title: An optimized transit peptide for effective targeting of diverse foreign proteins into chloroplasts in rice.
abstract: Various chloroplast transit peptides (CTP) have been used to successfully target some foreign proteins into chloroplasts, but for other proteins these same CTPs have reduced localization efficiencies or fail completely. The underlying cause of the failures remains an open question, and more effective CTPs are needed. In this study, we initially observed that two E.coli enzymes, EcTSR and EcGCL, failed to be targeted into rice chloroplasts by the commonly-used rice rbcS transit peptide (rCTP) and were subsequently degraded. Further analyses revealed that the N-terminal unfolded region of cargo proteins is critical for their localization capability, and that a length of about 20 amino acids is required to attain the maximum localization efficiency. We considered that the unfolded region may alleviate the steric hindrance produced by the cargo protein, by functioning as a spacer to which cytosolic translocators can bind. Based on this inference, an optimized CTP, named RC2, was constructed. Analyses showed that RC2 can more effectively target diverse proteins, including EcTSR and EcGCL, into rice chloroplasts. Collectively, our results provide further insight into the mechanism of CTP-mediated chloroplastic localization, and more importantly, RC2 can be widely applied in future chloroplastic metabolic engineering, particularly for crop plants.
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China.
authors.forename: Bo-Ran
authors.initials: BR
authors.lastname: Shen
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China.
authors.forename: Cheng-Hua
authors.initials: CH
authors.lastname: Zhu
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Yao
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China.
authors.forename: Li-Li
authors.initials: LL
authors.lastname: Cui
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China.
authors.forename: Jian-Jun
authors.initials: JJ
authors.lastname: Zhang
authors.affiliation: College of Life Sciences, South China Normal University, Guangzhou, China.
authors.forename: Cheng-Wei
authors.initials: CW
authors.lastname: Yang
authors.affiliation: Department of Biology, San Francisco State University, San Francisco, California, United States of America.
authors.forename: Zheng-Hui
authors.initials: ZH
authors.lastname: He
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China.
authors.forename: Xin-Xiang
authors.initials: XX
authors.lastname: Peng
mesh_terms: D000595:Amino Acid Sequence; D060365:Chloroplast Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D012275:Oryza / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D017510:Protein Folding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D059748:Proteolysis; D011523:Protoplasts / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D060365:Chloroplast Proteins; D029968:Escherichia coli Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C078737:chloroplast transit peptides; D012273:Ribulose-Bisphosphate Carboxylase