Journal of Biological Chemistry
Volume 270, Issue 34, 25 August 1995, Pages 19930-19935
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Nucleic Acids, Protein Synthesis, and Molecular Genetics
The Precursor of Pea Ferredoxin-NADP+ Reductase Synthesized in Escherichia coli Contains Bound FAD and Is Transported into Chloroplasts (∗)

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The precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase from pea was expressed in Escherichia coli as a carboxyl-terminal fusion to glutathione S-transferase. The fused protein was soluble, and the precursor could be purified in a few steps involving affinity chromatography on glutathione-agarose, cleavage of the transferase portion by protease Xa, and ion exchange chromatography on DEAE-cellulose. The purified prereductase contained bound FAD but displayed marginally low levels of activity. Removal of the transit peptide by limited proteolysis rendered a functional protease-resistant core exhibiting enzymatic activity. The FAD-containing precursor expressed in E. coli was readily transported into isolated pea chloroplasts and was processed to the mature size, both inside the plastid and by incubation with stromal extracts in a plastid-free reaction. Import was dependent on the presence of ATP and was stimulated severalfold by the addition of plant leaf extracts.

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This work was supported by Grants A-12830/1-000021 and A-13015/1-000018 from Fundación Antorchas (Buenos Aires, Argentina), Grant C/1354-2 from the International Foundation for Science (Stockholm, Sweden), and Grant CRP/AR688-15 from the International Centre for Genetic Engineering and Biotechnology (ICGEB/UNIDO) (Trieste, Italy). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

These authors are fellows of the Consejo Nacional de Investigaciones Cient&ıacute;ficas y Técnicas (CONICET), Argentina.

Present address: Centre d'Immunologie et de Biologie Parasitaire, Institut Pasteur, Lille 59800, France.

∗∗

These authors are staff members of the CONICET.

§§

Present address: IQUIFIB, Facultad de Farmacia y Bioqu&ıacute;mica y Farmacia, Universidad de Buenos Aires, Jun&ıacute;n 956, Buenos Aires 1113, Argentina.