Pea chloroplast DnaJ-J8 and Toc12 are encoded by the same gene and localized in the stroma

Abstract

Toc12 is a novel J domain-containing protein identified in pea (Pisum sativum) chloroplasts. It was shown to be an integral outer membrane protein localizing in the intermembrane space of the chloroplast envelope. Furthermore, Toc12 was shown to associate with an intermembrane space Hsp70, suggesting that Toc12 is important for protein translocation across the chloroplast envelope. Toc12 shares a high degree of sequence similarity with Arabidopsis (Arabidopsis thaliana) DnaJ-J8, which has been suggested to be a soluble protein of the chloroplast stroma. Here, we isolated genes encoding DnaJ-J8 from pea and found that Toc12 is a truncated clone of one of the pea DnaJ-J8s. Protein import analyses indicate that Toc12 and DnaJ-J8s possess a cleavable transit peptide and are localized in the stroma. Arabidopsis mutants with T-DNA insertions in the DnaJ-J8 gene show no defect in chloroplast protein import. Implications of these results in the energetics and mechanisms of chloroplast protein import are discussed.

Figure 1.

Toc12 is highly similar to the N-terminal two-thirds of J8 from various legume species. Sequence alignment of pea Toc12 and J8 from Medicago (MtJ8), soybean (GmJ8), castor bean (RcJ8), and Arabidopsis (AtJ8) is shown. The predicted transit peptide processing site of AtJ8 is indicated with an arrow. The conserved tripeptide HPD in the J domain is underlined. T-DNA insertion sites of Arabidopsis j8-1 and j8-2 mutants are indicated with white arrowheads.

Figure 2.

Deduced amino acid sequences and genomic structures of the two PsJ8 genes. A, Toc12 is part of PsJ8b. Sequence alignment of PsJ8a, PsJ8b, and Toc12 is shown. The additional Ala and Gly residues present in PsJ8b are indicated with the arrowhead. Thr residues at positions 34 and 36 of Toc12 substituted with Arg in PsJ8a and PsJ8b are boxed in red. Stop codons are marked with asterisks. B, TOC12 is a partially spliced or alternatively spliced RNA of PsJ8b. Exons and introns are shown as black arrows and blue lines, respectively. A unique 1,229-bp insertion in the second intron of PsJ8b is shown as a red line. TOC12 cDNA is represented by the pink line. Sequences of the beginning and end of the boxed region are shown in C. C, Detailed sequences of the beginning and end of the boxed region marked in B. Nucleotide sequences (same color code as in B) and deduced amino acid sequences of PsJ8b and TOC12 are shown.

Figure 3.

AtJ8, PsJ8a, and PsJ8b are synthesized as higher molecular mass precursors with cleavable transit peptides and imported into the chloroplast stroma. A, Import of AtJ8, PsJ8a, and PsJ8b into isolated pea chloroplasts. In vitro-translated, [³⁵S]Met-labeled precursor proteins (Ivt) and chloroplasts after import of the precursor proteins (Chpt) were treated with thermolysin or trypsin. Trypsin-treated chloroplasts were lysed hypotonically (hypo) or treated by alkaline extraction (alka) and then separated into membrane (M) and soluble (S) fractions. Samples were analyzed by SDS-PAGE followed by fluorography (for AtJ8 and PsJ8s) or immunoblotting (for Tic110, Toc75, and APS). pr, Precursor form; m, mature form. The Ivt lanes contained 1% of the in vitro-translated proteins used for the import reactions shown in the Chpt lanes. B, PsJ8b-M1x could not be imported into chloroplasts. In vitro-translated, [³⁵S]Met-PsJ8b-M1x (Ivt; lanes 2 and 3) and chloroplasts after import of PsJ8b-M1x (Chpt; lanes 4 and 5) were treated with thermolysin. All samples were analyzed by SDS-PAGE and fluorography. In vitro-translated PsJ8b (lane 1) was also analyzed for size comparison.

Figure 4.

A fraction of Toc12 is intrinsically resistant to thermolysin digestion. In vitro-translated [³⁵S]Met-Toc12 before import (Ivt) and chloroplasts after import of Toc12 (Chpt) were treated with 200, 300, or 400 μg mL⁻¹ thermolysin or with 200 μg mL⁻¹ thermolysin plus 0.1% Triton X-100. Samples were analyzed by SDS-PAGE followed by fluorography (for Toc12) or immunoblotting (for Toc159 and Tic110). Lanes 1 to 5 (Ivt) contained 3.6% of the in vitro-translated Toc12 used for chloroplast import shown in lanes 6 to 10. Toc159 is easily degraded into various fragments during chloroplast isolation. The full-length Toc159 is indicated by the arrow, and the major 86-kD degradation fragment is indicated with the asterisk.

Figure 5.

Toc12MM can be processed into a 7-kD protein after import into chloroplasts. In vitro-translated [³⁵S]Met-Toc12, [³⁵S]Met-Toc12MM, and [³⁵S]Met-prRBCS were incubated with isolated pea chloroplasts in the presence (lanes 2, 5, and 8) or absence (lanes 3, 6, and 9) of 5 mm ATP. In vitro-translated proteins (Ivt) and chloroplasts after the import reactions (Chpt) were analyzed by SDS-PAGE and fluorography. Lanes 1, 4, and 7 represent 0.8%, 0.24%, and 0.24% of the in vitro-translated proteins used for the corresponding import experiments, respectively.

Figure 6.

The 7-kD protein produced after import of Toc12MM is localized in the stroma. A, In vitro-translated [³⁵S]Met-Toc12MM (Ivt) and chloroplasts after import of Toc12MM (Chpt) were treated with trypsin. Samples were analyzed with SDS-PAGE followed by fluorography (for Toc12MM) or immunoblotting (for Toc75 and Tic110). The Ivt lanes contained 0.4% of the in vitro-translated Toc12MM used for the import reactions shown in the Chpt lanes. B, Fractionation of Toc12MM, Toc12, and prRBCS after import into chloroplasts. Chloroplasts after import of Toc12MM, Toc12, and prRBCS were lysed hypotonically (hypo) or treated by alkaline extraction (alka) and then separated into membrane (M) and soluble (S) fractions. Samples were analyzed by SDS-PAGE followed by fluorography (for imported proteins) or immunoblotting (for Toc75). For Toc12MM and Toc12 import reactions, 5 mm ATP was added. For the prRBCS import reaction, no additional ATP was supplied. Import reactions were performed under light, and the in vitro-translated protein products contained a low amount of ATP from the in vitro translation system.

Figure 7.

Characterizations of four Arabidopsis j8 mutants. A, Schematic representation of T-DNA insertion sites of the j8 mutants. Black and white boxes represent the translated and untranslated exon regions, respectively. LB, Left border of the T-DNA. Positions of primers used for RT-PCR shown in B, and real-time quantitative RT-PCR shown in C, are indicated by arrows. Primers R6 and Q-R1 span the junction between the second and the third exons. B, RT-PCR analyses of AtJ8 transcripts in the j8 mutants. The primers used are indicated in parentheses on the right. The parental ecotype of the j8-1, j8-2, and j8-4 alleles is Columbia (Col). The j8-3 allele is in the Wassilewskija-4 (Ws4) ecotype. The amount of UBQ10 transcripts was analyzed as a control. C, Real-time quantitative RT-PCR analyses of the AtJ8 transcript levels in the j8 mutants. The AtJ8 transcript level was first normalized to the level of UBQ10 in each sample, and the expression level in the wild type was then set as 100%. Primers for AtJ8 detection are Q-F1 and Q-R1 as shown in A. Data are means ± sd; n = 2. D, Phenotype of the j8 mutants. Arabidopsis seedlings were grown on MS medium under 16 h of light/8 h of dark for 14 d. [See online article for color version of this figure.]

Figure 8.

The Arabidopsis j8 mutants show no detectable defect in chloroplast protein import. A, In vitro-translated [³⁵S]Met-prRBCS, [³⁵S]Met-prL11, and [³⁵S]Met-prPC were imported into chloroplasts isolated from the j8 mutants and their corresponding wild types. Samples were analyzed by SDS-PAGE. The gel was first stained with Coomassie Brilliant Blue and then dried for fluorography. The amount of endogenous RBCS as revealed by Coomassie Brilliant Blue staining is shown below the fluorograph of the same gel. Col, Columbia; Ivt, in vitro-translated precursor protein; Ws4, Wassilewskija-4. B, Quantification of imported mature proteins shown in A. The amount of imported mature protein was first normalized to the amount of endogenous RBCS of the same sample. The import efficiency of the wild type was then set as 100%. Data are means ± sd; n = 3 to 6. [See online article for color version of this figure.]