Two Arabidopsis Chloroplast GrpE Homologues Exhibit Distinct Biological Activities and Can Form Homo- and Hetero-Oligomers.

Flowering plants have evolved two distinct clades of chloroplast GrpE homologues (CGEs), which are the nucleotide exchange factor for Hsp70. In Arabidopsis, they are named AtCGE1 (At5g17710) and AtCGE2 (At1g36390). Characterization of their corresponding T-DNA insertion mutants revealed that there is no visible change in phenotype except a defect in protein import in an AtCGE2-knockout mutant under normal growth conditions. However, the embryo development of an AtCGE1-knockout mutant was arrested early at the globular stage. An AtCGE1-knockdown mutant, harboring a T-DNA insertion in the 5'-UTR region, exhibited growth retardation and protein import defect, and its mutant phenotypes became more severe when AtCGE2 was further knocked out. Sub-organellar distribution implied that AtCGE2 might be important for membrane biology due to its preferential association with chloroplast membranes. Biochemical studies and complementation tests showed that only AtCGE1, but not AtCGE2, can effectively rescue the heat-sensitive phenotype of Escherichia coli grpE mutant and robustly stimulate the refolding of denatured luciferase by DnaK. Interestingly, AtCGE1 and AtCGE2 are tending to form heterocomplexes, which exhibit comparable co-chaperone activity to AtCGE1 homocomplexes. Our data indicate that AtCGE1 is the principle functional homologue of GrpE. The possibility that AtCGE2 has a subsidiary or regulatory function through homo- and/or hetero-oligomerization is discussed.

A Reliable and Non-destructive Method for Monitoring the Stromal pH in Isolated Chloroplasts Using a Fluorescent pH Probe.

The proton gradient established by the pH difference across a biological membrane is essential for many physiological processes, including ATP synthesis and ion and metabolite transport. Currently, ionophores are used to study proton gradients, and determine their importance to biological functions of interest. Because of the lack of an easy method for monitoring the proton gradient across the inner envelope membrane of chloroplasts (ΔpHenv), whether the concentration of ionophores used can effectively abolish the ΔpHenv is not proven for most experiments. To overcome this hindrance, we tried to setup an easy method for real-time monitoring of the stromal pH in buffered, isolated chloroplasts by using fluorescent pH probes; using this method the ΔpHenv can be calculated by subtracting the buffer pH from the measured stromal pH. When three fluorescent dyes, BCECF-AM [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester], CFDA-SE [5(6)-Carboxyfluorescein diacetate succinimidyl ester] and SNARF-1 carboxylic acid acetate succinimidyl ester were incubated with isolated chloroplasts, BCECF-AM and CFDA-SE, but not the ester-formed SNARF-1 were taken up by chloroplasts and digested with esterase to release high levels of fluorescence. According to its relatively higher pKa value (6.98, near the physiological pH of the stroma), BCECF was chosen for further development. Due to shielding of the excitation and emission lights by chloroplast pigments, the ratiometric fluorescence of BCECF was highly dependent on the concentration of chloroplasts. By using a fixed concentration of chloroplasts, a highly correlated standard curve of pH to the BCECF ratiometric fluorescence with an r-square value of 0.98 was obtained, indicating the reliability of this method. Consistent with previous reports, the light-dependent formation of ΔpHenv can be detected ranging from 0.15 to 0.33 pH units upon illumination. The concentration of the ionophore nigericin required to collapse the ΔpHenv was then studied. The establishment of a non-destructive method of monitoring the stromal pH will be valuable for studying the roles of the ΔpHenv in chloroplast physiology.

Chloroplast Hsp93 Directly Binds to Transit Peptides at an Early Stage of the Preprotein Import Process.

Three stromal chaperone ATPases, cpHsc70, Hsp90C, and Hsp93, are present in the chloroplast translocon, but none has been shown to directly bind preproteins in vivo during import, so it remains unclear whether any function as a preprotein-translocating motor and whether they have different functions during the import process. Here, using protein crosslinking followed by ionic detergent solubilization, we show that Hsp93 directly binds to the transit peptides of various preproteins undergoing active import into chloroplasts. Hsp93 also binds to the mature region of a preprotein. A time course study of import, followed by coimmunoprecipitation experiments, confirmed that Hsp93 is present in the same complexes as preproteins at an early stage when preproteins are being processed to the mature size. In contrast, cpHsc70 is present in the same complexes as preproteins at both the early stage and a later stage after the transit peptide has been removed, suggesting that cpHsc70, but not Hsp93, is important in translocating processed mature proteins across the envelope.

Evolution of chloroplast J proteins.

Hsp70 chaperones are involved in multiple biological processes and are recruited to specific processes by designated J domain-containing cochaperones, or J proteins. To understand the evolution and functions of chloroplast Hsp70s and J proteins, we identified the Arabidopsis chloroplast J protein constituency using a combination of genomic and proteomic database searches and individual protein import assays. We show that Arabidopsis chloroplasts have at least 19 J proteins, the highest number of confirmed J proteins for any organelle. These 19 J proteins are classified into 11 clades, for which cyanobacteria and glaucophytes only have homologs for one clade, green algae have an additional three clades, and all the other 7 clades are specific to land plants. Each clade also possesses a clade-specific novel motif that is likely used to interact with different client proteins. Gene expression analyses indicate that most land plant-specific J proteins show highly variable expression in different tissues and are down regulated by low temperatures. These results show that duplication of chloroplast Hsp70 in land plants is accompanied by more than doubling of the number of its J protein cochaperones through adding new J proteins with novel motifs, not through duplications within existing families. These new J proteins likely recruit chloroplast Hsp70 to perform tissue specific functions related to biosynthesis rather than to stress resistance.

Stromal Hsp70 is important for protein translocation into pea and Arabidopsis chloroplasts.

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.

Arabidopsis stromal 70-kD heat shock proteins are essential for plant development and important for thermotolerance of germinating seeds.

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 Deltacphsc70-1 and Deltacphsc70-2. No visible phenotype was observed in the Deltacphsc70-2 mutant under normal growth conditions. In contrast, Deltacphsc70-1 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 Deltacphsc70-1 mutant. After heat shock treatment of germinating seeds, root growth from Deltacphsc70-1 seeds was further impaired, indicating that cpHsc70-1 is important for thermotolerance of germinating seeds. No Deltacphsc70-1 Deltacphsc70-2 double mutant could be obtained, suggesting that the Deltacphsc70 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.

Toc GTPases.

Chloroplasts import more than 90% of their protein constituents from the cytosol. The import is mediated by translocon complexes located in the chloroplast envelope and the stroma. This review focuses on the two GTPases in the Toc (translocon at the outer envelope membrane of chloroplasts) complex. Hypotheses are presented about gating across the outer membrane and the possible functional states of the GTPases.