, Vtc1p is one of a few membrane organizers that determine the relative amounts of different membrane proteins in the various cellular membranes. We utilize the numerous yeast mutants generated in our laboratory to identify the specific organelle whose acidification is vital. The interaction between V-ATPase and the secretory pathway is investigated.

Structure and regulation of insect plasma membrane H(+)V-ATPase.

H(+) V-ATPases (V-ATPases) are found in two principal locations, in endomembranes and in plasma membranes. The plasma membrane V-ATPase from the midgut of larval Manduca sexta is the sole energizer of all transepithelial secondary transport processes. At least two properties make the lepidopteran midgut a model tissue for studies of general aspects of V-ATPases. First, it is a rich source for purification of the enzyme and therefore for structural studies: 20 larvae provide up to 0.5 mg of holoenzyme, and soluble, cytosolic V(1) complexes can be obtained in even greater amounts of up to 2 mg. Second, midgut ion-tranport processes are strictly controlled by the regulation of the V-ATPase, which is the sole energizer of all ion transport in this epithelium. Recent advances in our understanding the structure of the V(1) and V(o) complexes and of the regulation of the enzyme's biosynthesis and ion-transport activity will be discussed.

Vacuolar and plasma membrane proton-adenosinetriphosphatases.

The vacuolar H+-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. V-ATPases have similar structure and mechanism of action with F-ATPase and several of their subunits evolved from common ancestors. In eukaryotic cells, F-ATPases are confined to the semi-autonomous organelles, chloroplasts, and mitochondria, which contain their own genes that encode some of the F-ATPase subunits. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as ATP-dependent proton pumps. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. The mechanistic and structural relations between the two enzymes prompted us to suggest similar functional units in V-ATPase as was proposed to F-ATPase and to assign some of the V-ATPase subunit to one of four parts of a mechanochemical machine: a catalytic unit, a shaft, a hook, and a proton turbine. It was the yeast genetics that allowed the identification of special properties of individual subunits and the discovery of factors that are involved in the enzyme biogenesis and assembly. The V-ATPases play a major role as energizers of animal plasma membranes, especially apical plasma membranes of epithelial cells. This role was first recognized in plasma membranes of lepidopteran midgut and vertebrate kidney. The list of animals with plasma membranes that are energized by V-ATPases now includes members of most, if not all, animal phyla. This includes the classical Na+ absorption by frog skin, male fertility through acidification of the sperm acrosome and the male reproductive tract, bone resorption by mammalian osteoclasts, and regulation of eye pressure. V-ATPase may function in Na+ uptake by trout gills and energizes water secretion by contractile vacuoles in Dictyostelium. V-ATPase was first detected in organelles connected with the vacuolar system. It is the main if not the only primary energy source for numerous transport systems in these organelles. The driving force for the accumulation of neurotransmitters into synaptic vesicles is pmf generated by V-ATPase. The acidification of lysosomes, which are required for the proper function of most of their enzymes, is provided by V-ATPase. The enzyme is also vital for the proper function of endosomes and the Golgi apparatus. In contrast to yeast vacuoles that maintain an internal pH of approximately 5.5, it is believed that the vacuoles of lemon fruit may have a pH as low as 2. Similarly, some brown and red alga maintain internal pH as low as 0.1 in their vacuoles. One of the outstanding questions in the field is how such a conserved enzyme as the V-ATPase can fulfill such diverse functions.