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Identification and Functional Expression of Four Isoforms of ATPase II, the Putative Aminophospholipid Translocase

EFFECT OF ISOFORM VARIATION ON THE ATPase ACTIVITY AND PHOSPHOLIPID SPECIFICITY*
Open AccessPublished:July 28, 2000DOI:https://doi.org/10.1074/jbc.M910319199
      ATPase II, a vanadate-sensitive and phosphatidylserine-dependent Mg2+-ATPase, is a member of a subfamily of P-type ATPase and is presumably responsible for aminophospholipid translocation activity in eukaryotic cells. The aminophospholipid translocation activity plays an important physiological role in the maintenance of membrane phospholipid asymmetry that is observed in the plasma membrane as well as the membranes of certain cellular organelles. While the preparations of ATPase II from different sources share common fundamental properties, such as substrate specificity, inhibitor spectrum, and phospholipid dependence, they are divergent in several characteristics. These include specific ATPase activity and phospholipid selectivity. We report here the identification of four isoforms of ATPase II in bovine brain. These isoforms are formed by a combination of two major variations in their primary sequences and show that the structural variation of these isoforms has functional significance in both ATPase activity and phosholipid selectivity. Furthermore, studies with the phosphoenzyme intermediate of ATPase II and its recombinant isoforms revealed that phosphatidylserine is essential for the dephosphorylation of the intermediate. Without phosphatidylserine, ATPase II would be accumulated as phosphoenzyme in the presence of ATP, resulting in the interruption of its catalytic cycle.
      PM
      plasma membrane
      C12E9
      polyoxyethelene 9-lauryl ether
      PC
      phosphatidylcholine
      PE
      phosphatidylethanolamine
      PS
      phosphatidylserine
      PCR
      polymerase chain reaction
      DTT
      dithiothreitol
      PAGE
      polyacrylamide gel electrophoresis
      FPLC
      fast flow liquid chromatography
      NTA
      nitrolotriacetic acid
      aa
      amino acid(s)
      bp
      base pair(s)
      MES
      4-morpholineethanesulfonic acid
      The phospholipid distribution in the plasma membrane (PM)1 of eukaryotic cells is asymmetric. The outer leaflet consists predominantly of phosphatidylcholine (PC) and sphingomyelin, whereas aminophospholipids, including phosphatidylserine (PS) and phosphatidylethanolamine (PE), are present almost exclusively in the inner leaflet (
      • Devaux P.F.
      ,
      • Williamson P.
      • Schlegel R.A.
      ). The asymmetric nature of phospholipid composition is found in several other intracellular membrane systems as well, and the physiologic importance of phospholipid asymmetry is multifold. The asymmetric distribution of lipids provides the two sides of the membrane with different characteristics that are necessary for their respective physiologic function. For instance, the tight packing of the outer leaflet of the PM is important for membrane stability in circulating blood cells and the resistance of cells to attack by cytotoxic T lymphocytes (
      • Antia R.
      • Schlegel R.A.
      • Williamson P.
      ). On the other hand, the enrichment of aminophospholipid in the inner leaflet of the PM and the cytosolic leaflet of endocytotic and exocytotic vesicle membranes may contribute to these surfaces being in a fusion-competent state (
      • Schlegel R.A.
      • Williamson P.
      ). In addition, the regulated disruption of phospholipid asymmetry in the PM provides a pathway for cellular signaling in certain physiologic events. The surface exposure of PS, for example, has been demonstrated in activated platelet to promote the reaction cascade of blood coagulation (
      • Bevers E.M.
      • Comfurius P.
      • Van Rijn J.
      • Hemker C.
      • Zwaal R.F.
      ,
      • Bevers E.M.
      • Comfurius P.
      • Zwaal R.F.
      ) and in apoptotic cells to trigger the recognition between these cells and macrophages (
      • Fadok V.A.
      • Voelker D.R.
      • Campbell P.A.
      • cohen J.J.
      • Brantton D.L.
      • Henson P.M.
      ,
      • Bennett M.R.
      • Gibson D.F.
      • Schwartz S.M
      • Tait J.F.
      ). Furthermore, the dynamic process of phospholipid translocation may play important roles in cellular events such as membrane budding and endocytosis (
      • Williamson P.
      • Schlegel R.A.
      ).
      While a thorough understanding of the mechanism for membrane asymmetry and phospholipid translocation has yet to emerge, one of the enzymes that plays a major role in these processes has been identified as aminophospholipid translocase (
      • Seigneuret M.
      • Devaux P.F.
      ,
      • Zachowski A.
      • Favre E.
      • Cribier S.
      • Herve P.
      • Devaux P.F.
      ). This activity catalyzes an energy-dependent aminophospholipid translocation requiring Mg2+ and ATP (
      • Zachowski A.
      • Favre E.
      • Cribier S.
      • Herve P.
      • Devaux P.F.
      ), and it is sensitive to the sulfhydryl group reagent N-ethylmaleimide and vanadate, an inhibitor of P-type ATPases (
      • Seigneuret M.
      • Devaux P.F.
      ,
      • Daleke D.L.
      • Huestis W.J.
      ). The primary candidate protein for this activity is ATPase II, a PS-dependent and vanadate-sensitive Mg2+-ATPase. This ATPase has an apparent molecular mass of about 116 kDa and has been isolated and purified from several sources including chromaffin granules (
      • Moriyama Y.
      • Nelson N.
      ), clathrin-coated vesicles (
      • Xie X.-S.
      • Stone D.K.
      • Racker E.
      ), and the plasma membrane of erythrocytes (
      • Morrot G.
      • Zachowski A.
      • Devaux P.F.
      ). This enzyme is a P-type ATPase with characteristics strikingly similar to those of the aminophospholipid translocase activity measured in situ (
      • Seigneuret M.
      • Devaux P.F.
      ). Comparison of the sequence data of ATPase II from bovine chromaffin (
      • Tang X.
      • Halleck M.S.
      • Schlegel R.A.
      • Williamson P.
      ), mouse muscle,
      D. Pradhan, C. F. Blackman, P. Williamson, and R. A. Schlegel, GenBankTM accession numberU75321.
      2D. Pradhan, C. F. Blackman, P. Williamson, and R. A. Schlegel, GenBankTM accession numberU75321.
      and human tissues (
      • Mouro I.
      • Halleck M.S.
      • Schlegel R.A.
      • Mattei M.G.
      • Williamson P.
      • Zachowski A.
      • Devaux P.F.
      • Cartron J.P.
      • Colin Y.
      )
      Y. Nakanishi, GenBankTM accession number AB013452.
      3Y. Nakanishi, GenBankTM accession number AB013452.
      indicates that this enzyme contains 10 putative transmembrane domains and several P-type ATPase consensus sequences. An ATP binding site and a phosphoenzyme formation site are located within the largest cytosolic loop, whereas a sequence implicated in the coupling to transport activity was identified in another hydrophilic loop. The sequence of mammalian ATPase II is homologous to a yeast ATPase encoded by the DRS2 gene, of which the null mutant of a yeast strain lacks a specific PS internalization activity that is otherwise present in wild type yeast strains (
      • Tang X.
      • Halleck M.S.
      • Schlegel R.A.
      • Williamson P.
      ). This observation provides further evidence indicating the involvement of ATPase II in PS translocation. Furthermore, proteoliposomes that were reconstituted with purified ATPase II from erythrocytes were shown to transport fluorescent PS, and to a lesser extent PE, but not PC (
      • Auland M.E.
      • Roufogalis B.D.
      • Devaux P.F.
      • Zachowski A.
      ).
      The ATPase II from different mammalian sources, however, is divergent in several characteristics. The specific ATPase activity of these preparations, for instance, is reported to range from 0.8 (μmol Pi/min/mg of protein) for the enzyme isolated from human erythrocyte (
      • Morrot G.
      • Zachowski A.
      • Devaux P.F.
      ) to 8.0 for that from bovine chromaffin granules (
      • Moriyama Y.
      • Nelson N.
      ) and to 42 for the preparation from clathrin-coated vesicles (
      • Xie X.-S.
      • Stone D.K.
      • Racker E.
      ). Furthermore, the stimulating effect of PS over PE on these preparations varies from 2- to 20-fold. This disparity may be of physiological importance in fulfilling the requirement of different tissue- and/or organelle-specific processes in which aminophospholipid translocation is involved. Differences in the distribution of aminophospholipids in various cellular compartments may arise from intrinsic functional differences between isoforms of ATPase II, different co-factors that are required for the function of ATPase II, or entirely different enzymes. Alternatively, differences in the activity profile of ATPase II preparations from various sources may be due to variations in the purification protocols used to isolate these ATPases from various sources. Clarification of these issues would facilitate the study of aminophospholipid translocation system, a very important but poorly understood enzyme system.
      Although certain differences between the cDNA sequences of ATPase II from different sources have been found, their correlation to biochemical activity has not been established because of the limited characterization of these preparations at a protein level.
      ATPase II, when purified from clathrin-coated vesicles, appears as a doublet of 116 kDa by SDS-PAGE (
      • Xie X.-S.
      • Stone D.K.
      • Racker E.
      ). The two bands behave strikingly similar when either photoaffinity-labeled by [α-32P]ATP (
      • Xie X.-S.
      • Stone D.K.
      • Racker E.
      ) or analyzed for phosphoenzyme formation with [γ-32P]ATP.
      J. Ding and X.-S. Xie, unpublished observation.
      4J. Ding and X.-S. Xie, unpublished observation.
      These two proteins have proven difficult to separate biochemically, although gel filtration chromatography indicates that the two proteins do not exist as a complexed dimer (
      • Xie X.-S.
      • Stone D.K.
      • Racker E.
      ). These results, in composite, suggest the possibility that the two proteins represent different isozymes of ATPase II. The current studies were undertaken to characterize basic functional properties of the bovine brain form(s) of ATPase II and to determine if structurally and functionally distinct isoforms of the enzyme exist in brain.
      In this paper, we report the identification of four isoforms of ATPase II from bovine brain. These isoforms are characterized with respect to structural and functional differences in both ATPase activity and phospholipid selectivity. In addition, studies with the phosphoenzyme intermediate of ATPase II and its recombinant isoforms revealed that PS is essential for the dephosphorylation of the intermediate. The catalytic cycle of ATPase II would be stopped without PS or PE, resulting in the accumulation of its phosphoenzyme intermediate. This observation provides a biochemical explanation for the aminophospholipid dependence of this enzyme, as well as an important clue for further understanding of the mechanism of how aminophospholipid translocase transports PS across biological membranes.

      Acknowledgments

      We thank Drs. Dennis K. Stone, Shmuel Muallem, and Donald Hilgemann for helpful discussions and comments. Superb technical assistance in DNA sequence and tissue culture were provided by Jay Hunter and Sue Jean Tsai. We are grateful for the administrative assistance provided by Kay Martin and Pat Webb.

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