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Expression of a prokaryotic P-type ATPase in E. coli plasma membranes and purification by Ni2+-affinity chromatography

Biological Procedures Online volume 1, pages 70–80 (1998)Cite this article

Abstract

In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993) et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20–25% of the membrane protein. The purification of the Synechocystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC50= 119 μM) and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

References

  1. Møller J.V., Juul B., le Maire M. 1996. Structural organization, ion transport, and energy transduction of P-type ATPases. Biochim. Biophys. Acta 1286, 1–51.

    PubMed  Google Scholar 

  2. Tang X., Halleck M.S., Schlegel R.A., Williamson P. 1996. A subfamily of P-type ATPases with aminophospholipid transporting activity. Science 272, 1595–1497.

    Article  Google Scholar 

  3. Geisler M., Koenen W., Richter J., Schumann J. 1998. Expression and characterization of a Synechocystis PCC 6803 P-type ATPase in E. coli plasma membranes. Biochim. Biophys. Acta 1368, 267–275.

    Article  PubMed  CAS  Google Scholar 

  4. Jung K., Tjaden B., Altendorf K. (1997) Purification, Reconstitution, and Characterization of kdpD, the Turgor Sensor of Escherichia coli. J. Biol. Chem. 272, 10847–10852.

    Article  PubMed  CAS  Google Scholar 

  5. Berkelman T., Garret-Engele P., Hoffman N.E. 1994. The pacL gene of Synechococcus sp. Strain PCC 7942 encodes a Ca2+-Transporting ATPase. J. Bacteriol. 176, 4430–4436.

    PubMed  CAS  Google Scholar 

  6. Lichtner R., Wolf H.U. 1980. Phosphorylation of the isolated high-affinity (Ca2+ + Mg2+) ATPase of the human erythrocyte membrane. Biochim Biophys Acta 598, 472–85.

    Article  PubMed  CAS  Google Scholar 

  7. Hsieh W., Pierce W.S., Sze H. 1991. Calcium-pumping ATPases in vesicles from carrot cells. Stimulation by calmodulin or phosphatidylserine, and formation of a 120 kilodalton phosphoenzyme. Plant Physiol 97, 1535–1544.

    Article  PubMed  CAS  Google Scholar 

  8. Okorokov L.A., Tanner W., Lehle L. 1993. A novel primary Ca2+-transport system from Saccharomyces cerevisiae. FEBS Lett 216, 573–577.

    CAS  Google Scholar 

  9. Bowman E.M., Siebers A., Altendorf K. 1988. Bafilomycins: A class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc. Natl. Acad. Sci. USA 85, 7972–7976.

    Article  PubMed  CAS  Google Scholar 

  10. Lytton J., Westlin M., Burk S.E., Shull G.E., MacLennan D.H. 1992. Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps. J. Biol. Chem. 267, 14483–14489.

    PubMed  CAS  Google Scholar 

  11. Hochuli E., Döbeli H., Schacher A. 1987. New metal chelate adsorbenss selective for proteins and peptide containg neighbouring histidine residues. J. Chromatography 411, 177–184.

    Article  CAS  Google Scholar 

  12. Geisler M., Richter J., Schumann J. (1993) Molecular Cloning of a P-type ATPase Gene from the Cyanobacterium Synechocystis sp. PCC 6803. Homology to Eukaryotic Ca2+-ATPases. J. Mol. Biol 243, 1284–128913.

    Article  Google Scholar 

  13. Kaneko T., Sato S., Kotani H., Tanaka A., Azamizu E., Nakamura Y., Miyajima N., Hirosawa M., Sugiura M., Sasamoto S., Kimura T., Hosouchi T., Matsuno A., Muraki A., Nakazaki N., Naruo K., Okumura S., Shimpo S., Takeuchi C., Wada T., Watanabe A., Yamada M., Yasuda M., Tabata S. 1996. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 3, 109–13614.

    Article  PubMed  CAS  Google Scholar 

  14. Siebers A., Altendorf K. 1988. The K+-translocating Kdp-ATPase from Escherichia coli. Eur. J. Biochem 178, 131–140.

    Article  PubMed  CAS  Google Scholar 

  15. Geisler M., Jakobs B., Richter J., Schumann J. 1996. Expression and Characterization of a Synechocystis PCC 6803 P-type ATPase in E. coli Plasma Membranes. Biochim. Biophys. Acta 1309, 189–193.

    PubMed  CAS  Google Scholar 

  16. Clarke D.M., Loo T.W., MacLennan D.H. 1990. Functional Consequences of Mutations of Conserved Amino Acids in the β-Strand Domain of the Ca2+-ATPase of Sarcoplasmic Reticulum. J. Biol. Chem. 265, 14088–14092.

    PubMed  CAS  Google Scholar 

  17. Inesi G., Zhang Z., Sagara Y., Kirtley M.E. 1994. Intracellular signaling through long-range linked functions in the Ca2+ transport ATPase. Biophys. Chemistry 50, 129–138.

    Article  CAS  Google Scholar 

  18. Sarkadi B., Enyedi A., Földes-Papp Z., Gárdos G. 1986. Molecular Characterization of the in Situ Red Cell Membrane Calucium Pump by Limited Proteolysis. J. Biol. Chem. 261, 9552–9557.

    PubMed  CAS  Google Scholar 

  19. Carafoli E. 1991. Calcium Pump of the Plasma Membrane. Phys. Rev. 71 129–153.

    CAS  Google Scholar 

  20. Carafoli E. 1995. Biogenesis: Plasma membrane calcium ATPase: 15 years of work on the purified enzyme. FASEB 8, 993–1002.

    Google Scholar 

  21. Heim R., Iwata T., Zvaritch E., Adamo H.P., Rutishauer B., Strehler E.E., Guerini D., Carafoli E. 1992. Expression, Purification, and Properties of the Plasma Membrane Ca2+ Pump and of Its Nterminally Truncated 105-kDa Fragment. J. Biol. Chem. 267, 24476–24484.

    PubMed  CAS  Google Scholar 

  22. Lanfermeijer F.C., Venema K., Palmgren M.G. 1997. Purification of heterologous expressed plant plasma membrane H+-ATPase by Ni2+ affinity chromatography. Ann. NY Acad. Sci. 834, 139–141.

    Article  PubMed  CAS  Google Scholar 

  23. Papp B., Enyedi A., Kovács T., Sarkadi B., Wuytack F., Thastrup O., Gárdos G., Bredoux R., Levy-Toledano S., Enouf J. 1991. Demonstration of Two Forms of Calcium Pumps by Thapsigargin Inhibition and Radioimmunoblotting in Platelet Membrane Vesicles. J. Biol. Chem. 266, 14593–14596.

    PubMed  CAS  Google Scholar 

  24. Laemmli U.K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680–685.

    Article  PubMed  CAS  Google Scholar 

  25. Villalba J.M., Palmgren M.G., Berberián G.E., Ferguson C., Serrano R. 1992. Functional Expression of Plant Plasma Membrane H+-ATPase in Yeast Endoplamic Reticulum. J. Biol. Chem. 267, 12341–12349.

    PubMed  CAS  Google Scholar 

  26. Miroux B, Walker J.E. 1996. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298.

    Article  PubMed  CAS  Google Scholar 

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Author notes

  1. Markus Geisler

    Present address: Department of Plant Biology, Plant Physiology/Anatomy Laboratory, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Denmark

Authors and Affiliations

  1. Institut für Biochemie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, D-40225, Düsseldorf, Germany

    Markus Geisler

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Geisler, M. Expression of a prokaryotic P-type ATPase in E. coli plasma membranes and purification by Ni2+-affinity chromatography. Biol Proced Online 1, 70–80 (1998). https://doi.org/10.1251/bpo9

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Biological Procedures Online

ISSN: 1480-9222

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