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

RIAL, D.V.; ARAKAKI, A.K; ALMARÁ, A.M.; ORELLANO, E.G.; CECCARELLI, E.A.

Physiologia Plantarum

Blackwell Publishing

Año: 2006 vol. 128 p. 618 - 632

1399-3054

Heat-shock protein 70 (Hsp70) chaperones function as molecular motorspulling precursor proteins across membranes. Although several Hsp70s havebeen identified in chloroplasts, their participation in protein translocation isstill uncertain. A phylogenetic analysis of the peptide-binding domain fromplant Hsp70s shows that they can be classified into defined groups related totheir subcellular localizations, allowing differences in substrate specificities tobe inferred. Using an algorithm developed by Blond-Elguindi et al. we detectedthree regions in the transit peptide of the pea ferredoxin-NADP1 reductaseprecursor (preFNR) that are related to binding with immunoglobulin heavychainbinding protein (BiP), one of the members of the Hsp70 family resident inthe endoplasmic reticulum.We constructed a mutant transit peptide in whichprolines 18, 20 and 28 were substituted by serines. Thus, the theoreticalprobability of BiP-type binding of the peptidewas abolished without modifyingthe sites for Hsp70 with DnaK-type binding. The stromal Hsp70 homolog CSS1displayed lower affinity for this mutant transit peptide than for the wild-typepresequence. Nevertheless, preFNR containing the mutant transit peptide wasimported into isolated chloroplasts from pea with initial rates similar to thatobserved for the wild-type precursor, and only an 18% decrease in the totalnumber of imported molecules was observed after 20 min of reaction. Ourresults support an import model for the preFNR in which neither DnaK- norBiP-like Hsp70 molecular chaperones play a central role as motor of thetranslocation machinery in chloroplasts.