Protein Disulfide Isomerase as a Regulator of Chloroplast Translational Activation

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Science  12 Dec 1997:
Vol. 278, Issue 5345, pp. 1954-1957
DOI: 10.1126/science.278.5345.1954


Light-regulated translation of chloroplast messenger RNAs (mRNAs) requires trans-acting factors that interact with the 5′ untranslated region (UTR) of these mRNAs. Chloroplast polyadenylate-binding protein (cPABP) specifically binds to the 5′-UTR of the psbA mRNA and is essential for translation of this mRNA. A protein disulfide isomerase that is localized to the chloroplast and copurifies with cPABP was shown to modulate the binding of cPABP to the 5′-UTR of the psbA mRNA by reversibly changing the redox status of cPABP through redox potential or adenosine 5′-diphosphate–dependent phosphorylation. This mechanism allows for a simple reversible switch regulating gene expression in the chloroplast.

Synthesis of certain chloroplast photosynthetic proteins is activated 50- to 100-fold in response to light exposure without an increase in the corresponding mRNA levels, indicating that translation of chloroplast mRNAs is light-regulated (1). Genetic evidence has shown that nuclear-encoded trans-acting factors interact with the 5′-UTR of chloroplast mRNAs to activate translation of these mRNAs in a light-dependent manner (2, 3). A set of proteins (38, 47, 55, and 60 kD) was identified that binds as a complex to the 5′-UTR of the psbA mRNA, encoding the photosynthetic reaction center protein D1 from the green algae Chlamydomonas reinhardtii (3-6). Binding of this protein complex to the 5′-UTR of the psbA mRNA correlates with light-enhanced translation of this mRNA under a variety of environmental conditions and in mutations deficient in psbAmRNA translation (4-8). RNA binding activity of the protein complex for the 5′-UTR of the psbA mRNA can be regulated in vitro by at least two different mechanisms: adenosine 5′-diphosphate (ADP)–dependent phosphorylation and changes in redox potential (7, 9). Recently, a cDNA encoding the 47-kD RNA binding protein (RB47) was cloned that binds specifically to the 5′-UTR of psbA mRNA from C. reinhardtii chloroplast. The nuclear-encoded protein is homologous to PABP and is translocated to the chloroplast (8). Biochemical analysis of C. reinhardtii mutants lackingpsbA mRNA translation shows that both RB47 andpsbA-specific RNA binding activity are required forpsbA mRNA translation (8).

To clone the cDNA encoding the 60-kD psbA mRNA binding protein (RB60), we purified the psbA-specific RNA binding proteins from light-grown C. reinhardtii cells using heparin-agarose chromatography followed by psbA RNA affinity chromatography (RAC). RAC-purified proteins were separated by two-dimensional polyacrylamide gel electrophoresis (PAGE), the RB60 protein was digested with trypsin, and unambiguous amino acid sequences were obtained from two peptide fragments (10). The DNA corresponding to one peptide of 22 amino acid residues was amplified by polymerase chain reaction with degenerate oligonucleotides and used to screen a λ-gt10 cDNA library from C. reinhardtii. The predicted amino acid sequence of the cloned cDNA contained the complete amino acid sequences of the two tryptic peptides (Fig.1). The amino acid sequence of the encoded protein revealed that it has high sequence homology to both plant and mammalian protein disulfide isomerase (PDI) and contains the highly conserved thioredoxin-like domains with -Cys-Gly-His-Cys- (-CGHC-) catalytic sites in both the NH2- and COOH-terminal regions (Fig. 1) and the -Lys-Asp-Glu-Leu- (-KDEL-) endoplasmic reticulum (ER) retention signal at the COOH-terminus found in all PDIs. PDI is a mutifunctional protein possessing enzymatic activities for the formation, reduction, and isomerization of disulfide bonds during protein folding and is typically found in the ER (11-14). The first 30 amino acid residues of RB60 were shown to lack sequence homology with the NH2-terminal signal sequence of PDI from plants or mammalian cells. However, this region has characteristics of chloroplast transit peptides of C. reinhardtii, which have similarities with both mitochondrial and higher plant chloroplast presequences (15, 16). A transit peptide sequence should override the function of the -KDEL- ER retention signal and target the protein to the chloroplast because the -KDEL- signal acts only to retain the transported protein in the ER (11-13).

Figure 1

Alignment of amino acid sequences of the cDNA encoding RB60 from C. reinhardtii and protein disulfide isomerases from plants and mammals. The conserved amino acids are shaded in gray. The amino acid sequences of two tryptic peptides obtained from RB60 purified from C. reinhardtiicell extracts are indicated by black bars above the alignment. The -Cys-Gly-His-Cys catalytic sites are indicated by dots. The accession number for chloroplast PDI from C. reinhardtii is AF027727. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

To verify that RB60 is localized to the chloroplasts, we performed an immunoblot analysis of isolated pea chloroplasts using the C. reinhardtii antiserum to RB60 (anti-RB60) (17). To confirm that the isolated pea chloroplasts were free of cytoplasmic contamination, we performed immunoblot analysis with antiserum against the large subunit of ribulose bisphosphate carboxylase (RuBPCase, located in the chloroplast) and antiserum against the cytoplasmic protein tubulin. Anti-RuBPCase recognized proteins from both whole-leaf extracts (cytoplasm plus chloroplast) and from isolated chloroplasts (Fig. 2A, lanes 3 and 4). Anti-tubulin recognized a protein in whole-leaf extracts (lane 5, arrow), but not in the chloroplast fraction (lane 6), showing that the isolated chloroplasts were free of cytoplasmic proteins. The protein extracts from isolated pea chloroplasts were enriched by heparin-agarose chromatography; enrichment was required for immunoblot assays with anti-RB60 because RB60 is a minor component within the chloroplast (4). Immunoblot analysis was performed on proteins from purified pea chloroplasts, from C. reinhardtii cell extracts isolated by heparin-agarose chromatography, and on recombinant RB60. A specific signal immunochemically related to RB60 was detected at ∼63 kD in the pea chloroplast sample (Fig. 2B, lane 4). A signal of equal intensity was observed for C. reinhardtii proteins and for the recombinant RB60 (Fig. 2B, lanes 5 and 6).

Figure 2

Localization of RB60 to chloroplasts. (A) The protein samples from whole-leaf extracts (lanes 1, 3, and 5) and isolated pea chloroplast (lanes 2, 4, and 6) (23) were separated by SDS-PAGE and either stained with Coomassie blue (lanes 1 and 2) or analyzed by immunoblotting with antiserum to RuBPCase (lanes 3 and 4) or antiserum to cytoplasmic tubulin (lanes 5 and 6). (B) Pea chloroplasts (lane 1),C. reinhardtii proteins (lane 2) partially purified by heparin-agarose chromatography (4, 8), and r-RB60 (lane 3) were separated by SDS-PAGE and either stained with Coomassie blue (left) or subjected to immunoblot analysis with theC. reinhardtii anti-RB60 (right).

Chloroplast PDI (cPDI) contains the two -CGHC- catalytic sites that regulate the formation, reduction, and isomerization of disulfide bonds associated with protein folding (Fig. 1). The identification of these redox catalytic sites prompted us to investigate the role of RB60 in the redox-regulated binding of RB47 to the 5′-UTR of thepsbA mRNA (7). The precursor form of RB60 and the endogenous form of RB47, containing only the four RNA recognition motif domains (18), were expressed in Escherichia colias a fusion protein with a (His)10 tag, purified on a Ni–nitrilotriacetic acid (NTA) agarose affinity column, and used for subsequent RNA binding gel mobility-shift assays. We first investigated whether RNA binding activity of recombinant RB47 (r-RB47) could be altered by the addition of a reducing agent, dithiothreitol (DTT), in the presence of recombinant RB60 (r-RB60). r-RB47 was preincubated with 10 mM DTT, a fivefold excess of r-RB60 alone, or both DTT and r-RB60 before addition of a 32P-labeled 5′-UTR of thepsbA mRNA, followed by a gel mobility-shift assay (Fig.3A). These data showed that r-RB47 isolated from E. coli is in an active reduced form so that only a slight enhancement of RNA binding activity could be obtained with addition of DTT and r-RB60. To determine whether r-RB60 is able to reactivate r-RB47 that is in an inactive oxidized form, we incubated r-RB47 with the oxidant dithionitrobenzoic acid (DTNB) for 5 min and then dialyzed it against 104 volume of buffer to remove the oxidant. Oxidation of r-RB47 by DTNB completely abolished the binding activity of the protein (Fig. 3B). Addition of DTT to 1.0 mM partially restored the binding capacity of r-RB47 (lane 1), and the binding could be increased threefold by the addition of up to 25 mM DTT. With increasing amounts of r-RB60, the binding activity of r-RB47 was increased compared to the samples without r-RB60 at every concentration of DTT tested (lanes 2 and 3). When DTT was not present in the incubation medium, r-RB60 alone could not restore the binding of the oxidized r-RB47 (0 mM DTT), indicating that r-RB60 requires reducing equivalents to convert the inactive oxidized form of r-RB47 to an active reduced form.

Figure 3

r-RB60 regulates the binding of chloroplast PABP to the 5′-UTR of psbA mRNA through redox equivalents. (A) r-RB47 was incubated with 10 mM DTT and r-RB60 as indicated, and then subjected to gel mobility-shift assay with a 32P-labeled 5′-UTR of thepsbA mRNA. Only the portions of the gel containing the RNA-protein complexes are shown. (B) r-RB47 (0.2 μg) oxidized with DTNB was incubated with increasing concentrations of DTT in the presence (lanes 2 and 3) or absence (lane 1) of r-RB60 with a molar ratio of 1:1 (lane 2) or 5:1 (lane 3) over r-RB47. (C) r-RB47 (0.2 μg) was incubated with increasing concentrations of GSSG in the presence (lanes 6 to 8) or absence (lanes 3 to 5) of r-RB60. r-RB60 was added in a fivefold molar excess over r-RB47.

PDI catalyzes the formation of disulfide bonds by oxidation of the sulfhydryl groups of cysteine residues during protein folding. To examine whether r-RB60 is also capable of oxidative catalysis of the reduced form of r-RB47, we added GSSG, the oxidized form of the thiol tripeptide glutathionine, to the assay mixture. When GSSG alone was added to r-RB47 at up to 5 mM (Fig. 3C, lanes 3 to 5), binding activity of r-RB47 decreased by a factor of 2 compared with untreated protein (Fig. 3C, lane 2). Incubation of r-RB47 with both GSSG and r-RB60 reduced the binding activity of r-RB47 by a factor of 5 to 6 (Fig. 3C, lanes 6 to 8), indicating that r-RB60 can facilitate the conversion of the reduced form of r-RB47 to an inactive oxidized form under an oxidizing environment.

ADP-dependent phosphorylation of RB60 reduces binding of the protein complex to the 5′-UTR of the psbA mRNA (9). To identify if r-RB60 can be phosphorylated, we incubated r-RB60 with heparin-purified proteins from C. reinhardtii in the presence of [γ-32P]ATP (adenosine 5′-triphosphate). Phosphorylated r-RB60 was detected (Fig.4A, lane 2) among a number of phosphorylated proteins in the heparin-purified fraction. Purification of the incubation mixtures on Ni-NTA resin resulted in the isolation of phosphorylated r-RB60 (Fig. 4A, lane 4). Phosphorylated r-RB60 reduced the binding of r-RB47 to the 5′-UTR of the psbA mRNA (Fig. 4B, lane 4) (19), whereas phosphorylated C. reinhardtii proteins eluted from Ni-NTA resin (Fig. 4A, diamond) had little effect on r-RB47 RNA binding (Fig. 4B, lane 5).

Figure 4

Decrease in the binding of cPABP to the 5′-UTR ofpsbA mRNA by the addition of phosphorylated r-RB60. (A) Chlamydomonas reinhardtii proteins were incubated for 20 min at room temperature in the presence of [γ-32P]ATP with (lanes 2 and 4) or without (lanes 1 and 3) the purified r-RB60 protein. Phosphorylated proteins were separated by SDS-PAGE with (lanes 3 and 4) or without (lanes 1 and 2) purification by Ni-NTA resin and autoradiographed. (B) r-RB47 (lane 2) was incubated for 20 min at room temperature with r-RB60 (lane 3) or r-RB60 that had been phosphorylated with 5 mM ATP through use of the heparin-purified protein extracts and re-isolated by Ni-NTA resin (lane 4), followed by gel mobility-shift assay.

Thioredoxin can act as a transducer of redox potential to enhance the binding of a protein complex to the psbA mRNA (7). PDI fits well into this scheme because ferredoxin-thioredoxin reductase is capable of directly reducing PDI (20, 21). As shown in a schematic model (Fig.5), we propose that reducing equivalents, generated by photosynthesis, are donated to cPDI through ferredoxin and ferredoxin-thioredoxin reductase and act to catalyze the reduction of chloroplast polyadenylate-binding protein (cPABP). The reduced form of cPABP is then capable of binding to the 5′-UTR of the psbAmRNA to activate translation initiation of this mRNA, resulting in increased synthesis of the D1 protein (4, 8,9, 22). This mechanism provides a direct link in the chloroplast between the quantity of absorbed light and the rate of synthesis of the D1 protein, allowing the replacement of the photo-damaged D1 protein. PDI has an additional advantage in this scheme in that it has greater oxidation potential than thioredoxin (14), thus allowing the off switch (oxidation) when reducing potential is low. ADP-dependent phosphorylation of RB60, which might be triggered by the increased pool of ADP during dark growth, can act to reduce the RNA binding activity of cPABP by enhancing the oxidative catalysis of cPDI over the reductive catalysis, resulting in decreased translation of the psbA mRNA. The data presented here show that a PDI can act as a regulator of RNA binding activity and hence gene expression, and not just as a catalyst for protein folding.

Figure 5

A working model for light-regulated translation of chloroplast psbA mRNA inC. reinhardtii. See text for description. Thin arrows and thick arrows indicate the light pathway and the dark pathway, respectively. PSI and PSII, photosystem I and II; FD, ferredoxin; FDTR, ferredoxin-thioredoxin reductase; cPDI, chloroplast protein disulfide isomerase; cPABP, chloroplast polyadenylate-binding protein; ox, oxidized; red, reduced; Pi, inorganic phosphate group.


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