Abstract
A rapid method is described for measuring organelle-specific metabolite transport systems in crude homogenates from plants. The tissues were homogenized in liquid nitrogen, extracted with buffer and reconstituted into artificial membranes. The method allowed demonstration of the known different substrate specificities of chloroplast triose phosphate/phosphate translocators from C3- and C4-plants, of the triose phosphate/phosphate translocator from non-green tissue, and of the dicarboxylate translocator. It thus by-passes the necessity to isolate intact plant organelles and, in addition, only a low amount of tissue material is required for transport measurements.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- Chl:
-
chlorophyll
- TPT:
-
triose phosphate/phosphate translocator
References
Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1–15
Borchert, S., Große, H., Heldt, H.W. (1989) Specific transport of phosphate, glucose 6-phosphate, dihydroxyacetone phosphate and 3-phosphoglyerate into amyloplasts from pea roots. FEBS Lett. 253, 183–186
Fischer, K., Arbinger, B., Kammerer, B., Busch, C., Brink, S., Wallmeier, H., Sauer, N., Eckerskorn, C., Flügge, U.I. (1994) Cloning and in vivo expression of functional triose phosphate/phosphate translocators from C3- and C4-plants: Evidence for the putative participation of specific amino acid residues in the recognition of phosphoenolpyruvate. Plant J. 5, 215–226
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. (1992) Reaction mechanism and asymmetric orientation of the reconstituted chloroplast phosphate translocator. Biochim. Biophys. Acta 1110, 112–118
Flügge, U.I., Heldt, H.W. (1981) The phosphate translocator of the chloroplast envelope. Isolation of the carrier protein and reconstitution of transport. Biochim. Biophys. Acta 638, 296–304
Flügge, U.I., Heldt, H.W. (1991) Metabolite translocators of the chloroplast envelope. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 129–144
Gross, A., Bruckner, G., Heldt, H.W., Flügge, U.-I. (1990) Comparison of the kinetic properties, inhibition and labelling of the phosphate translocator from maize and spinach mesophyll chloroplasts. Planta 180, 262–271
Heineke, D., Kruse, A., Flügge, U.I., Frommer, W.B., Riesmeier, J.W., Willmitzer, L., Heldt, H.W. (1994) Effect of antisense repression of the chloroplast triose phosphate translocator on photosynthesis metabolism in transgenic potato plants. Planta, in press
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. (1992) Metabolite transport in plant cells. In: Plant organelles, pp. 21–47, Tobin, A.K., ed. Society for Experimental Biology Seminar Series 50, Cambridge University Press
Kasahara, M., Hinkle, P.C. (1977) Reconstitution and purification of the D-glucose transporter from human erythrocytes. J. Biol. Chem. 252, 7384–7390
Krämer, R., Klingenberg, M. (1977) Reconstitution of adenine nucleotide transport with purified ADP, ATP-carrier protein. FEBS Lett. 82, 363–367
Krämer, R., Palmieri, F. (1989) Molecular aspects of isolated and reconstituted carrier proteins from animal mitochondria. Biochim. Biophys. Acta 974, 1–23
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
Menzlaff, E., Flügge, U.I. (1993) Purification and functional reconstitution of the 2-oxoglutarate/malate translocator from spinach chloroplasts. Biochim. Biophys. Acta 1147, 13–18
Neuhaus, H.E., Henrichs, G., Scheibe, R. (1993) Characterization of glucose-6-phosphate incorporation into starch by isolated intact cauliflower-bud plastids. Plant Physiol 101, 573–578
Riesmeier, J.W., Flügge, U.I., Schulz, B., Heineke, D., Heldt, H.W., Willmitzer, L., Frommer, W.B. (1993) Antisense repression of the chloroplast triose phosphate translocator affects carbon partitioning in transgenic potato plants. Proc. Natl. Acad. Sci. USA 90, 6160–6164
Woo, K.C., Flügge, U.I., Heldt, H.W. (1987) A two-translocator model for the transport of 2-oxoglutarate and glutamate in chloroplasts during ammonia assimilation in the light. Plant Physiol. 84, 624–632
Author information
Authors and Affiliations
Additional information
Dedicated to Professor F.-C. Czygan on the occasion of his 60th birthday
This research was supported by the Bundesministerium für Forschung und Technologie. A.W. is a recipient of a Ph.D. fellowship from the Deutsche Forschungsgemeinschaft.
Rights and permissions
About this article
Cite this article
Flügge, UI., Weber, A. A rapid method for measuring organelle-specific substrate transport in homogenates from plant tissues. Planta 194, 181–185 (1994). https://doi.org/10.1007/BF00196386
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00196386