A fully active FAD-containing precursor remains folded up to its translocation across the chloroplast membranes.
The cytosolic and two recombinant precursors, containing 10 and 30 amino acid spacers between the transit peptide and the mature region of the chloroplast flavoprotein ferredoxin-NADP+ reductase (FNR), were expressed in Escherichia coli cells. These proteins were purified rendering fully active precursors that contained bound FAD. Neither the transit peptide nor the spacers affected the formation of the tightly folded enzyme structure. Protease treatment of the folded precursors resulted in a rapid removal of the transit sequence, rendering an enzymatically active resistant core, even at high protease concentration. All three preproteins could be efficiently imported by isolated pea chloroplasts. Addition of the enzyme substrate NADP+ to the import medium slightly decreased the polypeptide translocation. The precursor bound to isolated chloroplasts in the presence or absence of leaf extracts was as resistant to proteolysis as the folded precursor in solution. In contrast, the FNR precursor unfolded by urea was rapidly digested even at the lowest protease concentration. Together, our results indicate that precursor unfolding may take place during translocation but not during binding to chloroplast envelopes or by interaction with leaf extract soluble factors, and that this process is independent of the distance between the transit peptide and the folded mature region of the protein.
Ottado
J
J
Molecular Biology Division, PROMUBIE, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
Ceccarelli
E A
EA
D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active; D002736:Chloroplasts / Q000201:enzymology*; D017931:DNA Primers / Q000235:genetics; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D015870:Gene Expression; D007425:Intracellular Membranes / Q000201:enzymology; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D005287:Ferredoxin-NADP Reductase