Helical interactions and membrane disposition of the 16-kDa proteolipid subunit of the vacuolar H(+)-ATPase analyzed by cysteine replacement mutagenesis.

Theoretical mechanisms of proton translocation by the vacuolar H(+)-ATPase require that a transmembrane acidic residue of the multicopy 16-kDa proteolipid subunit be exposed at the exterior surface of the membrane sector of the enzyme, contacting the lipid phase. However, structural support for this theoretical mechanism is lacking. To address this, we have used cysteine mutagenesis to produce a molecular model of the 16-kDa proteolipid complex. Transmembrane helical contacts were determined using oxidative cysteine cross-linking, and accessibility of cysteines to the lipid phase was determined by their reactivity to the lipid-soluble probe N-(1-pyrenyl)maleimide. A single model for organization of the four helices of each monomeric proteolipid was the best fit to the experimental data, with helix 1 lining a central pore and helix 2 and helix 3 immediately external to it and forming the principal intermolecular contacts. Helix 4, containing the crucial acidic residue, is peripheral to the complex. The model is consistent not only with theoretical proton transport mechanisms, but has structural similarity to the dodecameric ring complex formed by the related 8-kDa proteolipid of the F(1)F(0)-ATPase. This suggests some commonality between the proton translocating mechanisms of the vacuolar and F(1)F(0)-ATPases.     

beta(1) integrin binds the 16-kDa subunit of vacuolar H(+)-ATPase at a site important for human papillomavirus E5 and platelet-derived growth factor signaling.

Integrins mediate adhesive interactions between cells and the extracellular matrix, and play a role in cell migration, proliferation, differentiation, cytoskeletal organization, and signal transduction. We have identified an interaction between the beta(1) integrin and the 16-kDa subunit of vacuolar H(+)-ATPase (16K). This interaction was first isolated in a yeast two-hybrid screen and confirmed by coimmunoprecipitation and in in vitro binding assays using bacterially expressed proteins. Immunofluorescent studies performed in L6 myoblasts expressing both native and epitope-tagged 16K demonstrate co-localization with beta(1) integrin in focal adhesions. Deletion of the fourth of four transmembrane helices in 16K results in loss of interaction with beta(1) integrin in vitro and in the two-hybrid system, and less prominent staining in focal adhesions. This helix is also required for ligand-independent activation of platelet-derived growth factor-beta receptor signaling by the human papillomavirus E5 oncoprotein. Overexpression of 16K or expression of 16K lacking this helix alters the morphology of myoblasts and fibroblasts, suggesting that the interaction of 16K with integrins could be important for cell growth control. We also discuss the possible role 16K might play in integrin movement.     

Defective assembly of a hybrid vacuolar H(+)-ATPase containing the mouse testis-specific E1 isoform and yeast subunits

Mammalian vacuolar-type proton pumping ATPases (V-ATPases) are diverse multi-subunit proton pumps. They are formed from membrane V(o) and catalytic V(1) sectors, whose subunits have cell-specific or ubiquitous isoforms. Biochemical study of a unique V-ATPase is difficult because ones with different isoforms are present in the same cell. However, the properties of mouse isoforms can be studied using hybrid V-ATPases formed from the isoforms and other yeast subunits. As shown previously, mouse subunit E isoform E1 (testis-specific) or E2 (ubiquitous) can form active V-ATPases with other subunits of yeast, but E1/yeast hybrid V-ATPase is defective in proton transport at 37 degrees C (Sun-Wada, G.-H., Imai-Senga, Y., Yamamoto, A., Murata, Y., Hirata, T., Wada, Y., and Futai, M., 2002, J. Biol. Chem. 277, 18098-18105). In this study, we have analyzed the properties of E1/yeast hybrid V-ATPase to understand the role of the E subunit. The proton transport by the defective hybrid ATPase was reversibly recovered when incubation temperature of vacuoles or cells was shifted to 30 degrees C. Corresponding to the reversible defect of the hybrid V-ATPase, the V(o) subunit a epitope was exposed to the corresponding antibody at 37 degrees C, but became inaccessible at 30 degrees C. However, the V(1) sector was still associated with V(o) at 37 degrees C, as shown immunochemically. The control yeast V-ATPase was active at 37 degrees C, and its epitope was not accessible to the antibody. Glucose depletion, known to dissociate V(1) from V(o) in yeast, had only a slight effect on the hybrid at acidic pH. The domain between Lys26 and Val83 of E1, which contains eight residues not conserved between E1 and E2, was responsible for the unique properties of the hybrid. These results suggest that subunit E, especially its amino-terminal domain, plays a pertinent role in the assembly of V-ATPase subunits in vacuolar membranes.     

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.     

Characterization of nucleotide binding sites of the isolated H(+)-ATPase from spinach chloroplasts, CF(0)F(1)

Soluble purified CF(0)F(1) from chloroplasts was either oxidized or reduced and then incubated with [alpha-(32)P]ATP in the presence or in the absence of Mg(2+). Depending on the conditions of incubation, the enzyme showed different tight-nucleotide binding sites. In the presence of EDTA, two sites bind [alpha-(32)P]ATP from the reaction medium at different rates. Both sites promote ATP hydrolysis, since equimolar amounts of [alpha-(32)P]ATP and [alpha-(32)P]ADP are bound to the enzyme. In the presence of Mg(2+), only one site appears during the first hour of incubation, with characteristics similar to those described in the absence of Mg(2+). However, after this time a third site appears also permitting binding of ATP from the reaction medium, but in this case the bound ATP is not hydrolyzed. Covalent derivatization by 2-azido-[alpha-(32)P]ATP was used to distinguish between catalytic and noncatalytic sites. In the presence of Mg(2+), there are at least three distinct nucleotide binding sites that bind nucleotide tightly from the reaction medium: two of them are catalytic and one is noncatalytic.     

Features of F(1)-ATPase catalytic and noncatalytic sites revealed by fluorescence lifetimes and acrylamide quenching of specifically inserted tryptophan residues

Catalytic and noncatalytic nucleotide sites of the F(1) sector of ATP synthase were characterized by tryptophan fluorescence techniques. Seven Trp residues inserted in varied microenvironments in the catalytic sites, and one in the noncatalytic sites, were studied in mutant F(1) enzymes which were otherwise devoid of Trp. Parameters measured were fluorescence lifetimes and dynamic and static quenching by acrylamide in the absence or presence of nucleotide. The results indicated that the solution structures of the mutant enzymes were consistent with reported crystal structures. In enzyme with three empty noncatalytic sites, all sites were relatively inaccessible to acrylamide, indicating a closed conformation. In contrast, when the three catalytic sites were empty, they were relatively and equally accessible to acrylamide, indicating an open conformation. This was the case in the presence or absence of Mg(2+). Residue beta-Trp-331 has been extensively used previously to determine nucleotide binding parameters in F(1). Results here showed that in betaY331W mutant F(1), each of the three beta-Trp-331 residues has an unusually long fluorescence lifetime, confirming that each contributes equally to the overall fluorescence signal.     

One-step purification of Escherichia coli H(+)-ATPase (F0F1) and its reconstitution into liposomes with neurotransmitter transporters.

About 30% of the protein in the inner membrane of Escherichia coli strain DK8/pBWU13 is H(+)-ATPase (F0F1), and practically homogeneous F0F1 could be obtained by gradient centrifugation after solubilization of these membranes. The recombinant plasmid pBWU13 carries the unc operon for F0F1. When reconstituted into liposomes, F0F1 formed an ATP-dependent proton gradient and membrane potential. Proteoliposomes reconstituted with F0F1 and solubilized transporters from chromaffin granules or synaptic vesicle membranes could transport serotonin, dopamine, and norepinephrine dependent on ATP hydrolysis. F0F1 can be obtained rapidly from DK8/pBWU13, and its reconstitution into liposomes with transporters may be useful for monitoring these transporters during their purification.     

The Na(+) cycle in Acetobacterium woodii: identification and characterization of a Na(+) translocating F(1)F(0)-ATPase with a mixed oligomer of 8 and 16 kDa proteolipids

The homoacetogenic bacterium Acetobacterium woodii relies on a sodium ion current across its cytoplasmic membrane for energy-dependent reactions. The sodium ion potential is established by a yet to be identified primary, electrogenic pump connected to the Wood-Ljungdahl pathway. Reactions possibly involved in Na(+) export are discussed. The electrochemical sodium ion potential generated is used to drive endergonic reactions such as flagellar rotation and ATP synthesis. Biochemical and molecular data identified the Na(+)-ATPase of A. woodii as a typical member of the F(1)F(0) class of ATPases. Its catalytic properties and the hypothetical sodium ion binding site in subunit c are discussed. The encoding genes were cloned and, surprisingly, the atp operon was shown to contain multiple copies of genes encoding subunit c. Two copies encode identical 8 kDa proteolipids, and a third copy arose by duplication and subsequent fusion of two genes. Furthermore, the duplicated subunit c does not contain the ion binding site in hair pin two. Biochemical and molecular data revealed that all three copies of subunit c constitute a mixed oligomer. The evolution of the structure and function of subunit c in ATPases from eucarya, bacteria, and archaea is discussed.     

Structural and conformational changes concomitant with the E1-E2 transition in H(+)K(+)-ATPase: a comparative protein modeling study

Comparative modeling studies on conserved regions of the gastric H(+)K(+)-ATPase reveal that the E1-E2 conformational transition induces significant tertiary structural changes while conserving the secondary structure. The residues 516-530 of the cytoplasmic domain and TM10 within the transmembrane (TM) regions undergo maximum tertiary structural changes. The luminal regions exhibit comparatively lesser tertiary structural deviations. Residues 249-304 show maximum secondary structural deviation in the conformational transition. The Cys-815 and Cys-323 residues involved in inhibitor binding are found to have smaller buried side chain areas in the E1 conformation compared to E2. Retention of activity correlates well with the buried side chain area when selected amino acid residues in TM6 are mutated using modeling techniques with bulkier amino acid residues. Conformational specificity for ion binding is corroborated with the fraction of side chains exposed to polar atoms of the residues E345, D826, V340, A341, V343, and E822.     

Kinetics of ATP synthesis catalyzed by the H(+)-ATPase from chloroplasts (CF0F1) reconstituted into liposomes and coreconstituted with bacteriorhodopsin.

The H(+)-ATPase from chloroplasts (CF0F1) was isolated, purified and reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. A transmembrane pH difference, delta pH, and a transmembrane electric potential difference, delta psi, were generated by an acid/base transition. The rate of ATP synthesis was measured at constant delta pH and constant delta psi as a function of temperature between 5 degrees C and 45 degrees C. The activation energy was 55 kJ mol-1. CF0F1 was coreconstituted with bacteriorhodopsin at a molar ratio of approximately 1:170 in the same type of liposomes. Illumination of the proteoliposomes leads to proton transport into the vesicles generating a constant delta pH = 1.8. The dependence of the rate of ATP synthesis on ADP concentration was measured with CF0F1 in the oxidized state, E(ox), and in the reduced state, E(red). The results can be described by Michaelis-Menten kinetics with the following parameters: Vmax = 0.5 s-1, Km = 8 microM for E(ox) and Vmax = 2.0 s-1, Km = 8 microM for E(red).     

H+-ATPase activity of Escherichia coli F1F0 is blocked after reaction of dicyclohexylcarbodiimide with a single proteolipid (subunit c) of the F0 complex

Dicyclohexylcarbodiimide (DCCD) specifically inhibits the F1F0-H+-ATP synthase complex of Escherichia coli by covalently modifying a proteolipid subunit that is embedded in the membrane. Multiple copies of the DCCD-reactive protein, also known as subunit c, are found in the F1F0 complex. In order to determine the minimum stoichiometry of reaction, we have treated E. coli membranes with DCCD, at varying concentrations and for varying times, and correlated inhibition of ATPase activity with the degree of modification of subunit c. Subunit c was purified from the membrane, and the degree of modification was determined by two methods. In the "specific radioactivity" method, the moles of [14C]DCCD per total mole of subunit c was calculated from the radioactivity incorporated per mg of protein, and conversion of mg of protein to mol of protein based upon amino acid analysis. In the "high performance liquid chromatography (HPLC) peak area" method, the DCCD-modified subunit c was separated from unmodified subunit c on an anion exchange AX300 HPLC column, and the areas of the peaks from the chromatogram quantitated. The shape of the modification versus inhibition curve indicated that modification of a single subunit c per F0 was sufficient to abolish ATPase activity. The titration data were fit by nonlinear regression analysis to a single hit mathematical model, A = Un(1 - r) + r, where A is the relative activity, U is the ratio of unmodified/total subunit c, n is the number of subunit c per F0, and r is a residual fraction of ATPase activity that was resistant to inhibition by DCCD. The two methods gave values for n equal to 10 by the specific radioactivity method and 14 by the HPLC peak area method, and values for r of 0.28 and 0.30, respectively. Most of the r value was accounted for by the observed dissociation of 15-20% of the F1-ATPase from the membrane under ATPase assay conditions. When the minimal, experimentally justified value of r = 0.15 was used in the equation above, the calculated values of n were reduced to 8 and 11, respectively. The value of n determined here, with a probable range of uncertainty of 8-14, is consistent with, and provides an independent type of experimental support for, the suggested stoichiometry of 10 +/- 1 subunit c per F1F0, which was determined by a more precise radiolabeling method (Foster, D. L., and Fillingame, R. H. (1982) J. Biol. Chem. 257, 2009-2015).     

Mapping of the Na+, K(+)-ATPase subunit alpha 2 (ATP1A2) and muscle phosphofructokinase (PFKM) genes in pig by somatic cell hybrid analysis

Two expressed sequence tags were isolated from a porcine skeletal muscle cDNA library and identified as the putative partial cDNAs of the porcine Na+, K(+)-ATPase subunit alpha 2 (ATP1A2) and muscle phosphofructokinase (PFKM) genes after sequencing and homology search. Results of analysis of a pig-rodent somatic cell hybrid panel by PCR allowed the assignments of ATP1A2 to porcine chromosome (chr) 4 and of PFKM to porcine chr 5. These assignments support previously observed conservation of syntenic relationships between human chr 1 and porcine chr 4 and between human chr 12 and porcine chr 5.     

Identification of Li(+) binding sites and the effect of Li(+) treatment on phospholipid composition in human neuroblastoma cells: a (7)Li and (31)P NMR study

Li(+) binding in subcellular fractions of human neuroblastoma SH-SY 5 Y cells was investigated using (7)Li NMR spin-lattice (T(1)) and spin-spin (T(2)) relaxation measurements, as the T(1)/T(2) ratio is a sensitive parameter of Li(+) binding. The majority of Li(+) binding occurred in the plasma membrane, microsomes, and nuclear membrane fractions as demonstrated by the Li(+) binding constants and the values of the T(1)/T(2) ratios, which were drastically larger than those observed in the cytosol, nuclei, and mitochondria. We also investigated by (31)P NMR spectroscopy the effects of chronic Li(+) treatment for 4--6 weeks on the phospholipid composition of the plasma membrane and the cell homogenate and found that the levels of phosphatidylinositol and phosphatidylserine were significantly increased and decreased, respectively, in both fractions. From these observations, we propose that Li(+) binding occurs predominantly to membrane domains, and that chronic Li(+) treatment alters the phospholipid composition at these membrane sites. These findings support those from clinical studies that have indicated that Li(+) treatment of bipolar patients results in irregularities in Li(+) binding and phospholipid metabolism. Implications of our observations on putative mechanisms of Li(+) action, including the cell membrane abnormality, the inositol depletion and the G-protein hypotheses, are discussed.     

Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine fluorophore into a phospholipid bilayer: complementary use of fluorescence quenching studies and molecular dynamics simulations

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed “light switch effect”. Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from − 6.5 to − 7.0 kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP.     

In-frame recombination between the yeast H(+)-ATPase isogenes PMA1 and PMA2: insights into the mechanism of recombination initiated by a double-strand break.

Chimeric PMA1::PMA2 sequences, placed under the control of the PMA1 promoter, were constructed by in vivo recombination between a gapped linearized plasmid containing the PMA2 gene and four different fragments of the PMA1 gene. Correct in-frame assembly of the PMA sequences was screened by the expression of the lacZ reporter gene fused to the PMA2 coding region. Restriction and sequencing analysis of 35 chimeras showed that in all cases, the hybrid sequences was obtained as fusions between continuous sequences specific to PMA1 and PMA2, separated by a region of identity. In all but three cases, the junction sequences were not located at regions of greatest identity. Strikingly, depending on the PMA1 fragment used, junction distribution fell into two categories. In the first, the junctions were scattered over several hundreds of nucleotides upstream of the extremity of the PMA1 fragment, while in the second, they were concentrated at this extremity. Analysis of the alignment of the PMA1 and PMA2 sequences suggests that the distribution is not related to the size of the region of identity at the PMA1-PMA2 boundary but depends on the degree of identity of the PMA genes upstream of the region of identity, the accumulation of successive mismatches leading to a clustered distribution of the junctions. Moreover, the introduction of seven closely spaced mismatches near the end of a PMA1 segment with an otherwise-high level of identity with PMA2 led to a significantly increased concentration of the junctions near this end. These data show that a low level of identity in the vicinity of the common boundary stretch is a strong barrier to recombination. In contrast, consecutive mismatches or regions of overall moderate identity which are located several hundreds of nucleotides upstream from the PMA1 end do not necessarily block recombination.     

Identification of a decidua-specific enhancer on the human prolactin gene with two critical activator protein 1 (AP-1) binding sites

Deletion analysis of the human PRL promoter in endometrial stromal cells decidualized in vitro revealed a 536-bp enhancer located between nucleotide (nt) -2,040 to -1,505 in the 5'-flanking region. The 536-bp enhancer fragment ligated into a thymidine kinase (TK) promoter-luciferase reporter plasmid conferred enhancer activity in decidual-type cells but not nondecidual cells. DNase I footprint analysis of decidualized endometrial stromal cells revealed three protected regions, FP1-FP3. Transfection of overlapping 100-bp fragments of the 536-bp enhancer indicated that FP1 and FP3 each conferred enhancer activity. Gel shift assays indicated that both FP1 and FP3 bind activator protein 1 (AP-1), and JunD and Fra-2 are components of the AP-1 complex in decidual fibroblasts. Mutation of the AP-1 binding site in either FP1 or FP3 decreased enhancer activity by approximately 50%, while mutation of both sites almost completely abolished activity. Coexpression of the 536-bp enhancer and A-fos, a dominant negative to AP-1, decreased enhancer activity by approximately 70%. Conversely, coexpression of Fra-2 in combination with JunD or c-Jun and p300 increased enhancer activity 6- to 10-fold. Introduction of JunD and Fra-2 into nondecidual cells is sufficient to confer enhancer activity. JunD and Fra-2 protein expression was markedly increased in secretory phase endometrium and decidua of early pregnancy (high PRL content) compared with proliferative phase endometrium (no PRL). These investigations indicate that the 5'-flanking region of the human PRL gene contains a decidua-specific enhancer between nt -2,040/-1,505 and AP-1 binding sites within this enhancer region are critical for activity.