Ovides 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 25033180 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 buy Sudan I 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 transFD&C Yellow 5 web membrane 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 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 TranslationCpLEPA is Associated with the Thylakoid MembraneTo investigate the localization of cpLEPA, intact chloroplasts were isolated and fractionated, and the proteins were subjected to immunoblot 
analysis with a specific cpLEPA antibody. Under normal growth conditions (120 mmol m22 s21), most of the cpLEPA protein was detected in the thylakoid fractions (Figure 2A), and the ratio of cpLEPA in the stroma to cpLEPA in the thylakoid membrane was approximately 0.25. These results indicate that cpLEPA is a membrane-associated protein. To further investigate the degree of membrane association of cpLEPA, we treated the thylakoid membrane with salts and chaotropic agents. Washing the membrane with 0.25 M NaCl did not release the cpLEPA from the membrane, but cpLEPA was barely detectable after washing the membrane with 0.2 M Na2CO3, 1 M CaCl2, or 6 M urea. As a control, the integral membrane protein CP47 was not released from the membranes by such treatments. RBcL, which is located in the stroma and thylakoid membrane, yielded results similar to those of cpLEPA (Figure 2B). CpLEPA is widely expressed in most Arabidopsis green tissues, including the seedlings, leaves, stems, siliques, flowers and cauline tissue (not in the roots), but the expression levels of cpLEPA in seedlings and cauline tissue are reduced compared w.Ovides 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 25033180 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 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 TranslationCpLEPA is Associated with the Thylakoid MembraneTo investigate the localization of cpLEPA, intact chloroplasts were isolated and fractionated, and the proteins were subjected to immunoblot analysis with a specific cpLEPA antibody. Under normal 
growth conditions (120 mmol m22 s21), most of the cpLEPA protein was detected in the thylakoid fractions (Figure 2A), and the ratio of cpLEPA in the stroma to cpLEPA in the thylakoid membrane was approximately 0.25. These results indicate that cpLEPA is a membrane-associated protein. To further investigate the degree of membrane association of cpLEPA, we treated the thylakoid membrane with salts and chaotropic agents. Washing the membrane with 0.25 M NaCl did not release the cpLEPA from the membrane, but cpLEPA was barely detectable after washing the membrane with 0.2 M Na2CO3, 1 M CaCl2, or 6 M urea. As a control, the integral membrane protein CP47 was not released from the membranes by such treatments. RBcL, which is located in the stroma and thylakoid membrane, yielded results similar to those of cpLEPA (Figure 2B). CpLEPA is widely expressed in most Arabidopsis green tissues, including the seedlings, leaves, stems, siliques, flowers and cauline tissue (not in the roots), but the expression levels of cpLEPA in seedlings and cauline tissue are reduced compared w.