A comparison of an ATPase from the archaebacterium Halobacterium saccharovorum with the F1 moiety from the Escherichia coli ATP Synthase

A purified ATPase associated with membranes from Halobacterium saccharovorum was compared with the F sub 1 moiety from the Escherichia coli ATP Synthase. The halobacterial enzyme was composed of two major (I and II) and two minor subunits (III and IV), whose molecular masses were 87 kDa, 60 kDa, 29 kDa, and 20 kDa, respectively. The isoelectric points of these subunits ranged from 4.1 to 4.8, which in the case of the subunits I and II was consistent with the presence of an excess of acidic amino acids (20 to 22 Mol percent). Peptide mapping of sodium dodecylsulfate-denatured subunits I and II showed no relationship between the primary structures of the individual halobacterial subunits or similarities to the subunits of the F sub 1 ATPase (EC 3.6.1.34) from E. coli. Trypsin inactivation of the halobacterial ATPase was accompanied by the partial degradation of the major subunits. This observation, taken in conjunction with molecular masses of the subunits and the native enzyme, was consistent with the previously proposed stoichiometry of 2:2:1:1. These results suggest that H. saccharovorum, and possibly, Halobacteria in general, possess an ATPase which is unlike the ubiquitous F sub o F sub 1 - ATP Synthase

Structure of the novel membrane-coating material in proton-secreting epithelial cells and identification as an H+ATPase.

Specialized proton-secreting cells known collectively as mitochondria-rich cells are found in a variety of transporting epithelia, including the kidney collecting duct (intercalated cells) and toad and turtle urinary bladders. These cells contain a population of characteristic tubulovesicles that are believed to be involved in the shuttling of proton pumps (H+ATPase) to and from the plasma membrane. These transporting vesicles have a dense, studlike material coating the cytoplasmic face of their limiting membranes and similar studs are also found beneath parts of the plasma membrane. We have recently shown that this membrane coat does not contain clathrin. The present study was performed to determine the structure of this coat in rapidly frozen and freeze-dried tissue, and to determine whether the coat contains a major membrane protein transported by these vesicles, a proton pumping H+ATPase. The structure of the coat was examined in proton-secreting, mitochondria-rich cells from toad urinary bladder epithelium by rapidly freezing portions of apical membrane and associated cytoplasm that were sheared away from the remainder of the cell using polylysine-coated coverslips. Regions of the underside of these apical membranes as large as 0.2 micron2 were decorated by studlike projections that were arranged into regular hexagonal arrays. Individual studs had a diameter of 9.5 nm and appeared to be composed of multiple subunits arranged around a central depression, possibly representing a channel. The studs had a density of approximately 16,800 per micron2 of membrane. Similar arrays of studs were also found on vesicles trapped in the residual band of cytoplasm that remained attached to the underside of the plasma membrane, but none were seen in adjacent granular cells. To determine whether these arrays of studs contained H+ATPase molecules, we examined a preparation of affinity-purified bovine medullary H+ATPase, using the same technique, after incorporation of the protein eluted from a monoclonal antibody affinity column into phospholipid liposomes. The affinity-purified protein was shown to be capable of ATP-dependent acidification. In such preparations, large paracrystalline arrays of studs identical in appearance to those seen in situ were found. The dimensions of the studs as well as the number per square micrometer of membrane were identical to those of toad bladder mitochondria-rich cells: 9.5 nm in diameter, 16,770 per micron2 of membrane.(ABSTRACT TRUNCATED AT 400 WORDS

Understanding the apparent stator-rotor connections in the rotary ATPase family using coarse-grained computer modeling

Advances in structural biology, such as cryo-electron microscopy (cryo-EM) have allowed for a number of sophisticated protein complexes to be characterized. However, often only a static snapshot of a protein complex is visualized despite the fact that conformational change is frequently inherent to biological function, as is the case for molecular motors. Computer simulations provide valuable insights into the different conformations available to a particular system that are not accessible using conventional structural techniques. For larger proteins and protein complexes, where a fully atomistic description would be computationally prohibitive, coarse-grained simulation techniques such as Elastic Network Modeling (ENM) are often employed, whereby each atom or group of atoms is linked by a set of springs whose properties can be customized according to the system of interest. Here we compare ENM with a recently proposed continuum model known as Fluctuating Finite Element Analysis (FFEA), which represents the biomolecule as a viscoelastic solid subject to thermal fluctuations. These two complementary computational techniques are used to answer a critical question in the rotary ATPase family; implicit within these motors is the need for a rotor axle and proton pump to rotate freely of the motor domain and stator structures. However, current single particle cryo-EM reconstructions have shown an apparent connection between the stators and rotor axle or pump region, hindering rotation. Both modeling approaches show a possible role for this connection and how it would significantly constrain the mobility of the rotary ATPase family

A proteomic and phosphoproteomic analysis of Oryza sativa plasma membrane and vacuolar membrane

Proteomic and phosphoproteomic analyses of rice shoot and root tonoplast-enriched and plasma membrane-enriched membrane fractions were carried out to look at tissue-specific expression, and to identify putative regulatory sites of membrane transport proteins. Around 90 unique membrane proteins were identified, which included primary and secondary transporters, ion channels and aquaporins. Primary H+ pumps from the AHA family showed little isoform specificity in their tissue expression pattern, whereas specific isoforms of the Ca2+ pump ECA/ACA family were expressed in root and shoot tissues. Several ABC transporters were detected, particularly from the MDR and PDR subfamilies, which often showed expression in either roots or shoots. Ammonium transporters were expressed in root, but not shoot, tissue. Large numbers of sugar transporters were expressed, particularly in green tissue. The occurrence of phosphorylation sites in rice transporters such as AMT1;1 and PIP2;6 agrees with those previously described in other species, pointing to conserved regulatory mechanisms. New phosphosites were found in many transporters, including H+ pumps and H+:cation antiporters, often at residues that are well conserved across gene families. Comparison of root and shoot tissue showed that phosphorylation of AMT1;1 and several further transporters may be tissue dependent

Intracellular proton pumps as targets in chemotherapy: V-ATPases and cancer

Cancer cells show a metabolic shift that makes them overproduce protons; this has the potential to disturb the cellular acid-base homeostasis. However, these cells show cytoplasmic alkalinisation, increased acid extrusion and endosome-dependent drug resistance. Vacuolar type ATPases (V-ATPases), toghether with other transporters, are responsible to a great extent for these symptoms. These multisubunit proton pumps are involved in the control of cytosolic pH and the generation of proton gradients (positive inside) across endocellular membrane systems like Golgi, endosomes or lysosomes. In addition, in tumours, they have been determined to play an important role in the acidification of the intercellular medium. This importance makes them an attractive target for control of tumour cells. In the present review we portray the major characteristics of this kind of proton pumps, we provide some recent insights on their in vivo regulation, an overview of the consequences that V-ATPase inhibition carries for the tumour cell, such as cell cycle arrest or cell death, and a brief summary of the studies related to cancer made recently with commercially available inhibitors for this kind of proton pump. Some new approaches to affect V-ATPase function are also suggested in the light of recent knowledge on the regulation of this proton pump.Junta de Andalucía PAIDI BIO-261 P07-CVI-3082Ministerio de Ciencia e Innovación BFU2007-61887 BFU2010-1562

Regulatory assembly of the vacuolar proton pump VOV1-ATPase in yeast cells by FLIM-FRET

We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for transport processes as well as for pH and Ca2+ homoeostasis in the cell. Activity of the VOV1-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.Comment: 8 pages, 3 figure

Alternative proton binding mode in ATP synthases

ATP synthases are rotary engines which use the energy stored in a transmembrane electrochemical gradient of protons or sodium ions to catalyze the formation of ATP by ADP and inorganic phosphate. Current models predict that protonation/deprotonation of specific amino acids of the rotating c-ring, extracting protons from one side and delivering them to the other side of the membrane, are at the core of the proton translocation mechanism of these enzymes. In this minireview, an alternative proton binding mechanism is presented, considering hydronium ion coordination as proposed earlier. Biochemical data and structural considerations provide evidence for two different proton binding modes in the c-ring of H+-translocating ATP synthases. Recent investigations in several other proton translocating membrane proteins suggest, that hydronium ion coordination by proteins might display a general principle which was so far underestimated in ATP synthase

Zinc, cadmium and lead resistance mechanisms in bacteria and their contribution to biosensing

In bacteria resistance to heavy metals is mainly achieved through active efflux, but also sequestration with proteins or as insoluble compounds is used. Although numerous studies have dealt with zinc, cadmium and lead resistance mechanisms in bacteria, it has still remained unclear how different transporters are integrated into an effective homeostasis/resistance network and whether specific mechanisms for lead sequestration exist. Furthermore, since metals are toxic not only to bacteria but to higher organisms as well, it is important to be able to estimate possible biological effects of heavy metals in the environment. This could be done by determining the bioavailable amount of the metals in the environment with bacterial bioreporters. That is, one can employ bacteria that respond to metal contamination by a measurable signal to assess the property of metals to cross biological membranes and to cause harmful effects in a possibly polluted environment. In this thesis a new lead resistance mechanism is described, interplay between CBA transporters and P-type ATPases in zinc and cadmium resistance is presented and finally the acquired knowledge is used to construct bacterial bioreporters for heavy metals with increased sensitivity and specificity. The new lead resistance model employs a P-type ATPase that removes Pb2+ ions from the cytoplasm and a phosphatase that produces inorganic phosphate for lead sequestration in the periplasm. This was the first study where the molecular mechanism of lead sequestration has been described. Characterization of two P-type ATPases and two CBA transporters showed that resistance mechanisms for Zn2+ and Cd2+ are somewhat different than for Pb2+ as these metals cannot be sequestered as insoluble compounds as easily. Resistance to Zn2+ was conferred merely by the CBA transporter that could export both cytoplasmic and periplasmic ions; whereas, full resistance to Cd2+ required interplay of a P-type ATPase that exported cytoplasmic ions to periplasm and a CBA transporter that further exported periplasmic ions to the outside. The knowledge on functionality of the transporters and metal-inducible promoters was exploited in bioreporter technology. A transporter-deficient bioreporter strain that lacked exporters for Zn2+/Cd2+/Pb2+ could detect up to 45-fold lower metal concentrations than its wild type counterpart due to the accumulation of metals in the cell. The broad specificity issue of bioreporters was overcome by using Zn-specific promoter as a sensor element, thus achieving Zn-specific bioreporter.Bakteereiden kyky sietää raskasmetalleja perustuu tyypillisesti metalli-ionien kuljettamiseen solusta ulos, mutta bakteerit pyrkivät myös saostamaan ioneja liukenemattomina yhdisteinä raskasmetallien toksisuuden vähentämiseksi. Vaikka puolustusmekanismit sinkille, kadmiumille ja lyijylle tunnetaan pääpiirteittäin, kokonaiskuva eri mekanismien yhteistoiminnasta on vielä jäänyt epäselväksi. Biologisen perustietämyksen lisäksi solun puolustusmekanismien ja niiden säätelyn ymmärtäminen on tärkeää bioreportteriteknologian kehittämisessä. Bioreporttereilla tarkoitetaan bakteereita, jotka tuottavat mitattavaa signaalia vastauksena esimerkiksi metallin esiintymiselle niiden kasvuympäristössä. Bioreporttereita käytetään biosaatavien metallien metallien, jotka pystyvät läpäisemään solukalvoja ja vaikuttamaan haitallisesti elintoimintoihin määrittelemiseen ympäristöstä. Tässä työssä karakterisoidaan uusi lyijyresistenssimekanismi, tutkitaan erilaisten metallitransporttereiden yhteistyötä sinkin ja kadmiumin uloskuljetuksessa sekä käytettään saatua tietoa herkempien ja spesifisimpien bioreporttereiden kehittämiseen. Uudessa lyijyresistenssimallissa kuljetusproteiini siirtää lyijyionit solusta ulos ja toinen proteiini, fosfataasientsyymi, tuottaa epäorgaanista fosfaattia lyijyn saostamiseen. Kyseessä on ensimmäinen kerta, kun lyijyn saostaminen on kuvattu molekyylitasolla. Muiden kuljetusproteiinien karakterisoinnin mukaan sinkin ja kadmiumin sietokyky bakteereissa perustuu pelkästään uloskuljetukseen eikä saostukseen. Sietokyky sinkille saavutetaan kuljetuskompleksilla, joka poistaa metalli-ioneja sekä solusta että solukalvojen välistä. Kadmiumin sietokyky puolestaan vaatii kahden eri kuljetusproteiinin yhteistyötä: ensin yksi proteiini kuljettaa metalli-ionit solusta solukalvojen väliin, josta toinen kuljetusproteiinin edelleen poistaa ne kokonaan ulos solusta. Kuljetusproteiinien toiminnasta saatua tietoa sovelletaan entistä herkempien bioreporttereiden rakentamisessa. Bakteerikanta, josta oli poistettu kaikki sinkin, kadmiumin ja lyijyn kuljetusproteiinit, pystyi detektoimaan ympäristöstä 45 kertaa pienempiä metallipitoisuuksia kun villityyppibakteeri. Parempi herkkyys johtui metalli-ionien kerääntymisestä soluun, kun kaikki poistomekanismit oli kytketty pois päältä. Uusien herkempien bioreporttereiden käyttöönotto mahdollistaa biosaatavien raskasmetallien tarkemman määrittelemisen ympäristössä ja helpottaa riskianalyysia sekä remediaatiokeinojen valintaa

Estimating the Rotation Rate in the Vacuolar Proton-ATPase in Native Yeast Vacuolar Membranes

The rate of rotation of the rotor of the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or the steady parts of enzyme, is estimated in native vacuolar membrane vesicles of Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are spontaneously formed after exposing purified yeast vacuoles to osmotic shock. The fraction of the total ATPase activity originating from V-ATPase is determined using the potent and specific inhibi-tor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during 10 min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit to the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data is incompatible with models assuming more binding sites) to the inhibitor titration curve determines the concentration of the enzyme. Combining it with the known rotation:ATP stoichiometry of V-ATPase and the assayed concentration of inorganic phosphate liberated by V-ATPase leads to an average rate of ~9.53 Hz of the 360 degrees rotation, which, according to the time-dependence of the activity, extrapolates to ~14.14 Hz for the beginning of the reaction. These are low limit estimates. To our knowledge this is the first report of the rotation rate in a V-ATPase that is not subjected to genetic or chemical modification and it is not fixed on a solid support, instead it is functioning in its native membrane environment

A cell-based assay for CD63-containing extracellular vesicles

Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion.</div