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Address correspondence to Jean-David Rochaix, Department of Molecular Biology, University of Geneva, 30, Quai Ernest Ansermet, Geneva 1211, Switzerland. Tel.: 41-22-702-6187. Fax: 41-22-702-6868. E-mail: Jean-David.Rochaix{at}molbio.unige.ch
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Abstract |
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Key Words: chloroplast; translation; Chlamydomonas; photosynthetic mutant; protein complex
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Introduction |
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Clues to the biochemical functions of some nucleus-encoded factors have come from identification of motifs or regions of homology these proteins share with enzymes known to be involved in RNA metabolism and other processes. For example, the splicing factor Raa2 (formerly called Maa2) resembles pseudouridine synthase and is required for the transsplicing of the second psaA intron in the chloroplast of C. reinhardtii (Perron et al., 1999). The splicing factor Crs2 of maize, required for the splicing of several plastid group II introns, is related to peptidyl-tRNA hydrolase enzymes (Jenkins and Barkan, 2001). Another splicing factor Raa3, which is involved in the splicing of the first psaA intron of C. reinhardtii, contains a short region of homology with pyridoxamine 5' phosphate oxidase (Rivier et al., 2001). In contrast, other factors do not resemble any known protein in the database. These include the Chlamydomonas Ac115 protein which is implicated in the translation elongation of the psbD mRNA (Rattanachaikunsopon et al., 1999).
The control of expression of the chloroplast genes encoding the major core photosystem (PS)*II subunits D1, D2, P5, and P6 of C. reinhardtii has been studied intensively in recent years. Biochemical approaches have identified two nucleus-encoded proteins that are involved in the light activation of the translation of the psbA mRNA of C. reinhardtii (Danon and Mayfield, 1991): one is a 47-kD protein resembling polyA-binding proteins (Yohn et al., 1998), and the other is a protein disulfide isomerase that both appear to be under redox control (Kim and Mayfield, 1997; Fong et al., 2000; Trebitsh et al., 2000, 2001). Analysis of several nuclear mutants of C. reinhardtii deficient in PSII activity has revealed that each of these mutations identifies a nuclear locus that is required for the translation of the mRNA of one specific PSII subunit (Goldschmidt-Clermont, 1998). For example, translation of the psbC mRNA requires functions encoded by two nuclear loci, TBC1 and TBC2. The psbC mRNA has the largest 5' untranslated region (UTR) of known chloroplast mRNAs in C. reinhardtii. It consists of 550 nucleotides, and acts as a target site for Tbc1 and Tbc2, strongly suggesting that these factors play a role in the initiation of translation (Zerges and Rochaix, 1994). A striking feature of the psbC 5'UTR is a large inverted repeat structure in its middle that is required for translation of the psbC mRNA. Mutations within this inverted repeat or deletion of the entire structure completely abrogate translation (Rochaix et al., 1989; Zerges and Rochaix, 1994; Zerges et al., 1997). A nuclear suppressor of these mutations has identified a third locus, TBC3, involved in the initiation of translation of psbC mRNA. This suppressor also reverses the translational defect caused by the tbc1, but not by the tbc2, mutation (Zerges et al., 1997). The two factors defined by TBC1 and TBC3 and the middle part of the psbC 5'UTR appear to interact functionally (Zerges et al., 1997). UV crosslinking studies with chloroplast extracts and the psbC 5'UTR have revealed an RNA binding activity in S100 fractions of a mutant strain carrying the tbc2-F64 mutation, but not in similar fractions prepared from wild-type strains (Zerges and Rochaix, 1994). To understand the exact role of these factors, and in particular their interactions with the psbC mRNA, it is important to examine their genes and the products that these genes encode. As a first step toward this goal, we have cloned the Tbc2 gene and characterized its product.
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Results |
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Complementation and cloning of TBC2 and its cDNA
In order to clone the Tbc2 gene, pools of genomic cosmid clones from an indexed cosmid library (Zhang et al., 1994) were tested for their ability to complement the photosynthesis deficiency produced by tbc2-B23. One pool yielded five photoautotrophic colonies. From this pool we isolated a cosmid that complements all three mutant tbc2 alleles (see Materials and methods).
To subclone the rescuing cosmid DNA, restriction fragments obtained from single, double, and triple digests of the cosmid DNA were used to transform the three tbc2 mutant strains. A Bam HI fragment of 9.5 kb was identified that was able to complement all three alleles. No subfragments of this Bam HI fragment were found to be able to complement the mutant phenotypes, suggesting that the DNA encodes a functional TBC2 gene with little extraneous sequence. This Bam HI fragment was cloned and partially sequenced. It was found to contain two Hind III sites and, upon digestion with Hind III, fragments of
0.9, 3.8, and 4.8 kb were generated. The two larger fragments were used to screen 6 x 105 phage of a cDNA library. Initially, only one 4.1-kb cDNA was isolated, and it hybridized to both fragments. However, this cDNA was unable to rescue the mutants. It was cloned and sequenced in its entirety. Analysis of its coding capacity (Genemark 2.4) indicated a probable frame shift around residue 1200. Indeed, fusions between the 5' genomic DNA (up to the Sac I site) and the cDNA downstream of the SacI site were able to complement the TBC2 mutations, indicating that the 3' half of the cDNA (downstream of the putative frameshift) encodes a functional protein (Fig. 2; unpublished data). Screening another 8 x 105 phage of the cDNA library yielded eight additional phage, two of which were able to complement. The 5' halves of the rescuing cDNAs were sequenced and found to contain an additional internal 179-bp DNA fragment relative to the first cDNA, also found in the genomic sequence, that restored the reading frame (nucleotides 11071285 of the cDNA). PCR analysis was performed on the other cDNAs unable to rescue the mutants. Three contain a similar deletion to that of the first cDNA, one is missing a different internal region, and the other two do not have any detectable rearrangements (unpublished data). It is not yet known whether the differences observed are due to alternative splicing or to cloning artifacts.
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Characterization of the predicted TBC2 amino acid sequence
The Tbc2 cDNA sequence predicts an ORF encoding a protein of 1,115 amino acids with a molecular mass of 114.8 kD (Fig. 4). The longest isolated cDNA has a 5'UTR of 174 bases, with an inframe stop codon 60 nucleotides upstream of the presumed initiation codon. This ATG probably encodes the translation initiation codon because the next ATG codon is 369 nucleotides downstream and would yield a protein that is significantly smaller than the observed size (see below). In the NH2 and COOH termini of the Tbc2 protein, there are stretches of the same amino acid repeated from 3 to 11 times (most often alanine, but also glutamine, threonine, and other residues; Fig. 4, A and B). The central section of the protein is free of these strings of repeated amino acids, and contains nine copies of a degenerate 3840 amino acid repeat which is neither a TPR (Lamb et al., 1995) nor a PPR repeat (Small and Peeters, 2000) (Fig. 4 C). Five of the repeats are arranged in tandem, whereas the last two are also consecutive in the COOH-terminal section of Tbc2. Whereas the NH2-terminal part of the repeats is poorly conserved, sequence identity is more apparent in the COOH-terminal part, especially the five residue PPPEW motif with the first P and the W present in all repeats. This motif is not repeated in any protein in the SWISS-PROT database (Release 40.4, 23 November, 2001; Bairoch and Apweiler, 2000), nor is it a previously characterized motif. The region of Tbc2 corresponding to the 179-bp deletion in the cDNAs that were unable to rescue the tbc2 mutations is underlined in Fig. 4, A and B. These cDNAs would give rise to a polypeptide of 598 residues that lacks the PPEW repeats and whose 227 COOH-terminal amino acids are read in a different phase relative to Tbc2. Whether such a protein exists and has any function is unknown.
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To further characterize the Tbc2 protein, its gene was tagged with a triple hemagglutinin (HA) epitope in either the middle of the coding sequence, at codon 593, or immediately before the stop codon (Fig. 4 A). The constructs were used separately for transformation of a C. reinhardtii strain carrying the tbc2-F64 mutation by selecting for growth on minimal medium. Several independent colonies from each transformation were screened by immunoblotting for expression of the HA epitopetagged Tbc2 protein. Although both constructs were able to rescue the mutant and expressed detectable HA-tagged protein of the same size, and are therefore functional, the signal from the construct with the epitope at the COOH terminus yielded a much stronger signal (unpublished data). It was used exclusively in the experiments that follow.
Characterization of the Tbc2 protein
The NH2 terminus of Tbc2 is enriched in basic and hydroxylated amino acids, but also contains a higher portion of acidic residues than expected for a typical chloroplast transit peptide. Analysis of this sequence with PSORT (http://psort.nibb.ac.jp/) or ChloroP (http://www.cbs.dtu.dk/ services/ChloroP/) predicts a mitochondrial rather than a chloroplast location. However, a particular feature of chloroplast transit peptides from C. reinhardtii is that they share features with both chloroplast and mitochondrial target sequences of vascular plants (Franzen et al., 1990).
In order to determine the subcellular location of Tbc2 directly, cells from the mutant tbc2-F64 were transformed with the HA epitopetagged Tbc2 construct by selecting for photoautotrophic growth. Cells were gently broken and chloroplasts were isolated by centrifugation on Percoll gradients and fractionated into a soluble and membrane fraction by high-speed centrifugation. The proteins were then separated on 8% polyacrylamide gels and probed with antisera raised against several proteins (Fig. 5). The monoclonal HA antibody detected a protein of 140 kD in extracts from whole cells (lane 2), chloroplasts (lane 7), and the chloroplast-derived soluble fraction (lane 9), but considerably less in the chloroplast membranes (lane 8). In other independent extractions, the signal in the chloroplast membrane fraction was undetectable, especially when sonication of the chloroplasts was omitted. The protein detected migrates as if it were
20 kD larger than expected (including 4.2 kD for the triple HA epitope), and is not detected in either nonrescued cells (lanes 1, 3, and 5) nor in cells rescued with a non-HAbearing Tbc2 gene (unpublished data). The protein is found in chloroplasts, and is detected mostly in the soluble fraction (Fig. 5, lane 9). In the experiment presented in Fig. 5, the chloroplast extracts were not treated with salt. However, a 0.5-M (NH4)2SO4 wash to remove loosely bound proteins from the membranes before centrifugation (Zerges and Rochaix, 1998) did not alter the fractionation results obtained with Tbc2 (unpublished data). As a control for the fractionation experiment, the distribution of a soluble chloroplast protein (ribulose-bis-phosphate carboxylase-oxygenase; Rubisco), D2, a PSII reaction center subunit in the thylakoid membrane, and thioredoxin-h, a cytosolic marker protein, were also determined (Fig. 5). Each of these proteins fractionated as expected, indicating that the chloroplasts used were not significantly contaminated with cytosolic proteins. Thus, despite the absence of an identifiable chloroplast transit peptide, the Tbc2 protein is targeted to the chloroplast. Similar results were obtained using cells complemented with Tbc2 HA-epitope tagged in the middle of the protein (in the Sac I site, Fig. 2 B; unpublished data). This substantiates the chloroplast localization of the protein.
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Discussion |
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Other loci involved in PSII biogenesis have been mapped previously. They include the AC-114 locus on linkage group III and AC-115 on linkage group I. Analysis of the progeny from crosses between the tbc2-F64 mutant and the ac-114 and ac-115 mutants revealed that the TBC2 locus is unlinked to these two loci. Thus, there is no evidence for the clustering of nuclear genes involved in posttranscriptional steps of the expression of plastid genes encoding PSII subunits.
Structural features of Tbc2
The Tbc2 gene encodes a protein of 1,115 amino acids. A noticeable feature of this protein is that it contains nine degenerate repeats of 3840 amino acids that extend from the middle to the COOH-terminal region. Although the Tbc2 protein is not obviously related to any protein in the data bases, it is particularly interesting that the maize Crp1 protein is found among the proteins that displays low sequence identity with Tbc2. Crp1 is required for the processing and translation of the plastid petA and petD RNAs (Fisk et al., 1999). Crp1 contains PPR repeats (Small and Peeters, 2000) that are similar to TPR repeats, except that the repeat consists of 35 residues. The region of sequence similarity between Tbc2 and Crp1 includes the repeated motifs of these two proteins, although the PPR motifs are distinct from the internal repeats of Tbc2 aside from sharing three consensus residues in their COOH termini. The presence of TPR motifs has been found in a large set of proteins involved in many different activities such as cell cycle control, transcription, protein import into mitochondria, and chloroplast RNA metabolism (Blatch and Lassle, 1999). In particular, the Nac2 and Mbb1 proteins containing 9 to 10 TPR-like repeats are required for the stable accumulation of the chloroplast psbD and psbB mRNAs in C. reinhardtii (Boudreau et al., 2000; Vaistij et al., 2000). Changing one conserved residue of one of the Nac2 TPR repeats abolishes the activity of the protein, indicating that these repeats have an important functional role (Boudreau et al., 2000).
All of these proteins appear to belong to a large family of helical repeat proteins. The atomic structure of several representatives of these proteins has been determined. They include ß catenin with its 12 Arm repeats of 42 amino acids (Huber et al., 1997), the A subunit of protein phosphatase 2A with its 15 HEAT repeats of 39 amino acids (Groves et al., 1999), Pumilio with its 8 Puf repeats of 36 amino acids (Edwards et al., 2001), and protein phosphatase 5 with its 3 TPR repeats of 34 amino acids (Das et al., 1998). These tandem helical repeats form an extended surface of the protein that is thought to be involved in proteinprotein interactions. In the case of Pumilio, a translational regulator of the hunchback mRNA in Drosophila, this surface can also be used for recognizing RNA (Edwards et al., 2001). Although Tbc2 does not contain any known repeat, it is predicted to contain a considerable amount of -helical structures and a novel repeated sequence. It is not yet known whether or not Tbc2 has RNA binding activity.
Further searches for sequence similarity revealed that Tbc2 shares a short stretch of 42 amino acids with selenophosphate synthase (52% sequence identity). Other unusual structural features of Tbc2 include the presence of long stretches of Ser, Ala, or Gln mostly in the NH2- and COOH-terminal regions that have also been noticed in other nucleus-encoded proteins from C. reinhardtii (Boudreau et al., 2000; Vaistij et al., 2000).
Tbc2 is a chloroplast stromal protein
The NH2-terminal region of Tbc2 does not resemble typical chloroplast transit peptides but is recognized as a mitochondrial presequence using PSORT or ChloroP subcellular localization programs. This finding is not necessarily incompatible with a chloroplast location, because the analysis of chloroplast transit peptides from C. reinhardtii has revealed that they share features with both mitochondrial and higher plant chloroplast presequences (Franzen et al., 1990). They contain both the potential amphiphilic -helix of mitochondrial presequences and the amphiphilic ß-strand of higher plant chloroplast transit peptides. It is also possible that the chloroplast targeting sequence is located elsewhere in the protein.
To determine the location of Tbc2, the protein was tagged with an HA epitope and was localized mainly in the stromal compartment of the chloroplast by cell fractionation and immunoblotting. It is interesting to note that among the nucleus-encoded factors known to be involved in chloroplast post-transcriptional steps, some, like Tbc2, Crp1 (Fisk et al., 1999), Nac2 (Boudreau et al., 2000), Mbb1 (Vaistij et al., 2000), and Raa3 (Rivier et al., 2001), are found in the soluble chloroplast phase, whereas others, like RB47 and Raa2, are associated with a low-density membrane system (Zerges and Rochaix, 1998; Perron et al., 1999). RB60 appears to be partitioning both with the stroma (Boudreau et al., 2000) and thylakoids (Trebitsh et al., 2001). The functional significance of this different compartmentalization remains to be determined. It is possible that several of these factors interact only transiently with the chloroplast membrane during some of the steps leading to the integration of the newly synthesized polypeptides into the membranes. Although the HA-tagged Tbc2 protein is functional and rescues the tbc2 mutations, we cannot exclude that it may not behave exactly the same way as the authentic Tbc2 protein.
Tbc2 appears to be part of a high molecular weight complex of 400 kD. It could represent a homomeric complex, or Tbc2 could be associated with other factors. Possible candidates include Tbc1 and Tbc3, which are known to be involved in the initiation of translation of the psbC mRNA (Zerges et al., 1997). The Tbc2 complex does not appear to contain RNA based on the observation that its size is not altered by RNase treatment. In addition, as for Crp1, no stable association of Tbc2 with polysomes could be detected, suggesting that this factor interacts only transiently or indirectly with the translational machinery. As Tbc2 has a specific role in the translation of the psbC mRNA and its action is mediated through the psbC 5'UTR, it has to functionally interact with this region. Whether this interaction occurs directly or indirectly through other RNA-binding factors remains to be determined. Most factors involved in translation have been identified by biochemical approaches. This study, and others described here, reveal that genetic approaches can identify new translational regulators. The culmination of these approaches will be a comprehensive understanding of the molecular mechanisms underlying translation in vivo.
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Materials and methods |
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Genetic analysis
Mating, germination, and tetrad analysis were performed according to Harris (1989). Reversion and recombination tests were performed as described (Kuras et al., 1997), and complementation tests were done according to Goldschmidt-Clermont et al. (1990).
DNA isolation
Total Chlamydomonas DNA was prepared as described (Boudreau et al., 1997). Gels were transferred to Hybond N+ or Hybond C-extra (Amersham Pharmacia Biotech), and hybridized and washed as described (Church and Gilbert, 1984). PCRs were performed with a 5-ng/µl template and 0.5-µM primers in 50 mM Tris-Cl, pH 8.3, 1 mM MgCl2, 250 µg/ml BSA, 200 µM dNTP, and 5% DMSO using Pfu polymerase (Stratagene).
Pulse labeling of proteins
Pulse labeling of cells with [14C]acetate was performed as described (Rochaix et al., 1989). At the end of the of the labeling period, 200 ml of culture was used for thylakoid membrane purification, and the extract was fractionated by electrophoresis on 7.515% SDS polyacrylamide gels (Chua and Bennoun, 1975).
Complementation and cloning of the Tbc2 gene and cDNA
An indexed cosmid library (Zhang et al., 1994) of C. reinhardtii genomic DNA was used to transform tbc2-B23;cw15 essentially as described (Kindle, 1990). 2.5 µg of pooled DNA from each microtiter plate was used to transform 3 x 107 cells in the presence of 100 µl 20% PEG 8000 (in 5 mM Tris-Cl, pH 8.0) using sterile 0.4-mm glass beads (Thomas Scientific). Cells were plated on high-salt minimal medium and immediately placed under medium intensity light (60 µE/m2/s). Positive plates were generally detected after 1418 d. Library plate number 64 yielded 02 colonies per plate after transformation. DNA was prepared from each column and row to identify the well containing the cosmid that complements the tbc2 mutation (F5). 1 µg of purified cosmid DNA yielded six colonies in tbc2-B2;cw15 (no DNA control yielded 0 colonies); 93 transformants in tbc2-F64;cw15 (no DNA control yielded 19 colonies); and 315 colonies in tbc2-G314;cw15 (no DNA control yielded two colonies per plate).
To identify which region of the cosmid rescued the tbc2 mutants, the cosmid DNA was digested with different restriction enzymes. The smallest fragment that was able to complement was a 9.5-kb BamHI fragment. This fragment was cloned into pBluescriptKS- (Stratagene) in which the SacI, SalI, HindIII, and NotI polylinker sites had been deleted, and was partially sequenced. This BamHI fragment contains two HindIII sites (Fig. 2). The two largest HindIII fragments (4.8 and 3.8 kb in size) were used separately to screen 6 x 105 phage of a cDNA library constructed by H. Sommer (Max Planck Institute für Züchtungsforschung, Cologne, Germany). One cDNA that hybridized to both fragments was sequenced in its entirety by making exonucleaseIII deletions of the cDNA (Sambrook et al., 1989). A subsequent round of screening of 8 x 105 phage using a 5'-terminal region of the cDNA (nucleotides 8931208 of the first cDNA) identified another eight cDNA clones. The 5'-terminal ends of two were sequenced. The sequences were assembled using AssemblyLIGN software (Oxford Molecular Ltd).
Epitope tagging of Tbc2
The triple HA epitope was inserted into the SacI site in the middle of the genomic coding sequence of Tbc2 (Fig. 2). The triple HA epitope was also inserted at the COOH-terminal end of the protein. A SalI/HincII site was first introduced upstream of the stop codon of the genomic Tbc2 DNA to allow for the insertion of the tag. The HA epitopecontaining DNAs were introduced into tbc2-F64;cw15 by selecting for restoration of photosynthesis, and the transformants were screened for expression of the HA epitope by immunoblotting. HA epitopetagged strains were maintained on HSM to ensure continued expression of the tagged protein.
Immunoblotting and cell fractionation
Total protein extracts obtained from cell pellets of 10-ml cultures were resuspended in 250 µl HKM (Hepes-KOH 20 mM, pH 7.8, KCl 50 mM, MgCl2 10 mM) containing protease inhibitors: 5 mM -amino caproic acid, 1 mM benzamidine HCl, 25 µg/ml pepstatin A, and 10 µg/ml leupeptin, and then sonicated on ice. A portion of this extract was centrifuged at 100,000 g for 30 min at 4°C, giving rise to a pellet and supernatant fraction. The pellet was washed with 1 ml of STN buffer (0.4 M sucrose, 100 mM Tris, pH 8.0, 10 mM NaCl) and centrifuged again for 15 min at 50,000 g at 4°C to remove soluble contaminating proteins. The pellet was resuspended in lysis buffer (50 mM Tris, pH 6.8, 2% SDS, 10 mM EDTA, and protease inhibitors), incubated for 1 h at room temperature, and centrifuged for 5 min in a microfuge. The resulting supernatant was used as total insoluble protein extract.
Chloroplasts were isolated from a strain carrying the tbc2-F64 and cw15 alleles, which had been transformed with Tbc2:HA. A-500 ml culture of cells grown to mid-log phase was centrifuged and resuspended in 10 ml of breaking buffer (300 mM sorbitol, 50 mM Hepes-KOH, pH 7.8, 5 mM MgCl2) with protease inhibitors. Saponin (S-4521; Sigma-Aldrich) was added to a final concentration of 0.25% (from a freshly made solution of 10% saponin in H2O), incubated for 1 min on ice, and then centrifuged for 3 min at 3,000 rpm (Sorvall, HB6) at 4°C. The pellet containing intact chloroplasts was resuspended carefully in 10 ml of breaking buffer with protease inhibitors, and loaded on a discontinuous 4575% Percoll gradient prepared in breaking buffer with protease inhibitors as described (Zerges and Rochaix, 1998). Chloroplasts were collected at the 4575% interface, washed once in breaking buffer, and osmotically lysed in chloroplast lysis buffer (10 mM Tricine, 5 mM MgCl2, 2 mM DTT, and protease inhibitors). The soluble and insoluble fractions were prepared as described above.. 50 µg of protein were loaded on a 1018% linear gradient SDS-PAGE gel and electroblotted to nitrocellulose membranes, and reacted with antisera against the following proteins or epitopes: HA, D2, SSU Rubisco, and cytosolic thioredoxin-h.
Alternatively, chloroplasts were isolated from tbc2-F64;cw15 cells transformed with HA epitopetagged Tbc2 essentially as described (Zerges and Rochaix, 1998) from continuous 1080% Percoll gradients. Intact chloroplasts were collected, diluted in 0.3 M sorbitol, 50 mM Hepes-KOH, pH 7.8, and 5 mM MgCl2, and centrifuged for 5 min at 4°C. After adding protease inhibitors, the chloroplasts were broken by repeated pipetting in 50 mM Hepes-KOH, pH 7.8, and 5 mM MgCl2, and frozen at -80°C. The lysate was thawed on ice, KCl was added to 50 mM, MgCl2 to 10 mM, (NH4)2SO4 to 0.5 M, and was left to precipitate on ice for 45 min. The lysate was centrifuged at 100,000 g for 30 min to yield a membrane pellet, and a soluble chloroplast extract that was concentrated in a Centricon C-30 (Amicon). 8% gels were loaded with 50 µg protein (to detect Tbc2:HA and eIF4a) or with 10 µg protein (to detect Rubisco and PsaA).
Whole-cell extracts were used for size fractionation. Cells were grown and harvested as above and resuspended in either HMK (20 mM Hepes-KOH, pH 7.8, 50 mM KCl, 10 mM MgCl2) or HEK (20 mM Hepes-KOH, pH 7.8, 50 mM KCl, 10 mM EDTA) plus protease inhibitors (as above) in the presence or absence of 1 mg/ml heparin. Cells were broken by passing once through a small French Press cell (SLM-Aminco; Spectronic Instruments) at 1,380 lb/in2. RNase was added to 10 µg/ml when necessary, and incubated for 10 min at room temperature. The lysate was centrifuged at 35,000 g for 45 min, then at 100,000 g for another 45 min to yield a soluble extract that was concentrated to 1520 mg/ml protein. An aliquot of 50 µl was loaded onto a Superose 6 PC 3.2/30 column using the SMART system (Amersham Pharmacia Biotech), run with either HMK or HEK at 40 µl/min, and 1 1.2 MPa at 4°C. 50-µl fractions were collected, of which 20 µl were loaded on an 8% gel. The molecular weight range of the column was estimated using the MW-GF-1000 size standard kit (Sigma-Aldrich). Polysomes were prepared as described (Boudreau et al., 2000).
Protein samples were incubated with an equal volume of 80 mM Tris-Cl, pH 6.8, 1.6% SDS, 80 mM DTT, 30% glycerol, 8 mM -NH2 caproic acid, 1.6 mM benzamidine, 1.6 µg/ml leupeptin, 1.6 µM E64, 32 µg/ml pepstatin A, 2.6 µM PMSF, 1.6 µM 1,10 o-phenanthroline, and a 1:100 dilution of Sigma protease inhibitors P 8849 for 20 min at ambient temperature before loading on polyacrylamide gels. Gels were transferred to Protran nitrocellulose membranes (Schleicher and Schuell), blocked in a Tris-buffered salt solution containing 0.1% Triton X-100 and 4% nonfat milk for 1 h, and incubated with primary antibody overnight at 4°C. Primary antisera used were a mouse
-HA (1:1,000, Eurogentec),
-thioredoxin-h (1:1,000),
-Rubisco (1:5,000),
-PsbD (D2) (1:10,000), and
-RB60 is rabbit (1:1,000). Horseradish peroxidasecoupled secondary antibody at 1:10,000 was used to detect binding by chemiluminescence (Durrant, 1990).
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Footnotes |
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W. Zerges's present address is Biology Department, Concordia University, 1445 Maisonneuve W., Montreal, Quebec, H3G 1M8, Canada.
* Abbreviations used in this paper: HA, hemagglutinin; PS, photosystem; UTR, untranslated region.
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Acknowledgments |
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This work was supported by grant 3100-050895.97 from the Swiss National Fund.
Submitted: 15 January 2002
Revised: 30 April 2002
Accepted: 30 April 2002
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References |
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