V-ATPase

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Michael Forgac - One of the best experts on this subject based on the ideXlab platform.

  • vacuolar atpases rotary proton pumps in physiology and pathophysiology
    Nature Reviews Molecular Cell Biology, 2007
    Co-Authors: Michael Forgac
    Abstract:

    The acidity of intracellular compartments and the extracellular environment is crucial to various cellular processes, including membrane trafficking, protein degradation, bone resorption and sperm maturation. At the heart of regulating acidity are the vacuolar (V-)ATPases--large, multisubunit complexes that function as ATP-driven proton pumps. Their activity is controlled by regulating the assembly of the V-ATPase complex or by the dynamic regulation of V-ATPase expression on membrane surfaces. The V-ATPases have been implicated in a number of diseases and, coupled with their complex isoform composition, represent attractive and potentially highly specific drug targets.

  • vacuolar atpases rotary proton pumps in physiology and pathophysiology
    Nature Reviews Molecular Cell Biology, 2007
    Co-Authors: Michael Forgac
    Abstract:

    The vacuolar ATPases are proton pumps that have a central role in maintaining the pH of intracellular compartments and in proton transport across the plasma membrane. Their activity is controlled at many different levels and, increasingly, their dysregulation is being linked to specific diseases. The acidity of intracellular compartments and the extracellular environment is crucial to various cellular processes, including membrane trafficking, protein degradation, bone resorption and sperm maturation. At the heart of regulating acidity are the vacuolar (V-)ATPases — large, multisubunit complexes that function as ATP-driven proton pumps. Their activity is controlled by regulating the assembly of the V-ATPase complex or by the dynamic regulation of V-ATPase expression on membrane surfaces. The V-ATPases have been implicated in a number of diseases and, coupled with their complex isoform composition, represent attractive and potentially highly specific drug targets.

  • arg 735 of the 100 kda subunit a of the yeast v atpase is essential for proton translocation
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Shoko Kawasakinishi, Tsuyoshi Nishi, Michael Forgac
    Abstract:

    The vacuolar (H+)-ATPases (V-ATPases) are ATP-dependent proton pumps that acidify intracellular compartments and pump protons across specialized plasma membranes. Proton translocation occurs through the integral V0 domain, which contains five different subunits (a, d, c, c′, and c"). Proton transport is critically dependent on buried acidic residues present in three different proteolipid subunits (c, c′, and c"). Mutations in the 100-kDa subunit a have also influenced activity, but none of these residues has proven to be required absolutely for proton transport. On the basis of previous observations on the F-ATPases, we have investigated the role of two highly conserved arginine residues present in the last two putative transmembrane segments of the yeast V-ATPase a subunit (Vph1p). Substitution of Asn, Glu, or Gln for Arg-735 in TM8 gives a V-ATPase that is fully assembled but is totally devoid of proton transport and ATPase activity. Replacement of Arg-735 by Lys gives a V-ATPase that, although completely inactive for proton transport, retains 24% of wild-type ATPase activity, suggesting a partial uncoupling of proton transport and ATP hydrolysis in this mutant. By contrast, nonconservative mutations of Arg-799 in TM9 lead to both defective assembly of the V-ATPase complex and decreases in activity of the assembled V-ATPase. These results suggest that Arg-735 is absolutely required for proton transport by the V-ATPases and is discussed in the context of a revised model of the topology of the 100-kDa subunit a.

  • structure mechanism and regulation of the clathrin coated vesicle and yeast vacuolar h atpases
    The Journal of Experimental Biology, 2000
    Co-Authors: Michael Forgac
    Abstract:

    The vacuolar H(+)-ATPases (or V-ATPases) are a family of ATP-dependent proton pumps that carry out acidification of intracellular compartments in eukaryotic cells. This review is focused on our work on the V-ATPases of clathrin-coated vesicles and yeast vacuoles. The coated-vesicle V-ATPase undergoes trafficking to endosomes and synaptic vesicles, where it functions in receptor recycling and neurotransmitter uptake, respectively. The yeast V-ATPase functions to acidify the central vacuole and is necessary both for protein degradation and for coupled transport processes across the vacuolar membrane. The V-ATPases are multisubunit complexes composed of two functional domains. The V(1) domain is a 570 kDa peripheral complex composed of eight subunits of molecular mass 73-14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V(o) domain is a 260 kDa integral complex composed of five subunits of molecular mass 100-17 kDa (subunits a, d, c, c' and c") that is responsible for proton translocation. To explore the function of individual subunits in the V-ATPase complex as well as to identify residues important in proton transport and ATP hydrolysis, we have employed a combination of chemical modification, site-directed mutagenesis and in vitro reassembly. A central question concerns the mechanism by which vacuolar acidification is controlled in eukaryotic cells. We have proposed that disulfide bond formation between conserved cysteine residues at the catalytic site of the V-ATPase plays an important role in regulating V-ATPase activity in vivo. Other regulatory mechanisms that are discussed include reversible dissociation and reassembly of the V-ATPase complex, changes in the tightness of coupling between proton transport and ATP hydrolysis, differential targeting of V-ATPases within the cell and control of the Cl(-) conductance that is necessary for vacuolar acidification.

  • structure and properties of the clathrin coated vesicle and yeast vacuolar v atpases
    Journal of Bioenergetics and Biomembranes, 1999
    Co-Authors: Michael Forgac
    Abstract:

    The V-ATPases are a family of ATP-dependent proton pumps responsible foracidification of intracellular compartments in eukaryotic cells. This reviewfocuses on the the V-ATPases from clathrin-coated vesicles and yeastvacuoles. The V-ATPase of clathrin-coated vesicles is a precursor to thatfound in endosomes and synaptic vesicles, which function in receptorrecycling, intracellular membrane traffic, and neurotransmitter uptake. Theyeast vacuolar ATPase functions to acidify the central vacuole and to drivevarious coupled transport processes across the vacuolar membrane. TheV-ATPases are composed of two functional domains. The V1 domain isa 570-kDa peripheral complex composed of eight subunits of molecular weight70—14 kDa (subunits A—H) that is responsible for ATP hydrolysis.The V0 domain is a 260-kDa integral complex composed of fivesubunits of molecular weight 100—17 kDa (subunits a, d, c, c8 and c9)that is responsible for proton translocation. Using chemical modification andsite-directed mutagenesis, we have begun to identify residues that play arole in ATP hydrolysis and proton transport by the V-ATPases. A centralquestion in the V-ATPase field is the mechanism by which cells regulatevacuolar acidification. Several mechanisms are described that may play a rolein controlling vacuolar acidification in vivo. One mechanisminvolves disulfide bond formation between cysteine residues located at thecatalytic nucleotide binding site on the 70-kDa A subunit, leading toreversible inhibition of V-ATPase activity. Other mechanisms includereversible assembly and dissociation of V1 and V0domains, changes in coupling efficiency of proton transport and ATPhydrolysis, and regulation of the activity of intracellular chloride channelsrequired for vacuolar acidification.

Masamitsu Futai - One of the best experts on this subject based on the ideXlab platform.

  • subunit rotation of vacuolar type proton pumping atpase relative rotation of the g and c subunits
    Journal of Biological Chemistry, 2003
    Co-Authors: T Hirata, Yoh Wada, Gehong Sunwada, Atsuko Iwamotokihara, Toshihide Okajima, Masamitsu Futai
    Abstract:

    Abstract Vacuolar-type ATPases V1V0 (V-ATPases) are found ubiquitously in the endomembrane organelles of eukaryotic cells. In this study, we genetically introduced a His tag and a biotin tag onto the c and G subunits, respectively, of Saccharomyces cerevisiae V-ATPase. Using this engineered enzyme, we observed directly the continuous counter-clockwise rotation of an actin filament attached to the G subunit when the enzyme was immobilized on a glass surface through the c subunit. V-ATPase generated essentially the same torque as the F-ATPase (ATP synthase). The rotation was inhibited by concanamycin and nitrate but not by azide. These results demonstrated that the V- and F-ATPase carry out a common rotational catalysis.

  • diversity of mouse proton translocating atpase presence of multiple isoforms of the c d and g subunits
    Gene, 2003
    Co-Authors: Gehong Sunwada, Yoh Wada, Yoko Imaisenga, Takao Yoshimizu, Masamitsu Futai
    Abstract:

    Abstract Vacuolar-type proton-translocating ATPases (V-ATPases), multimeric proton pumps, are involved in a wide variety of physiological processes. For their diverse functions, V-ATPases utilize a specific subunit isoform(s). Here, we reported the molecular cloning and characterization of three novel subunit isoforms, C 2, d 2 and G 3, of mouse V-ATPase. These isoforms were expressed in a tissue-specific manner, in contrast to the ubiquitously expressed C 1, d 1 and G 1 isoforms. C 2 was expressed predominantly in lung and kidney, and d 2 and G 3 specifically in kidney. We introduced these isoforms into yeasts lacking the corresponding genes. Although the G 3 and d 2 did not rescue the vmaΔ phenotype, d 1 and the two C isoforms functionally complemented the Δvma6 and Δvma5 , respectively, indicating that they are bona fide subunits of V-ATPase.

  • a proton pump atpase with testis specific e1 subunit isoform required for acrosome acidification
    Journal of Biological Chemistry, 2002
    Co-Authors: Gehong Sunwada, Yoh Wada, Yoko Imaisenga, Akitsugu Yamamoto, Yoshiko Murata, Tomoyuki Hirata, Masamitsu Futai
    Abstract:

    Abstract The vacuolar-type H+-ATPases (V-ATPases) are a family of multimeric proton pumps involved in a wide variety of physiological processes. We have identified two novel mouse genes, Atp6e1 and Atp6e2, encoding testis-specific (E1) and ubiquitous (E2) V-ATPase subunit E isoforms, respectively. TheE1 transcript appears about 3 weeks after birth, corresponding to the start of meiosis, and is expressed specifically in round spermatids in seminiferous tubules. Immunohistochemistry with isoform-specific antibodies revealed that the V-ATPase withE1 and a2 isoforms is located specifically in developing acrosomes of spermatids and acrosomes in mature sperm. In contrast, the E2 isoform was expressed in all tissues examined and present in the perinuclear compartments of spermatocytes. The E1 isoform exhibits 70% identity with theE2, and both isoforms functionally complemented a null mutation of the yeast counterpart VMA4, indicating that they are bona fide V-ATPase subunits. The chimeric enzymes showed slightly lower K m ATP than yeast V-ATPase. Consistent with the temperature-sensitive growth of Δvma4-expressing E1 isoform, vacuolar membrane vesicles exhibited temperature-sensitive coupling between ATP hydrolysis and proton transport. These results suggest thatE1 isoform is essential for energy coupling involved in acidification of acrosome.

  • vacuolar type h atpase in mouse bladder epithelium is responsible for urinary acidification
    FEBS Letters, 1997
    Co-Authors: Ken Ichi Tomochika, Yoshinori Moriyama, Sumio Shinoda, Hiromi Kumon, Masaharu Mori, Masamitsu Futai
    Abstract:

    Abstract The urine in the mouse bladder was found to be acidic, ranging from pH 5.3 to 5.5 in the daytime and pH 6.0 to 6.3 at night. Administration of bafilomycin A1 or concanamycin A, specific inhibitors of vacuolar-type H+-ATPase, into bladder lumen caused neutralization of urinary pH at least for 36 h, whereas inhibitors of mitochondrial ATP synthase (F-type H+-ATPase) or P-type H+-ATPases did not. Bafilomycin A1-sensitive proton secretion from isolated inside-out bladder was also observed. Immuno-electron microscopy with antibodies against vacuolar H+-ATPase revealed that vacuolar-type H+-ATPase is rich in luminal plasma membrane and endosomes of superficial cells of the bladder epithelium. These results indicate that vacuolar-type H+-ATPases present in luminal plasma membrane of the superficial epithelial cells secrete protons so as to acidify the urine in mouse bladder. © 1997 Federation of European Biochemical Societies.

  • vanadate sensitive atpase from chromaffin granule membranes formed a phosphoenzyme intermediate and was activated by phosphatidylserine
    Archives of Biochemistry and Biophysics, 1991
    Co-Authors: Yoshinori Moriyama, Nathan Nelson, Masatomo Maeda, Masamitsu Futai
    Abstract:

    Abstract Vanadate-sensitive ATPase (115 kDa molecular weight) in adrenal chromaffin granules is an intrinsic membrane enzyme with its catalytic site located at the outer surface of the granules. Upon incubation with [γ-32P]ATP, the purified ATPase formed an alkaline-labile phosphoenzyme intermediate, which was inhibited by vanadate but not by Na+ or K+. Ratio of ATPase or phosphatase activity and formation of phosphoenzyme intermediate was constant during purification after the first glycerol density gradient centrifugation. Phosphatidyl-serine specifically activated the enzyme about three-fold by increasing the Vmax value without changing the Km for ATP. Other phospholipids, including phosphatidyl-glycerol, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, as well as lysophospholipids and detergents, had no effect. These results indicated that the vanadate-sensitive ATPase belongs to the P-type ATPases, which differ from known cation-translocating P-type ATPases.

Yoshimi Kakinuma - One of the best experts on this subject based on the ideXlab platform.

  • rotation mechanism of enterococcus hirae v 1 atpase based on asymmetric crystal structures
    Nature, 2013
    Co-Authors: Yoshimi Kakinuma, Satoshi Arai, Shinya Saijo, Kano Suzuki, Kenji Mizutani, Yoshiko Ishizukakatsura, Noboru Ohsawa
    Abstract:

    Several crystal structures of the rotary motor of bacterial V-ATPase are solved at high resolution, representing different asymmetric structures and enabling the prediction of a model for the rotational mechanism of V1-ATPase. Vacuolar-type H+-ATPases (V-ATPases) are biomolecular rotary motors that couple ATP hydrolysis to the transport of a proton across intracellular and plasma membranes of eukaryotic cells. V-ATPase functions in many cellular processes and is an important drug target for diseases such as osteoporosis and cancer. This paper reports several X-ray crystal structures of a V1-ATPase from Enterococcus hirae, which reveal the conformational changes that occur when ATP binds the protein. On the basis of these structures the authors propose a model for the rotation mechanism of this membrane protein. In various cellular membrane systems, vacuolar ATPases (V-ATPases) function as proton pumps, which are involved in many processes such as bone resorption and cancer metastasis, and these membrane proteins represent attractive drug targets for osteoporosis and cancer1. The hydrophilic V1 portion is known as a rotary motor, in which a central axis DF complex rotates inside a hexagonally arranged catalytic A3B3 complex using ATP hydrolysis energy, but the molecular mechanism is not well defined owing to a lack of high-resolution structural information. We previously reported on the in vitro expression, purification and reconstitution of Enterococcus hirae V1-ATPase from the A3B3 and DF complexes2,3. Here we report the asymmetric structures of the nucleotide-free (2.8 A) and nucleotide-bound (3.4 A) A3B3 complex that demonstrate conformational changes induced by nucleotide binding, suggesting a binding order in the right-handed rotational orientation in a cooperative manner. The crystal structures of the nucleotide-free (2.2 A) and nucleotide-bound (2.7 A) V1-ATPase are also reported. The more tightly packed nucleotide-binding site seems to be induced by DF binding, and ATP hydrolysis seems to be stimulated by the approach of a conserved arginine residue. To our knowledge, these asymmetric structures represent the first high-resolution view of the rotational mechanism of V1-ATPase.

  • structure and mechanism of vacuolar na translocating atpase from enterococcus hirae
    Journal of Bioenergetics and Biomembranes, 2005
    Co-Authors: Takeshi Murata, Ichiro Yamato, Yoshimi Kakinuma
    Abstract:

    V-type Na+-ATPase from Entercoccus hirae consists of nine kinds of subunits (NtpA3, B3, C1, D1, E1−3, F1−3, G1, I1, and K10) which are encoded by the ntp operon. The amino acid sequences of the major subunits, A, B, and K (proteolipid), were highly similar to those of A, B, and c subunits of eukaryotic V-ATPases, and those of β, α, and c subunits of F-ATPases. We modeled the A and B subunits by homology modeling using the structure of β and α subunits of F-ATPase, and obtained an atomic structure of NtpK ring by X-ray crystallography. Here we briefly summarize our current models of the whole structure and mechanism of the E. hirae V-ATPase.

  • catalytic properties of na translocating v atpase in enterococcus hirae
    Biochimica et Biophysica Acta, 2001
    Co-Authors: Takeshi Murata, Ichiro Yamato, Kazuei Igarashi, Miyuki Kawano, Yoshimi Kakinuma
    Abstract:

    V-ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. We found a variant of this family, a Na a -translocating ATPase, in a Gram-positive bacterium, Enterococcus hirae. The Na a -ATPase was encoded by nine ntp genes from F to D in an ntp operon (ntpFIKECGABDHJ): the ntpJ gene encoded a K a transporter independent of the Na a -ATPase. Expression of this operon, encoding two transport systems for Na a and K a ions, was regulated at the transcriptional level by intracellular Na a as the signal. Structural aspects and catalytic properties of purified Na a -ATPase closely resembled those of other V-type H a -ATPases. Interestingly, the E. hirae enzyme showed a very high affinity for Na a at catalytic reaction. This property enabled the measurement of ion binding to this ATPase for the first time in the study of V- and F-ATPases. Properties of Na a binding to V-ATPase were consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. We propose here a structure model of Na a binding sites of the enzyme. fl 2001 Elsevier Science B.V. All rights reserved.

  • cloning and sequencing of the genes coding for the a and b subunits of vacuolar type na atpase from enterococcus hirae coexistence of vacuolar and f0f1 type atpases in one bacterial cell
    Journal of Biological Chemistry, 1993
    Co-Authors: Kazuma Takase, Ichiro Yamato, Yoshimi Kakinuma
    Abstract:

    The eubacterium Enterococcus hirae ATCC 9790 possesses a H(+)-translocating ATPase, and the deduced amino acid sequences of the genes coding for this enzyme have indicated that it is a typical F0F1-type ATPase (Shibata, C., Ehara, T., Tomura, K., Igarashi, K., and Kobayashi, H. (1992) J. Bacteriol. 174, 6117-6124). We cloned the ntpA and ntpB genes coding for the A and B subunits, respectively, of Na(+)-translocating ATPase from the same bacterium, and the full amino acid sequences of the two subunits were deduced from the nucleotide sequence. The A (593 amino acid residues) and B (458 amino acid residues) subunits were highly homologous (48-60% identical) to the A (large or alpha) and the B (small or beta) subunits, respectively, of vacuolar-type H(+)-ATPases which have been found in eukaryotic endomembrane systems (Neurospora crassa, Saccharomyces cerevisiae, Arabidopsis thaliana, and carrot) and archaebacterial cell membranes (Sulfolobus acidocaldarius and Methanosarcina barkeri). The A and B subunits of Na(+)-ATPase showed about 23-28% identities with the beta and alpha subunits of E. hirae F1-ATPase and of Escherichia coli F1-ATPase, respectively. These results indicate that E. hirae Na(+)-ATPase belongs to the vacuolar-type ATPase. This is the first demonstration that both genes for V- and F-type ATPases are functionally expressed in one bacterial cell.

Todd R Graham - One of the best experts on this subject based on the ideXlab platform.

  • directed evolution of a sphingomyelin flippase reveals mechanism of substrate backbone discrimination by a p4 atpase
    Proceedings of the National Academy of Sciences of the United States of America, 2016
    Co-Authors: Bartholomew P Roland, Todd R Graham
    Abstract:

    Phospholipid flippases in the type IV P-type ATPase (P4-ATPases) family establish membrane asymmetry and play critical roles in vesicular transport, cell polarity, signal transduction, and neurologic development. All characterized P4-ATPases flip glycerophospholipids across the bilayer to the cytosolic leaflet of the membrane, but how these enzymes distinguish glycerophospholipids from sphingolipids is not known. We used a directed evolution approach to examine the molecular mechanisms through which P4-ATPases discriminate substrate backbone. A mutagenesis screen in the yeast Saccharomyces cerevisiae has identified several gain-of-function mutations in the P4-ATPase Dnf1 that facilitate the transport of a novel lipid substrate, sphingomyelin. We found that a highly conserved asparagine (N220) in the first transmembrane segment is a key enforcer of glycerophospholipid selection, and specific substitutions at this site allow transport of sphingomyelin.

  • reconstitution of phospholipid translocase activity with purified drs2p a type iv p type atpase from budding yeast
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: Xiaoming Zhou, Todd R Graham
    Abstract:

    Type-IV P-type ATPases (P4-ATPases) are putative phospholipid translocases, or flippases, that translocate specific phospholipid substrates from the exofacial to the cytosolic leaflet of membranes to generate phospholipid asymmetry. In addition, the activity of Drs2p, a P4-ATPase from Saccharomyces cerevisiae, is required for vesicle-mediated protein transport from the Golgi and endosomes, suggesting a role for phospholipid translocation in vesicle budding. Drs2p is necessary for translocation of a fluorescent phosphatidylserine analogue across purified Golgi membranes. However, a flippase activity has not been reconstituted with purified Drs2p or any other P4-ATPase, so whether these ATPases directly pump phospholipid across the membrane bilayer is unknown. Here, we show that Drs2p can catalyze phospholipid translocation directly through purification and reconstitution of this P4-ATPase into proteoliposomes. The noncatalytic subunit, Cdc50p, also was reconstituted in the proteoliposome, although at a substoichiometric concentration relative to Drs2p. In proteoliposomes containing Drs2p, a phosphatidylserine analogue was actively flipped across the liposome bilayer to the outer leaflet in the presence of Mg2+-ATP, whereas no activity toward the phosphatidylcholine or sphingomyelin analogues was observed. This flippase activity was mediated by Drs2p, because protein-free liposomes or proteoliposomes reconstituted with a catalytically inactive form of Drs2p showed no translocation activity. These data demonstrate for the first time the reconstitution of a flippase activity with a purified P4-ATPase.

  • linking phospholipid flippases to vesicle mediated protein transport
    Biochimica et Biophysica Acta, 2009
    Co-Authors: Babyperiyanayaki Muthusamy, Paramasivam Natarajan, Xiaoming Zhou, Todd R Graham
    Abstract:

    Type IV P-type ATPases (P4-ATPases) are a large family of putative phospholipid translocases (flippases) implicated in the generation of phospholipid asymmetry in biological membranes. P4-ATPases are typically the largest P-type ATPase subgroup found in eukaryotic cells, with five members in Saccharomyces cerevisiae, six members in Caenorhabditis elegans, 12 members in Arabidopsis thaliana and 14 members in humans. In addition, many of the P4-ATPases require interaction with a noncatalytic subunit from the CDC50 gene family for their transport out of the endoplasmic reticulum (ER). Deficiency of a P4-ATPase (Atp8b1) causes liver disease in humans, and studies in a variety of model systems indicate that P4-ATPases play diverse and essential roles in membrane biogenesis. In addition to their proposed role in establishing and maintaining plasma membrane asymmetry, P4-ATPases are linked to vesicle-mediated protein transport in the exocytic and endocytic pathways. Recent studies have also suggested a role for P4-ATPases in the nonvesicular intracellular trafficking of sterols. Here, we discuss the physiological requirements for yeast P4-ATPases in phospholipid translocase activity, transport vesicle budding and ergosterol metabolism, with an emphasis on Drs2p and its noncatalytic subunit, Cdc50p.

Jose M Arguello - One of the best experts on this subject based on the ideXlab platform.

  • structure of the atp binding domain from the archaeoglobus fulgidus cu atpase
    Journal of Biological Chemistry, 2006
    Co-Authors: Matthew H. Sazinsky, Jose M Arguello, Atin K. Mandal, Amy C Rosenzweig
    Abstract:

    Abstract The P-type ATPases translocate cations across membranes using the energy provided by ATP hydrolysis. CopA from Archaeoglobus fulgidus is a hyperthermophilic ATPase responsible for the cellular export of Cu+ and is a member of the heavy metal P1B-type ATPase subfamily, which includes the related Wilson and Menkes diseases proteins. The Cu+-ATPases are distinct from their P-type counter-parts in ion binding sequences, membrane topology, and the presence of cytoplasmic metal binding domains, suggesting that they employ alternate forms of regulation and novel mechanisms of ion transport. To gain insight into Cu+-ATPase function, the structure of the CopA ATP binding domain (ATPBD) was determined to 2.3 A resolution. Similar to other P-type ATPases, the ATPBD includes nucleotide binding (N-domain) and phosphorylation (P-domain) domains. The ATPBD adopts a closed conformation similar to the nucleotide-bound forms of the Ca2+-ATPase. The CopA ATPBD is much smaller and more compact, however, revealing the minimal elements required for ATP binding, hydrolysis, and enzyme phosphorylation. Structural comparisons to the AMP-PMP-bound form of the Escherichia coli K+-transporting Kdp-ATPase and to the Wilson disease protein N-domain indicate that the five conserved N-domain residues found in P1B-type ATPases, but not in the other families, most likely participate in ATP binding. By contrast, the P-domain includes several residues conserved among all P-type ATPases. Finally, the CopA ATPBD structure provides a basis for understanding the likely structural and functional effects of various mutations that lead to Wilson and Menkes diseases.

  • identification of ion selectivity determinants in heavy metal transport p1b type atpases
    The Journal of Membrane Biology, 2003
    Co-Authors: Jose M Arguello
    Abstract:

    P1B-type ATPases transport a variety of metals (Cd2+, Zn2+, Pb2+, Co2+, Cu2+, Ag+, Cu+) across biomembranes. Characteristic sequences CP[C/H/S] in transmembrane fragment H6 were observed in the putative transporting metal site of the founding members of this subfamily (initially named CPx-ATPases). In spite of their importance for metal homeostasis and biotolerance, their mechanisms of ion selectivity are not understood. Studies of better-characterized PII-type ATPases (Ca-ATPase and Na,K-ATPase) have identified three transmembrane segments that participate in ion binding and transport. Testing the hypothesis that metal specificity is determined by conserved amino acids located in the equivalent transmembrane segments of P1B-type ATPases (H6, H7, and H8), 234 P1B-ATPase protein sequences were analyzed. This showed that although H6 contains characteristic CPX or XPC sequences, conserved amino acids in H7 and H8 provide signature sequences that predict the metal selectivity in each of five P1B-ATPase subgroups identified. These invariant amino acids contain diverse side chains (thiol, hydroxyl, carbonyl, amide, imidazolium) that can participate in transient metal coordination during transport and consequently determine the particular metal selectivity of each enzyme. Each subgroup shares additional structural characteristics such as the presence (or absence) of particular amino-terminal metal-binding domains and the number of putative transmembrane segments. These differences suggest unique functional characteristics for each subgroup in addition to their particular metal specificity.

  • characterization of a thermophilic p type ag cu atpase from the extremophile archaeoglobus fulgidus
    Journal of Biological Chemistry, 2002
    Co-Authors: Atin K. Mandal, Win D Cheung, Jose M Arguello
    Abstract:

    Abstract The thermophilic, sulfur metabolizingArchaeoglobus fulgidus contains two genes,AF0473 and AF0152, encoding for PIB-type heavy metal transport ATPases. In this study, we describe the cloning, heterologous expression, purification, and functional characterization of one of these ATPases, CopA (NCB accession number AAB90763), encoded by AF0473. CopA is active at high temperatures (75 °C; E a = 103 kJ/mol) and inactive at 37 °C. It is activated by Ag+(ATPase V max = 14.82 μmol/mg/h) and to a lesser extent by Cu+ (ATPase V max = 3.66 μmol/mg/h). However, Cu+ interacts with the enzyme with higher apparent affinity (ATPase stimulation, Ag+ K = 29.4 μm; Cu+ K = 2.1 μm). This activation by Ag+ or Cu+ is dependent on the presence of millimolar amounts of cysteine. In the presence of ATP, these metals drive the formation of an acid-stable phosphoenzyme with apparent affinities similar to those observed in the ATPase activity determinations (Ag+, K = 23.0 μm; Cu+, K = 3.9 μm). However, comparable levels of phosphoenzyme are reached in the presence of both cations (Ag+, 1.40 nmol/mg; Cu+, 1.08 nmol/mg). The stimulation of phosphorylation by the cations suggests that CopA drives the outward movement of the metal. CopA presents additional functional characteristics similar to other P-type ATPases. ATP interacts with the enzyme with two apparent affinities (ATPase K m = 0.25 mm; phosphorylation K m = 4.81 μm), and the presence of vanadate leads to enzyme inactivation (IC50= 24 μm). This is the first Ag+/Cu+-ATPase expressed and purified in a functional form. Thus, it provides a model for structure-functional studies of these transporters. Moreover, its characterization will also contribute to an understanding of thermophilic ion transporters.