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.

The 70-kD heat shock proteins (Hsp70s) have been shown to be important for protein folding, protein translocation, and stress responses in almost all organisms and in almost all subcellular compartments. However, the function of plastid stromal Hsp70s in higher plants is still uncertain. Genomic surveys have revealed that there are two putative stromal Hsp70s in Arabidopsis thaliana, denoted cpHsc70-1 (At4g24280) and cpHsc70-2 (At5g49910). In this study, we show that cpHsc70-1 and cpHsc70-2 could indeed be imported into the chloroplast stroma. Their corresponding T-DNA insertion knockout mutants were isolated and designated as Dcphsc70-1 and Dcphsc70-2. No visible phenotype was observed in the Dcphsc70-2 mutant under normal growth conditions. In contrast, Dcphsc70-1 mutant plants exhibited variegated cotyledons, malformed leaves, growth retardation, and impaired root growth, even though the protein level of cpHsc70-2 was up-regulated in the Dcphsc70-1 mutant. After heat shock treatment of germinating seeds, root growth from Dcphsc70-1 seeds was further impaired, indicating that cpHsc70-1 is important for thermotolerance of germinating seeds. No Dcphsc70-1 Dcphsc70-2 double mutant could be obtained, suggesting that the Dcphsc70 double knockout was lethal. Genotype analyses of F 1 seedlings from various crosses indicated that double-knockout mutation was lethal to the female gametes and reduced the transmission efficiency of the male gametes. These results indicate that cpHsc70s are essential for plant development and the two cpHsc70s most likely have redundant but also distinct functions.

An Arabidopsis thaliana mutant defective in chloroplast protein import was isolated and the mutant locus, cia5, identified by map-based cloning. CIA5 is a 21-kD integral membrane protein in the chloroplast inner envelope membrane with four predicted transmembrane domains, similar to another potential chloroplast inner membrane protein-conducting channel, At Tic20, and the mitochondrial inner membrane counterparts Tim17, Tim22, and Tim23. cia5 null mutants were albino and accumulated unprocessed precursor proteins. cia5 mutant chloroplasts were normal in targeting and binding of precursors to the chloroplast surface but were defective in protein translocation across the inner envelope membrane. Expression levels of CIA5 were comparable to those of major translocon components, such as At Tic110 and At Toc75, except during germination, at which stage At Tic20 was expressed at its highest level. A double mutant of cia5 At tic20-I had the same phenotype as the At tic20-I single mutant, suggesting that CIA5 and At Tic20 function similarly in chloroplast biogenesis, with At Tic20 functioning earlier in development. We renamed CIA5 as Arabidopsis Tic21 (At Tic21) and propose that it functions as part of the inner membrane protein-conducting channel and may be more important for later stages of leaf development.

The copper chaperone for superoxide dismutase (CCS) has been identified as a key factor integrating copper into copper/zinc superoxide dismutase (CuZnSOD) in yeast (Saccharomyces cerevisiae) and mammals. In Arabidopsis (Arabidopsis thaliana), only one putative CCS gene (AtCCS, At1g12520) has been identified. The predicted AtCCS polypeptide contains three distinct domains: a central domain, flanked by an ATX1-like domain, and a C-terminal domain. The ATX1-like and C-terminal domains contain putative copper-binding motifs. We have investigated the function of this putative AtCCS gene and shown that a cDNA encoding the open reading frame predicted by The Arabidopsis Information Resource complemented only the cytosolic and peroxisomal CuZnSOD activities in the Atccs knockout mutant, which has lost all CuZnSOD activities. However, a longer AtCCS cDNA, as predicted by the Munich Information Centre for Protein Sequences and encoding an extra 66 amino acids at the N terminus, could restore all three, including the chloroplastic CuZnSOD activities in the Atccs mutant. The extra 66 amino acids were shown to direct the import of AtCCS into chloroplasts. Our results indicated that one AtCCS gene was responsible for the activation of all three types of CuZnSOD activity. In addition, a truncated AtCCS, containing only the central and C-terminal domains without the ATX1-like domain failed to restore any CuZnSOD activity in the Atccs mutant. This result indicates that the ATX1-like domain is essential for the copper chaperone function of AtCCS in planta.