Abstract Leishmania encode six paralogs of the cap‐binding protein eIF4E and five eIF4G candidates, forming unique complexes. Two cap‐binding proteins, LeishIF4E1 and LeishIF4E2, do not bind any identified LeishIF4Gs, thus their roles are intriguing. Here, we combine structural prediction, proteomic analysis, and interaction assays to shed light on LeishIF4E2 function. A nonconserved C‐terminal extension was identified through structure prediction and sequence alignment. m 7 GTP‐binding assays involving both recombinant and transgenic LeishIF4E2 with and without the C‐terminal extension revealed that this extension functions as a regulatory gate, modulating the cap‐binding activity of LeishIF4E2. The interactomes of the two LeishIF4E2 versions were investigated, highlighting the role of the C‐terminal extension in binding to SLBP2. SLBP2 is known to interact with a stem‐loop structure in the 3′ UTRs of histone mRNAs. Consistent with the predicted inhibitory effect of SLBP2 on histone expression in Xenopus laevis , a hemizygous deletion mutant of LeishIF4E2, exhibited an upregulation of several histones. We therefore propose that LeishIF4E2 is involved in histone expression, possibly through its interaction between SLBP2 and LeishIF4E2, thus affecting cell cycle progression. In addition, cell synchronization showed that LeishIF4E2 expression decreased during the S‐phase, when histones are known to be synthesized. Previous studies in T. brucei also highlighted an association between TbEIF4E2 and SLBP2, and further reported on an interaction between TbIF4E2 and S‐phase‐abundant mRNAs. Our results show that overexpression of LeishIF4E2 correlates with upregulation of cell cycle and chromosome maintenance proteins. Along with its effect on histone expression, we propose that LeishIF4E2 is involved in cell cycle progression.
ABSTRACT Background Parkinson’s disease (PD) is associated with dysbiosis, proinflammatory gut microbiome, disruptions to intestinal barrier functions, and immunological imbalance. Microbiota-produced short-chain fatty acids promote gut barrier integrity and immune regulation, but their impact on PD pathology remains mostly unknown. Objectives To evaluate supplementation with short-chain fatty acids as an add-on intervention in PD. Methods In a randomized double-blind prospective study, 72 PD patients received short-chain fatty acids and/or the prebiotic fiber 2′-fucosyllactose supplementation over 6 months. Results We observed improvement in motor and nonmotor symptoms, in addition to modulation of peripheral immunity and improved mitochondrial respiration in immunocytes. The supplementation had no effect on microbiome diversity or composition. Finally, multiobjective analysis and comprehensive immunophenotyping revealed parameters associated with an optimal response to short-chain fatty acids and/or 2′-fucosyllactose supplementation. Conclusion Short-chain fatty acids ameliorate clinical symptoms in Parkinson’s disease patients and modulate mitochondrial function and peripheral immunity.
The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their gut microbiomes. While experiments with well-characterized bacteria or defined bacterial communities can facilitate the analysis of molecular mechanisms, studying nematodes in their natural microbial context is essential for exploring the diversity of such mechanisms. At the same time, the isolation of worms from the wild is not always feasible, and, even when possible, sampling from the wild restricts the use of the genetic toolkit otherwise available for C. elegans research. The following protocol describes a method for microbiome studies utilizing compost microcosms for the in-lab growth in microbially diverse and natural-like environments. Locally sourced soil can be enriched with produce to diversify the microbial communities in which worms are raised and from which they are harvested, washed, and surface-sterilized for subsequent analyses. Representative experiments demonstrate the ability to modulate the microbial community in a common soil by enriching it with different produce and further demonstrate that worms raised in these distinct environments assemble similar gut microbiomes distinct from their respective environments, supporting the notion of a species-specific core gut microbiome. Overall, compost microcosms provide natural-like in-lab environments for microbiome research as an alternative to synthetic microbial communities or to the isolation of wild nematodes.
Previous work showed a transient but dramatic arrest in the synthesis of Rubisco large subunit (LSU) upon transfer ofChlamydomonas reinhardtii cells from low light (LL) to high light (HL). Using dichlorofluorescin, a short-term increase in reactive oxygen species (ROS) was demonstrated, suggesting that their excessive formation could signal LSU down-regulation. A decrease in LSU synthesis occurred at LL in the presence of methyl viologen and was prevented at HL by ascorbate. Interfering with D1 function by mutations or by incubation with DCMU prevented the increase in ROS formation at HL and the concomitant down-regulation of LSU synthesis. If the electron transport was blocked further downstream, by mutation in the cytochromeb 6/f or by incubation with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, ROS formation increased, and LSU synthesis ceased. The elevation of ROS occurred concurrently with a change in the redox state of the glutathione pool, which shifted toward its oxidized form immediately after the transfer to HL and returned to its original value after 6 h. The decrease in the reduced/oxidized glutathione ratio at HL was prevented by ascorbate and could be induced at LL by methyl viologen. We suggest that excess ROS mediate a decrease in the reduced/oxidized glutathione ratio that in turn signals the translational arrest of the rbcL transcript. Previous work showed a transient but dramatic arrest in the synthesis of Rubisco large subunit (LSU) upon transfer ofChlamydomonas reinhardtii cells from low light (LL) to high light (HL). Using dichlorofluorescin, a short-term increase in reactive oxygen species (ROS) was demonstrated, suggesting that their excessive formation could signal LSU down-regulation. A decrease in LSU synthesis occurred at LL in the presence of methyl viologen and was prevented at HL by ascorbate. Interfering with D1 function by mutations or by incubation with DCMU prevented the increase in ROS formation at HL and the concomitant down-regulation of LSU synthesis. If the electron transport was blocked further downstream, by mutation in the cytochromeb 6/f or by incubation with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, ROS formation increased, and LSU synthesis ceased. The elevation of ROS occurred concurrently with a change in the redox state of the glutathione pool, which shifted toward its oxidized form immediately after the transfer to HL and returned to its original value after 6 h. The decrease in the reduced/oxidized glutathione ratio at HL was prevented by ascorbate and could be induced at LL by methyl viologen. We suggest that excess ROS mediate a decrease in the reduced/oxidized glutathione ratio that in turn signals the translational arrest of the rbcL transcript. reactive oxygen species 1,5-ribulose biphosphate carboxylase-oxygenase large subunit(s) low light high light minimal medium lacking sulfate [3-(3,4-dichlorophenyl)-1,1-dimethyl urea 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone methyl viologen dichlorofluorescin dichlorofluorescin hydrolyzed reduced glutathione oxidized glutathione polyacrylamide gel electrophoresis Exposure of photosynthetic organisms to light intensities that exceed the limits of photosynthesis saturation can cause severe damage to the photosynthetic machinery, referred to as photoinhibition (1.Barber J. Andersson B. Trends Biochem. Sci. 1992; 17: 61-66Abstract Full Text PDF PubMed Scopus (832) Google Scholar, 2.Osmond C.B. What is photoinhibition: Some Insights from Comparisons of Shade and Sun Plants. Bios Scientific Publishers, Oxford, UK1994: 1-24Google Scholar, 3.Prásil O. Adir N. Ohad I. Barber J. The Photosystems: Structure, Function and Molecular Biology. Elsevier Science Publishers B.V., Amsterdam1992: 295-348Crossref Google Scholar). Most plants and algae have the capacity to recover from light stress through photoacclimation, which normally involves a reduction in either the number or size of the light harvesting complexes and increased synthesis of the photodamaged D1, the core protein of photosystem II (PSII) (4.Anderson J.M. Osmond C.B. Shade-Sun Responses: Compromises between Acclimation and Photoinhibition. Elsevier Science Publishers B.V., Amsterdam1987: 1-38Google Scholar, 5.Falkowski P. Laroche J. J. Phycol. 1991; 27: 8-14Crossref Scopus (599) Google Scholar). Excess light energy generates reactive oxygen species (ROS),1 which in turn lead to the induction of antioxidant photoprotective mechanisms enabling the plant to combat the danger posed by the presence of ROS. Ribulose biphosphate carboxylase-oxygenase (Rubisco) is the key enzyme in carbon assimilation during photosynthesis. In Chlamydomonas reinhardtii and in land plants the enzyme is composed of eight large subunits (LSU) encoded by the chloroplast rbcL gene and eight small subunits encoded by the nuclear rbcS gene family (6.Spreitzer R.J. Annu. Rev. Plant. Phusiol. Plant. Mol. Biol. 1993; 44: 1-49Crossref Google Scholar, 7.Gutteridge S. Gatenby A.A. Plant Cell. 1995; 7: 809-819Crossref PubMed Scopus (139) Google Scholar). Assembly of the Rubisco holoenzyme is driven by the chloroplast Cpn60 and Cpn10, encoded by groEL andgroES, respectively. Previously we observed unique and opposite patterns for translational regulation of the chloroplast LSU and D1 polypeptides in response to changes in light intensity. Within minutes of shifting cells of C. reinhardtii from low light to higher light intensities, LSU synthesis was down-regulated dramatically for a period that did not exceed 4–6 h, whereas that of D1 was gradually up-regulated. Translation of other genes was hardly affected, including photosynthesis-related genes such as the chloroplast encoded ATPase β-subunit, the nuclear encoded small subunit of Rubisco (small subunits), or nonphotosynthetic genes, such as tubulin. The observed changes in D1 and LSU synthesis could not be correlated with changes in the steady state levels of their corresponding mRNAs, implying that translational regulation was involved. Primer extension analysis of rbcL mRNA revealed two transcripts that differed in their 5′ ends and in their abundance at LL and after transfer to HL. The appearance of the longer transcript correlated with the down-regulation in LSU synthesis, but its involvement in arresting LSU translation was unresolved. These several distinct effects of temporary light stress were correlated with a rapid, sustained increase in the reduction state of QA, a transient decline in the photosynthetic efficiency, a less rapid drop in total chlorophyll content, and a delay in cell division (8.Shapira M. Lers A. Heifetz P. Irihimoritch V. Osmond B.C. Gillham N.W. Boynton J.E. Plant Mol. Biol. 1997; 33: 1001-1011Crossref PubMed Scopus (70) Google Scholar). This study attempts to decipher the mechanism that controls translation of LSU and signals the immediate and short-term down-regulation observed upon transferring low light grown C. reinhardtiicells to higher light intensities. We propose that modulation of the glutathione redox potential by changes in the level of ROS regulate the synthesis of Rubisco LSU in the chloroplast. C. reinhardtiiwild type strain CC-125 was used in all experiments. Cultures in High Salt Reduced Sulfate (HSRS) (300 ml) (9.Harris E.H. Burkhart B.D. Gillham N.W. Boynton J.C. Genetics. 1989; 123: 281-292Crossref PubMed Google Scholar) were grown with 5% CO2 bubbling and constant rotary shaking at 22 °C. Cultures were illuminated with low light (LL; 70 μmol m−2 s−1) using cool white fluorescent lamps. LL grown cells were adapted for low irradiance and were not permitted to attain densities greater that 0.2–0.3 A 750. The D1 mutant strains were CC-741 (FUD7), a D1 deletion mutant, and CC-3376 (A251R*), which carries a point mutation in D1 (10.Lardans A. Gillham N.W. Boynton J.E. J. Biol. Chem. 1997; 272: 210-216Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The cytochrome b 6/f-deficient mutant was CC-2910 (F2D8) (11.Howe G. Merchant S. EMBO J. 1992; 11: 2789-2801Crossref PubMed Scopus (61) Google Scholar). In vivo labeling of plastid and nuclear encoded proteins with [35S]H2SO4 was performed essentially as described (12.Lers A. Heifetz P.B. Boynton J.E. Gillham N.W. Osmond C.B. J. Biol. Chem. 1992; 267: 17494-17497Abstract Full Text PDF PubMed Google Scholar), with the following modifications. Cells were grown under LL in HSRS (13.Schmidt R.J. Gillham N.W. Boynton J.E. Mol. Cell. Biol. 1985; 5: 1093-1099Crossref PubMed Google Scholar), until their biomassA 750 reached 0.2–0.3. The cells were then harvested by centrifugation for 5 min (4500 × g, 20 °C) and resuspended at 0.4 A 750 in minimal medium lacking sulfate (HS-S; Ref. 13.Schmidt R.J. Gillham N.W. Boynton J.E. Mol. Cell. Biol. 1985; 5: 1093-1099Crossref PubMed Google Scholar) and containing 10 mmbicarbonate to ensure carbon availability for photosynthesis. The cells were equilibrated for 1 h under LL in HS-S. Aliquots (4 ml) of the HS-S cell suspension were placed in 30-ml Corex tubes containing magnetic stirring bars and illuminated at LL or at high light (HL; 700 μmol m−2 s−1) for the designated time periods. Anisomycin (Sigma) was then added to a concentration of 250 μg ml−1 for 15 min to reduce labeling of cytoplasmic proteins (14.Chua N.-H. Gillham N.W. J. Cell Biol. 1977; 74: 441-452Crossref PubMed Scopus (124) Google Scholar). [35S]H2SO4 (125 μCi; carrier free; NEN Life Science Products) was then added to each aliquot of cells for additional 15 min. To terminate labeling, 3 ml of the cell aliquot were added rapidly to 10 ml of ice-cold acetone incubated on ice for 1–2 h and centrifuged for 10 min, and the protein pellets were dried. Samples were resuspended in 100 μl of H2O and 100 μl of denaturing solution (4% SDS, 5 mm EDTA, 40 mm Tris-HCl, pH 7.4), briefly vortexed, and boiled for 5 min. Incorporation of the radiolabel was measured by trichloroacetic acid precipitation, and the protein content was determined with the BCA reagent (Pierce). Samples containing equal protein quantities were loaded and verified by Coomassie staining. The gels were dried, exposed to XAR5 film (Kodak), and also analyzed by a Fuji phosphorimager. Wild type cells were grown photoautotrophically and processed for labeling as described above. Following transfer to HS-S for 1 h, the cells were incubated at LL or at HL in the presence of DCMU (10−7m), DBMIB (10−6m), or ascorbic acid (5 and 10 mm) for 2 h and then labeled as described for wild type cells. Following cell harvest, proteins were extracted and analyzed as described above. Labeling of LSU and D1 was evaluated by phosphorimaging. The nonphotosynthetic mutant strains CC-741 (FUD7), CC-3376 (A251R*), and CC-2910 (F2D8) were grown heterotrophically in TAP medium (9.Harris E.H. Burkhart B.D. Gillham N.W. Boynton J.C. Genetics. 1989; 123: 281-292Crossref PubMed Google Scholar) under LL conditions to early log phase. Acetate was depleted by transfer of the cells to minimal growth conditions (HSRS/5% CO2) 16 h before transfer to HL. The cells were then resuspended in HS-S for 1 h and transferred to LL and HL for 2.5 h. Labeling and SDS-PAGE analysis were performed as described for wild type cells. Early log cells grown photoautotrophically at LL on HSRS with bubbling of 5% CO2. Aliquots (4 ml) were transferred to HS-S medium for 1 h at LL in presence of MeV (10−6, 6 × 10−6, and 10−5m) for 45 min and radiolabeled. Early log cells grown photoautotrophically on minimal medium with 5% CO2 at LL were transferred to HS-S for 1 h. A sample of the cell suspension (10 ml) was placed in 30-ml Corex tubes containing magnetic stirring bars and illuminated with LL or HL irradiance. Samples (2 ml) from LL and HL were removed after 0.5, 1.5, and 2.5 h and added to 8 ml of loading buffer (10 mmTris-HCl, pH 7.2, 50 mm KCl) containing 0.05 mmDCFH (5 μl from a 100 mm stock solution in Me2SO; Ref. 15.Cathcart R. Schwiers E. Ames B.N. Anal. Biochem. 1983; 134: 111-116Crossref PubMed Scopus (722) Google Scholar). The samples were maintained for 20 min in the dark, and relative fluorescence was monitored in a Perkin-Elmer LS50B Spectrofluorometer, set at an excitation wavelength of 488 nm and emission wavelength of 525 nm with a slit width of 7.5 nm. DCF fluorescence at LL and after the shift to HL was performed in wild type CC-125 and in the nonphotosynthetic mutants (CC-741, CC-3376, and CC-2910) as well as in wild type cells in the presence of DCMU (10−7m), DBMIB (10−6m), and ascorbic acid (10 mm). Measurement of DCF fluorescence at LL in the presence of MeV (10−4–10−6m) was performed with samples that were collected after 0.5 and 1.5 h. Fluorometry measurements were performed in triplicate and expressed as relative fluorescence units. GSH and GSSG were measured using a modified method for glutathione measurement in microtiter plates (16.Anderson M.E. Methods Enzymol. 1985; 113: 548-555Crossref PubMed Scopus (2380) Google Scholar, 17.Baker M.A. Cerniglia G.J. Zaman A. Anal. Biochem. 1990; 190: 360-365Crossref PubMed Scopus (787) Google Scholar). Cells were grown under LL in HSRS up to a density of 0.2–0.3 A 750, harvested by centrifugation for 5 min (4500 × g, 20 °C), and resuspended at a density of 0.4 A 750 in HS-S. Cultures were then illuminated at LL and at HL for different time periods, and cell samples (12 ml) were removed, washed once in phosphate buffer (10 mm, pH 7.0), and centrifuged. The wet weight of the washed cell pellet was measured, and 5% sulfosalicylic acid (Sigma) was added, 150 μl/50 mg pellet. The mixture was agitated on a Vortex mixer and stored at −70 °C until their analysis, at which time the cells were thawed, and the insoluble debris was removed by centrifugation (13,000 × g, 4 °C, 20 min). The supernatant was collected, and samples of 100 μl were diluted 1:1 with distilled water and distributed into two aliquots, for individually measuring GSSG and total glutathione. To conjugate GSH, 2-vinylpyridine (Fluka) was added to one of the aliquots to a final concentration of 0.35 m. The mixture was neutralized to pH 6.7–7 with triethanolamine (diluted 1:2 with double distilled H2O). GSSG concentrations in these extracts were determined using the enzymatic recycling assay (16.Anderson M.E. Methods Enzymol. 1985; 113: 548-555Crossref PubMed Scopus (2380) Google Scholar, 17.Baker M.A. Cerniglia G.J. Zaman A. Anal. Biochem. 1990; 190: 360-365Crossref PubMed Scopus (787) Google Scholar) involving the color development at 412 nm of 0.15 mm 5,5-dithiobis 2-nitro-benzoic acid (Sigma) in the presence of 0.2 mmNADPH and 1 unit ml−1 of GSH reductase (Fluka). Total glutathione (GSH and GSSG) concentrations were determined in the second aliquot using the same assay without adding 2-vinylpyridine. GSSG standards (Calbiochem) were used for calibration, and all measurements were performed in triplicate. GSH and GSSG were measured also in cells incubated with ascorbic acid at LL and at HL and in cells incubated with MeV at LL. To test whether the redox state of specific components along the electron transport chain affected the down-regulation of LSU, labeling experiments of wild type cells at LL and after transfer to HL were performed in the presence of herbicides that inhibit the electron transfer at different sites along the electron transfer chain. DCMU inhibits the oxidation of the plastoquinone pool, and DBMIB reduces its oxidation by competitively binding to cytochromeb 6/f complex. Wild type C. reinhardtii cells were labeled at LL and after transfer to HL (2 h) in the presence of DCMU (10−7m) and DBMIB (10−6m), which were added when the cells were transferred to HL. Although synthesis of LSU decreased 2-fold in control cells shifted to HL, it was almost unaffected in the presence of DCMU (Fig. 1 A). However, addition of DBMIB did not prevent the down-regulation of LSU synthesis after transfer from LL to HL (Fig. 1 B). In addition, LSU synthesis decreased already at LL in the presence of DBMIB (10−6m). To complement the experiments using herbicides, C. reinhardtii mutants in which D1 was completely absent (FUD-7) or inactivated by a point mutation (A251R*), and a mutant defective in cytochrome b 6/f (F2D8) were labeled at LL and after shifting to HL (2 h). The FUD-7 mutant does not synthesize any D1 and is thus deficient of functional PSII. In the D1 mutant A251R*, Ala at position 251 was substituted with Arg. Ala251 is located in the quinone binding loop connecting the IV–V helices of D1 (10.Lardans A. Gillham N.W. Boynton J.E. J. Biol. Chem. 1997; 272: 210-216Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). This mutation does not alter the size (32 kDa) or amount of the D1 protein. The mutant synthesizes D1 up to 80% of its level in wild type when grown under HL but has a nonphotosynthetic phenotype because electron transfer between QA and QB is completely blocked (10.Lardans A. Gillham N.W. Boynton J.E. J. Biol. Chem. 1997; 272: 210-216Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Labeling experiments of both D1 mutants indicate that in the absence of a functional D1, synthesis of LSU is unaffected by shifting the cells from LL to HL (Fig.2, B and C). However, inactivation of cytochromeb 6/f in the mutant strain F2D8 did not prevent the down-regulation of LSU synthesis (Fig.2 D). The effect of mutations in the photosynthetic electron carriers that prevent oxidation or reduction of the plastoquinone (Fig. 2) corroborated with that of the herbicides (Fig. 1). In the presence of DCMU or in mutants defective in D1, the plastoquinone pool remained oxidized in wild type cells, despite transfer to HL. Thus LL conditions are mimicked, and LSU down-regulation was not observed. Preventing oxidation of the plastoquinone pool by incubation with DBMIB or by mutagenesis of the cytochrome b 6/fcomplex did not alter the pattern of LSU down-regulation. Although these results could indicate that the redox state of the PQ has a regulatory role in LSU synthesis during LL to HL shifts, previous fluorimetric measurements ruled out this possibility. The plastoquinone was reduced immediately after transfer from LL to HL and was maintained in a reduced state in HL (8.Shapira M. Lers A. Heifetz P. Irihimoritch V. Osmond B.C. Gillham N.W. Boynton J.E. Plant Mol. Biol. 1997; 33: 1001-1011Crossref PubMed Scopus (70) Google Scholar), whereas LSU synthesis initially declined and then recovered. The labeling pattern of D1 indicated that in control cells synthesis of this protein increased 9-fold upon transfer from LL to HL, in line with previous observations (18.Schuster G. Timberg R. Ohad I. Eur. J. Biochem. 1988; 177: 403-410Crossref PubMed Scopus (173) Google Scholar). This increase was inhibited by DCMU, DBMIB, and a mutation in cytochrome b 6/f. These data are in line with the redox control of D1 synthesis in the chloroplast (19.Danon A. Mayfield S.P. Science. 1994; 266: 1717-1719Crossref PubMed Scopus (278) Google Scholar). However, it was difficult to explain why D1 synthesis increased (by 5.5-fold) in A251R*, because electron transport is blocked in this nonphotosynthetic mutant. Different forms of D1 that vary in their half lives were shown to coexist in the thylakoids of this mutant, and these could be reflected in the pattern of labeling (10.Lardans A. Gillham N.W. Boynton J.E. J. Biol. Chem. 1997; 272: 210-216Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Excess energy not trapped by the photosynthetic electron transport chain can increase the formation of ROS at specific sites of the PSII and PSI reaction centers. In PSII, singlet oxygen is formed by the reaction of the triplet state of P680 with oxygen. Hydrogen peroxide is generated both in PSI and in PSII, and can be converted to hydroxyradicals by interaction with non-heme iron (20.Bowler C. Montagu V. Inze D. Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1992; 43: 83-116Crossref Scopus (2218) Google Scholar, 21.Okada K. Ikeuchi M. Yamamoto N. Ono T.A. Miyao M. Biochim. Biophys. Acta. 1996; 1274: 73-79Crossref Scopus (63) Google Scholar). As photoprotective mechanisms come into play (4.Anderson J.M. Osmond C.B. Shade-Sun Responses: Compromises between Acclimation and Photoinhibition. Elsevier Science Publishers B.V., Amsterdam1987: 1-38Google Scholar, 5.Falkowski P. Laroche J. J. Phycol. 1991; 27: 8-14Crossref Scopus (599) Google Scholar), the free radical density should decline, and LSU synthesis would normalize once again. We therefore hypothesized that the increase in ROS could signal the down-regulation of LSU synthesis, either directly or indirectly. To test this possibility, ascorbic acid, which acts as an antioxidant by removing hydrogen peroxide (22.Alcher R.G. Donahue J.L. Cramer C.L. Physiol. Plant. 1997; 100: 224-233Crossref Google Scholar) was added to cells labeled at HL. Synthesis of LSU was not interrupted if the cells were transferred to HL in the presence of ascorbic acid (5 and 10 mm), whereas in its absence, LSU translation decreased at HL (2 h). Addition of ascorbic acid increased the synthesis of D1 already at LL, thus reducing the difference between incorporation of radiolabel into D1 at LL and at HL (Fig.3 A). To examine whether accumulation of excess ROS indeed signaled the translational arrest of LSU, their level was measured using the oxidatively sensitive DCFH (15.Cathcart R. Schwiers E. Ames B.N. Anal. Biochem. 1983; 134: 111-116Crossref PubMed Scopus (722) Google Scholar). DCFH enters the cells in its diacetate form (DCFH-DA), becomes hydrolyzed and remains trapped intracellularly. Oxidation of DCFH by H2O2 generates DCF, a highly fluorescent compound. Although DCFH reacts mainly with H2O2, it is useful for monitoring other ROS species, because singlet oxygen is converted to superoxide anion resulting in the formation of H2O2 by superoxide dismutase activity (20.Bowler C. Montagu V. Inze D. Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1992; 43: 83-116Crossref Scopus (2218) Google Scholar, 23.Foyer C.H. Lelandais M. Kuner K.J. Physiol. Plant. 1994; 92: 696-717Crossref Google Scholar). The results of DCF fluorescence indicate that the level of ROS increased by almost 2-fold at 0.5 and 1.5 h after transfer from LL to HL and decreased after 2.5 h back to the level measured at LL (Fig. 3 B). Addition of ascorbic acid (5 and 10 mm) to wild type cells prevented the ROS increase at HL, in line with its antioxidant activity. To establish the correlation between down-regulation in LSU synthesis and the increase in ROS levels, DCF fluorescence was measured at LL and after transfer to HL in the presence of DCMU (10−7m) and DBMIB (10−6m) and in mutants defective in D1 and in cytochromeb 6/f. DCF fluorescence in cells transferred from LL to HL hardly changed in the presence of DCMU and in mutants deficient or defective in D1 (Fig.4, A–C), indicating that the level of ROS at HL did not increase. However, addition of DBMIB or inactivation of the cytochrome b 6/fcomplex did not prevent the increase in ROS at HL (Fig. 4, Dand E). With DBMIB (10−6m) the basal level of ROS was higher already at LL than that measured in the absence of this herbicide (Fig. 4 D). This result was in correlation to the already reduced synthesis of LSU observed with DBMIB at LL (10−6m, Fig. 1). To establish the direct involvement of ROS in signaling the down-regulation of LSU synthesis, cells were incubated at LL with MeV. MeV accepts an electron from ferredoxin and reacts with molecular O2, forming a superoxide radical anion that is transformed in subsequent reactions to ROS such as hydrogen peroxide and hydroxy radicals (24.Härtel H. Haseloff R.F. Ebert B. Rank B. J. Photochem. Photobiol. B Biology. 1992; 12: 375-387Crossref Scopus (18) Google Scholar). The free radical pool can therefore be increased by MeV, similar to what occurs when cells are transferred from LL to HL. Cells were labeled at LL in the presence of MeV, (10−6–10−5m). Labeling was performed in the absence of anisomycin to maintain the synthesis of cytoplasmic proteins and to ensure that MeV did not cause a general decrease in protein synthesis, which could be masked in the presence of anisomycin. Increasing concentrations of MeV exclusively reduced the synthesis of LSU at LL (by 79% after 1 h), whereas synthesis of other proteins was unaffected. DCF fluorescence at LL in the presence of MeV (10−6–10−4m) shows an increase in the level of ROS in a dose-dependent manner (Fig.5 B). Although the highest ROS level was measured with 10−4m, this concentration was too high for labeling, because it inhibited protein synthesis nonspecifically (data not shown). These results in combination with the labeling data indicate the role of ROS in controlling the translation of Rubisco LSU. Glutathione is a low molecular mass thiol that has a key regulatory role in plants and algae. Most of it is present in the reduced form (GSH), and only a minor fraction is oxidized and exists as two molecules of GSH linked by a disulfide bond (GSSG). An increase in ROS during stress could affect the GSH/GSSG ratio, shifting the balance toward oxidation. As shown in our DCF based fluorescence assay, a "light shock" causes a rapid increase in the level of ROS that subsequently returns to the original value measured at LL. We therefore examined whether the transient increase in ROS after the LL to HL shift is coupled to parallel changes in the GSH/GSSG ratio. Transfer ofC. reinhardtii cells from LL to HL resulted in a transient decrease in the GSH/GSSG ratio (Fig.6 A) which dropped 2-fold after 1.5 h and recovered its original LL level within 6 h. The accumulation of excess ROS thus changed the redox state of glutathione, increasing its relative oxidized fraction. The changes in the GSH/GSSG ratio paralleled the down-regulation and the subsequent recovery of LSU synthesis. The translational arrest of LSU was maximal at 2 h and recovered after 4–6 h (8.Shapira M. Lers A. Heifetz P. Irihimoritch V. Osmond B.C. Gillham N.W. Boynton J.E. Plant Mol. Biol. 1997; 33: 1001-1011Crossref PubMed Scopus (70) Google Scholar). The GSH/GSSG ratio returned to its original value also after 6 h, only after the level of ROS decreased. Modulation of the GSH/GSSG ratio by changes in the ROS level is further supported by the opposite effects observed for ascorbate and MeV. Addition of ascorbic acid to cells transferred to HL prevented the transient decrease in the GSH/GSSG ratio (Fig. 6 B), and addition of MeV to cells grown at LL increased the relative fraction of oxidized glutathione decreasing the GSH/GSSG ratio, mimicking the transfer to HL (Fig. 6 C). Changes in light intensities play a key role in regulation of photosynthetic genes. Recent studies assigned a regulatory role for the redox state of components in the photosynthetic electron pathway in controlling expression of chloroplastic proteins (19.Danon A. Mayfield S.P. Science. 1994; 266: 1717-1719Crossref PubMed Scopus (278) Google Scholar, 25.Escoubas J.-M. Lomas M. LaRoche J. Falkowski P.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10237-10241Crossref PubMed Scopus (536) Google Scholar). Regulation of the nuclear encoded cab genes, and the chloroplast encoded psbA is thought to be controlled directly by the redox potential of specific components in the electron transport chain. Expression of cab genes is reversibly repressed by a phosphorylatable factor coupled to the redox status of PQ through a chloroplast protein kinase (25.Escoubas J.-M. Lomas M. LaRoche J. Falkowski P.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10237-10241Crossref PubMed Scopus (536) Google Scholar). Translation of D1 is subject to regulation by the redox state of thioredoxin and ferredoxin (19.Danon A. Mayfield S.P. Science. 1994; 266: 1717-1719Crossref PubMed Scopus (278) Google Scholar) by affecting the thiol groups on proteins that bind to the 5′-untranslated region (26.Kim J. Mayfield S.P. Science. 1997; 278: 1954-191957Crossref PubMed Scopus (196) Google Scholar). Unlike D1, whose synthesis gradually increases in response to elevation of light intensities, LSU synthesis follows a unique regulatory pattern, displayed by its transient arrest in cells transferred from LL to HL and a subsequent recovery upon photoacclimation (8.Shapira M. Lers A. Heifetz P. Irihimoritch V. Osmond B.C. Gillham N.W. Boynton J.E. Plant Mol. Biol. 1997; 33: 1001-1011Crossref PubMed Scopus (70) Google Scholar). The mechanism that regulates the synthesis of these two proteins should therefore differ. In this study we propose that translational arrest of rbcLis signaled by the increased generation of ROS that can modulate the redox potential of the glutathione pool and thus inhibit the translation of this protein. The labeling pattern of LSU in the presence of DCMU and DBMIB or in mutants defective at different sites of the electron transport chain could imply that the redox state of plastoquinone is involved in signaling the down-regulation in LSU synthesis. However, because the measured value of 1-qP (the index of QA reduction state) was high and remained unaltered during the first hours after transfer to HL, whereas LSU translation initially declined and then recovered (8.Shapira M. Lers A. Heifetz P. Irihimoritch V. Osmond B.C. Gillham N.W. Boynton J.E. Plant Mol. Biol. 1997; 33: 1001-1011Crossref PubMed Scopus (70) Google Scholar), changes in the redox state of PQ were not likely to be the direct cause for the down-regulation of LSU, although indirect effects of the chloroplast redox state could be involved. Alternatively, these results could be explained on the basis of ROS formation upon transfer to HL. The down-regulation of therbcL gene encoding LSU could occur in response to a signal generated by the imbalance that takes place when cells grown in LL, with their extensive chlorophyll antenna complexes, are shifted to HL. These antennae would trap more light quanta than can be processed by the photosynthetic electron transport system, resulting in the rapid elevation of ROS. As the antenna size is adjusted downward the imbalance would be dissipated, the level of ROS would decrease, and LSU synthesis would increase once again. Quantitative estimation of ROS correlates with this hypothesis, with their level transiently increasing upon transfer from LL to HL and then returning to the basal level. Incubation with ascorbic acid prevented the increase in ROS at HL and prevented the translational arrest of LSU. In accordance with these data, incubation of cells at LL in the presence of MeV, an inducer of ROS in the chloroplast, led to the increase of ROS and the down-regulation of LSU synthesis, whereas translation of other proteins was unaffected. The results of labeling wild type cells in the presence of herbicides or labeling of mutants defective at different sites of the electron transport chain could also be interpreted by ROS formation. DCMU protects cells from the oxidative stress experienced during high intensity illumination by preventing the increase in ROS and the light induced breakdown of D1 (27.Jegerschöld C. Virgin I. Styring S. Biochemistry. 1990; 29: 6179-6186Crossref PubMed Scopus (140) Google Scholar, 28.Gong H. Ohad I. Biochim. Biophys. Acta. 1995; 1228: 181-188Crossref Scopus (20) Google Scholar). The DCF-based measurements of ROS in the presence of DCMU confirmed this observation, which correlated with the continued synthesis of LSU at HL in the presence of DCMU. Likewise, ROS levels in nonphotosynthetic D1 mutants did not show a marked increase at HL. DBMIB or mutations in the cytochromeb 6/f complex did not prevent the elevation of ROS levels nor the translational arrest of LSU. The increase in ROS in the presence of DBMIB is relatively short-term for reasons not completely clear to us; however, the basal level of ROS is high already at LL, possibly because of the block in the electron pathway. Previous reports indicated that ROS can function as second messengers in mediating stress responses in plants. Plants attacked by pathogens respond by elevating ROS levels, leading to the induction of pathogen response genes (29.Chen Z. Silva H. Klessig D.F. Science. 1993; 262: 1883-1886Crossref PubMed Scopus (971) Google Scholar, 30.Green R. Fluhr R. Plant Cell. 1995; 7: 203-212Crossref PubMed Scopus (221) Google Scholar). Transcriptional activation of pathogen response genes can also be obtained by elicitors of ROS (31.Allan A. Fluhr R. Plant Cell. 1997; 9: 1559-1572Crossref PubMed Scopus (461) Google Scholar), by direct application of H2O2 (32.Bei Y.M. Kenton P. Mur L. Darby R. Draper J. Plant J. 1995; 8: 235-245Crossref PubMed Scopus (216) Google Scholar), or by suppression of catalase activity resulting in increased levels of H2O2 (33.Chamnongpol S. Willekens H. Langebartels C. Van M.M. Inze D. Van C.W. Plant J. 1996; 10: 491-503Crossref Scopus (172) Google Scholar, 34.Chen Z. Malamy J. Henning J. Conrath U. Sanchez C.P. Silva H. Ricigliano J. Klessig D.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4134-4137Crossref PubMed Scopus (146) Google Scholar). Modulation of gene expression by ROS can be mediated by changes in the redox state of the glutathione pool (23.Foyer C.H. Lelandais M. Kuner K.J. Physiol. Plant. 1994; 92: 696-717Crossref Google Scholar, 35.Wingate V.P.M. Lawton M.A. Lamb C.J. Plant Physiol. 1988; 87: 206-210Crossref PubMed Google Scholar). Elevation of reduced glutathione in transformed plants overexpressing glutathione reductase increased resistance to oxidative stress (36.Foyer C.H. Souriau N. Perret S. Lelandais-M Kunert K.J. Pruvost C. Jouanin L. Plant Physiol. 1995; 109: 1047-1057Crossref PubMed Scopus (371) Google Scholar), and changes in the redox status of glutathione regulated the expression of copper, zinc-superoxide dismutase and of ascorbate peroxidase (37.Karpinski S. Escobar C. Karpinska B. Creissen G. Mullineaux P.M. Plant Cell. 1997; 9: 627-640Crossref PubMed Scopus (497) Google Scholar). Here we show that transfer of cells from LL to HL causes a transient increase in ROS that correlates with the reduction in the GSH/GSSG ratio. Addition of ascorbic acid prevented the increase in ROS, and thus the GSH/GSSG ratio remained unaltered at HL, whereas addition of MeV at LL increased the formation of ROS and decreased the GSH/GSSG ratio. Concomitantly, ascorbic acid prevented the down-regulation of LSU translation at HL and MeV induced it at LL. Thus the decrease in the GSH/GSSG ratio in the chloroplast could serve as a signal for the translational arrest of LSU. We hypothesize that translational arrest of the rbcL transcript could occur because of oxidation of sulfhydryl groups in one or more of the components of the translational initiation complex that assembles on the rbcL5′-untranslated region. During photoacclimation the intrachloroplastic glutathione pool shifts to its reduced form, oxidation of the sulfhydryl groups on the target protein is reversed, and translation of LSU can proceed. At this stage the nature of the putative protein that modulates translation of the rbcL transcript possibly by oxidation of its sulfhydryl groups is yet unclear. In addition to its role in translational control, redox changes induced during photoinhibitory stress and senescence have been implicated in Rubisco breakdown (38.Moreno J. Penarrubia L. Garcia-Ferris C. Plant Physiol. Biochem. 1995; 33: 121-127Google Scholar, 39.Ishida H. Nishimori Y. Sugisawa M. Makino A. Mae T. Plant Cell Physiol. 1997; 38: 471-479Crossref PubMed Scopus (132) Google Scholar), and oxidation of sulfhydryl groups in critical Cys residues has been demonstrated to play a key role in LSU degradation (40.Moreno J. J. S.R. J. Biol. Chem. 1999; 274: 26789-26793Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The time period required for recovery of the GSH/GSSG ratio is similar to that observed for restoration of LSU translation (4–6 h), suggesting that the two processes are associated. However, the level of ROS returned to its original level already within 2.5 h. This difference can be explained by a lag period that is required for the cell to overcome the oxidative damage induced by the transfer to HL. The delay in restoring the GSH/GSSG ratio could be due to a requirement for synthesis of new proteins. The synthesis of subunits that compose organellar multimeric protein complexes is coordinated, even when they are encoded by the different genomes of the cell (41.Schmidt G.W. Mishkind M.L. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2632-2636Crossref PubMed Scopus (162) Google Scholar, 42.Choquet Y. Stern D.B. Wostrikoff-K Kuras R. Girard-Bascou J. F. A. W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4380-4385Crossref PubMed Scopus (104) Google Scholar). Translation of the chloroplast-encoded Rubisco LSU was inhibited when the two genes encoding the small subunits in C. reinhardtii were deleted (43.Khrebtukova I. Spreitzer R.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13689-13693Crossref PubMed Scopus (85) Google Scholar) or if their synthesis was inhibited by the antisense approach (44.Rodermel S. Haley J. Jiang C.Z. Tsai C.H. Bogorad L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3881-3885Crossref PubMed Scopus (107) Google Scholar). Unlike these effects, the transient arrest in LSU translation that we observe during a light shock in C. reinhardtii did not lead to a coordinated down-regulation of small subunit synthesis, possibly because the down-regulation of LSU was short-term and not long enough to affect the steady state of this protein (8.Shapira M. Lers A. Heifetz P. Irihimoritch V. Osmond B.C. Gillham N.W. Boynton J.E. Plant Mol. Biol. 1997; 33: 1001-1011Crossref PubMed Scopus (70) Google Scholar). Uncoordinated synthesis of Rubisco subunits has been previously reported, but only during a short term (45.Barraclough R. Ellis J. Eur. J. Biochem. 1979; 94: 165-177Crossref PubMed Scopus (35) Google Scholar). Although the current study reveals a novel signaling mechanism that controls translation of Rubisco LSU, the physiological significance of its down-regulation during a light shock remains to be elucidated. We suggest that the arrest in LSU synthesis ceases Rubisco assembly, thus releasing the Cpn60/10 chaperonins from mediating assembly of this highly abundant complex and transiently recruiting them for overcoming the damaging effects of oxidative stress. We thank Prof. Boynton and Prof. Gillham from Duke University for support and for valuable discussions, Dr. E. Harris from the Chlamydomonas Duke Center for the various algal strains, and Dr. Irit Dahan for technical assistance.
ABSTRACT Assembly of photosynthetic complexes is sensitive to changes in light intensities, drought, and pathogens that induce a redox imbalance, and require a variety of substrate-specific chaperones to overcome the stress. Proteins with cysteine (C) residues and disulfide bridges are more responsive to the redox changes. This study reports on a thylakoid membrane-associated DnaJ-like protein, ZnJ6 (ZnJ6.g251716.t1.2) in Chlamydomonas reinhardtii . The protein has four CXXCX(G)X(G) motifs that form a functional zinc-binding domain. Site-directed mutagenesis (Cys to Ser) in all the CXXCX(G)X(G) motifs eliminates its zinc-binding ability. In vitro chaperone assays using recombinant ZnJ6 confirm that it is a chaperone that possesses both holding and oxidative refolding activities. Although mutations (Cys to Ser) do not affect the holding activity of ZnJ6, they impair its ability to promote redox-controlled reactivation of reduced and denatured RNaseA, a common substrate protein. The presence of an intact zinc-binding domain is also required for protein stability at elevated temperatures, as suggested by a single spectrum melting curve. Pull-down assays with recombinant ZnJ6 revealed that it interacts with oxidoreductases, photosynthetic proteins (mainly PSI), and proteases. Our in vivo experiments with Chlamydomonas reinhardtii insertional mutants (ΔZnJ6) expressing a low level of ZnJ6, suggested that the mutant is more tolerant to oxidative stress. In contrast, the wild type has better protection at elevated temperature and DTT induced stress. We propose that DnaJ-like chaperone ZnJ6 assists in the prevention of protein aggregation, stress endurance, and maintenance of redox balance. One-sentence summary ZnJ6 is a redox-regulated DnaJ-like chaperone associated with the thylakoid membrane and involved in the prevention of protein aggregation and stress endurance.
Delayed postpneumonectomy empyema is uncommon. The condition is usually elusive and diagnosed late in the course of the disease, leading to increased morbidity. New air-fluid level on chest x-ray film or appearance of empyema necessitatis may enhance the index of suspicion and lead to early diagnosis, but in many cases no clinical or laboratory clues are apparent. We describe the case of a 60-year-old man with high fever and dyspnea 3(1/2) years after pneumonectomy. Diagnosis of postpneumonectomy empyema was delayed and finally suggested by the lack of expected mediastinal shift on chest film. Computed tomography (CT) of the chest showed a large quantity of fluid, which later proved to be empyema. The patient was treated successfully by continuous cavity irrigation with neomycin and systemic antibiotics. We conclude that in postpneumonectomy patients with septic fever, the only clue to diagnosis of delayed postpneumonectomy empyema may be hemithorax opacification without mediastinal shift, confirmed by CT-guided thoracocentesis. Therapy with cavity irrigation and systemic antibiotics seems appropriate.
Innate immunity is an ancient and conserved defense mechanism. Although host responses toward various pathogens have been delineated, how these responses are orchestrated in a whole animal is less understood. Through an unbiased genome-wide study performed in Caenorhabditis elegans, we identified a conserved function for endodermal GATA transcription factors in regulating local epithelial innate immune responses. Gene expression and functional RNAi-based analyses identified the tissue-specific GATA transcription factor ELT-2 as a major regulator of an early intestinal protective response to infection with the human bacterial pathogen Pseudomonas aeruginosa. In the adult worm, ELT-2 is required specifically for infection responses and survival on pathogen but makes no significant contribution to gene expression associated with intestinal maintenance or to resistance to cadmium, heat, and oxidative stress. We further demonstrate that this function is conserved, because the human endodermal transcription factor GATA6 has a protective function in lung epithelial cells exposed to P. aeruginosa. These findings expand the repertoire of innate immunity mechanisms and illuminate a yet-unknown function of endodermal GATA proteins.
Transfer of the green algae Chlamydomonas reinhardtii from low light to high light generated an oxidative stress that led to a dramatic arrest in the synthesis of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The translational arrest correlated with transient changes in the intracellular levels of reactive oxygen species and with shifting the glutathione pool toward its oxidized form (Irihimovitch, V., and Shapira, M. (2000) J. Biol. Chem. 275, 16289–16295). Here we examined how the redox potential of glutathione affected the RNA-protein interactions with the 5′-untranslated region of rbcL. This RNA region specifically binds a group of proteins with molecular masses of 81, 62, 51, and 47 kDa in UV-cross-linking experiments under reducing conditions. Binding of these proteins was interrupted by exposure to oxidizing conditions (GSSG), and a new protein of 55 kDa was shown to interact with the RNA. The 55-kDa protein comigrated with Rubisco LSU in one- and two-dimensional gels, and its RNA binding activity was further verified by using the purified protein in UV-cross-linking experiments under oxidizing conditions. However, the LSU of purified and oxidized Rubisco bound to RNA in a sequence-independent manner. A remarkable structural similarity was found between the amino-terminal domain of Rubisco LSU in C. reinhardtii and the RNA binding domain, a highly prevailing motif among RNA-binding proteins. It appears from the crystal structure of Rubisco that the amino terminus of LSU is buried within the holoenzyme. We propose that under oxidizing conditions it is exposed to the surface and can, therefore, bind RNA. Accordingly, a recombinant form of the polypeptide domain that corresponds to the amino terminus of LSU was found to bind RNA in vitro with or without GSSG. Transfer of the green algae Chlamydomonas reinhardtii from low light to high light generated an oxidative stress that led to a dramatic arrest in the synthesis of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The translational arrest correlated with transient changes in the intracellular levels of reactive oxygen species and with shifting the glutathione pool toward its oxidized form (Irihimovitch, V., and Shapira, M. (2000) J. Biol. Chem. 275, 16289–16295). Here we examined how the redox potential of glutathione affected the RNA-protein interactions with the 5′-untranslated region of rbcL. This RNA region specifically binds a group of proteins with molecular masses of 81, 62, 51, and 47 kDa in UV-cross-linking experiments under reducing conditions. Binding of these proteins was interrupted by exposure to oxidizing conditions (GSSG), and a new protein of 55 kDa was shown to interact with the RNA. The 55-kDa protein comigrated with Rubisco LSU in one- and two-dimensional gels, and its RNA binding activity was further verified by using the purified protein in UV-cross-linking experiments under oxidizing conditions. However, the LSU of purified and oxidized Rubisco bound to RNA in a sequence-independent manner. A remarkable structural similarity was found between the amino-terminal domain of Rubisco LSU in C. reinhardtii and the RNA binding domain, a highly prevailing motif among RNA-binding proteins. It appears from the crystal structure of Rubisco that the amino terminus of LSU is buried within the holoenzyme. We propose that under oxidizing conditions it is exposed to the surface and can, therefore, bind RNA. Accordingly, a recombinant form of the polypeptide domain that corresponds to the amino terminus of LSU was found to bind RNA in vitro with or without GSSG. When plants and algae absorb light energy that exceeds the level of electron carrier saturation they generate reactive oxygen species (ROS), 1The abbreviations used are: ROS, reactive oxygen species; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; RB, RNA binding; EMSA, electrophoretic mobility shift assays; SK, pBluescript; RBD, RNA binding domain; LSU, large subunit; UTR, untranslated region; DTT, dithiothreitol; aa, amino acids. that cause a variety of cellular and molecular damage. This phenomenon is referred to as photoinhibition and is common to all photosynthetic organisms (1Barber J. Andersson B. Trends Biochem. Sci. 1992; 17: 61-66Abstract Full Text PDF PubMed Scopus (843) Google Scholar, 2Osmond C.B. Baker N.R. Boyer J.R. Photoinhibition of Photosynthesis, from Molecular Mechanisms to the Field. BIOS Scientific Publishers Ltd., Oxford1994: 1-24Google Scholar, 3Prásil O. Adir N. Ohad I. Barber J. The Photosystems: Structure, Function, and Molecular Biology. Elsevier Science Publishers B. V., Amsterdam1992: 295-348Crossref Google Scholar). Recovery from photoinhibition can be achieved by decreasing the chlorophyll content and by activating a variety of antioxidant pathways that involve ascorbate and glutathione (4Foyer C.H. Lopez-Delgado H. Dat J.F. Scott I.M. Physiol. Plant. 1997; 100: 241-254Crossref Google Scholar, 5Noctor G. Gomez L. Vanacker H. Foyer C.H. J. Exp. Bot. 2002; 53: 1283-1304Crossref PubMed Scopus (689) Google Scholar). Ribulose-1,5-bisphosphate carboxylase (Rubisco) is the key enzyme in photosynthetic carbon assimilation. In Chlamydomonas reinhardtii and in land plants the enzyme is composed of eight large subunits (LSU) encoded by the chloroplast rbcL gene and eight small subunits encoded by the nuclear rbcS gene family. Assembly of the holoenzyme is mediated by the chloroplast chaperonins cpn60 and cpn10 (6Gutteridge S. Gatenby A.A. Plant Cell. 1995; 7: 809-819Crossref PubMed Scopus (139) Google Scholar, 7Thirumalai D. Lorimer G.H. Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 245-269Crossref PubMed Scopus (329) Google Scholar). We previously showed that transfer of the green algae C. reinhardtii from low light (70 μmol m–2 s–1) to high light (700 μmol m–2 s–1) generates an oxidative stress that leads to photoinhibition and a dramatic arrest in the synthesis of the LSU of Rubisco (8Shapira M. Lers A. Heifetz P. Yrihimovitz V. Osmond B.C. Gillham N.W. Boynton J.E. Plant Mol. Biol. 1997; 33: 1001-1011Crossref PubMed Scopus (70) Google Scholar). These light-induced effects were found to be transient, with cell recovery taking place within 6–12 h once chlorophyll levels were reduced and ROS levels were decreased. It was further found that translation of Rubisco LSU varies with the changes in ROS production and correlates with alterations in the ratio between oxidized and reduced glutathione. Upon transfer to high light the glutathione pool shifts to its oxidized form, and LSU synthesis stops almost completely. When the cells recover from light stress, the glutathione pool shifts back to its reduced form, and LSU translation resumes (9Irihimovitch V. Shapira M. J. Biol. Chem. 2000; 275: 16289-16295Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Rubisco holoenzyme is highly susceptible to oxidative stress in vivo. Excess ROS caused a rapid translocation of the soluble enzyme complex into the chloroplast membrane and the formation of intermolecular cross-linking between the large subunits via disulfide bonds (10Mehta R.A. Fawcett T.W. Porath D. Mattoo A.K. J. Biol. Chem. 1992; 267: 2810-2816Abstract Full Text PDF PubMed Google Scholar). Furthermore, oxidative stress can cause direct fragmentation of Rubisco LSU at Gly-329 into 37- and 16-kDa polypeptides in illuminated intact chloroplasts in chloroplast extracts and in its purified form when exposed to a hydroxyl radical-generating system (11Ishida H. Makino A. Mae T. J. Biol. Chem. 1999; 274: 5222-5226Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Translational regulation allows plants to respond quickly to environmental changes such as light intensities and is, therefore, a predominant mechanism in chloroplasts. Upstream UTRs are expected to play a key role in translation of chloroplast genes via interaction with regulatory proteins. A group of RNA binding (RB) proteins with molecular masses of 60, 55, 47, and 38 kDa assemble on the 5′-UTR of the psbA RNA (12Danon A. Mayfield S.P.Y. EMBO J. 1991; 10: 3993-4002Crossref PubMed Scopus (165) Google Scholar). RB47 shows a high homology with the eukaryotic poly(A)-binding protein (13Yohn C.B. Cohen A. Danon A. Mayfield S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2238-2243Crossref PubMed Scopus (86) Google Scholar), and RB60 shows high homology to protein disulfide isomerase (14Kim J. Mayfield S.P. Science. 1997; 278: 1954-1957Crossref PubMed Scopus (198) Google Scholar, 15Trebitsh T. Meiri E. Ostersetzer O. Adam Z. Danon A. J. Biol. Chem. 2001; 276: 4564-4569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Binding of the protein complex is mediated by specific thiol-containing proteins, and light-induced reduction of thioredoxin enhances this binding. It was proposed that RB60 serves as a redox sensor that activates binding of RB47 (16Trebitsh T. Levitan A. Sofer A. Danon A. Mol. Cell. Biol. 2000; 20: 1116-1123Crossref PubMed Scopus (105) Google Scholar). Proteins with similar molecular masses assemble on the 5′-UTR of psbC and other chloroplast leaders (17Hauser C.R. Gillham N.W. Boynton J.E. J. Biol. Chem. 1996; 271: 1486-1497Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 18Zerges W. Rochaix J.D. J. Cell Biol. 1998; 140: 101-110Crossref PubMed Scopus (100) Google Scholar), although it is possible that different RNA-binding proteins of size 47 kDa have altered target specificities (19Zerges W. Wang S. Rochaix J.D. Plant Mol. Biol. 2002; 50: 573-585Crossref PubMed Scopus (12) Google Scholar). In an attempt to examine the mechanism that underlies the unique pattern of regulation observed for Rubisco LSU, we examined how the redox state of RNA-binding proteins affected their interaction with the rbcL leader. We show that the interaction between RNA-binding proteins and the rbcL leader is interrupted by oxidative stress and that Rubisco LSU can bind RNA in its oxidized form, although in a nonspecific manner. Strains and Growth Conditions—C. reinhardtii wild type CC-125 cells were grown in high salt reduced sulfate medium with bubbling of 5% CO2 and constant rotary shaking at 25 °C. Cultures were illuminated with medium light (150 μmol m–2 s–1) using cool white fluorescent lamps. The photosynthesis-deficient mutant CC-2653 has an amber mutation at the 5′ end that terminates translation of Rubisco LSU at position 65 (20Spreitzer R. Goldschnidt-Clermont M. Rahire M. Rochaix J.-D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5460-5464Crossref PubMed Google Scholar) and was grown on Tris acetate phosphate medium at 25 °C under dark conditions. For Rubisco purification CC-125 cells were grown in Tris acetate phosphate medium under low light conditions (70 μmol m–2 s–1). Plasmids Used for in Vitro RNA Synthesis—The 5′-UTR of rbcL from C. reinhardtii was cloned from P-266, a plasmid that contains the 4-kilobase EcoRI-BamHI fragment of chloroplast DNA (21Dron M. Rahire M. Rochaix J.D. J. Mol. Biol. 1982; 162: 775-793Crossref PubMed Scopus (179) Google Scholar). An EcoRI-XmnI fragment of the insert was inserted into pBluescript at the EcoRI-SmaI site, resulting in pVI1. The complete 5′-UTR of rbcL (between positions –93 to +24 relative to the ATG start codon) was amplified from pVI1 using the primers 5′-TAAATGTATTTAAAATTTTTCAACAAT-3′ (forward) and 5′-TTTAGTTTCTGTTTGTGGAACCAT-3′ (reverse). The resulting PCR fragment was cloned into the pGEM-T vector (Promega), and sequences between the PstI and NsiI sites were removed, resulting in pΔVI3. The plasmid encoding rbcL was linearized by NcoI and NotI for synthesis of the sense and antisense strands, respectively. In vitro synthesis of the 5′-UTRs derived from psbA, atpB (chloroplast genes), and α-tubulin (a nuclear gene) was performed from plasmids D1-HA, P-419, and P-654, respectively. Plasmid D1-HA was the generous gift of A. Danon; plasmids P-419 and P-654 were obtained from the Chlamydomonas Center at Duke University. Plasmid D1-HA was linearized with EcoRI, and P-419 and P-654 were linearized with BamHI. In vitro synthesis of the sense strands derived from the corresponding 5′-UTRs was performed with T7 RNA polymerase. Synthesis of a non-related RNA fragment was performed using pBluescript (SK) as a template. The plasmid was linearized by NotI, and RNA was synthesized with T7 RNA polymerase. Preparation of Protein Extracts—C. reinhardtii wild type CC-125 cells were grown at medium light, and protein extracts were prepared essentially as described previously (12Danon A. Mayfield S.P.Y. EMBO J. 1991; 10: 3993-4002Crossref PubMed Scopus (165) Google Scholar, 17Hauser C.R. Gillham N.W. Boynton J.E. J. Biol. Chem. 1996; 271: 1486-1497Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Cells (3 × 1 liter) were grown in high salt reduced sulfate and harvested at a concentration of 5–7 × 106 cell/ml. The pellets were frozen in liquid nitrogen and stored at –70 °C until use. Cell pellets (8 g fresh weight) were thawed in 25 ml of low salt buffer (10 mm Tris-HCl, pH 7.5, 10 mm NaCl, 10 mm MgCl2, and 10 mm DTT) in the presence of protease inhibitors (2 μg/ml aprotinin, 10 μg/ml benzamidine, 5 μg/ml leupeptin, and 76 μg/ml phenylmethylsulfonyl fluoride). The cells were disrupted by triple passage through a French press (Sim-Amico, Spectronic Instruments) at 4000 p.s.i. The broken cells were centrifuged for 10 min at 20,000 × g (SS-34 rotor, 10,000 rpm in an RC-2 Sorvall). The supernatants were collected and further centrifuged at 200,000 × g for 1 h at 4 °C (TI50 rotor, 50,000 rpm in an L8–55 Beckman ultracentrifuge). The 200,000 × g supernatant was collected and applied immediately onto a 5-ml heparin-Actigel column (Amersham Biosciences) at a flow rate of 1 ml/min. The column was first prewashed with 3 volumes of high salt buffer (10 mm Tris-HCl, pH 7.5, 10 mm NaCl, 10 mm MgCl2, 10 mm DTT, and 2 m potassium acetate) and equilibrated with extraction buffer containing (20 mm Tris-HCl, pH 7.5, 3 mm MgCl2, 0.1 mm EDTA, and 2 mm DTT). The bound proteins were eluted with a continuous gradient of potassium acetate concentrations (0–1.6 m) in low salt buffer. Fractions (500 μl) were collected, dialyzed against dialysis buffer (20 mm Tris-HCl, pH 7.5, 100 mm potassium acetate, 0.2 mm EDTA, 2 mm DTT, and 20% glycerol), and stored at –70 °C. The fractions were analyzed by SDS-PAGE (12%), and their protein content was evaluated by Coomassie Blue staining. In Vitro RNA Synthesis—Radiolabeled RNA transcripts (described above) were synthesized in vitro using 0.5–1 μg of DNA in 40 mm Tris-HCl, pH 7.5, 6 mm MgCl2, 2 mm spermidine, 12.5 mm NaCl, 10 mm DTT, 20 units of RNasin, 0.5 mm ATP, GTP, and CTP, 12 μm UTP, 50 μCi of [α-32P]UTP (800 Ci/mmol, Amersham Biosciences), and 20 units of SP6 (Roche Applied Science) or T7 (Promega) RNA polymerase in a reaction volume of 20 μl. The reactions were performed at 37 °C for 40 min followed by the addition of 1 unit of DNase I (RNase-free, Promega) and were then incubated for an additional 30 min at 37 °C. The labeled RNAs were separated from the unincorporated ribonucleotides on a spun-down mini-column of Sephadex G-50 in double-distilled H20. Under these conditions transcripts were labeled to specific activities that ranged between 5 × 108 and 2 × 109 cpm/μg of RNA. Unlabeled transcripts that were used for competition assays were synthesized as described above, except that the reactions were scaled up to 100 μl, and all the four ribonucleotides were included at equal concentrations (0.5 mm). RNA products were analyzed on 7 m urea, 6% polyacrylamide gels to verify production of a single transcript and to evaluate its size and concentration. Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift assays (EMSA) were performed essentially as described previously (17Hauser C.R. Gillham N.W. Boynton J.E. J. Biol. Chem. 1996; 271: 1486-1497Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Samples from the heparin-Actigel column fractions (2 μl containing approximately 9 μg of protein) were preincubated for 10 min at room temperature with 5 units of RNasin (Promega) in 3 mm MgCl2 in a total volume of 5 μl. The mixtures were then added to the RNA probes (10,000 cpm) in the presence of 20 μg of Escherichia coli tRNA (Sigma) in a final volume of 15 μl. After an incubation of 15 min at room temperature, 2 μl of loading buffer (0.25 μg/μl xylene cyanol, 0.25 μg/μl bromphenol blue, and 6% (v/v) glycerol) were added, and the reactions were separated on a native 5% polyacrylamide gel (acrylamide:bisacrylamide 49:1) in 1× TBE (89 mm Tris, 89 mm boric acid, and 2 mm EDTA, pH 8). Running conditions were 25 mA for 2–3 h. The gels were then fixed in a solution of 20% methanol and 10% acetic acid, dried, and subjected to autoradiography. In competition experiments unlabeled RNA transcripts were added in varying amounts of mass excess and preincubated with the protein samples before the addition of the radio-labeled probe. UV Cross-linking Assays—Binding assays were performed as previously described (17Hauser C.R. Gillham N.W. Boynton J.E. J. Biol. Chem. 1996; 271: 1486-1497Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Samples of proteins eluted from the heparin-Actigel column (2 μl containing approximately 9 μg of protein), purified Rubisco (25 ng), or the recombinant polypeptides that corresponded to Rubisco LSU (amino acids 1–475) or its sub-fragments (amino acids 1–150 and 151–475) after purification over a nickel nitrilotriacetic acid column (25 ng) were preincubated for 10 min at room temperature with 0.5 units of RNasin (Promega) in 3 mm MgCl2 in a volume of 5 μl. Radiolabeled RNA (100,000 cpm) was then added to the protein solution along with 0.2 μg of E. coli tRNA (Sigma) in a final volume of 15 μl. After 15 min of incubation at room temperature the binding reactions were placed on ice and cross-linked by UV irradiation at 254 nm in a UV cross-linker (Hoefer) for 90 s. RNA transcripts were then digested with 20 μg of RNase A (Sigma) for 40 min at 37 °C. The samples were separated over 15% SDS-PAGE. Gels were stained by Coomassie Blue, dried, and subjected to autoradiography or analyzed by phosphorimaging. In competition experiments unlabeled RNA transcripts were added in varying mass excess and preincubated with the protein samples before the addition of the radiolabeled probe. To examine how the redox state affected the binding proteins, GSSG or GSH was added to the protein extracts or to the purified Rubisco (25 ng) 10 min before the addition of the labeled 5′-UTR. Two-dimensional Gel Electrophoresis—Protein separation on two-dimensional gel electrophoresis was performed as previously described (22O'Farrel P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar). Separation on the first dimension was performed by isoelectric focusing using ampholytes (Bio-Rad) that ranged between pH 3 and 9. Separation on the second dimension was preformed by SDS-PAGE over 12% polyacrylamide gels. Antisera—Polyclonal rabbit antisera raised against Rubisco holoenzyme from tobacco were a generous gift of T. J. Andrews from the Australian National University, Canberra, Australia. Western Blot Analysis—Proteins were separated over one- and two-dimensional SDS-polyacrylamide gels and electroblotted onto a nitrocellulose membrane (Schleicher & Schuell). Western blot analysis was performed using anti-Rubisco antibodies (1:4000) and a conjugate of protein A with alkaline phosphatase (1:2000). Antibody binding was detected with nitro blue tetrazolium and 5′-bromo-4-chloro-3-indolylphosphate (Sigma) dissolved in a buffer containing 100 mm Tris-HCl, pH 9.5, 100 mm NaCl, and 5 mm MgCl2. Rubisco Purification—Wild type C. reinhardtii cells (CC-125) were grown in Tris acetate phosphate medium (3 liters), harvested to yield 8 g fresh weight, and disrupted in a French press as describe above. The cell extract was loaded on a heparin-Actigel column pre-equilibrated in low salt buffer (see above) and was eluted by a single step of 1.6 m potassium acetate in low salt buffer. The eluate (5 ml) was dialyzed against low salt buffer and loaded (1 ml) on a linear 10–30% sucrose gradient in low salt buffer. The gradient was centrifuged for 16 h at 164,000 × g (SW40 rotor, 40,000 RPM in a Beckman LE-80K ultracentrifuge) at 4 °C, and fractions (1 ml) were collected. Samples of each fraction were examined by Western blot analysis using antibodies raised against Rubisco. Expression of Recombinant LSU and Its Subdomains in Bacteria— The DNA region that encodes for Rubisco LSU was amplified using primers derived from both ends of the rbcL gene; the LSU-fwd-(1–21) primer was 5′-ATCCATGGTTCCACAAACAGAAACT-3′, and the LSU-rev-(1506–1482) primer was 5′-TTAGGAATTCAACGTAAACACCATA-3′. The DNA region which encodes the amino terminus of Rubisco LSU (aa 1–150) was amplified by PCR using the LSU-fwd-(1–21) primer; and the reverse primer, LSU-rev-(450–432), 5′-GGAATTCTTAACCTACGAATGTTTTAACG-3′. The DNA region that encodes the TIM barrel domain of Rubisco (aa 151–475) was amplified by PCR using the primer LSU-fwd-(451–466), 5′-ATCCATGGAACCTCCACACGGTATTC-3′, and the LSU-rev-(1506–1482) primer. Anchors that introduced restriction sites, NcoI (fwd primers) and EcoRI (rev primers), were added and are marked in bold letters. The resulting fragments were cloned in-frame into the parallel pHisII expression vector (23Sheffield P. Garard S. Derewenda Z. Protein Expression Purif. 1999; 15: 34-39Crossref PubMed Scopus (530) Google Scholar). Expression was induced in BL21 cells by incubation of the transgenic bacteria with 0.3 mm isopropyl-1-thio-β-d-galactopyranoside at 20 °C for 2.5 h. The bacteria were harvested, washed once in double-distilled H2O, resuspended in a buffer containing 30 mm Tris-HCl, 300 mm NaCl, and 5 mm imidazole, and lysed in a French press using 20,000 p.s.i. The lysate was centrifuged at 10,000 × g at 4 °C for 25 min. The expressed protein present in the soluble bacterial fraction was affinity-purified over a nickel nitrilotriacetic acid column (Qiagen). Because the recombinant TIM barrel domain was insoluble, it was denatured after a previously described procedure (24Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual.3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001: 15.49-15.54Google Scholar) and renatured by dialysis against 50 mm Tris-HCl, pH 8.5, 10 mm MgCl2,10 mm NaHCO3, 1 mm DTT. The soluble recombinant polypeptides were subjected to Western blot analysis using antibodies against Rubisco. Structural Characterization of Rubisco Subdomains—Subdomains of the large subunit of Rubisco were identified based on the SCOP data base that provides a structural classification of proteins (25Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5595) Google Scholar). RNA Binding Activity of Protein Extracts to the rbcL 5′-UTR; Specificity and Sensitivity to the Redox State—To test whether translational arrest of the rbcL transcript at high light and during oxidative stress correlated with changes in the redox state of proteins that bind to its 5′-UTR, electrophoretic mobility shift assays were performed. The fraction of proteins that bind nucleic acids was enriched by purification of cell extracts over a heparin-Actigel affinity column followed by elution with a gradient (0–1.6 m) of potassium acetate concentrations. Radiolabeled RNA extending from positions –93 to +24 relative to the translational start site of the rbcL gene was synthesized in vitro and incubated with the protein fractions. Binding of proteins to the radiolabeled RNA was monitored by their inhibitory effect on migration of the RNA in native polyacrylamide gels as compared with control unbound RNA. RNA binding activity was observed with the radiolabeled 5′-UTR of rbcL (Fig. 1A). Migration of the RNA-protein complexes resulted in multiple bands, suggesting that the binding involved more than a single protein. Binding to the labeled 5′-UTR of rbcL was interrupted in a dose-response manner by adding increasing amounts of the corresponding unlabeled rbcL 5′-UTR (Fig. 1A, lanes b–f). Binding was not affected by the addition of increasing amounts of SK RNA (lanes h–l) or by a large excess of E. coli tRNA (lanes m and n). Thus, only the homologous non-radioactive RNA fragment could efficiently compete-out the RNA-protein interaction. In addition, no binding was observed with a nonspecific labeled RNA fragment of comparable size, derived from pBluescript (SK RNA, data not shown). To strengthen the association between the shift of glutathione to its oxidized form during light stress and the translational arrest of Rubisco LSU (9Irihimovitch V. Shapira M. J. Biol. Chem. 2000; 275: 16289-16295Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), binding assays were carried out under oxidizing (GSSG) and reducing (GSH, DTT) conditions. Fractionated proteins capable of binding to the 5′-UTR of rbcL in EMSA were preincubated with increasing concentrations of GSSG, GSH, and DTT (5, 10, and 25 mm) before the addition of the radiolabeled rbcL 5′-UTR. Band shifting of the RNA was inhibited already in the presence of 5 mm GSSG and was completely abolished by 10 and 25 mm GSSG, whereas GSH and DTT had no effect on the binding activity (Fig. 1B). These results indicate that mediating the redox of thiol groups on RNA-binding protein(s) affects their ability to interact with the 5′-UTR of the rbcL transcript. Binding Specificity of Proteins to the 5′-UTR of rbcL—A biochemical characterization of the proteins that bind to the rbcL leader was initiated using UV cross-linking. Radiolabeled RNA corresponding to the 5′-UTR of rbcL was incubated with proteins eluted from the heparin column and subjected to UV irradiation followed by RNase digestion. Using this approach we found that proteins with a molecular mass of 81, 62, 51, 47, 38, 36, and 34 kDa bind to the 5′-UTR of rbcL. To establish that the binding was specific to the 5′-UTR of rbcL, UV cross-linking was performed in the presence of non-radioactive RNA competitors. Binding of the 81-, 62-, 51-, and 47-kDa proteins was inhibited by the homologous rbcL –93/+24 fragment (Fig. 2A, lanes a–g) and was hardly affected by the SK RNA or tRNA controls (Fig. 2A, lanes h–q), indicating a sequence specificity for the RNA-protein interactions. Because binding of the 38-, 34-, and 32-kDa proteins was not competed-out by the cold rbcL RNA, their interaction with the RNA was most probably nonspecific. UV Cross-linking of Proteins to the 5′-UTR of rbcL Is Sensitive to the Redox State of Glutathione—To test how the redox state of thiol groups modulated binding of proteins to the 5′-UTR of rbcL, UV-cross-linking assays were performed in the presence of reduced and oxidized glutathione. Protein extracts eluted from the heparin-Actigel column were preincubated with increasing concentrations of GSSG before the addition of the labeled rbcL 5′-UTR. Under oxidizing conditions binding of the 81-, 62-, 51-, and 47-kDa proteins decreased (Fig. 2B); however, cross-linking of a new 55-kDa protein was observed. In view of the similar molecular weights of this newly UV-cross-linked protein and the LSU of Rubisco, the possibility that an autoregulatory pathway exists was considered. Rubisco LSU Is an RNA-binding Protein—The 55-Da protein was characterized on one- and two-dimensional polyacrylamide gels combined with Western blot analysis. Proteins were preincubated with GSSG (7.5 mm) before their UV cross-linking with the radiolabeled 5′-UTR of rbcL. The cross-linked proteins were separated by two-dimensional SDS-PAGE and blotted onto nitrocellulose membranes. The blots were exposed to a film (Fig. 3, C and D) and then reacted with an antibody raised against Rubisco LSU (Fig. 3, A and B). Alignment of the autoradiograms and films indicated that the 55-kDa protein that cross-linked to the 5′-UTR of rbcL under oxidizing conditions co-migrated with Rubisco LSU on two-dimensional gels. Direct evidence for binding of Rubisco LSU to the rbcL leader under oxidizing conditions was obtained by using the purified enzyme in UV-cross-linking experiments. As shown in Fig. 4, A and B, the purified protein cross-linked with the rbcL leader in direct correlation with the GSSG concentration, whereas no binding was observed in the absence of GSSG. In addition, the 55-kDa protein was absent in UV-cross-linking experiments performed under oxidizing conditions using CC 2653 (20Spreitzer R. Goldschnidt-Clermont M. Rahire M. Rochaix J.-D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5460-5464Crossref PubMed Google Scholar), a mutant that fails to express Rubisco LSU due to a point mutation that pre-terminates translation (data not shown). To examine the binding specificity between Rubisco LSU and its corresponding rbcL leader, competition assays were performed with unlabeled RNAs that corresponded to the homologous rbcL leader in its sense and antisense orientations. Equal inhibition of binding to the labeled rbcL leader was observed with similar amounts of either fragment, suggesting that in vitro the binding of Rubisco LSU to its leader was sequence-independent (Fig. 5A). A similar conclusion was drawn from experiments that compared the competition between non-labeled SK RNA and the sense rbcL fragment. Both of these RNA fragments competed out the binding between Rubisco LSU and its RNA leader with comparable efficiencies (Fig. 5B). It, therefore, appears that under oxidizing conditions Rubisco LSU binds non-specifically to RNA. In the presence of a large excess of cold competitor RNA, a higher band was observed. Its appearance could result from incomplete RNase digesti
