Interaction of the targeting sequence of chloroplast precursors with Hsp70 molecular chaperones.
We have analyzed the interaction of DnaK and plant Hsp70 proteins with the wild-type ferredoxin-NADP+ reductase precursor (preFNR) and mutants containing amino-acid replacements in the targeting sequence. Using an algorithm already developed [Rüdiger, S., Germeroth, L., Schneider-Mergener, J. & Bukau, B. (1997) EMBO J. 16, 1501-1507] we observed that 75% of the 727 plastid precursor proteins analyzed contained at least one site with high likelihood of DnaK binding in their transit peptides. Statistical analysis showed a decrease of DnaK binding site frequency within the first 15 amino-acid residues of the transit peptides. Using fusion proteins we detected the interaction of DnaK with the transit peptide of the folded preFNR but not with the mature region of the protein. Discharge of DnaK from the presequence was favored by addition of MgATP. When a putative DnaK binding site was artificially added at the N-terminus of the mature protein, we observed formation of complexes with bacterial and plant Hsp70 molecular chaperones. Reducing the likelihood of DnaK binding by directed mutagenesis of the presequence increased the release of bound DnaK. The Hsp70 proteins from plastids and plant cell cytosol also interacted with the preFNR transit peptide. Overall results are discussed in the context of the proposed models to explain the organelle protein import.
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Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
Arakaki
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Ceccarelli
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EA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002151:Calorimetry; D002736:Chloroplasts / Q000378:metabolism*; D004792:Enzyme Precursors / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000378:metabolism*; D014644:Genetic Variation; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000378:metabolism*; D018087:Plastids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D004792:Enzyme Precursors; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C098699:pre-ferredoxin-NADP-oxidoreductase; D005287:Ferredoxin-NADP Reductase; C063842:dnaK protein, E coli