Abstract
Protein import into chloroplasts is postulated to occur with the involvement of molecular chaperones. We have determined that the transit peptide of ferredoxin-NADP(H) reductase precursor binds preferentially to an Hsp70 from chloroplast stroma. To investigate the role of Hsp70 molecular chaperones in chloroplast protein import, we analyzed the import into pea chloroplasts of preproteins with decreased Hsp70 binding affinity in their transit peptides. Our results indicate that the precursor with the lowest affinity for Hsp70 molecular chaperones in its transit peptide was imported to chloroplasts with similar apparent Km as the wild type precursor and a 2-fold increase in Vmax. Thus, a strong interaction between chloroplast stromal Hsp70 and the transit peptide seems not to be essential for protein import. These results indicate that in chloroplasts the main unfolding force during protein import may be applied by molecular chaperones other than Hsp70s. Although stromal Hsp70s undoubtedly participate in chloroplast biogenesis, the role of these molecular chaperones in chloroplast protein translocation differs from the one proposed in the mechanisms postulated up to date.
Highlights
Plastids accomplish a great variety of metabolic functions and developmental roles in plants and eukaryotic algae
Our results indicate that the precursor with the lowest affinity for Hsp70 molecular chaperones in its transit peptide was imported to chloroplasts with similar apparent Km as the wild type precursor and a 2-fold increase in Vmax
Hsp70 molecular chaperones located in the inner side of the organelle bind to sites distributed along the polypeptide chain hampering the reverse movement of the polypeptide, promoting an unidirectional movement of the chain into the mitochondrial matrix [13, 14]
Summary
Plasmid Constructions, Expression, and Purification of preFNRs— The cDNAs coding for preFNR variants (preFNR-14, preFNR-23, and preFNR-1234) were inserted into a modified pET32 vector (Novagen) and expressed in E. coli as thioredoxin N-terminal fusion proteins The construction of this modified vector was previously described [29]. Radioactive precursor proteins were precipitated with ammonium sulfate at 66% final concentration and centrifuged for 10 min at 4 °C and 10,000 ϫ g, and the pellet was dissolved in import buffer. After a 30-min treatment on ice, EDTA was added to a final concentration of 10 mM, and chloroplasts were recovered by centrifugation through silicone oil layers (AR200). For studying the import of unfolded precursors, the preproteins were incubated with 8 M urea for 10 min after the precipitation with ammonium sulfate Prior to their incubation with intact chloroplasts, the precursors were diluted 20 times in import buffer. FNR-dependent diaphorase activity was determined by published methods [35] using ferricyanide reduction at 420 nm (⑀420 ϭ 1 mMϪ11⁄7cmϪ1)
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