Abstract
Hsp70 family proteins function as motors driving protein translocation into mitochondria and the endoplasmic reticulum. Whether Hsp70 is involved in protein import into chloroplasts has not been resolved. We show here Arabidopsis thaliana knockout mutants of either of the two stromal cpHsc70s, cpHsc70-1 and cpHsc70-2, are defective in protein import into chloroplasts during early developmental stages. Protein import was found to be affected at the step of precursor translocation across the envelope membranes. From solubilized envelope membranes, stromal cpHsc70 was specifically coimmunoprecipitated with importing precursors and stoichiometric amounts of Tic110 and Hsp93. Moreover, in contrast with receptors at the outer envelope membrane, cpHsp70 is important for the import of both photosynthetic and nonphotosynthetic proteins. These data indicate that cpHsc70 is part of the chloroplast translocon for general import and is important for driving translocation into the stroma. We further analyzed the relationship of cpHsc70 with the other suggested motor system, Hsp93/Tic40. Chloroplasts from the cphsc70-1 hsp93-V double mutant had a more severe import defect than did the single mutants, suggesting that the two proteins function in parallel. The cphsc70-1 tic40 double knockout was lethal, further indicating that cpHsc70-1 and Tic40 have an overlapping essential function. In conclusion, our data indicate that chloroplasts have two chaperone systems facilitating protein translocation into the stroma: the cpHsc70 system and the Hsp93/Tic40 system.
Highlights
Most chloroplast proteins are nucleus encoded and cytosolically synthesized as a precursor form with an N-terminal targeting signal called the transit peptide
Protein import defects were not observed in our previous study because in 30-d-old plants, import efficiency in the wild type had dropped to the same level as that in the mutants
We show here that we observed the same import defects in mutants of Arabidopsis cpHsc70s
Summary
Most chloroplast proteins are nucleus encoded and cytosolically synthesized as a precursor form with an N-terminal targeting signal called the transit peptide. Import of these precursor proteins into chloroplasts is mediated by a translocon complex located at the chloroplast envelope. Toc159 (translocon of the outer envelope membrane of chloroplast 159 kD) and Toc are the initial receptors for precursors, Toc is the protein-translocating channel across the outer membrane, and Tic110 (translocon of the inner envelope membrane of chloroplast 110 kD) most likely functions as the stroma-side receptor for transit peptides during precursor translocation across the inner membrane and as the scaffold for assembling other stromal translocon components (Inaba and Schnell, 2008; Jarvis, 2008). It has been shown that photosynthetic proteins are preferentially imported through at-Toc159, at-Toc, and at-Toc, whereas nonphotosynthetic proteins are preferentially imported through at-Toc132, at-Toc120, and at-Toc (Kubis et al, 2003, 2004; Smith et al, 2004; Kessler and Schnell, 2009)
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