In of domain I of EF-G are absent. LEPA has a special C-terminal domain called CTD with an unusual fold which might interact with tRNA or 23S rRNA [2]. Although the overall structure of LEPA has been described in great detail, the physiological functions involved in translation have not yet been resolved. In E. coli, LEPA is located upstream of the LEP gene, which encodes nonspecific signal peptidase I [3]. Deletion of LEPA does not cause any apparent phenotype under optimal growth conditions [4,5]. These observations are difficult to reconcile with the ubiquity of LEPA and its extreme conservation. Other results have demonstrated that, although E. coli LEPAdefective cells grown in rich medium have no phenotype [4], under several stress conditions, including high salt, low pH, and low temperature, the LEPA mutant is overgrown by wild-type bacterial cells [6]. In bacteria, DLEPA strains have been shown to be hypersensitive to potassium tellurite and penicillin [7] and to enhance the production of the calcium-dependent antibiotic in Streptomyces bacteria [8]. Recent studies get Licochalcone A suggested that LEPA may react with both the PRE and POST ribosome complexes, leading to the formation of an intermediate complex that effectively sequesters a catalytically active ribosome, resulting in a transientinhibition of elongation that provides a mechanism for the optimization of functional protein synthesis [9,10]. The physiological function of the chloroplast homologs of LEPA (cpLEPA) in vivo has not been characterized. In this study, we report the identification of an Arabidopsis DLEPA mutant, which was termed cplepa-1. A slightly high chlorophyll fluorescence and pale green phenotype are detected in the cplepa-1mutant when grown under normal growth conditions. Physiological and biochemical analyses of the mutant revealed that cpLEPA has an important function in chloroplast biogenesis and plays an essential role in chloroplast translation.Results Chloroplast LEPA in Arabidopsis is a Highly Conserved Homolog of EF-GDatabase searches and protein UKI 1 sequence alignments revealed that cpLEPA shares significant sequence identity with its homologs, from bacteria to eukaryotes (64 ?7 ) (Figure 1). CpLEPA encodes a 681-amino acid protein with a calculated molecular mass of 75 kD. This protein was predicted to be localized to the chloroplast, and the N-terminal 51 amino acids were predicted to be a chloroplast transit peptide by the programs TargetP 1.1 and ChloroP 1.1 (Figure 1). Analysis by the TMHMM program suggests that cpLEPA does not contain a transmembrane domain (data not shown). Four out of the five CpLEPA domains share strong similarity to the counterpart of EF , except for domain IV, whereas the CTD is unique to cpLEPA (Figure 1).cpLEPA in Chloroplast TranslationFigure 1. CpLEPA Protein Sequence Alignment. The amino acid sequence of cpLEPA was compared 15857111 with the sequences of homologous proteins from mitochondria in Arabidopsis, Oryza sativa, Glycine max, Physcomitrella patens, Hordeum vulgare, Micromonas pusilla, Synechococcus, Microcystis aeruginosa, and Bacillus cereus. The black boxes indicate strictly conserved amino acids, and the gray boxes indicate closely related residues. The predicted chloroplast transmembrane peptides are underlined in green, The LEPA domains are underlined in red, and the LEPA-II domain is underlined in blue. LEPA-C is underlined in purple, and the CTD is underlined in yellow. doi:10.1371/journal.pone.0049746.gcpLEPA in Chloroplast.In of domain I of EF-G are absent. LEPA has a special C-terminal domain called CTD with an unusual fold which might interact with tRNA or 23S rRNA [2]. Although the overall structure of LEPA has been described in great detail, the physiological functions involved in translation have not yet been resolved. In E. coli, LEPA is located upstream of the LEP gene, which encodes nonspecific signal peptidase I [3]. Deletion of LEPA does not cause any apparent phenotype under optimal growth conditions [4,5]. These observations are difficult to reconcile with the ubiquity of LEPA and its extreme conservation. Other results have demonstrated that, although E. coli LEPAdefective cells grown in rich medium have no phenotype [4], under several stress conditions, including high salt, low pH, and low temperature, the LEPA mutant is overgrown by wild-type bacterial cells [6]. In bacteria, DLEPA strains have been shown to be hypersensitive to potassium tellurite and penicillin [7] and to enhance the production of the calcium-dependent antibiotic in Streptomyces bacteria [8]. Recent studies suggested that LEPA may react with both the PRE and POST ribosome complexes, leading to the formation of an intermediate complex that effectively sequesters a catalytically active ribosome, resulting in a transientinhibition of elongation that provides a mechanism for the optimization of functional protein synthesis [9,10]. The physiological function of the chloroplast homologs of LEPA (cpLEPA) in vivo has not been characterized. In this study, we report the identification of an Arabidopsis DLEPA mutant, which was termed cplepa-1. A slightly high chlorophyll fluorescence and pale green phenotype are detected in the cplepa-1mutant when grown under normal growth conditions. Physiological and biochemical analyses of the mutant revealed that cpLEPA has an important function in chloroplast biogenesis and plays an essential role in chloroplast translation.Results Chloroplast LEPA in Arabidopsis is a Highly Conserved Homolog of EF-GDatabase searches and protein sequence alignments revealed that cpLEPA shares significant sequence identity with its homologs, from bacteria to eukaryotes (64 ?7 ) (Figure 1). CpLEPA encodes a 681-amino acid protein with a calculated molecular mass of 75 kD. This protein was predicted to be localized to the chloroplast, and the N-terminal 51 amino acids were predicted to be a chloroplast transit peptide by the programs TargetP 1.1 and ChloroP 1.1 (Figure 1). Analysis by the TMHMM program suggests that cpLEPA does not contain a transmembrane domain (data not shown). Four out of the five CpLEPA domains share strong similarity to the counterpart of EF , except for domain IV, whereas the CTD is unique to cpLEPA (Figure 1).cpLEPA in Chloroplast TranslationFigure 1. CpLEPA Protein Sequence Alignment. The amino acid sequence of cpLEPA was compared 15857111 with the sequences of homologous proteins from mitochondria in Arabidopsis, Oryza sativa, Glycine max, Physcomitrella patens, Hordeum vulgare, Micromonas pusilla, Synechococcus, Microcystis aeruginosa, and Bacillus cereus. The black boxes indicate strictly conserved amino acids, and the gray boxes indicate closely related residues. The predicted chloroplast transmembrane peptides are underlined in green, The LEPA domains are underlined in red, and the LEPA-II domain is underlined in blue. LEPA-C is underlined in purple, and the CTD is underlined in yellow. doi:10.1371/journal.pone.0049746.gcpLEPA in Chloroplast.
