Involvement of molecular chaperones in chloroplast protein import.

RIAL, D; CECCARELLI, EDUARDO A.

Budapest, Hungría

Congreso; 30th FEBS Congress – 9th IUBMB Conference; 2005

IUBMB

Plastids accomplish a great variety of metabolic functions in plants and eukaryotic algae. Although these organelles contain their own genomes, about 90% of chloroplast proteins are synthesized in the cytosol and post-translationally routed to their final destination. This process is carried out by a coordinate action of the chloroplast import machinery, molecular chaperones, and sequences present in precursor proteins, largely in the amino-terminal regions called transit peptides. We searched for Escherichia coli (DnaK) and endoplasmic reticulum (BiP) Hsp70 molecular chaperones binding sites, and plant Hsp70 interactions along the transit peptide of the ferredoxin-NADP+ reductase precursor (preFNR). We determined that this transit peptide binds preferentially to CSS1, one of the Hsp70 isoforms present in the chloroplast stroma. Reduction of DnaK-binding tendency by mutagenesis in the transit peptide strongly affected the interaction with DnaK and CSS1. Surprisingly, the precursor with the lowest affinity for DnaK in its transit peptide was imported into chloroplasts with similar apparent Km as the wild-type precursor and a twofold increase in Vmax. The inclusion of chloroplast stroma during in vitro protein import experiments decreased the import rate of wild-type preFNR and did not affect the import of the mutant precursor. The reduction of BiP-binding tendency by mutagenesis only showed a slight decrease both in the interaction with CSS1 and in the rate of import of this mutant precursor. Thus, the interactions analyzed in this work seem not to be the main motor for protein import, suggesting that other factors may be responsible for unfolding and import of chloroplast precursors.