The import of ferredoxin-NADP+ reductase precursor into chloroplasts is modulated by the region between the transit peptide and the mature core of the protein.
Protein transport across organelles' membranes requires that precursor proteins adopt an unfolded structure in order to be translocated by the import machinery. Ferredoxin-NADP+ reductase precursor, as well as many others, acquires a tightly folded structure that needs to be unfolded before or during its import. Several steps of chloroplast protein import are not fully understood. In particular, the role of different regions of the precursor protein has not been completely elucidated. In this work, we have studied the import into chloroplasts of precursor proteins with inclusions of amino acid spacers between the transit peptide and the mature protein, and with deletions in the N-terminal region of the mature enzyme. We measured the import rate constants for these precursors and the results indicate that the distance between the transit peptide and the core of the mature protein determines the import kinetics. The longer precursors were imported into the organelle faster than the wild type form. Precursors with deletions in the N-terminal region of the mature protein also showed increased import rates compared to the wild type. Homology studies amongst all family members reveal that only chloroplastic proteins possess this region. We suggest that even if the first amino acids of the mature protein do not contribute to its overall structural stability, they condition the kinetic parameters of the import reaction. Besides, the distance between the transit peptide and the mature protein core may be modulating the import rate at which the chloroplast incorporates this protein from the cytosol.
Rial
Daniela V
DV
Molecular Biology Division, IBR (Instituto de Biología Molecular y Celular de, Rosario), CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
Lombardo
Verónica A
VA
Ceccarelli
Eduardo A
EA
Ottado
Jorgelina
J
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D010455:Peptides; D011994:Recombinant Proteins; D014508:Urea; D005287:Ferredoxin-NADP Reductase