Oxidative Folding in Chloroplasts

Abstract

Significance: Disulfide-bonded proteins in chloroplasts from green plants exist in the envelope and the thylakoid membrane, and in the stroma and the lumen. The formation of disulfide bonds in proteins is referred to as oxidative folding and is linked to the import and folding of chloroplast proteins as well as the assembly and repair of thylakoid complexes. It is also important in the redox regulation of enzymes and signal transfer. Recent Advances: Green-plant chloroplasts contain enzymes that can form and isomerize disulfide bonds in proteins. In Arabidopsis thaliana, four proteins are identified that are relevant for the catalysis of disulfide bond formation in chloroplast proteins. The proteins' low quantum yield of Photosystem II 1 (LQY1, At1g75690) and snowy cotyledon 2 (SCO2, At3g19220) exhibits protein disulfide isomerase activity and is suggested to function in the assembly and repair of Photosystem II (PSII), and the biogenesis of thylakoids in cotyledons, respectively. The thylakoid-located Lumen thiol oxidoreductase 1 (LTO1, At4g35760) can catalyze the formation of the disulfide bond of the extrinsic PsbO protein of PSII. In addition, the stroma-located protein disulfide isomerase PDIL1-3 (At3g54960) may have a role in oxidative folding. Critical Issues: Research on oxidative folding in chloroplasts plants is in an early stage and little is known about the mechanisms of disulfide bond formation in chloroplast proteins. Future Directions: The close link between the import and folding of chloroplast proteins suggests that Hsp93, a component of the inner envelope's import apparatus, may have co-chaperones that can catalyze disulfide bond formation in newly imported proteins. Antioxid. Redox Signal. 19, 72–82.

Introduction

T he chloroplasts of green plants are well known for their ability to perform oxygenic photosynthesis and are established models for plastid function and evolution. It is widely accepted that chloroplasts evolved from a single cyanobacterial ancestor that was acquired by a host organism through endosymbiosis more than 1 billion years ago (60, 70).

A chloroplast from a plant leaf has a complex ultrastructure (Fig. 1A). Inside, there is the thylakoid membrane that performs the primary events of oxygenic photosynthesis. The thylakoid membrane forms cylindrical stacks known as grana that are connected by the circular stroma lamellae (6). It is surrounded by a compartment known as the stroma, where many metabolic processes occur. These include the Calvin-Benson cycle for carbon dioxide fixation (39). Both the stroma and the thylakoids are enclosed by the envelope membrane, which consists of an inner and an outer membrane separated by an intermembrane space. The envelope has a complex system of transporters for transferring proteins and metabolites between the cytoplasm and the chloroplast (30). The thylakoid membrane encloses a compartment called the lumen, which is essential for photosynthetic electron transport and for proton gradient formation during photosynthesis. However, the functions of most of the lumen proteins are currently unknown (48).

FIG. 1.

Ultrastructure of chloroplasts. (A) This electron micrograph of a chloroplast from the leaf mesophyll cells of Arabidopsis thaliana shows the envelope and the thylakoid membrane, and the stroma between them. The center of this chloroplast contains a large starch granule. The enlarged section shows the regularly stacked thylakoid grana, which are linked by the stroma thylakoids. The thylakoid lumen is enclosed by the thylakoid membrane. The envelope membrane is a double membrane that consists of an outer and an inner envelope, which is not visible in this electron micrograph. The compartment between the inner and the outer envelope is called the intermembrane space. (B) The box shows some biochemical properties of a chloroplast from the mesophyll cells of plant leaves. The chloroplast stroma has a pH of about 7.8 to 8 (51), and the presence of ascorbate, GSH, and NADPH (66) creates a reducing environment. During photosynthesis, the NADPH/NADP+ ratio increases and the stroma becomes more reducing (41).The thylakoid lumen is acidified during photosynthesis and its pH changes from about 7.5 in darkness or under low light to about 5.7 under saturating light (81). Like the stroma, the lumen contains ascorbate (31) but GSH is not detectable (90). The protein concentrations of the lumen and the stroma are similar, at around 10 mg/ml (47).

Different lines of research have suggested that chloroplasts can catalyze the formation of disulfide bonds in proteins, a process that is also referred to as oxidative folding. Notably, photosynthetic activity regulates the activity of enzymes in the chloroplast stroma due to the reduction of disulfide bonds via a cascade involving ferredoxin, ferredoxin thioredoxin (Trx) reductase, and Trxs m and f (75). During the last decade, more than 100 new potential Trx targets have been discovered in chloroplasts (57). Although the chloroplast stroma is reducing (Fig. 1B), the disulfides of these proteins are at least partially oxidized. Some of these proteins were also identified during in vivo screens for disulfide-bonded proteins in Arabidopsis thaliana (2, 53). In addition, chloroplasts from transgenic plants can produce functional therapeutic proteins that have correctly formed disulfide bonds (24, 80, 86). Overall, these findings indicate that chloroplasts can catalyze disulfide bond formation in proteins (88).

While many studies have addressed the function of disulfide bonds in the redox regulation of chloroplast enzymes and signal transduction (13, 54), relatively few have examined the mechanisms of disulfide bond formation in chloroplast proteins (42). This review provides an overview of the current knowledge in this field, with particular emphasis on oxidative folding in the chloroplasts of green plants. The mechanisms of disulfide bond formation in chloroplast proteins are not well understood. However, there is evidence that most chloroplast members of the Trx superfamily from the model plant A. thaliana are known, and some of them probably have functions related to oxidative folding. These include the protein disulfide isomerase PDIL1-3 (At3g54960) and the Lumen thiol oxidoreductase 1 (LTO1, At4g35760), which may be a useful starting point for future studies.

Chloroplast Protein Import Pathways Indicate that Disulfides Are Formed at Different Subcellular Sites

The folding of chloroplast proteins is closely linked to their biosynthesis and subsequent transport to their subcellular locations. Although chloroplasts have retained their own small genomes, most chloroplast proteins are nuclear-encoded and synthesized in the cytoplasm. They reach their subcellular destination by importation into the organelle (Fig. 2) (45).

FIG. 2.

Protein import into chloroplasts. Most chloroplast proteins are nuclear encoded. They are synthesized on the ribosomes in the cytoplasm and imported into the organelle. The import of chloroplast proteins is controlled by transit peptides that have a targeting sequence for chloroplasts. The proteins of the outer envelope are an exception—most of them insert directly into the membrane from the cytoplasm. The scheme shows the major pathways of protein import into the chloroplast (45). The cytoplasmic protein precursors bind to the translocon outer complex (TOC) in a process that is driven by GTP and the low concentrations of ATP (∼0.1 mM) in the intermembrane space. Subsequently, the precursor proteins are pulled through the translocon inner complex (TIC) by Hsp93 chaperones in the stroma (76). Transport through the inner envelope is energy-intensive and requires high concentrations of ATP (∼1 mM). On the stroma side of the envelope, the chloroplast-targeting sequence is removed by a processing protease that is symbolized by scissors. Stromal proteins can now fold and form complexes. Most thylakoid proteins move through the stroma to the thylakoid membrane and insert into the membrane spontaneously without apparent assistance. However, the light-harvesting complexes use a different pathway that is assisted by the chloroplast signal recognition particle (cpSRP). Lumenal proteins reach their final destinations via either the Sec or the Tat pathways of the thylakoid membrane. They have bipartite transit peptides that consist of a chloroplast and a lumenal-targeting sequence. When the chloroplast-targeting sequence is cleaved off after importation into the stroma, the lumenal-targeting sequence directs these proteins to either the Sec or the Tat translocation system (1). After importation into the thylakoid lumen, the lumenal-targeting sequence is cleaved off by a processing protease (symbolized by scissors). While the Tat pathway can import folded proteins, the proteins that enter the lumen using the Sec machinery are unfolded and must be refolded after importation. Finally, two different pathways have been proposed to explain the import of proteins into the inner envelope. Some of these proteins have a stop sequence and exit the TIC complex laterally to insert into the membrane of the inner envelope. Others are imported into the stroma and then exported to the inner envelope (45).

Most outer envelope proteins can be directly inserted into the membrane from the cytoplasm, but proteins with subcellular locations inside the chloroplasts are imported via the translocation systems of the envelope (Fig. 2). Usually, the cytoplasmic protein precursors have cleavable transit peptides with a chloroplast-targeting sequence that binds to receptors in the outer envelope. The protein precursors are then unfolded and enter the pore of the translocon outer complex. Finally, Hsp93 chaperones in the stroma pull the unfolded protein precursors through the translocon inner complex (TIC) (76). On the stromal side of the inner envelope, the stromal processing protease removes the chloroplast transit peptides of the imported precursors. At this point, the imported proteins may fold or be delivered to one of the chloroplast membranes (45).

Proteins of the inner envelope are exported to the inner envelope membrane from the stroma. Alternatively, they can exit the TIC laterally and insert into the inner membrane of the envelope from the intermembrane space (45). Proteins that are targeted to the thylakoid membrane usually insert into the membrane without apparent assistance by other factors in a so-called spontaneous way. However, the light-harvesting complexes are inserted into the membrane by the chloroplast signal recognition particle (45). The thylakoid membrane has two distinct import systems for lumen proteins (1). The Sec pathway, which resembles the bacterial SecYEG channel, imports unfolded precursors and requires ATP for the translocation. The twin-arginine translocation pathway imports folded proteins and is driven by the pH gradient across the thylakoid membrane. Lumenal protein precursors are tagged with a transit peptide that determines which of these import pathways will be used (1).

Both the envelope and the thylakoid membrane have the ability to unfold proteins for translocation. Although the envelope can import small folded proteins (18), protein precursors usually translocate the envelope in an unfolded conformation (15, 74, 87). It is likely that disulfide bonds are reduced when this happens because the precursors enter the chloroplast from the reducing environment of the cytoplasm. That implies that the disulfide bonds of newly imported chloroplast proteins are formed at their final subcellular location.

Proteins containing disulfide bonds have been observed in most of the compartments and membranes of the chloroplast, including the stroma (2, 53, 75), the thylakoid membrane (62, 64), and the lumen (2, 34, 53, 82). There is also evidence that proteins with disulfide bonds are present in the inner (9) and outer envelopes (10). The only chloroplast compartment that is not known to contain proteins with disulfide bonds is the intermembrane space. It is not currently known whether disulfide bonds are formed in the intermembrane space, but it is likely that both the stroma and the thylakoids contain enzymes that catalyze disulfide bond formation. The heat shock protein Hsp93 is a component of the import apparatus of the inner envelope that promotes the correct folding of imported proteins. Because the importation and folding of chloroplast proteins are closely related, Hsp93 may have co-chaperones that assist in the formation of disulfide bonds in newly imported proteins. Such co-chaperones would represent a sort of missing link, but they have not yet been observed in plant chloroplasts.

The folding of proteins that have multiple disulfide bonds requires both disulfide bond-forming activity and also isomerase activity to reshuffle incorrect disulfide bonds and establish the required connectivity. A representative disulfide-bonded protein whose folding requires both of these activities is the basic pancreatic trypsin inhibitor BPTI (aprotinin), which has three disulfide bonds. The chloroplasts of transgenic tobacco lines can synthesize BPTI in the stroma and export it to the lumen, where it is folded with the correct disulfide bonds (86). This suggests that the lumen has an enzymatic activity that can form and isomerize protein disulfide bonds, which raises a question: Do any known plant chloroplast enzymes have such functions?

The Chloroplast Members of the Trx Superfamily in A. thaliana

Many enzymes that catalyze disulfide bond formation in proteins are members of the Trx superfamily. A survey of the Trx superfamily from Arabidopsis shows that it has 34 members with a confirmed location in chloroplasts (Fig. 3). Most of these proteins are located in the stroma and/or peripherally associated with the envelope and the thylakoid membrane. This group includes 10 Trxs (4, 14, 19, 20) and 9 Trx-like proteins (14, 23, 61, 71). In addition, there are four glutaredoxins (8, 16, 21, 22) and peroxiredoxins (7, 11, 43). Finally, there are four protein disulfide isomerases (3, 14, 44) as well as the proteins low quantum yield of Photosystem II 1 (LQY1) and snowy cotyledon 2 (SCO2), which lack Trx active sites but have protein disulfide isomerase activity (58, 78). Only three known members of the chloroplast Trx superfamily are located in the thylakoid membrane or the lumen. These proteins include the Trx-like protein Hcf164 (55), the LTO1 (29, 46), and the lumen-located peroxiredoxin Q (48, 68). No Trx-like proteins have been reported in the envelope.

FIG. 3.

Chloroplast-located members of the thioredoxin superfamily of chloroplasts from A. thaliana . The chloroplasts of Arabidopsis contain many members of the thioredoxin superfamily. This has been demonstrated by studies using green fluorescent protein (GFP) fusion proteins to determine proteins' subcellular locations, in vitro protein import assays, and biochemical assays of subcellular fractions. The references for the subcellular locations of individual proteins from the stroma and the stroma side of the thylakoids are as follows. Thioredoxins: Trx-f1 (At3g02730) (14, 19), Trx-f2 (At5g16400) by high homology to Trx-f1, Trx-m1 (At1g03680) (14, 19), Trx-m2 (At4g03520) inferred by high homology to Trx-m1, Trx-m3 (At2g15570) (19), Trx-m4 (At3g15360) inferred by high homology to Trx-m1 and Trx-m3, Trx-x (At1g50320) (14, 19), Trx-y1 (At1g76760) (14, 20), Trx-y2 (At1g43560) (14, 20), Trx-z (At3g06730) (4). Thioredoxin-like proteins: NTRC (At2g41680) (61), CDSP32 (At1g76080) (14, 71), WCRKC1 (At5g06690) (14), WCRKC2 (At5g04260) (14), Lilium 1, AtACHT4 (At1g08570) (23), Lilium 2, AtACHT2 (At4g29670) (23), Lilium 3, AtACHT5 (At5g61440) (23), Lilium 4, AtACHT3 (At2g33270) (23), Lilium 5, AtACHT1 (At4g26160) (14, 23). Glutaredoxins: GrxC5 (At4g28730) (22), GrxS12 (At2g20270) (21), GrxS14 (At3g54900) (8, 16), GrxS16 (At2g38270) (8). Peroxiredoxins: 2-Cys peroxiredoxin BAS1 (At3g11630) (7), 2-Cys peroxiredoxins BAS1-like (At5g06290) (43), PrxIIE (At3g52960) (11). Protein disulfide isomerases: PDIL1-3, PDI1 (At3g54960) (3), APR1 (At4g04610) (14), APR2 (At1g62180) (14), APR3 (At4g21990) inferred by high homology to APR1 and APR2, LQY1 (At1g75690) (58), SCO2 (CYO1) (At3g19220) (78, 83). The references for the individual proteins of the thylakoid membrane and the thylakoid lumen are as follows. Thioredoxin-like proteins: Hcf164 (At4g37200) (55); vitamin K₁ epoxide reductase (VKOR)-thioredoxin-like: LTO1 (At4g35760) (29, 32, 46); peroxiredoxins: PrxQ (At3g26060) (48, 68). As for PrxQ, a subcellular location in the stroma was also reported (52), but quantitative proteomics suggests that most of PrxQ is located in thylakoids (30).

Some of the chloroplast members of the Trx superfamily have been studied in detail. For instance, the impact of Trxs and peroxiredoxins in redox regulation of chloroplast enzymes and in antioxidative defense has been highlighted in many excellent articles and some recent reviews (13, 26, 50, 54, 75). However, no functions have been identified for some other members of the Trx superfamily, including the WCRKC proteins (14), and the lilium proteins (23). The only chloroplast proteins for which disulfide bond-forming activity has been reported are the LTO1 (29, 46), and the thylakoid proteins LQY1 (58) and SCO2 (CYO1) (78). The soluble Trx-like domain of LTO1 (starting at L231) was determined to have a midpoint potential of −180 mV (32), which matches that for typical protein disulfide isomerases (5). Can midpoint potentials help to elucidate which other proteins of the Trx superfamily of Arabidopsis chloroplasts might function in disulfide bond formation? While the biological function of a dithiol-disulfide oxidoreductase can certainly not be inferred from its in vitro midpoint potential alone, it may provide a helpful suggestion concerning a protein's function in vivo.

A survey of midpoint potentials reported in the literature for the chloroplast-located members of the Trx super family from Arabidopsis (Fig. 4) revealed values ranging from −368 mV for stromal Trx-m2 (19) to −180 mV for the soluble Trx-like domain of LTO1 (32). After LTO1, the highest midpoint potentials in this group are those of the soluble part of the thylakoid protein Hcf164 (residues 116 to 261; −224 mV) and the stroma-located lilium 5 (AtACHT1; −237 mV) (23, 62). These values are lower than those of protein disulfide isomerase (−180 mV) and DsbA (−124 mV), which are both known to be involved in disulfide formation (5). This suggests that the chloroplast Trxs, the Trx-like lilium proteins, CDSP32, and the peroxiredoxins have no direct roles in the formation of disulfide bonds in proteins. The midpoint potential of NADPH-dependent thioredoxin reductase (NTRC) has not been reported, but NTRC can efficiently reduce the peroxiredoxin Bas1 (67), and must therefore have a lower midpoint potential. Some of the midpoint potentials for the glutaredoxins GrxC5, GrxS12, GrxS14, and GrxS16 have also not yet been reported. However, glutaredoxins are reduced by glutathione, which has a midpoint potential of −240 mV (5). This suggests that they do not have any direct function in the formation of protein disulfide bonds.

FIG. 4.

Reported midpoint potentials of chloroplast located members of the thioredoxin superfamily of A. thaliana . All midpoint potentials were determined at pH 7.0 unless a different pH value is indicated. Thioredoxins: Trx-f1 (−351 mV), Trx-m1 (−357 mV), Trx-m2 (−368 mV), Trx-m3 (−358 mV), Trx-m4 (−358 mV), and Trx-x (−336 mV) (19); Trx-z from poplar (−251 mV) (17). Thioredoxin-like proteins: CDSP32 (−337 mV at pH 7.9) (84), lilium 1 (AtACHT4) (−240 mV), lilium 2 (AtACHT2) (−239 mV), and lilium 5 (AtACHT1) (−237 mV) (23); −224 mV for the soluble part of Hcf164 (Hcf164-sol: residues 116 to 261) (−224 mV) (62). Glutaredoxins: GrxS12 from poplar (−315 mV for the mixed disulfide with GSH) (89). Peroxiredoxins: 2-Cys Prx BAS1 (−315 mV) (49), PrxIIE (−307 mV) (43), PrxQ from poplar (−325 mV) (73). Protein disulfide isomerases: −180 mV for the soluble Trx-like domain of LTO1 (LTO1-sol: residues 231–376) (32), −159 mV for the protein disulfide isomerase from Oldenlandia affinis (35), which has 58% sequence identity to PDIL1-3 from Arabidopsis.

Although these proteins are not able to form protein disulfide bonds on their own, they might participate in alternative pathways. It has been suggested that chloroplast peroxiredoxins might catalyze peroxide-mediated disulfide bond formation in target proteins (50). This reaction is believed to proceed in a similar fashion to the Orp1-Yap1 redox relay in yeast (37). In that reaction, the peroxiredoxin Orp1 oxidizes the transcription factor Yap1 by forming a disulfide bond. The proposed mechanism for this reaction involves the oxidation of a thiol group in the catalytic site of Orp1 to a sulfonic acid (Cys-OH) by hydrogen peroxide. The sulfenic acid then forms an intermediate mixed disulfide bond with a target thiol (Cys598) of YAP1, after which the mixed disulfide bond between Orp1 and Yap1 is reduced by a nucleophilic attack from a second thiol group (Cys303) of Yap1. The final reaction products are oxidized Yap1, which migrates to the nucleus to induce a transcriptional response, and reduced Orp1 (37).

Finally, there is a group of protein disulfide isomerases and related proteins that includes the protein PDIL1-3, and the 5′-adenylylsulfate reductases APR1, APR2, and APR3. The presence of three 5′-adenylylsulfate reductases in this group was unexpected. However, these proteins were identified by a phylogenetic analysis, as members of the protein disulfide isomerase family of Arabidopsis (44) and in vitro import assays indicated that they are located in the chloroplast stroma (14). The enzyme 5′-adenylylsulfate reductase has an important function in the biosynthesis of cysteine (59), but it is not known whether it also catalyzes disulfide bond formation in proteins. The protein disulfide isomerase PDIL1-3 (3) has domains that are very similar to protein disulfide isomerase from yeast and mammalian protein disulfide isomerases (Fig. 5) (36). PDIL1-3 has not yet been reported to have a role in the formation of disulfide bonds of chloroplast proteins, but it is very similar to the protein disulfide isomerase OaPDI from Oldenlandia affinis (35). OaPDI catalyzes the formation of disulfide bonds in cyclotides—cyclic peptides that have three disulfide bonds and are involved in defense against insects. The protein disulfide isomerase and chaperone activity of OaPDI are about 70% and 140% as strong as those of human protein disulfide isomerase (PDI), respectively. The midpoint potential of OaPDI is −159 mV, which is similar to that of mammalian protein disulfide isomerases (77). The amino acid sequences of OaPDI and PDIL1-3 have 58% identity, which suggests that PDIL1-3 from Arabidopsis has functions similar to those of OaPDI. It is thus likely that PDIL1-3 can catalyze disulfide bond formation and shuffle non-native disulfide bonds into their correct connectivity.

FIG. 5.

Domain structure of the chloroplast-located protein disulfide isomerase PDIL1-3 (At3g54960) of A. thaliana . The sequence of PDIL1-3 displays typical features of a protein disulfide isomerase, including the thioredoxin-like domains a, b, b′ and a′. The domains a and a′ contain the thioredoxin motifs WCGAC and WCGHC. The domains b′ and a′ are connected by a linker domain, and subsequent to domain a′ there is an acidic C-terminal extension with the ER retention signal KDEL. PDIL1-3 is a noncanonical chloroplast protein that lacks a cleavable transit peptide. The chloroplast location of PDIL1-3 from Arabidopsis has been demonstrated by in vitro import and by using a GFP fusion protein (3). The subcellular location of PDIL1-3 within the chloroplasts has not been determined, but it is probably located in the stroma because it is a soluble protein and lacks a lumenal transit peptide. PDIL1-3 is also targeted to the endoplasmic reticulum (ER) (28) and may be targeted to chloroplasts via this organelle, although passage though the ER is not required for its import into chloroplasts in vitro (3).

Finally, the proteins LQY1 (58) and SCO2 (78) have been suggested to assist in the repair and assembly of photodamaged Photosystem II (PSII) (58) and the integration of light-harvesting complexes into the thylakoid membranes in the chloroplasts of cotyledons (83). LQY1 and SCO2 have protein disulfide isomerase activity (58, 63, 78) but lack the CXXC sequence motif that is found in the active site of Trx and is characteristic of the Trx superfamily. Instead, these proteins resemble the chaperone DnaJ, although they do not have its N-terminal domain for binding to DnaK. It was shown that DnaJ of Escherichia coli has protein disulfide isomerase activity in vitro, and it was proposed that this activity is based on its four CXXCXGXG Zn-binding motifs in the C-terminal region (25). This Zn-binding domain is also present in the C-terminal regions of the proteins LQY1 and SCO2, and may be important in their function as protein disulfide isomerases.

Disulfide Formation in Different Chloroplast Compartments

The above survey of the chloroplast-located members of the Trx superfamily in Arabidopsis identified the protein disulfide isomerase PDIL1-3 in the stroma and the LTO1 in the thylakoid membrane as potentially being involved in catalyzing disulfide bond formation in other proteins. These two proteins can be studied to investigate the roles of disulfide bond formation in the folding, regulation, and signal transduction of chloroplast proteins. In addition, the proteins LQY1 and SCO2 demonstrate that the ability to catalyze disulfide formation in proteins is not limited to members of the Trx superfamily. At present, relatively little is known about the biochemical reactions involved in the formation of disulfide bonds in chloroplast proteins. However, the chloroplasts of green plants contain numerous potential Trx targets that have functions in transcription and translation, and many metabolic pathways (57). This suggests that the network of biochemical reactions involving disulfide bond formation in chloroplast proteins is probably very complex.

Two well-known cases of redox control of chloroplast enzymes by Trxs are the Calvin-Benson cycle enzyme fructose 1,6 bisphosphatase and the pentose phosphate pathway enzyme glucose 6-phosphate dehydrogenase. Fructose 1,6 bisphosphatase is light-dependently activated by the reduction of a disulfide bond by Trx f, whereas glucose 6-phosphate dehydrogenase is light-dependently inactivated by reduction mediated by Trxs m and f (75). It has been suggested that reactive oxygen species (ROS) and hydrogen peroxide might be involved in the inactivation of fructose 1,6 bisphosphatase (75) and the activation of glucose 6-phosphate dehydrogenase (12), respectively. While ROS and hydrogen peroxide may indeed be involved, enzymatic catalysis of disulfide bond formation might also be required to ensure the rapid inactivation of fructose 1,6 bisphosphatase and activation of glucose 6-phosphate dehydrogenase during the transition from light to darkness.

The biosynthesis and repair of the photosynthetic complexes of the thylakoid membrane is another area where disulfide bond formation may play an important role. The cotyledons of Arabidopsis seedlings in mutants lacking the protein SCO2 have small chloroplasts with a deviant shape (78) and partially aberrant ultrastructures (83). SCO2 can bind to LHCB1 and has been proposed to have a role in the assembly of PSII in germinating seedlings (83). Arabidopsis mutants that lack the LQY1 protein are more sensitive to high light stress than wild-type plants and exhibit reduced levels of the PSII-LHCII supercomplex when grown under high light conditions. LQY1 binds to different PSII complexes, and has been suggested to aid in the repair and reassembly of photodamage to PSII (58). These examples demonstrate that enzymes that can catalyze disulfide bond formation in other proteins probably play important roles in the biogenesis and function of the thylakoid membrane.

The ability of the LTO1 to catalyze the formation of a disulfide bond in the extrinsic PSII subunit PsbO (46) highlights the importance of disulfide bond formation in the thylakoid lumen, and provides a link between oxidative folding and the import of lumen-located proteins. The PsbO protein binds to the lumen side of PSII and stabilizes the water-oxidizing complex. As discussed in a recent review, reductions in the abundance of PsbO have severe effects on photosynthesis (69). Knockdown plants that express reduced levels of LTO1 show retarded growth and an impaired function of PSII (46). In addition, the levels of PsbO and other PSII subunits are lower than in wild-type plants (46). How is the ability of LTO1 to form a disulfide bond in PsbO linked to the assembly of PSII and the import of lumenal proteins? Shortly after the import of PsbO into the thylakoids, it adopts a transient form that is sensitive to proteolysis (40). Mature PsbO with a correctly folded disulfide bridge is stable, but it is destabilized when the disulfide bond is reduced (65) and becomes sensitive to proteolysis (38). Taken together, these observations suggest that the disulfide bond of newly imported PsbO proteins is reduced. Fast folding and disulfide bond formation would be needed to ensure that PsbO assumes a stable fold and can bind to PSII. The disulfide bond might form spontaneously due to the oxidizing environment in the lumen during photosynthesis, but spontaneous disulfide formation is relatively slow. Therefore, enzymatic assistance is probably required to minimize losses of newly imported unstable PsbO via proteolysis. LTO1 would thus be very important for the correct folding of PsbO and the assembly of PSII.

The sequence of LTO1 from Arabidopsis contains an N-terminal vitamin K₁ epoxide reductase (VKOR) domain that is followed by a C-terminal Trx-like domain (Fig. 6). The VKOR domain is related to mammalian vitamin K₁ epoxide reductase complex subunit 1 (VKORC1) of the vitamin K cycle (79). VKORC1 regenerates active vitamin K₁ by reducing the inactive epoxide of vitamin K₁ that is produced during the maturation of blood-clotting proteins (79). To understand the function of LTO1, it is important to know its membrane topology and the subcellular location of its C-terminal Trx-like domain. Two studies (29, 46) showed that the C-terminal Trx-like domain is located in the thylakoid lumen (Fig. 7A, B), which is consistent with a role in the formation of disulfide bonds in lumenal proteins. It is also important to determine the mechanism by which the reduced Trx-like domain becomes oxidized. It has been proposed that the electrons used to reduce the vitamin K₁ epoxide are provided by a Trx that reduces the cysteine residues C43 and C51 in the cytoplasmic loop of VKORC1, as occurs in human VKORC1 (Fig. 7C). These cysteine residues in turn reduce C132 and C135 in the third transmembrane helix, where vitamin K₁ epoxide is reduced (72). In agreement with this model, the recombinant VKOR domain of LTO1 can oxidize the reduced Trx-like domain in vitro, which suggests that it also can serve as an acceptor for electrons from the reduced Trx-like domain in vivo (32). However, unlike VKCORC1, LTO1 cannot reduce vitamin K₁ epoxide. The VKOR domain of LTO1 needs vitamin K₁ (phylloquinone) as an electron acceptor (32). The mechanism for the re-oxidation of reduced vitamin K₁ in the thylakoid membrane has yet to be determined, and doing so will be an important objective for future studies in this area.

FIG. 6.

Domain structure of Lumen thiol oxidoreductase 1 (LTO1) (At4g35760) of A. thaliana . LTO1 (Uniprot Q8L540) incorporates a chloroplast transit peptide (residues 1 to 55) (29), and a membrane-located VKOR domain (Interpro IPR012932) along with a soluble thioredoxin-like domain (Interpro IPR012336) (29, 46). The VKOR domain is similar to that of the human VKORC1 protein (Uniprot Q9BQB6) (33) and has four putative transmembrane helices (29) that are indicated as predicted by TMHMM v2.0 (www.cbs.dtu.dk/services/TMHMM-2.0/). Both the VKOR and the Trx-like domain contain four conserved cysteine residues that are marked by triangles and needed for the oxidoreductase function of LTO1 (29). The C-terminal cysteine residues 195 and 198 of the VKOR domain correspond to Cys132 and Cys135 in human VKORC1, which reduce vitamin K₁ epoxide to vitamin K₁ quinone and then to vitamin K₁ hydroquinone. The N-terminal cysteine residues 109 and 116 correspond to Cys43 and Cys51 in human VKORC1, which are believed to mediate electron transfer from a thioredoxin to the residues Cys132 and Cys135 to facilitate the reduction of vitamin K₁ epoxide (72). The LTO1 protein from Arabidopsis is a hybrid in which the N-terminal VKOR domain is linked to a C-terminal thioredoxin-like moiety. Like the VKOR domain, the thioredoxin-like domain has four highly conserved cysteine residues located at positions 293, 296, 316 and 331. The Cys293 and Cys296 residues of the thioredoxin-like domain are part of the WCSHC motif, which resembles the CXHC motif of human protein disulfide isomerases (27). VKOR proteins that have a C-terminal thioredoxin-like domain and resemble LTO1 are present in many other plants, algae and cyanobacteria.

FIG. 7.

Membrane topology of Lumen thiol oxidoreductase 1 (At4g35760) of A. thaliana . Two models have been suggested for the membrane topology of the protein LTO1 (29, 46). In both models the C-terminal thioredoxin-like domain is located on the lumen side of the thylakoid membrane. However, the models differ with respect to the membrane topology of the VKOR domain. (A) In one (29), LTO1 has four transmembrane helices, and both the N- and the C-termini of the protein face the lumen of the thylakoid membrane. Cysteine residues 64 and 71 of the VKOR domain face the lumen, while cysteine residues 150 and 154 of the CXXC motif are located in the third helix. (B) In the alternative model (46), the VKOR domain has one additional membrane-spanning helix in the N-terminus of LTO1 that is located in the stroma and not in the lumen. This topology model corresponds to the X-ray structure of a VKOR-Trx-like protein from Synechococcus sp. (56). The membrane-embedded part of this protein contains a VKOR domain with four membrane-spanning helices that is linked to the cytoplasmic thioredoxin-like domain through the fifth transmembrane helix (56). (C) The orientation of human VKORC1 in the membrane of the endoplasmic reticulum (85) is different from that of the VKOR domain of LTO1. The VKORC1 protein has three membrane spanning helices, and Cys43 and Cys51 are located in the cytoplasm rather than the lumen. The C-terminus of VKORC1 is also located in the cytoplasm rather than the lumen (85). One more difference between the human VKORC1 protein and LTO1 is the presence of an additional transmembrane helix between the VKOR domain and the thioredoxin-like domain of LTO1. This helix is one of the putative transmembrane helices predicted using TMHMM (Fig. 6), and its function is unknown.

Conclusions

Four chloroplast proteins from A. thaliana that are likely to have important functions in the formation of disulfide bonds in target proteins have been identified. The thylakoid proteins LQY1 and SCO2 exhibit protein disulfide isomerase activity and function in the assembly and repair of PSII, and the biogenesis of thylakoids in cotyledons, respectively. The disulfide oxidoreductase LTO1 is important for the disulfide bond formation and folding of the extrinsic PsbO subunit of PSII. It is probably also involved in disulfide bond formation in other lumenal disulfide proteins and might control the transfer of thiol-mediated signals. Finally, the protein disulfide isomerase PDIL1-3 is a likely candidate for the catalysis of oxidative folding in the chloroplast stroma. At this point it is premature to discuss the evolution of the protein folding system of chloroplasts from green plants. However, many cyanobacteria have proteins that are related to LTO1 from Arabidopsis, which suggests a bacterial origin for this protein. Conversely, PDIL1-3 appears to have a eukaryotic origin. Many details of the mechanisms of oxidative folding in green plant chloroplasts are currently unknown, but foundations for future studies in this area have been established. Given the close link between import and folding of chloroplast proteins, it is reasonable to suggest that there may be a set of as-yet unidentified components of the import apparatus of the envelope that function as co-chaperones for Hsp93 and that catalyze disulfide bond formation in newly imported chloroplast proteins.

Footnotes

Acknowledgment

T.K. thanks Wolfgang P. Schröder for critical reading of the manuscript.

Abbreviations Used

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