Precursors with Altered Affinity for Hsp70 in Their Transit Peptides Are Efficiently Imported into Chloroplasts

Abstract

Protein import into chloroplasts is postulated to occur with the involvement of molecular chaperones. We have determined that the transit peptide of ferredoxin-NADP(H) reductase precursor binds preferentially to an Hsp70 from chloroplast stroma. To investigate the role of Hsp70 molecular chaperones in chloroplast protein import, we analyzed the import into pea chloroplasts of preproteins with decreased Hsp70 binding affinity in their transit peptides. Our results indicate that the precursor with the lowest affinity for Hsp70 molecular chaperones in its transit peptide was imported to chloroplasts with similar apparent K m as the wild type precursor and a 2-fold increase in V max . Thus, a strong interaction between chloroplast stromal Hsp70 and the transit peptide seems not to be essential for protein import. These results indicate that in chloroplasts the main unfolding force during protein import may be applied by molecular chaperones other than Hsp70s. Although stromal Hsp70s undoubtedly participate in chloroplast biogenesis, the role of these molecular chaperones in chloroplast protein translocation differs from the one proposed in the mechanisms postulated up to date.

A 38-Amino-Acid Sequence Encompassing the Arm Domain of the Cucumber Necrosis Virus Coat Protein Functions as a Chloroplast Transit Peptide in Infected Plants

“…These finding are therefore consistent with a role of the CNV chloroplast TP-like region in the CNV infection process. Chloroplast TPs have been postulated to interact with the chloroplast outer membrane as part of the initial stages of the import process (6,30). Consistent with the role of the CNV chloroplast TP-like region in chloroplast targeting is the presence of a predicted transmembrane binding region (Fig.…”

“…on May 9, 2018 by guest http://jvi.asm.org/ to the chloroplast stroma, where proteolytic removal of the TP occurs (2,22,30,37). Based on this and the other observations made thus far, we suggest that the CNV arm(ϩSVRI) sequence can function as a chloroplast TP that targets proteins to the stroma.…”

Non-native, N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation

“…10E. Both FNRtp and its mutant (FNRtp-1234) having all of the H70BSs mutated were still able to support import (84). FNRtp was recently found to interact with Hsp93 only when it is located at the N-ter of preprotein (85).…”

Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts

“…Our data also showed that import defects of cphsc70 mutants are observed only in young true leaves. The presence of two systems may also explain why precursors with altered affinities for Hsp70 in their transit peptides are still efficiently imported into chloroplasts (Rial et al, 2003(Rial et al, , 2006.…”

“…In vitro binding assays have shown interactions between cpHsc70 and a recombinant transit peptide of the precursor to the small subunit of RuBP carboxylase (prRBCS; Ivey et al, 2000). However, precursors with altered affinities for Hsp70 in their transit peptides were still efficiently imported into pea (Pisum sativum) chloroplasts (Rial et al, 2003(Rial et al, , 2006. Using an antibody against the pea stromal cpHsc70, S78 (also named CSS1; Marshall and Keegstra, 1992;Nielsen et al, 1997), it was shown that no, or very little, importing precursors were precipitated by the anti-S78 antibody from the soluble fraction of solubilized total chloroplast membranes Nielsen et al, 1997), suggesting that cpHsc70 was not associated with importing precursors in vivo.…”