The Protein Disulfide Isomerase-like RB60 Is Partitioned between Stroma and Thylakoids in Chlamydomonas reinhardtiiChloroplasts*

Translation of psbA mRNA inChlamydomonas reinhardtii chloroplasts is regulated by a redox signal(s). RB60 is a member of a protein complex that binds with high affinity to the 5′-untranslated region of psbAmRNA. RB60 has been suggested to act as a redox-sensor subunit of the protein complex regulating translation of chloroplastpsbA mRNA. Surprisingly, cloning of RB60 identified high homology to the endoplasmic reticulum-localized protein disulfide isomerase, including an endoplasmic reticulum-retention signal at its carboxyl terminus. Here we show, by in vitro import studies, that the recombinant RB60 is imported into isolated chloroplasts of C. reinhardtii and pea in a transit peptide-dependent manner. Subfractionation of C. reinhardtii chloroplasts revealed that the native RB60 is partitioned between the stroma and the thylakoids. The nature of association of native RB60, and imported recombinant RB60, with thylakoids is similar and suggests that RB60 is tightly bound to thylakoids. The targeting characteristics of RB60 and the potential implications of the association of RB60 with thylakoids are discussed.

The chloroplast contains a small circular genome that encodes about 5-10% of the chloroplast proteins (1). The rest are encoded by the cell nucleus. This two-compartment gene organization dictates a close coordination between nuclear and organellar gene expression (2)(3)(4). Chloroplast mRNAs accumulate in fully developed chloroplasts to relatively high levels in both light-and dark-grown plants and algae. Translation of these mRNAs occurs at a much higher rate during the lightgrowth phase, thus identifying translation as a key regulatory point (reviewed in Refs. 2 and 4 -6). The molecular basis of light-regulated translation in the chloroplast has been shown to be dependent on the function of a growing list of nucleusencoded proteins (7)(8)(9)(10)(11)(12)(13)(14). These factors are thought to mediate translational regulation by interacting with the 5Ј-untranslated region (5Ј-UTR) 1 of chloroplast mRNAs (11,12,(15)(16)(17). Nucleus-encoded chloroplast proteins are typically directed to the chloroplast by a transit peptide located at the amino terminus of the protein (18).
A set of mRNA-binding proteins that bind to the chloroplastic psbA mRNA 5Ј-UTR with high affinity and specificity has been identified and purified from Chlamydomonas reinhardtii cells (19). psbA mRNA 5Ј-UTR-binding proteins are composed of four major proteins, RB38, RB47, RB55, and RB60. These form a complex (psbA 5Ј-PC) that appears to bind the mRNA via the RB47 protein. The level of binding of psbA 5Ј-PC to the mRNA parallels the level of psbA mRNA translation and its association with polyribosomes in light-and dark-grown wildtype C. reinhardtii (19). Moreover, several nuclear mutants have been isolated in which the loss of RB47 is accompanied by the absence of D1 synthesis due to a block in the association of psbA mRNA with polyribosomes (10,20). This suggests that light regulates polyribosome association and translation of psbA mRNA by modulating the binding of psbA 5Ј-PC to the 5Ј-UTR. Cloning of RB47 revealed its high homology with poly(A)-binding proteins (10). Characterization of the intrachloroplast localization of RB47 showed that it is associated with thylakoids in the C. reinhardtii chloroplast (21).
RB60 has been implicated as a regulatory subunit of psbA 5Ј-PC which is subject to light control via phosphorylation and redox signals in the chloroplast (22,23). Cloning of RB60 identified high homology to protein disulfide isomerase (PDI) (24). PDI-like proteins typically catalyze the formation, reduction, and isomerization of disulfide bonds during protein folding in the endoplasmic reticulum (ER). However, in addition to their enzymatic role, PDI-like proteins have also been found to be indispensable subunits in protein complexes such as prolyl hydroxylase and triacylglycerol transfer protein (25). Furthermore, PDI-like proteins have recently been implicated in the regulation of E2A transcription factor (26) and the shedding of L-selectin (27). PDIs are most abundant in the lumen of the ER. They are directed to the ER by a signal peptide at the amino terminus and then are retained there by virtue of a second signal, -(K/H)DEL, at the carboxyl terminus (25,28). The open reading frame of the recombinant RB60 (rRB60) contains an amino-terminal extension, the targeting identity of which has yet to be determined. Notably, despite the implicated function of rRB60 in the chloroplast, its open reading frame contains a carboxyl-terminal signal for ER retention (24). Therefore, we first set out to determine whether the cloned rRB60 gene product is targeted to chloroplasts, and thereafter to study the subchloroplast localization of the native RB60. We show that rRB60 is imported into isolated C. reinhardtii and pea chloroplasts in a transit peptide-dependent fashion. Sub-fractionation of chloroplasts showed that whereas a portion of the native RB60 is present in the stroma, it is also found tightly bound to thylakoids. Moreover, following uptake by chloroplasts, the recombinant rRB60 associates with thylakoids in a manner similar to the native RB60. The association of RB60 to thylakoids was resistant to EDTA and RNase treatments, indicating that it is probably not mediated by binding to polysome-associated psbA mRNA.

EXPERIMENTAL PROCEDURES
Preparation of Intact Chloroplasts-C. reinhardtii cw15 cells were grown in Tris/acetate/phosphate medium (29), under a 12-h light/12-h dark period at 25°C, to a density of ϳ1 ϫ 10 7 cells/ml. Intact chloroplasts were collected from the 45/70% interface of discontinuous Percoll gradient according to a protocol based on Goldschmidt-Clermont et al. (30) and Belknap (31).
Chlorophyll concentration was determined spectrophotometrically according to method of Arnon (34).
In Vitro Import Assays-In vitro protein synthesis reactions were performed with a T 3 TNT-coupled reticulocyte lysate system according to manufacturer's instructions (Promega) using 2 g of DNA from RB60, ⌬28RB60, LHCII, or SSU, or pea PPO cDNA containing plasmid constructs. Translation products were fractionated by SDS-PAGE, and protein incorporation of [ 35 S]methionine was determined by trichloroacetic acid precipitation.
In vitro import assays into intact chloroplasts were performed as described previously (32,35). The import assay was conducted for 30 min at 25°C in the light in the presence of 10 mM ATP, unless otherwise indicated. Competition import assays were performed in the presence of nonlabeled pea OEE1 protein, which was expressed and purified as described by Betts et al. (36). Following import, chloroplasts were pelleted by centrifugation and then resuspended in HS buffer (50 mM Hepes-KOH, pH 8, 0.33 M sorbitol). Each import reaction was divided into 3 aliquots treated with or without thermolysin (0.3 mg/ml) or with thermolysin (0.3 mg/ml) containing 1% Triton X-100 for 45 min at 4°C. Intact chloroplasts were then re-isolated by centrifugation through a 40% Percoll cushion, washed with HS buffer containing 5 mM EDTA, and resuspended in HS buffer.
Localization of Imported rRB60 and Native RB60 -To localize RB60, we used isolated chloroplasts (10 g of chlorophyll) or isolated chloroplasts containing imported radioactive rRB60 that were re-isolated using a 40% Percoll cushion and washed with HS buffer containing 5 mM EDTA. Stromal and thylakoid fractions were obtained by freezing and thawing, followed by a 1-min centrifugation at 15,000 ϫ g at 4°C (37). The resulting supernatant contained the stroma proteins, and the pellet contained the thylakoid fraction. The pellet was washed with 10 mM Hepes-KOH, pH 7.6, and centrifuged as above. Thylakoids were resuspended in 50 l of wash solution (HS with or without 1 M NaCl or 1 M NaCl, 0.05% Triton X-100, or 0.3 mg/ml thermolysin, or 0.1 M sodium carbonate pH 11, or 10 mM EDTA, or 10 mM DTT) and incubated on ice for 30 min. Membranes were then centrifuged as above for 5 min. The supernatant was saved, and thylakoids were washed with HS buffer, centrifuged, and resuspended in 50 l of HS.

RESULTS
rRB60 Is Imported into C. reinhardtii Chloroplasts-The targeting of proteins synthesized by cytoplasmic ribosomes to the chloroplast is typically determined by an amino-terminal transit peptide. The amino-terminal sequence of RB60, preceding the conserved sequence of PDIs, is quite different from other PDIs in both length and composition ( Fig. 1) and contains a putative cleavage sequence in position 26 -28 (39). To test whether the amino-terminal sequence of rRB60 could direct import into chloroplasts, radiolabeled rRB60 was synthesized in vitro and incubated with C. reinhardtii chloroplasts isolated by Percoll step gradient. Since in contrast to import by the ER, protein uptake by chloroplasts occurs post-translationally, we added the radiolabeled rRB60 only after termination of in vitro translation. Following incubation, protein that had not entered the chloroplast was degraded by treatment with the protease thermolysin. Intact chloroplasts were then repurified on a Percoll cushion and lysed with denaturing buffer, and the protein extracts were fractionated by SDS-gel electrophoresis. As seen in Fig. 2, rRB60 was imported into the chloroplasts and was protected from protease treatment (Fig. 2, lane 3), similar to a control import reaction containing in vitro synthesized chloroplast LHCII protein (Fig. 2, lane 11). Disruption of chloroplast membranes by treatment with nonionic detergent resulted in the degradation of both imported rRB60 (Fig. 2, lane 4) and LHCII proteins (Fig. 2, lane 12). These results verified the effectiveness of the protease treatment and that the radiolabeled protein was indeed protected from degradation by being taken up into chloroplasts.
To assay whether the import into chloroplasts was determined by its amino-terminal sequence, a leaderless version of rRB60 (lacking the first 28 amino acids, ⌬28RB60) was prepared and subjected to chloroplast import assays. The leaderless rRB60 was not imported by isolated chloroplasts (Fig. 2,  lane 7), corroborating the proposed function of the amino-terminal sequence of RB60 as a chloroplast transit peptide required for import into chloroplasts. The mobility of the chloroplast-imported rRB60 was similar to that of its in vitro translated precursor (Fig. 2, lanes 1 and 3), and it corresponded to the mobility of the native RB60 in immunoblot assays (data not shown). In contrast, the mobility of the ⌬28RB60 was slightly higher (Fig. 2, lane 5), suggesting that the transit peptide may not be cleaved after import as shown for the thylakoid-associated kinase, TAK1 (40).
The Targeting of rRB60 Is Conserved in Chloroplasts of Higher Plants-Next, we assayed whether rRB60 would also be taken up by the well established system of highly purified pea chloroplasts. We chose pea polyphenol oxidase (Ps-PPO) as a control for the import activity of the isolated pea chloroplasts and C. reinhardtii small subunit of ribulose-1,5-bisphosphate carboxylase (Cr-SSU) as a control for import of a heterologous protein. All proteins were incubated with chloroplasts post-in vitro translation. As seen in the autoradiogram in Fig. 3A, both pea PPO and C. reinhardtii SSU were imported into the chloroplasts and were protected from protease treatment (Fig. 3A,   lanes 3 and 7). Disruption of chloroplast membranes by treating with nonionic detergent resulted in degradation of the imported Ps-PPO and Cr-SSU proteins (Fig. 3A, lanes 4 and 8). These results demonstrated the intactness and capacity of the isolated pea chloroplasts to import C. reinhardtii chloroplasttargeted proteins in vitro. In a manner comparable to Cr-SSU, rRB60 of C. reinhardtii was imported into the isolated pea chloroplasts, as reflected by its protection from protease degradation in the presence of intact chloroplast membranes (Fig.  3B, compare lanes 3 and 4). As with rRB60 imported into C. reinhardtii chloroplasts (Fig. 2), the mobility of rRB60 imported into pea chloroplasts corresponded to that of its precursor (Fig. 3B, lanes 1 and 3). The import of rRB60 into pea chloroplasts was dependent upon its amino terminus, as removal of the rRB60 leader abolished import (Fig. 3B, lanes 6  and 7).
To reaffirm the import of rRB60 into chloroplasts, we tested whether its import exhibits additional characteristics typical of chloroplast import. The findings that the uptake of rRB60 was enhanced in the presence of ATP (Fig. 3C) and was competed by the pea chloroplast protein OEE1 (Fig. 3D) corroborate this. To rule out the possibility of contaminating ER in the import reactions, we assayed the purity of the isolated chloroplasts. Fig. 3E shows that antibodies raised against the tobacco ER protein BiP (41) reacted against pea BiP in total protein extract of leaves but not of isolated chloroplasts. Similar results were obtained for C. reinhardtii chloroplasts using antibodies raised against the yeast BiP (Fig. 3E). This indicates that the isolated chloroplasts used were devoid of ER and that rRB60 was taken up by the chloroplasts. The import of rRB60 into both C. reinhardtii and pea chloroplasts suggests that it is directed to  lanes 1-3) and antibodies specific to yeast BiP and C. reinhardtii OEE2 (lanes 4 and 5).  1 and 4) were lysed by freezing and thawing. Stromal (St, lanes 2 and 5) and thylakoid fractions were separated by centrifugation. Thylakoid membranes were washed with 10 mM Hepes-KOH, pH 7.6 (T, lanes 3 and 6). Immunoblot analysis of C. reinhardtii (C.r. panel) and pea (Pea panel) chloroplast proteins was performed using antibodies directed against C. reinhardtii RB60 (23), OEE2 and CF1 proteins, and against pea OEE1 (52) and ClpC protein (53), and spinach CF1 (54). B, autoradiogram of recombinant RB60 imported into C. reinhardtii (C.r. panel) or pea (Pea panel) chloroplasts. Thermolysin-protected import into chloroplasts (Chlps, lanes 1 and 4) was performed as in Fig. 2, lane 3. Stromal (St, lanes 2 and 5) and thylakoid fractions (T, lanes 3 and 6) were obtained as in A. Imported radiolabeled proteins were visualized as in Fig. 2. chloroplasts in vivo and that this capacity is conserved in higher plants.
RB60 Is Partitioned between Stroma and Thylakoids-Import to C. reinhardtii and pea chloroplasts, in a transit peptidedependent manner, indicated that the nucleus-encoded RB60 contains a chloroplast targeting signal. To identify the subchloroplast localization of native RB60, we lysed isolated C. reinhardtii chloroplasts, and we separated the thylakoid fraction from the supernatant containing the stroma proteins by centrifugation. The purity of the stromal fraction was determined by the absence of thylakoid-associated OEE2 and CF1 proteins, and the purity of the thylakoid fraction was verified by the lack of stromal ClpC protein (Fig. 4A). RB60 was present in both the stroma and thylakoid fractions of C. reinhardtii chloroplasts (Fig. 4A, lanes 2 and 3). The proportion of RB60 in these fractions fluctuated in several replications of this assay (data not shown). However, we routinely observed about 50% of the pool of C. reinhardtii chloroplast RB60 to be associated with thylakoids.
To check whether following import rRB60 is also directed toward the thylakoid membranes, we assayed the association of imported rRB60 with thylakoids. Both C. reinhardtii and pea chloroplasts containing imported radiolabeled rRB60 were disrupted and fractionated as in Fig. 4A. Most of the imported rRB60 was found associated with thylakoids of C. reinhardtii chloroplasts, whereas the proportion of soluble rRB60 was higher in pea chloroplasts (Fig. 4B). Together, these results suggest that at least some of the pool of chloroplast RB60 is associated with thylakoids.
The Nature of the Association of RB60 with Thylakoids-Next, we studied the nature of the association between native RB60 and C. reinhardtii thylakoids by washing purified thylakoids with high salt (1 M NaCl) with or without 0.05% Triton X-100, or with alkali buffer (0.1 M Na 2 CO 3 , pH 11), or by treating with the protease thermolysin (0.1 mg/ml) (Fig. 5). Thylakoid membranes were then isolated by centrifugation, and the resistance of thylakoid-associated RB60 to each of these treatments was determined by immunoblot assay comparing the amounts of thylakoid-associated and released soluble RB60. The nature of the association of RB60 with thylakoids was compared with that of the peripheral thylakoid membrane proteins CF1 and OEE2. Similar to CF1 and OEE2, most of the RB60 remained bound to the thylakoids after a wash with 1 M NaCl or 0.1 M Na 2 CO 3 , pH 11 (Fig. 5, lanes 2, 3,  6, and 7), suggesting that RB60 is tightly bound to the thylakoids. The inclusion of 0.05% Triton X-100 in the salt wash completely removed RB60 from the thylakoids, whereas it had only a mild impact on CF1 and OEE2 (Fig. 5, lanes 4 and 5). These results suggest that RB60 is a peripheral protein and that its association with thylakoids may be mediated, at least in part, by hydrophobic interactions. The protease treatment removed RB60 exclusively from the thylakoids leaving the CF1 and OEE2 proteins intact (Fig. 5, lanes 8 and 9), suggesting that RB60 is located on the stromal face of the thylakoids.
Furthermore, we tested whether imported rRB60 displays the same type of thylakoid association as the native RB60. Following import of radiolabeled rRB60 to C. reinhardtii chloroplasts, thylakoid membranes were challenged with the same treatments as in Fig. 5. Similar to the native RB60 (Fig. 5), most of the imported thylakoid-associated rRB60 was resistant to high salt and alkali washes (Fig. 6, lanes 2, 3, 6, and 7) and sensitive to a wash with 1 M NaCl including 0.05% Triton X-100 (Fig. 6, lanes 4 and 5) and to treatment with thermolysin ( Fig.  6, lanes 8 and 9). The same type of association with thylakoids was observed with rRB60 imported into pea chloroplasts (Fig.  6, lanes 1-9). Together, these data further corroborate the authenticity of rRB60 and suggest that at least a portion of chloroplast RB60 is tightly bound to the stromal face of the thylakoids.
RB60 has been shown to contain reactive thiols (23). This raises the possibility that the thylakoid association of RB60 is mediated by formation of a mixed disulfide bond with a thylakoid protein. In this case, the association of RB60 with thylakoids would be expected to be sensitive to reduction. To test this, we washed isolated thylakoids with buffer containing 10 mM dithiothreitol (DTT) (Fig. 7, lanes 2 and 3). The resistance of thylakoid-associated RB60 to reduction by DTT did not corroborate the assumption. RB60 was initially isolated and characterized as a component of a protein complex showing high affinity to the 5Ј-UTR of psbA mRNA (19). Because translation of psbA mRNA is by thylakoid-bound polyribosomes (42,43), RB60 may associate with thylakoids by binding to the 5Ј-UTR of polysomes-associated psbA mRNA. The resistance of the RB60-thylakoid association to a wash with 10 mM EDTA (which dissociates ribosomes) (Fig. 7, lanes 4 and 5) and to treatment with RNase A (data not shown) indicates otherwise. Therefore, it is conceivable that RB60 associates with thylakoids either directly or as part of a protein complex. DISCUSSION RB60 was first isolated as a component of a protein complex (psbA 5Ј-PC) which assembles with high affinity on the 5Ј-UTR of C. reinhardtii chloroplast psbA mRNA (19). Consistent with the predicted location of RB60, antisera raised against RB60 cross-reacted with a single protein in C. reinhardtii chloroplasts (23). However, the cloning of rRB60 depicted a PDI-like protein containing a putative amino-terminal leader sequence, FIG. 5. Association of native RB60 with C. reinhardtii thylakoid membranes. Thylakoid membranes (obtained as in Fig. 4A) were washed with buffers containing 1 M NaCl, 1 M NaCl, and 0.05% Triton X-100 or 0.1 M Na 2 CO 3 , pH 11, or subjected to a protease treatment (thermolysin (Therm), 0.1 mg/ml) and each separated into membraneassociated (P) and soluble (S) fractions. Immunoblot analysis of each fraction was performed with antibodies against C. reinhardtii RB60, OEE2, and CF1.
FIG. 6. Association of imported rRB60 with C. reinhardtii and pea thylakoid membranes. Thermolysin-protected import into C. reinhardtii (C.r panel) and pea (Pea panel) chloroplasts was performed as in Fig. 2, lane 3. Following import, thylakoid membranes were isolated and treated as in Fig. 4 producing membrane-associated (P) and soluble (S) fractions. Radiolabeled proteins were detected by autoradiography. whose targeting information was unknown, and an ER-retention signal, -KDEL, at the carboxyl-end of the protein (24). This prompted us to investigate the authenticity of the recombinant rRB60 by testing whether its amino terminus can direct import by chloroplasts. We showed that rRB60 is imported into both C. reinhardtii and pea chloroplasts in a transit peptide-dependent process (Figs. 2 and 3), indicating that RB60 is directed by its amino-terminal sequence to chloroplasts in vivo. The import of rRB60 displayed several characteristics typical of chloroplast import, such as post-translational import, ATP-dependent import, and sensitivity to a competing import of a nonlabeled chloroplast protein (Fig. 3 and Fig. 4, B and C). The authenticity of rRB60 was further substantiated by the similar intrachloroplast localization of the native RB60 and the imported rRB60 (Fig. 4). The reason for the presence of a -KDEL signal in the carboxyl terminus of rRB60 is yet unknown. It may be cryptic and may represent a potential signature of the evolution of RB60 from an ancestral PDI gene. Alternatively, RB60 may have dual functions as follows: one in the chloroplast and a second in the ER. Such polytopic proteins, present in both the mitochondrion and an additional cell compartment, have been described (44). Polytopic targeting of mitochondrial proteins has been suggested to arise either from export mechanisms from the mitochondria (44) or, alternatively, via dual targeting by a unique amino terminus signal peptide (45). We are currently studying whether RB60 is also an ER protein.
In the chloroplast, RB60 is partitioned between the stroma and the thylakoids (Figs. 4 -6). A second component of psbA 5Ј-PC, the RB47 protein, has also been shown to be tightly bound to thylakoids (21). The finding of RB60 and RB47 as membrane-associated proteins suggests a regulatory membrane-associated step in the expression of psbA mRNA. Translation of chloroplast mRNAs encoding integral thylakoid proteins is via membrane-bound polyribosomes (42,43). Therefore, it is possible that RB60 attaches to thylakoids by binding to the 5Ј-UTR of polysome-associated psbA mRNA. However, the association of RB60 with thylakoids is resistant to washes containing EDTA and treatment with RNase A, suggesting that the binding is not mediated by membrane-bound ribosomes. This, however, does not rule out the possibility that some amount of RB60 was associated with psbA mRNA and was washed away from the thylakoids in our Mg 2ϩ -free washes.
An alternative explanation for the membrane association of 5Ј-UTR-interacting proteins has been invoked by genetic studies showing many parallels in translational regulation in C. reinhardtii chloroplast and Saccharomyces cerevisiae mitochondrion (2,3,46). In the latter, the cox3 mRNA translation activator proteins, PET54, PET122, PET494, form a complex that has the capacity for three-way interaction with the 5Ј-UTR, the small ribosomal subunit, and the inner mitochondrial membrane (47). This three-way association suggests that the yeast translational activators tether the mitochondrion mRNA to the inner membrane (46). Similar function has been proposed for the proteins binding the 5Ј-UTR of chloroplast mRNAs, encoding thylakoid membrane proteins (2,3). The detection of tight binding of RB60 (Figs. 4 -6) and RB47 (21) to thylakoids corroborates this model. Furthermore, the characterization of RB60 as a peripheral protein localized on the stromal face of the thylakoids (Fig. 5) is consistent with this proposed function of 5Ј-UTR-binding proteins in the chloroplast. However, thylakoid association is not common to all translational regulators: CRP1, a regulator of petA and petD mRNAs in maize, was found to be part of a soluble high molecular weight complex and not to be associated with chloroplast membranes (14).
The finding of specific oxidizing activity of RB60 (23) suggests an additional reason for the RB60-thylakoid association. Recently, a novel yeast protein, ERO1, whose function is to oxidize PDI, has been identified and shown to be membranelocalized (48,49). Likewise, in Escherichia coli, the source of oxidizing equivalents necessary for protein disulfide catalysis has been shown to be membranal electron transport (50,51). By analogy with these studies, one may hypothesize that the photosynthetic electron transport of the thylakoid is the source of oxidizing equivalents for RB60. If so, this might necessitate the close association of RB60 with thylakoids.  4 and 5) producing membrane-associated (P) and soluble (S) fractions. Immunoblot analysis of each fraction was performed with antibodies against C. reinhardtii RB60, OEE2, and CF1.