Abstract
The kinetic properties of the phosphate translocator from maize (Zea mays L.) mesophyll chloroplasts have been determined. We have used a double silicone-oil-layer centrifugation system in order to obtain true initial uptake rates in forward-reaction experiments. In addition, it was possible to perform back-exchange experiments and to study the effects of illumination and of preloading the chloroplasts with different substrates on transport. It is shown that the phosphate translocator from mesophyll chloroplasts of maize, a C4 plant, transports inorganic phosphate and phosphorylated C3 compounds in which the phosphate group is linked to the C3 atom (e.g. 3-phosphoglycerate and triose phosphate). The affinities of the transported metabolites towards the translocator protein are about one order of magnitude higher than in mesophyll chloroplasts from the C3 plant, spinach. In contrast to the phosphate translocator from C3-mesophyll chloroplasts, that of C4-mesophyll chloroplasts catalyzes in addition the transport of C3 compounds where the phosphate group is attached to the C2 atom (e.g. 2-phosphoglycerate, phosphoenolpyruvate). The phosphate translocator from both chloroplast types is strongly inhibited by pyridoxal-5′-phosphate (PLP), 2,4,6-trinitrobenzenesulfonic acid and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). In the case of the spinach translocator protein these inhibitors were shown to react with the same amino-acid residue at the substrate binding site, and one molecule of either DIDS or PLP is obviously required per substrate binding site for the inactivation of the translocation process. In the functionally active dimeric translocator protein only one substrate-binding site appears to be accessible at a particular time, indicating that the site might be exposed to each side of the membrane in turn. Using [3H]-H2DIDS for the labelling of maize mesophyll envelopes the radioactivity was found to be associated with two polypeptides of about 29 and 30 kDa. Since Western-blot analysis showed that only the 30 kDa polypeptide reacted with an antiserum directed against the spinach phosphate translocator protein it is suggested that this polypeptide presumably represents the phosphate translocator from maize mesophyll chloroplasts.
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Abbreviations
- DIDS:
-
4,4′-diisothiocyanostilbene-2,2′-disulfonic acid
- PEP:
-
phosphoenolpyruvate
- 2-,3-PGA:
-
2-,3-phosphoglycerate
- PLP:
-
pyridoxal-5′-phosphate
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- TNBS:
-
2,4,6-trinitrobenzenesulfonic acid
- triose P:
-
triose phosphate
References
Basch, R.S. (1968) An improved method for counting tritum and carbon-14 in acrylamide gels. Anal. Biochem. 26, 184–188
Bonner, W.M., Lasky, R.A. (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 84–88
Cockburn, W., Walker, D.A., Baldry, C.W. (1968) The isolation of spinach chloroplasts in pyrophosphate media. Plant Physiol. 43, 1415–1418
Day, D.A., Hatch, M.D. (1981) Transport of 3-phosphoglyceric acid, phosphoenolpyruvate, and inorganic phosphate in maize mesophyll chloroplasts, and the effect of 3-phosphoglyceric acid on malate and phosphoenolpyruvate production. Arch. Biochem. Biophys. 211, 743–749
Douce, R., Holtz, B.R., Benson, A.A. (1973) Isolation and properties of the envelope of spinach chloroplasts. J. Biol. Chem. 248, 7215–7222
Fliege, R., Flügge, U.-I., Werdan, K., Heldt, H.W. (1978) Specific transport of inorganic phosphate, 3-phosphoglycerate and triosephosphates across the inner membrane of the envelope in spinach chloroplasts. Biochim. Biophys. Acta 502, 232–247
Flügge, U.-I. (1985) Hydrodynamic properties of the Triton X-100-solubilized chloroplast phosphate translocator. Biochim. Biophys. Acta 815, 299–305
Flügge, U.-I., Heldt, H.W. (1977) Specific labelling of a protein involved in phosphate transport of chloroplasts by pyridoxal-5′phosphate. FEBS Lett. 82, 29–33
Flügge, U.-I., Heldt, H.W. (1978) Specific labelling of the active site of the phosphate translocator in spinach chloroplasts by 2,4,6-trinitrobenzenesulfonate. Biochem. Biophys. Res. Commun. 84, 37–44
Flügge, U.-I., Heldt, H.W. (1979) Phosphate translocator in chloroplasts: identification of the functional protein and characterization of its binding site. In: Function and molecular aspects of biomembrane transport, pp. 373–382, Quagliariello, E., Palmieri, F., Papa, S., Klingenberg, E.M., eds. Elsevier/North Holland Biomedical, Amsterdam
Flügge, U.-I., Heldt, H.W. (1984) The phosphate-triose phosphate-phosphoglycerate translocator of the chloroplast. Trends Biochem. Sci. 9, 530–533
Flügge, U.-I., Wessel, D. (1984) Cell-free synthesis of putative precursors for envelope membrane polypeptides of spinach chloroplasts. FEBS Lett. 168, 255–259
Flügge, U.-I., Stitt, M., Heldt, H.W. (1985) Light-driven uptake of pyruvate into mesophyll chloroplasts from maize. FEBS Lett. 183, 335–339
Flügge, U.-I., Fischer, K., Gross, A., Sebald, W., Lottspeich, F. and Eckerskorn, C. (1989) The triose phosphate-3-phosphoglycerate-phosphate translocator from spinach chloroplasts: nucleotide sequence of a full-length cDNA clone and import of the in vitro synthesized precursor protein into chloroplasts. EMBO J. 8, 39–46
Hampp, R., Ziegler, J. (1977) Sulfate and sulfite translocation via phosphate translocator of inner envelope membrane of chloroplasts. Planta 137, 309–312
Heldt, H.W. (1980) Measurement of metabolite movement across the envelope and of the pH in the stroma and the thylakoid space in intact chloroplasts. Methods Enzymol. 69, 604–613
Heldt, H.W., Flügge, U.-I., Fliege, R. (1978) The influence of illumination on the transport of 3-phosphoglycerate across the chloroplast envelope. In: Mechanism of proton and calcium pumps, pp. 105–114, Avron, M., Azzone, G.F., Metcalfe, J., Quagliariello, E., Siliprandi, N., eds. Elsevier/North Holland, Amsterdam
Howitz, K.T., McCarty, R.E. (1985) Kinetic characteristics of the chloroplasts envelope glycolate transporter. Biochemistry 24, 2645–2652
Huber, S.C., Edwards, G.E. (1977a) Transport in C4 mesophyll chloroplasts. Characterization of the pyruvate carrier. Biochim. Biophys. Acta 462, 583–602
Huber, S.C., Edwards, G.E. (1977b) Transport in C4 mesophyll chloroplasts. Evidence for an exchange of inorganic phosphate and phosphoenolpyruvate. Biochem. Biophys. Acta 462, 603–612
Jenkins, C.L.D., Russ, V.J. (1984) Large scale, rapid preparation of functional mesophyll chloroplasts from Zea mays and other C4 species. Plant Sci. Lett. 35, 19–24
Kampmann, L., Lepke, S., Fasold, H., Fritsch, G. Passow, H. (1982) The kinetics of intramolecular cross-linking of the band 3 protein in the red blood cell membrane by 4,4′-diisothiocyano-dihydrostilbene-2,2′-disulfonic acid (H2DIDS). J. Membr. Biol. 70, 199–216
Klingenberg, M. (1981) Membrane protein oligomeric structure and transport function. Nature 290, 449–454
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
Lilley, R.McC., Walker, D.A. (1974) The reduction of 3-phosphoglycerate by reconstituted chloroplasts and by chloroplast extracts. Biochim. Biophys. Acta 368, 269–278
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275
Mourioux, G., Douce, R. (1981) Slow passive diffusion of orthophosphate between intact isolated chloroplasts and suspending medium. Plant Physiol. 67, 470–473
Murray, E.E., Lotzer, J., Eberle, M. (1989) Codon usage in plant genes. Nucleic Acids Res. 17, 477–492
Ribaud, O., Goldberg, M.E. (1974) A revised method for the tritium labelling of pyridoxal-5′-phosphate. FEBS Lett. 40, 41–44
Rumpho, M.E., Edwards, G.E. (1985) Characterization of 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene inhibition of 3-phosphoglycerate dependent O2 evolution in isolated chloroplasts. Plant Physiol. 78, 537–544
Rumpho, M.E., Edwards, G.E., Yousif, A.E., Keegstra, K. (1988) Specific labelling of the phosphate translocator in C3 and C4 mesophyll chloroplasts by tritiated dihydro-DIDS (1,2-ditritio-1,2-[2,2′-disulfo-4,4′-diisothiocyano]-diphenylethane). Plant Physiol. 86, 1193–1198
Stock, A., Ortanderl, F., Pfleiderer, G. (1966) Darstellung von radioaktiv markiertem Pyridoxal-5′-phosphat. Biochem. Z. 344, 353–360
Thompson, A.G., Brailsford, M.A., Beechey, R.B. (1987) Identification of the phosphate-translocator from maize mesophyll chloroplasts. Biochem. Biophys. Res. Commun. 143, 164–169
Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354
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This work was supported by the Deutsche Forschungsgemeinschaft
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Gross, A., Brückner, G., Heldt, H.W. et al. Comparison of the kinetic properties, inhibition and labelling of the phosphate translocators from maize and spinach mesophyll chloroplasts. Planta 180, 262–271 (1990). https://doi.org/10.1007/BF00194006
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DOI: https://doi.org/10.1007/BF00194006