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The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts

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Abstract

The NADPH-protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) is the major protein in the prolamellar bodies (PLBs) of etioplasts, where it catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide during chlorophyll synthesis in higher plants. The suborganellar location in chloroplasts of light-grown plants is less clear. In vitro assays were performed to characterize the assembly process of the pchlide reductase protein in pea chloroplasts. Import reactions employing radiolabelled precursor protein of the pchlide reductase showed that the protein was efficiently imported into fully matured green chloroplasts of pea. Fractionation assays following an import reaction revealed that imported protein was targeted to the thylakoid membranes. No radiolabelled protein could be detected in the stromal or envelope compartments upon import. Assembly reactions performed in chloroplast lysates showed that maximum amount of radiolabelled protein was associated to the thylakoid membranes in a thermolysin-resistant conformation when the assays were performed in the presence of hydrolyzable ATP and NADPH, but not in the presence of NADH. Furthermore, membrane assembly was optimal at pH 7.5 and at 25°C. However, further treatment of the thylakoids with NaOH after an assembly reaction removed most of the membrane-associated protein. Assembly assays performed with the mature form of the pchlide reductase, lacking the transit peptide, showed that the pre-sequence was not required for membrane assembly. These results indicate that the pchlide reductase is a peripheral protein located on the stromal side of the membrane, and that both the precursor and the mature form of the protein can act as substrates for membrane assembly.

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Authors and Affiliations

  1. Dept. of Plant Physiology, Botanical Institute, Göteborg University, Carl Skottsbergs Gata 22, S-413 19, Göteborg, Sweden

    Clas Dahlin & Christer Sundqvist

  2. Dept. of Biology, University of Virginia, 22901, Charlottesville, VA, USA

    Michael P. Timko

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  1. Clas Dahlin
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  2. Christer Sundqvist
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  3. Michael P. Timko
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Dahlin, C., Sundqvist, C. & Timko, M.P. The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts. Plant Mol Biol 29, 317–330 (1995). https://doi.org/10.1007/BF00043655

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  • DOI: https://doi.org/10.1007/BF00043655

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