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Chloroplast Hsp70s are not involved in the import of ferredoxin-NADP⁺ reductase precursor

Author:
Rial, Daniela V., Arakaki, Adrián K., Almará, Adriana M., Orellano, Elena G., Ceccarelli, Eduardo A.
Source:
Physiologia plantarum 2006 v.128 no.4 pp. 618-632
ISSN:
0031-9317
Subject:
algorithms, binding proteins, chloroplasts, endoplasmic reticulum, ferredoxin-NADP reductase, immunoglobulins, membrane proteins, models, molecular chaperones, peas, phylogeny, probability, protein transport, substrate specificity
Abstract:
Heat-shock protein 70 (Hsp70) chaperones function as molecular motors pulling precursor proteins across membranes. Although several Hsp70s have been identified in chloroplasts, their participation in protein translocation is still uncertain. A phylogenetic analysis of the peptide-binding domain from plant Hsp70s shows that they can be classified into defined groups related to their subcellular localizations, allowing differences in substrate specificities to be inferred. Using an algorithm developed by Blond-Elguindi et al. we detected three regions in the transit peptide of the pea ferredoxin-NADP⁺ reductase precursor (preFNR) that are related to binding with immunoglobulin heavy-chain binding protein (BiP), one of the members of the Hsp70 family resident in the endoplasmic reticulum. We constructed a mutant transit peptide in which prolines 18, 20 and 28 were substituted by serines. Thus, the theoretical probability of BiP-type binding of the peptide was abolished without modifying the sites for Hsp70 with DnaK-type binding. The stromal Hsp70 homolog CSS1 displayed lower affinity for this mutant transit peptide than for the wild-type presequence. Nevertheless, preFNR containing the mutant transit peptide was imported into isolated chloroplasts from pea with initial rates similar to that observed for the wild-type precursor, and only an 18% decrease in the total number of imported molecules was observed after 20 min of reaction. Our results support an import model for the preFNR in which neither DnaK- nor BiP-like Hsp70 molecular chaperones play a central role as motor of the translocation machinery in chloroplasts.
Agid:
422496