Control of protein life-span by N-terminal methionine excision
2002; Springer Nature; Volume: 22; Issue: 1 Linguagem: Inglês
10.1093/emboj/cdg007
ISSN1460-2075
AutoresCarmela Giglione, Olivier Vallon, Thierry Meinnel,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoArticle2 January 2003free access Control of protein life-span by N-terminal methionine excision Carmela Giglione Carmela Giglione Protein Maturation, Trafficking and Signaling, UPR2355, Centre National de la Recherche Scientifique, Institut des Sciences du Végétal, Bâtiment 23, 1 avenue de la Terrasse, F-91198 Gif-sur-Yvette, cedex, France Search for more papers by this author Olivier Vallon Olivier Vallon Laboratoire de Physiologie Membranaire et Moléculaire du Chloroplaste, UPR1261, Centre National de la Recherche Scientifique, Institut de Biologie Physico-Chimique, 13, rue Pierre et Marie Curie, F-75005 Paris, France Present address: Department of Plant Biology, The Carnegie Institution of Washington, 260 Panama Street, Stanford, CA, 94305 USA Search for more papers by this author Thierry Meinnel Corresponding Author Thierry Meinnel Protein Maturation, Trafficking and Signaling, UPR2355, Centre National de la Recherche Scientifique, Institut des Sciences du Végétal, Bâtiment 23, 1 avenue de la Terrasse, F-91198 Gif-sur-Yvette, cedex, France Search for more papers by this author Carmela Giglione Carmela Giglione Protein Maturation, Trafficking and Signaling, UPR2355, Centre National de la Recherche Scientifique, Institut des Sciences du Végétal, Bâtiment 23, 1 avenue de la Terrasse, F-91198 Gif-sur-Yvette, cedex, France Search for more papers by this author Olivier Vallon Olivier Vallon Laboratoire de Physiologie Membranaire et Moléculaire du Chloroplaste, UPR1261, Centre National de la Recherche Scientifique, Institut de Biologie Physico-Chimique, 13, rue Pierre et Marie Curie, F-75005 Paris, France Present address: Department of Plant Biology, The Carnegie Institution of Washington, 260 Panama Street, Stanford, CA, 94305 USA Search for more papers by this author Thierry Meinnel Corresponding Author Thierry Meinnel Protein Maturation, Trafficking and Signaling, UPR2355, Centre National de la Recherche Scientifique, Institut des Sciences du Végétal, Bâtiment 23, 1 avenue de la Terrasse, F-91198 Gif-sur-Yvette, cedex, France Search for more papers by this author Author Information Carmela Giglione1, Olivier Vallon2,3 and Thierry Meinnel 1 1Protein Maturation, Trafficking and Signaling, UPR2355, Centre National de la Recherche Scientifique, Institut des Sciences du Végétal, Bâtiment 23, 1 avenue de la Terrasse, F-91198 Gif-sur-Yvette, cedex, France 2Laboratoire de Physiologie Membranaire et Moléculaire du Chloroplaste, UPR1261, Centre National de la Recherche Scientifique, Institut de Biologie Physico-Chimique, 13, rue Pierre et Marie Curie, F-75005 Paris, France 3Present address: Department of Plant Biology, The Carnegie Institution of Washington, 260 Panama Street, Stanford, CA, 94305 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:13-23https://doi.org/10.1093/emboj/cdg007 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Peptide deformylases (PDFs) have been discovered recently in eukaryotic genomes, and it appears that N-terminal methionine excision (NME) is a conserved pathway in all compartments where protein synthesis occurs. This work aimed at uncovering the function(s) of NME in a whole proteome, using the chloroplast-encoded proteins of both Arabidopsis thaliana and Chlamydomonas reinhardtii as model systems. Dis ruption of PDF1B in A.thaliana led to an albino phenotype, and an extreme sensitivity to the PDF- specific inhibitor actinonin. In contrast, a knockout line for PDF1A exhibited no apparent phenotype. Photosystem II activity in C.reinhardtii cells was substantially reduced by the presence of actinonin. Pulse–chase experiments revealed that PDF inhibi tion leads to destabilization of a crucial subset of chloroplast-encoded photosystem II components in C.reinhardtii. The same proteins were destabilized in pdf1b. Site-directed substitutions altering NME of the most sensitive target, subunit D2, resulted in similar effects. Thus, plastid NME is a critical mechanism specifically influencing the life-span of photosystem II polypeptides. A general role of NME in modulating the half-life of key subsets of proteins is suggested. Introduction N-terminal methionine excision (NME) is the major pathway causing diversity of N-terminal amino acids. As a result of NME, Gly, Ala, Pro, Cys, Ser, Thr or Val residues may be found at the N-terminus of proteins, in addition to Met (Meinnel et al., 1993). NME was originally described as a cytoplasmic co-translational pathway involving about two of every three proteins in any proteome. NME requires the sequential action of two enzymes: (i) peptide deformylase (PDF), the activity initially described as required for specifically removing the N-formyl group present on all nascent polypeptides synthesized in eubacteria (Giglione et al., 2000a), and (ii) methionine aminopeptidase (MAP), which removes methionine specifically in all organisms (Bradshaw et al., 1998). The removal of the N-formyl group is a prerequisite for the subsequent action of MAP (Solbiati et al., 1999). In contrast with PDF, which acts on almost all polypeptides, MAP activity depends on the nature of the second residue in the target chain. If it is Gly, Ala, Pro, Cys, Ser, Thr or Val, the methionine is cleaved; other wise it is retained. Recently, nuclear-encoded organelle-targeted PDF and MAP have been identified in most genomes, including those of lower eukaryotes, mammals and insects (Giglione et al., 2000a; Meinnel, 2000; Bracchi-Ricard et al., 2001; Giglione and Meinnel, 2001b). At the same time, the basis of NME in higher and lower plants has been described and shown to involve three organellar MAPs, one mitochondria-targeted PDF (PDF1A) and one chloroplast- and mitochondria-targeted PDF (PDF1B) (Giglione et al., 2000b; Giglione and Meinnel, 2001a). These unexpected findings indicate that NME is a conserved pathway in all compartments where protein synthesis occurs. There is a variety of evidence that NME is essential. First, NME is the target of several natural anti-cellular drugs such as actinonin, which is active in bacteria (Chen et al., 2000), and fumagillin, which is active in angiogenic cells, ameba and other human parasites (McCowen et al., 1951; Griffith et al., 1997; Sin et al., 1997; Liu et al., 1998; Zhang et al., 2002). Secondly, PDF and MAP are part of the ∼300-gene minimal genome requirement of eubacteria (Hutchison et al., 1999). Similarly, MAP is present in the extremely reduced genome of the eukaryotic parasite Encephalitozoon cuniculi (∼2000 proteins; Katinka et al., 2001). NME is such a highly conserved function that it is present even in organelles although organellar-encoded proteomes all include 300%) PDF1A than the wild type (Figure 2A), suggesting that the overexpression of the second PDF gene compensated for the defect of the first. In sucrose-minus medium, in which the photosynthetic function of the plastid is required for plant growth, some pdf1b lines grew very slowly and displayed a pronounced albino phenotype without any rapid recovery, whereas others recuperated and greened (Figure 2B). Western blot analysis showed that individuals with a long-lasting albino phenotype contained an amount of PDF1A comparable with the wild type. In contrast, the fast-growing greener plantlets reproducibly had a higher content of PDF1A (Figure 2B). Thus the severe consequences associated with pdf1b disruption could be compensated epigenetically by overexpression of PDF1A. Figure 1.Characterization of the A.thaliana pdf1b line. (A) Schematic representation of pdf1b gene disruption in A.thaliana line pdf1b. The exon–intron structure is shown. Translation initiation and termination codons are indicated. The T-DNA inserts are represented with the 5′ border sequences of the insert (LB) labeled to indicate the orientation of the insertion. A three-base-pair deletion was observed at the site of the insertion as indicated. (B) Presence of PDF1B in wild-type and line pdf1b. Arabidopsis seeds were synchronized in the dark at 4°C for 2 days before sowing. Four hundred milligrams of 2-week-old shoots were homogenized and total proteins were extracted as described. Aliquots (250 μg) of protein were analyzed by SDS–PAGE; 250 ng of cPDF1B, the purified catalytic domain of PDF1B (Serero et al., 2001a), was run in parallel. The gels were blotted and analyzed by western blotting with anti-PDF1B and anti-NMT1 as a control. (C) Albino phenotype of 2-day-old pdf1b plantlet compared with wild type. (D) Intermediate phenotype of 3-day-old pdf1b plantlet compared with wild type. (E) Forty-five-day-old wild-type, pdf1b+/− and pdf1b plantlets, seen from the top. (F) Forty-five-day-old wild-type and pdf1b plantlets, seen from the side. Download figure Download PowerPoint Figure 2.KO pdf1b plants counteract the absence of PDF1B by increasing the level of PDF1A. (A) Expression of PDF1A in wild-type and pdf1b plants grown in 1% sucrose medium. One hundred and fifty micrograms of total protein was separated by SDS–PAGE, transferred to a nitrocellulose membrane and stained with Ponceau S red stain. The membrane was destained and probed using anti-PDF1A and anti-NMT1 antibodies. (B) Wild-type and pdf1b seeds were sown in a sucrose-minus growth medium, synchronized in the dark at 4°C for 2 days and then incubated in a growth cabinet. Seedlings were photographed 2 weeks later and each phenotype was collected separately (I, albino; II, greening). Aliquots (150 μg) of total protein extract were analyzed by 14% SDS–PAGE and western blotting as in (A). Download figure Download PowerPoint Isolation and characterization of an A.thaliana pdf1a line: the PDF1A deficiency is fully compensated by PDF1B The albino phenotype of line pdf1b and its reversibility indicate that the deformylation process is essential in the chloroplast by affecting the biogenesis of the photosynthetic apparatus. Moreover, this process seems to be so essential that the absence of PDF1B in KO pdf1b plants is counteracted by overproduction of PDF1A. This is consistent with PDF1A being routed to the chloroplast when overproduced, as observed in transient expression studies (Giglione and Meinnel, 2001a). Therefore, we investigated the effect of pdf1a inactivation. Various T-DNA mutant collections were screened for insertions in pdf1a. Two independent mutant lines were identified (Figure 3A). Homozygous lines were obtained in each case and western blotting experiments, performed to check for effective gene knockout, showed that the line named pdf1a was a true PDF1A KO mutant (Figure 3B). Line CS15–187 still produced the PDF1A protein, probably because the T-DNA insertion was located in the promoter region too far (∼300 bases) from the transcribed sequence. Mutant pdf1a behaved similarly to the wild type and displayed no visible phenotype under standard growth conditions (data not shown). This was not unexpected, as both PDF1A and PDF1B are present in the mitochondria (Giglione et al., 2000b). These data suggest that PDF1B can fully compensate for the absence of PDF1A from mitochondria despite their biochemical differences (Serero et al., 2001a). Finally, PDF1B was not overproduced in line pdf1a (Figure 3B) unlike PDF1A in line pdf1b (Figure 2B). Figure 3.Characterization of the A.thaliana pdf1a line. (A) Schematic representation of PDF1A gene disruption in A.thaliana lines CS1813–43 and CS15–187. The exon–intron structure is shown as in Figure 1. Translation initiation and termination codons are indicated. The T-DNA inserts are represented with the 5′ border sequences of the insert (LB) labeled to indicate the orientation. (B) PDF1A protein in A.thaliana wild-type, pdf1a and CS15–187 lines. Aliquots (250 μg) of total protein extracts were analyzed by 14% SDS–PAGE. Western blots were performed with anti-PDF1A, anti-PDF1B and anti-NMT1 antibodies. Download figure Download PowerPoint Deformylase activity is essential in the chloroplast: plastid PDF1B is the main target of actinonin in plants Actinonin specifically inhibits bacterial PDF (Chen et al., 2000). In A.thaliana, it induces an albino phenotype at concentrations higher than 100 μM, indicating an alteration of plastid biogenesis (Giglione and Meinnel, 2001a; Serero et al., 2001b). Both plant PDFs are highly sensitive to actinonin in vitro (Serero et al., 2001a). Moreover, A.thaliana plantlets treated with actinonin only grow from germination to flowering on media containing a reduced carbon source; under these conditions no deleterious effects on development are observed. Thus it has been suggested that mitochondria were unaffected by the drug possibly because at submillimolar concentrations in the growth medium it could not penetrate these organelles (Serero et al., 2001a). To determine whether the albino phenotype induced by actinonin is due to inhibition of plastid PDF in vivo and which PDF is the target of the drug, lines pdf1a and pdf1b were grown in the presence of various concentrations of actinonin. The sensitivity of the pdf1a line was the same as that of the wild type (Figure 4). Thus inhibition of PDF1A contributed little to the effect of actinonin in the wild type. In contrast, pdf1b was about three orders of magnitude more sensitive than the wild type to the drug (Figure 4). Pronounced bleaching was observed when pdf1b was challenged after the recovery stage with submicro molar concentrations of actinonin, whereas 100 μM was necessary to obtain the same effect in the wild type or in the pdf1a line. Thus actinonin treatment mimics PDF1B inactivation, indicating that plastid PDF1B is the major and primary target of actinonin in plants. The residual sensitivity of pdf1b to actinonin also indicated that the PDF1A protein overexpressed in line pdf1b was fully accessible and sensitive to the drug, consistent with a plastid localization. Figure 4.Line pdf1b is hypersensitive to actinonin. Plants were grown for 5 days in the presence of the indicated (top) concentration of actinonin (μM): (A) wild type; (B) pdf1a line; (C) pdf1b line. Download figure Download PowerPoint These experiments showed that actinonin treatment at high concentrations in the wild-type line or at low concentrations in line pdf1b affected plastid development via complete inhibition of plastid PDF activity. This demonstrates (i) that NME is essential in plastids, and (ii) that actinonin is a suitable specific drug to block plastid NME. Absence of plastid deformylation leads to a reduction of PSII efficiency in C.reinhardtii We next investigated why deformylation is so important for plastid function, and whether blocking chloroplastic deformylation affects the behavior of all or only a subset of plastid proteins. The protein targets of PDF are restricted to the ∼80 plastid-encoded proteins, most of which under go N-deformylation (Giglione and Meinnel, 2001a). C.reinhardtii has dual PDF apparatus similar to that of land plants (Giglione and Meinnel, 2001a). This unicellular green alga features a single large plastid and has long been exploited for molecular studies of chloroplast function, as it allows powerful molecular genetics techniques to be used (Dent et al., 2001). These techniques include plastid transformation, which is not currently possible in Arabidopsis. Moreover, studies of photosynthesis in vivo, and particularly those of the light reactions, have benefited from a vast array of biophysical techniques used to examine specific components of the photosynthetic apparatus. To test C.reinhardtii sensitivity to deformylation inhibition, cells were grown in the presence and the absence of actinonin, and the fluorescence induction kinetics was followed. There was a strong dose-dependent decrease of the variable fluorescence Fv/Fmax (Figure 5A), an indicator of PSII function (Lavergne and Briantais, 1995). When the culture reached stationary phase, PSII activity was reduced by >70%. In contrast, the other fluorescence parameters indicative of photosystem I or cytochrome b6f functions were unaffected, suggesting that the other components of the photosynthetic apparatus were insensitive to the block of deformylation. Figure 5.Actinonin leads to a reduction of PSII efficiency in C.reinhardtii. (A) Measurement of the variable fluorescence parameter Fv/Fmax upon actinonin addition (circles, 0 μM; triangles, 50 μM; squares, 500 μM) at time zero in early exponential-phase cultures. (B) Photo-inhibition experiments were carried out for 30 min when the variable fluorescence reached zero. The time-course of recovery in the absence (circles) or the presence (squares) of 0.5 mM actinonin was followed. To visualize only the fraction of recovery that depends on de novo protein synthesis (70%), we subtracted the Fv/Fmax measured at each time point in a duplicate sample incubated with 20 μg/ml chloramphenicol. Download figure Download PowerPoint PSII activity is sensitive to high light treatment (photo-inhibition) (Aro et al., 1993). PSII recovery after photo-inhibition depends on two components, one involving post-translational repair of photodamaged subunits and the other relying on the synthesis of new proteins. To investigate the molecular effect of actinonin on PSII biogenesis, recovery experiments were followed after high light exposure in the presence of the drug. Each sample was analyzed in the presence and the absence of chloramphenicol, a specific inhibitor of organelle protein synthesis. PSII recovery was measured by monitoring the variable fluorescence Fv/Fmax (Figure 5B). Actinonin did not impair the protein-synthesis-independent (i.e. chloramphenicol-insensitive) recovery of PSII. In contrast, de novo synthesis of chloroplast-encoded PSII components appeared to be significantly reduced by the presence of the drug. We concluded that the inhibition of PSII in the presence of actinonin was due to a defect limiting the accumulation of one or several PSII components. Inhibition of plastid deformylation in C.reinhardtii leads to rapid degradation of newly synthesized PSII core subunits Pulse–chase experiments were carried out either immediately or 48 h after addition of actinonin. Labeling was performed in the presence of cycloheximide, so that only chloroplast-encoded proteins were labeled. In the sample treated for 48 h (Figure 6A), [14C]acetate incorporation into proteins was indistinguishable from that in untreated controls. This indicated that the plastid protein synthesis machinery, i.e. the components of the plastid RNA polymerases or those of the plastid ribosomes, was not affected by PDF inhibition. Therefore the inhibitory effect of actinonin was not due to a general block of protein synthesis. SDS–PAGE analysis of the various plastid-encoded (i.e. labeled) proteins showed that the large subunit of Rubisco (RbcL), the components of photo system I (PsaA and PsaB), ATPase (AtpA and AtpB) and cytochrome b6/f (Cytf) accumulated normally. These data were confirmed by western blotting analysis and chase experiments (data not shown). In contrast, the signals corresponding to several components of PSII, including D1, D2, CP43 and CP47 were significantly lower than in the control (Figure 6A). Figure 6.Inhibition of PDF induces a rapid degradation of plastid-encoded PSII subunits in C.reinhardtii. The experiments were performed in the presence (+) or the absence (−) of 0.5 mM actinonin. The location of several plastid proteins identified by western blot is indicated. (A) Samples were 45 min pulse-labeled with [14C]acetate 48 h after addition of actinonin. A phosphoimage of the urea-SDS–PAGE (12%–18% acrylamide) is shown. (B) Time-course pulse-labeling with [14C]acetate was performed 10 min after addition of actinonin. The contrast in the lower part of the gel was intensified to show PsbH and its derivatives, PsbH′ and PsbH″, more clearly. (C) Quantification of the data reported in (B) for CP43, CP47, D1, D2 (D2.1 + D2.2), RbcL and AtpB. (D) Pulse–chase labeling of whole cells with [35S]sulfate. A close-up of the phosphoimage of the SDS–PAGE (7.5%–15% acrylamide) in the region of the RbcL band is shown. AtpA and AtpB are not separated under these conditions and co-migrate just below RbcL. (E) Phosphoimage of [35S]sulfate-labeled membrane fractions analyzed by two-dimensional PAGE involving native (horizontal; top right) followed by denaturing conditions of separation (vertical). D1 and D2 were localized on the gels by western blotting analysis with specific antibodies (data not shown). Download figure Download PowerPoint Figure 7.The accumulation of PSII components is affected in A.thaliana pdf1b lines as in actinonin-treated C.reinhardtii cells. Plants were grown for 15 days. Proteins were analyzed by SDS–PAGE. Western blot analysis was performed using anti-D2, anti-CP43 and anti-CP47 antibodies. (A) Wild-type and pdf1b lines were grown in the absence of sucrose. Plantlets were collected as described in Figure 2. Aliquots (150 μg) of total proteins were loaded on the gel, transferred onto a nitrocellulose membrane and stained with Ponceau S red stain. The position of RbcL is indicated. (B) The destained nitrocellulose membrane from (A) was analyzed by western blotting. (C) Wild-type and pdf1b lines were grown in the absence (−) or the presence (+) of 1 μM actinonin. One-hundred-milligram samples of membrane proteins were analyzed by western blotting. Download figure Download PowerPoint Time-course pulse-labeling experiments performed immediately after actinonin addition (Figure 6B and C) showed that the accumulation of the same set of PSII components was affected. At t = 5 min, the whole plastid protein set was synthesized normally in the presence of actinonin, except for a reduction of D2 labeling. After longer labeling times, the accumulation of each D1, D2, CP43 and CP47 was very much lower than in the wild type. This effect was dose dependent (data not shown). Despite the general reduction of D2 labeling in the actinonin-treated sample, the D2.2 band was converted to D2.1 by phosphorylation of the mature N-terminal threonine (Thr2) residue (Vener et al., 2001). This indicated that D2 accumulation, but not its capacity to be phosphorylated, was inhibited by actinonin. In another experiment, D2 synthesized in the presence of actinonin was completely degraded during 10 min chase experiments in the absence of the drug, in contrast with D1, CP43 and CP47, which were stable for >40 min (data not shown; see also Figure 8, T2T). Figure 8.Inhibiting D2 N-terminal methionine excision by changing its second residue leads to effects similar to those induced by actinonin. Pulse–chase experiments with the four C.reinhardtii psbD mutant strains were performed in the absence (−) or the presence (+) of 0.5 mM actinonin. Cells were pulse-labeled with [14C]acetate for 5 min; chase duration was 40 min. Chloroplast proteins were analyzed by urea-SDS–PAGE. Pulse experiments were carried out for 40 min. The positions of several plastid proteins are indicated. The relative position of D2 in various lanes of interest is labeled with an asterisk on the left-hand side of the corresponding band. Download figure Download PowerPoint In addition to the PSII components D1, D2, CP43 and CP47, PsbH, a more peripheral component of the PSII complex, was also destabilized by actinonin. As described previously (de Vitry et al., 1991), the control experiment showed the appearance of two bands (PsbH′ and PsbH″) due to the phosphorylation of residues Thr2 and Thr4 (Gomez et al., 2002). In the presence of actinonin, the mobility of the unphosphorylated form was affected. The electrophoretic mobility of such small bacterial proteins is similarly shifted following actinonin treatment inhibiting PDF and thus inhibiting processing of the N-terminus (Apfel et al., 2001; Solbiati et al., 2002). Interestingly, only one additional band, of intermediate mobility, was detected (Figure 6B, bottom), suggesting an effect on one of the two phosphorylation events. In addition, the RbcL newly synthesized in the presence of actinonin had a mobility lower than normal, most clearly evidenced in a urea-free gel system (Figure 6D). This shift probably reflects an actinonin-induced block of the multiple pro cessing events that RbcL undergoes at its N-terminus. This processing, which is unique to RbcL in the plastid, is initiated by the PDF-catalyzed removal of N-formyl (see references in Giglione and Meinnel, 2001a). It is remarkable that the block of this processing is not accompanied by a reduction in protein accumulation. Hence, of all the chloroplast-encoded proteins analyzed, only the five PSII proteins described above showed abnormally low accumulation. Since actinonin has no general effect on protein synthesis, this strongly suggests specific destabilization of these components. The stability of polypeptides engaged in multi-subunit protein complexes is largely a function of their ability to assemble with their partners (Choquet and Vallon, 2000). To analyze the effect of actinonin on PSII assembly more directly, we used two-dimensional native gel electrophoresis after [35S]sulfate pulse-labeling. A large proportion of the PSII-associated label was incorporated into a complex of ∼360 kDa (Figure 6E). This is consistent with the co-translational assembly of D1 into PSII centers. D2 is also incorporated directly into this complex, as expected from the comparable synthesis rates of the two polypeptides. In addition, lower molecular weight complexes containing various amounts of D1 and D2 (pre-PSII) were evidenced. That these latter are true assembly intermediates, originating from de novo assembled complexes, was supported by western blotting analysis and chase experiments (data not shown). The intensity of labeling associated with these assembly intermediates was reduced by actinonin treatment, probably due to enhanced sensitivity to degradation. We conclude that NME inhibition acts primarily by committing specific sensitive PSII subunits to the proteolytic pathway. In contrast, despite the alteration of its N-terminal processing, Rubisco assembly was found to proceed normally and all intermediary complexes were detected in normal amounts in the soluble fraction (data not shown). ClpP is a major ATP-dependent protease involved in the N-end rule degradation mechanism of bacterial proteins (Tobias et al., 1991). We tested whether PSII degradation occurred via the plastid ClpP protease as in the case of cytochrome b6f (Majeran et al., 2000). ClpP is an essential gene in C.reinhardtii, and therefore we used a ClpP mutant with significantly reduced expression of the protein (Majeran et al., 2000). The rate of actinonin-dependent protein degradation in this Clp mutant was identical with that in the control (data not shown). Thus D2 and the other components of PSII were presumably degraded via a ClpP-independen
Referência(s)