Over 95% of plastid proteins are nuclear-encoded as their precursors containing an N-terminal extension known as the transit peptide （TP）. Although highly variable, TPs direct the precursors through a conserved, pos...Over 95% of plastid proteins are nuclear-encoded as their precursors containing an N-terminal extension known as the transit peptide （TP）. Although highly variable, TPs direct the precursors through a conserved, posttranslational mechanism involving translocons in the outer （TOC） and inner envelope （TOC）. The organelle import specificity is mediated by one or more components of the Toc complex. However, the high TP diversity creates a paradox on how the sequences can be specifically recognized. An emerging model of TP design is that they contain multiple loosely conserved motifs that are recognized at different steps in the targeting and transport process. Bioinformatics has demonstrated that many TPs contain semiconserved physicochemical motifs, termed FGLK. In order to characterize FGLK motifs in TP recognition and import, we have analyzed two well-studied TPs from the precursor of RuBisCO small subunit （SStp） and ferredoxin （Fdtp）. Both SStp and Fdtp contain two FGLK motifs. Analysis of large set mutations （-85） in these two motifs using in vitro, in organello, and in vivo approaches support a model in which the FGLK domains mediate interaction with TOC34 and possibly other TOC components. In vivo import analysis suggests that multiple FGLK motifs are functionally redundant. Furthermore, we discuss how FGLK motifs are required for efficient precursor protein import and how these elements may permit a convergent function of this highly variable class of targeting sequences.展开更多
Chloroplast transit peptides(CTPs) can be used to transport non-chloroplastic proteins into the chloroplasts. Here, we studied the CTPs of three rice(Oryza sativa L.) chloroplast-localized proteins and found that thei...Chloroplast transit peptides(CTPs) can be used to transport non-chloroplastic proteins into the chloroplasts. Here, we studied the CTPs of three rice(Oryza sativa L.) chloroplast-localized proteins and found that their CTPs could be used to transport non-chloroplast-localized proteins into the chloroplasts. Fusion proteins lacking the CTP remained located in the cytoplasm. Furthermore, we constructed green fluorescent protein fusion vectors with the three CTPs and three non-chloroplast-localized proteins, Ghd10, MULTI-FLORET SPIKELET1(MFS1), and SHORTENED UPPERMOST INTERNODE 1(SUI1). After transforming these constructs into rice protoplasts, the fusion proteins all localized in the chloroplasts. Collectively, our results showed that these CTPs can transport non-chloroplast-localized proteins into the chloroplasts, and more importantly, these CTPs can be applied to engineer chloroplast metabolism.展开更多
Transglutaminases (TGases, EC 2.3.2.13) catalyse posttranslational modification of proteins by establishing ε-(γ-glutamyl) links and covalent conjugation of polyamines. In plants, the functionality of these enzymes ...Transglutaminases (TGases, EC 2.3.2.13) catalyse posttranslational modification of proteins by establishing ε-(γ-glutamyl) links and covalent conjugation of polyamines. In plants, the functionality of these enzymes is scarcely known. The maize transglutaminase gene (tgz), the only cloned plant TGase, produces major alterations in thylakoid membrane architecture when the transglutaminase (chlTGZ) protein was over-expressed in tobacco chloroplasts, significantly increasing the number of grana stacked layers. Here we demonstrate that nuclear transformation of rice plants starting from a tgz gene truncated in 17 N-terminal aas (tgzt) non altered chloroplast thylakoid structures. F3 transformed plants were analysed for TGase activity, chlTGZ presence and tgzt transcription levels. Transformed plants exhibited double the in vitro TGase activity of the non-transformed plants. Immunoblot and quantitative RT-PCR analysis results of tgzt-rice plants grown under different illumination periods revealed that chlTGZ maintains its differential expression depending on the light regime. Nevertheless, the maize protein was localised by confocal microscopy in the cell wall of transformed rice cells. TEM analyses of the transformed cells showed normal, non-altered chloroplast thylakoid structures with the maize protein preferentially located in the cell walls. The results confirmed that the tgz eliminated sequence is essential for chloroplast targeting, being its absence sufficient to the lack of protein expression in its original plastidal compartment. Interestingly, the immunolocalization of a putative endogenous rice TGase protein is also showed. These data give further information on plant TGase functionality and its relationship to photosynthetic membranes.展开更多
文摘Over 95% of plastid proteins are nuclear-encoded as their precursors containing an N-terminal extension known as the transit peptide （TP）. Although highly variable, TPs direct the precursors through a conserved, posttranslational mechanism involving translocons in the outer （TOC） and inner envelope （TOC）. The organelle import specificity is mediated by one or more components of the Toc complex. However, the high TP diversity creates a paradox on how the sequences can be specifically recognized. An emerging model of TP design is that they contain multiple loosely conserved motifs that are recognized at different steps in the targeting and transport process. Bioinformatics has demonstrated that many TPs contain semiconserved physicochemical motifs, termed FGLK. In order to characterize FGLK motifs in TP recognition and import, we have analyzed two well-studied TPs from the precursor of RuBisCO small subunit （SStp） and ferredoxin （Fdtp）. Both SStp and Fdtp contain two FGLK motifs. Analysis of large set mutations （-85） in these two motifs using in vitro, in organello, and in vivo approaches support a model in which the FGLK domains mediate interaction with TOC34 and possibly other TOC components. In vivo import analysis suggests that multiple FGLK motifs are functionally redundant. Furthermore, we discuss how FGLK motifs are required for efficient precursor protein import and how these elements may permit a convergent function of this highly variable class of targeting sequences.
基金supported by the National Natural Science Foundation of China(Grant Nos.31601284 and 31661143006)the Transgenic Plant Research and Commercialization Project of the Ministry of Agriculture of China(Grant No.2016ZX08001003-002)+1 种基金Zhejiang Province Outstanding Youth Fund(Grant No.LR16C130001)the Collaborative Innovation Project of the Chinese Academy of Agricultural Sciences(Grant No.Y2016XT05)
文摘Chloroplast transit peptides(CTPs) can be used to transport non-chloroplastic proteins into the chloroplasts. Here, we studied the CTPs of three rice(Oryza sativa L.) chloroplast-localized proteins and found that their CTPs could be used to transport non-chloroplast-localized proteins into the chloroplasts. Fusion proteins lacking the CTP remained located in the cytoplasm. Furthermore, we constructed green fluorescent protein fusion vectors with the three CTPs and three non-chloroplast-localized proteins, Ghd10, MULTI-FLORET SPIKELET1(MFS1), and SHORTENED UPPERMOST INTERNODE 1(SUI1). After transforming these constructs into rice protoplasts, the fusion proteins all localized in the chloroplasts. Collectively, our results showed that these CTPs can transport non-chloroplast-localized proteins into the chloroplasts, and more importantly, these CTPs can be applied to engineer chloroplast metabolism.
文摘Transglutaminases (TGases, EC 2.3.2.13) catalyse posttranslational modification of proteins by establishing ε-(γ-glutamyl) links and covalent conjugation of polyamines. In plants, the functionality of these enzymes is scarcely known. The maize transglutaminase gene (tgz), the only cloned plant TGase, produces major alterations in thylakoid membrane architecture when the transglutaminase (chlTGZ) protein was over-expressed in tobacco chloroplasts, significantly increasing the number of grana stacked layers. Here we demonstrate that nuclear transformation of rice plants starting from a tgz gene truncated in 17 N-terminal aas (tgzt) non altered chloroplast thylakoid structures. F3 transformed plants were analysed for TGase activity, chlTGZ presence and tgzt transcription levels. Transformed plants exhibited double the in vitro TGase activity of the non-transformed plants. Immunoblot and quantitative RT-PCR analysis results of tgzt-rice plants grown under different illumination periods revealed that chlTGZ maintains its differential expression depending on the light regime. Nevertheless, the maize protein was localised by confocal microscopy in the cell wall of transformed rice cells. TEM analyses of the transformed cells showed normal, non-altered chloroplast thylakoid structures with the maize protein preferentially located in the cell walls. The results confirmed that the tgz eliminated sequence is essential for chloroplast targeting, being its absence sufficient to the lack of protein expression in its original plastidal compartment. Interestingly, the immunolocalization of a putative endogenous rice TGase protein is also showed. These data give further information on plant TGase functionality and its relationship to photosynthetic membranes.
