Successful import of hundreds of nucleus-encoded proteins is essential for chloroplast biogenesis. The import of cytosolic precursor proteins relies on the Toc- （translocon at the outer chloroplast membrane） and Ti...Successful import of hundreds of nucleus-encoded proteins is essential for chloroplast biogenesis. The import of cytosolic precursor proteins relies on the Toc- （translocon at the outer chloroplast membrane） and Tic- （translocon at the inner chloroplast membrane） complexes. In Arabidopsis thaliana, precursor recognition is mainly mediated by outer membrane receptors belonging to two gene families： Toc34/33 and Toc159/132/120/90. The role in import and precursor selectivity of these receptors has been intensively studied, but the function of Toc90 still remains unclear. Here, we report the ability of Toc90 to support the import of Toc159 client proteins. We show that the overexpression of Toc90 partially complements the albino knockout of Toc159 and restores photoautotrophic growth. Several lines of evidence including proteome profiling demonstrate the import and accumulation of proteins essential for chloroplast biogenesis and functionality.展开更多
Chloroplast-located proteins which are encoded by the nuclear genome have to be imported from the cytosol into the organelle in a posttranslational manner. Among these nuclear-encoded chloroplast proteins are the ligh...Chloroplast-located proteins which are encoded by the nuclear genome have to be imported from the cytosol into the organelle in a posttranslational manner. Among these nuclear-encoded chloroplast proteins are the light- harvesting chlorophyll a/b-binding proteins （LHCPs）. After translation in the cytosol, precursor proteins of LHCPs are imported via the TOC/TIC translocase, processed to their mature size to insert into thylakoid membranes where they recruit chlorophylls a and b to form pigment-protein complexes. The translocation of proteins is a highly regulated process which employs several regulators. To analyze whether CAO （chlorophyll a oxigenase） which converts chlorophyll a to chlorophyll b at the inner chloroplast membrane, is one of these regulators, we performed import reactions utilizing a homozygous loss-of-function mutant （cao-1）. We imported in vitro translated and 35S-labeled precursor proteins of light- harvesting proteins of photosystem II LHCB1, LHCB4, and LHCB5 into chloroplasts isolated from cao-1 and show that import of precursor proteins and their processing to mature forms are not impaired in the mutant. Therefore, regulation of the import machinery cannot be responsible for the decreased steady-state levels of light-harvesting complex （LHC） proteins. Regulation does not take place at the transcriptional level either, because Lhcb mRNAs are not down-regulated. Additionally, reduced steady-state levels of LHCPs also do not occur due to posttranslational turnover of non-functional LHCPs in chloroplasts. Taken together, our data show that plants in the absence of CAO and therefore devoid of chlorophyll b are not influenced in their import behavior of LHC proteins.展开更多
Protein translocation across membranes is a fundamental cellular process. The majority of the proteins of organelles such as mitochondria and chloroplasts is synthesized in the cytosol and subsequently imported in a p...Protein translocation across membranes is a fundamental cellular process. The majority of the proteins of organelles such as mitochondria and chloroplasts is synthesized in the cytosol and subsequently imported in a posttranslational manner. The precursor proteins have to be unfolded at least for translocation, but it has also been assumed that they are unfolded during transport to the organelle in the cytosol. Unfolding is governed by chaperones and the translocon itself. At the same time, chaperones provide the energy for the import process. The energetic properties of the chloroplast translocon were studied by import of the Ig-like module of the muscle protein titin fused to the transit peptide of the chloroplast targeted oxygen evolving complex subunit of 33 kDa （OE33）. Our results suggest that p（OE33）titin is folded prior to import and that translocation is initiated by unfolding after having bound to the translocon at the chloroplast surface. Using a set of stabilizing and destabilizing mutants of titin previously analyzed by atomic force microscopy and as passenger for mitochondrial translocation, we studied the unfolding force provided by the chloroplast translocon. Based on these results, a model for translocation is discussed.展开更多
The TicS5 （Translocon at the inner envelope membrane of chloroplasts, 55 kDa） protein was identified in pea as a putative regulator, possibly linking chloroplast protein import to the redox state of the photosynthet...The TicS5 （Translocon at the inner envelope membrane of chloroplasts, 55 kDa） protein was identified in pea as a putative regulator, possibly linking chloroplast protein import to the redox state of the photosynthetic machinery. Two Tic55 homologs have been proposed to exist in Arabidopsis： atTic55-11 and AtPTC52 （Protochlorophyllide-dependent Trans- Iocon Component, 52 kDa; has also been called atTic55-1V）. Our phylogenetic analysis shows that attic55-11 is an ortholog of psTic55 from pea （Pisum sativurn）, and that AtPTC52 is a more distant homolog of the two. AtPTC52 was included in this study to rule out possible functional links between the proteins in Arabidopsis. No detectable mutant phenotypes were found in two independent T-DNA knockout mutant plant lines for each Arabidopsis protein, when compared with wild- type： visible appearance, chlorophyll content, photosynthetic performance, and chloroplast protein import, for example, were all normal. Both wild-type and tic55-11 mutant chloroplasts exhibited deficient protein import when treated with diethylpyrocarbonate, indicating that Tic55 is not the sole target of this reagent in relation to protein import. Furthermore, ptc52 mutant chloroplasts were not defective with respect to pPORA import, which was previously reported to involve PTC52 in barley. Thus, we conclude that atTic55-11 and AtPTC52 are not strictly required for functional protein import in Arabidopsis.展开更多
Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide （TP） directing them specifically to a correct organelle. However, th...Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide （TP） directing them specifically to a correct organelle. However, there is a group of proteins that are dually targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide （dTP）. Here, we have investigated pattern properties of import determinants of organelle-specific TPs and dTPs combining mathematical multivariate data analysis （MVDA） with in vitro organellar import studies. We have used large datasets of mitochondrial and chloroplastic proteins found in organellar proteomes as well as manually selected data sets of experimentally confirmed organelle-specific TPs and dTPs from Arabidopsis thaliana. Two classes of organelle-specific TPs could be distinguished by MVDA and potential patterns or periodicity in the amino acid sequence contributing to the separation were revealed, dTPs were found to have intermediate sequence features between the organelle-specific TPs. Interestingly, introducing positively charged residues to the dTPs showed clustering towards the mitochondrial TPs in silico and resulted in inhibition of chloroplast, but not mitochondrial import in in vitro organellar import studies. These findings suggest that positive charges in the N-terminal region of TPs may function as an ＇avoidance signal＇ for the chloroplast import.展开更多
Most chloroplast and mitochondrial proteins are synthesized in the cytosol of the plant cell and have to be imported into the organelles post-translationally. Molecular chaperones play an important role in preventing ...Most chloroplast and mitochondrial proteins are synthesized in the cytosol of the plant cell and have to be imported into the organelles post-translationally. Molecular chaperones play an important role in preventing protein aggregation of freshly translated preproteins and assist in maintaining the preproteins in an import competent state. Pre- proteins can associate with HSP70, HSP90, and 14-3-3 proteins in the cytosol. In this study, we analyzed a large set of wheat germ-translated chloroplast preproteins with respect to their chaperone binding. Our results demonstrate that the formation of distinct 14-3-3 or HSP90 containing preprotein complexes is a common feature in post-translational protein transport in addition to preproteins that seem to interact solely with HSP70. We were able to identify a diverse and extensive class of preproteins as HSP90 substrates, thus providing a tool for the investigation of HSP90 client protein association. The analyses of chimeric HSP90 and 14-3-3 binding preproteins with exchanged transit peptides indicate an involvement of both the transit peptide and the mature part of the proteins, in HSP90 binding. We identified two partner components of the HSP90 cycle, which were present in the preprotein containing high-molecular-weight complexes, the HSP70/HSP90 organizing protein HOP, as well as the immunophilin FKBP73. The results establish chloroplast preproteins as a general class of HSP90 client proteins in plants using HOP and FKBP as novel cochaperones.展开更多
Protein import into chloroplasts has been a focus of research for several decades. The first publications dealing with this fascinating topic appeared in the 1970s. From the initial realization that many plastid prote...Protein import into chloroplasts has been a focus of research for several decades. The first publications dealing with this fascinating topic appeared in the 1970s. From the initial realization that many plastid proteins are being encoded for in the nucleus and require transport into their target organelle to the identification of import components in the cytosol, chloroplast envelopes, and stroma, as well as elucidation of some mechanistic details, more fascinating aspects are still being unraveled. With this overview, we present a survey of the beginnings of chloroplast protein import research, the first steps on this winding road, and end with a glimpse into the future.展开更多
The chloroplast is surrounded by a double-membrane envelope at which proteins, ions, and numerous metabolites including nucleotides, amino acids, fatty acids, and carbohydrates are exchanged between the two aqueous ph...The chloroplast is surrounded by a double-membrane envelope at which proteins, ions, and numerous metabolites including nucleotides, amino acids, fatty acids, and carbohydrates are exchanged between the two aqueous phases, the cytoplasm and the chloroplast stroma. The chloroplast envelope is also the location where the biosynthesis and accumulation of various lipids take place. By contrast to the inner membrane, which contains a number of specific transporters and acts as the permeability barrier, the chloroplast outer membrane has often been considered a passive compartment derived from the phagosomal membrane. However, the presence of galactoglycerolipids and β-barrel membrane proteins support the common origin of the outer membranes of the chloroplast envelope and extant cyanobacteria. Furthermore, recent progress in the field underlines that the chloroplast outer envelope plays important roles not only for translocation of various molecules, but also for regulation of metabolic activities and signaling processes. The chloroplast outer envelope membrane offers various interesting and challenging questions that are relevant to the understanding of organelle biogenesis, plant growth and development, and also membrane biology in general.展开更多
文摘Successful import of hundreds of nucleus-encoded proteins is essential for chloroplast biogenesis. The import of cytosolic precursor proteins relies on the Toc- （translocon at the outer chloroplast membrane） and Tic- （translocon at the inner chloroplast membrane） complexes. In Arabidopsis thaliana, precursor recognition is mainly mediated by outer membrane receptors belonging to two gene families： Toc34/33 and Toc159/132/120/90. The role in import and precursor selectivity of these receptors has been intensively studied, but the function of Toc90 still remains unclear. Here, we report the ability of Toc90 to support the import of Toc159 client proteins. We show that the overexpression of Toc90 partially complements the albino knockout of Toc159 and restores photoautotrophic growth. Several lines of evidence including proteome profiling demonstrate the import and accumulation of proteins essential for chloroplast biogenesis and functionality.
文摘Chloroplast-located proteins which are encoded by the nuclear genome have to be imported from the cytosol into the organelle in a posttranslational manner. Among these nuclear-encoded chloroplast proteins are the light- harvesting chlorophyll a/b-binding proteins （LHCPs）. After translation in the cytosol, precursor proteins of LHCPs are imported via the TOC/TIC translocase, processed to their mature size to insert into thylakoid membranes where they recruit chlorophylls a and b to form pigment-protein complexes. The translocation of proteins is a highly regulated process which employs several regulators. To analyze whether CAO （chlorophyll a oxigenase） which converts chlorophyll a to chlorophyll b at the inner chloroplast membrane, is one of these regulators, we performed import reactions utilizing a homozygous loss-of-function mutant （cao-1）. We imported in vitro translated and 35S-labeled precursor proteins of light- harvesting proteins of photosystem II LHCB1, LHCB4, and LHCB5 into chloroplasts isolated from cao-1 and show that import of precursor proteins and their processing to mature forms are not impaired in the mutant. Therefore, regulation of the import machinery cannot be responsible for the decreased steady-state levels of light-harvesting complex （LHC） proteins. Regulation does not take place at the transcriptional level either, because Lhcb mRNAs are not down-regulated. Additionally, reduced steady-state levels of LHCPs also do not occur due to posttranslational turnover of non-functional LHCPs in chloroplasts. Taken together, our data show that plants in the absence of CAO and therefore devoid of chlorophyll b are not influenced in their import behavior of LHC proteins.
文摘Protein translocation across membranes is a fundamental cellular process. The majority of the proteins of organelles such as mitochondria and chloroplasts is synthesized in the cytosol and subsequently imported in a posttranslational manner. The precursor proteins have to be unfolded at least for translocation, but it has also been assumed that they are unfolded during transport to the organelle in the cytosol. Unfolding is governed by chaperones and the translocon itself. At the same time, chaperones provide the energy for the import process. The energetic properties of the chloroplast translocon were studied by import of the Ig-like module of the muscle protein titin fused to the transit peptide of the chloroplast targeted oxygen evolving complex subunit of 33 kDa （OE33）. Our results suggest that p（OE33）titin is folded prior to import and that translocation is initiated by unfolding after having bound to the translocon at the chloroplast surface. Using a set of stabilizing and destabilizing mutants of titin previously analyzed by atomic force microscopy and as passenger for mitochondrial translocation, we studied the unfolding force provided by the chloroplast translocon. Based on these results, a model for translocation is discussed.
文摘The TicS5 （Translocon at the inner envelope membrane of chloroplasts, 55 kDa） protein was identified in pea as a putative regulator, possibly linking chloroplast protein import to the redox state of the photosynthetic machinery. Two Tic55 homologs have been proposed to exist in Arabidopsis： atTic55-11 and AtPTC52 （Protochlorophyllide-dependent Trans- Iocon Component, 52 kDa; has also been called atTic55-1V）. Our phylogenetic analysis shows that attic55-11 is an ortholog of psTic55 from pea （Pisum sativurn）, and that AtPTC52 is a more distant homolog of the two. AtPTC52 was included in this study to rule out possible functional links between the proteins in Arabidopsis. No detectable mutant phenotypes were found in two independent T-DNA knockout mutant plant lines for each Arabidopsis protein, when compared with wild- type： visible appearance, chlorophyll content, photosynthetic performance, and chloroplast protein import, for example, were all normal. Both wild-type and tic55-11 mutant chloroplasts exhibited deficient protein import when treated with diethylpyrocarbonate, indicating that Tic55 is not the sole target of this reagent in relation to protein import. Furthermore, ptc52 mutant chloroplasts were not defective with respect to pPORA import, which was previously reported to involve PTC52 in barley. Thus, we conclude that atTic55-11 and AtPTC52 are not strictly required for functional protein import in Arabidopsis.
文摘Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide （TP） directing them specifically to a correct organelle. However, there is a group of proteins that are dually targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide （dTP）. Here, we have investigated pattern properties of import determinants of organelle-specific TPs and dTPs combining mathematical multivariate data analysis （MVDA） with in vitro organellar import studies. We have used large datasets of mitochondrial and chloroplastic proteins found in organellar proteomes as well as manually selected data sets of experimentally confirmed organelle-specific TPs and dTPs from Arabidopsis thaliana. Two classes of organelle-specific TPs could be distinguished by MVDA and potential patterns or periodicity in the amino acid sequence contributing to the separation were revealed, dTPs were found to have intermediate sequence features between the organelle-specific TPs. Interestingly, introducing positively charged residues to the dTPs showed clustering towards the mitochondrial TPs in silico and resulted in inhibition of chloroplast, but not mitochondrial import in in vitro organellar import studies. These findings suggest that positive charges in the N-terminal region of TPs may function as an ＇avoidance signal＇ for the chloroplast import.
文摘Most chloroplast and mitochondrial proteins are synthesized in the cytosol of the plant cell and have to be imported into the organelles post-translationally. Molecular chaperones play an important role in preventing protein aggregation of freshly translated preproteins and assist in maintaining the preproteins in an import competent state. Pre- proteins can associate with HSP70, HSP90, and 14-3-3 proteins in the cytosol. In this study, we analyzed a large set of wheat germ-translated chloroplast preproteins with respect to their chaperone binding. Our results demonstrate that the formation of distinct 14-3-3 or HSP90 containing preprotein complexes is a common feature in post-translational protein transport in addition to preproteins that seem to interact solely with HSP70. We were able to identify a diverse and extensive class of preproteins as HSP90 substrates, thus providing a tool for the investigation of HSP90 client protein association. The analyses of chimeric HSP90 and 14-3-3 binding preproteins with exchanged transit peptides indicate an involvement of both the transit peptide and the mature part of the proteins, in HSP90 binding. We identified two partner components of the HSP90 cycle, which were present in the preprotein containing high-molecular-weight complexes, the HSP70/HSP90 organizing protein HOP, as well as the immunophilin FKBP73. The results establish chloroplast preproteins as a general class of HSP90 client proteins in plants using HOP and FKBP as novel cochaperones.
文摘Protein import into chloroplasts has been a focus of research for several decades. The first publications dealing with this fascinating topic appeared in the 1970s. From the initial realization that many plastid proteins are being encoded for in the nucleus and require transport into their target organelle to the identification of import components in the cytosol, chloroplast envelopes, and stroma, as well as elucidation of some mechanistic details, more fascinating aspects are still being unraveled. With this overview, we present a survey of the beginnings of chloroplast protein import research, the first steps on this winding road, and end with a glimpse into the future.
文摘The chloroplast is surrounded by a double-membrane envelope at which proteins, ions, and numerous metabolites including nucleotides, amino acids, fatty acids, and carbohydrates are exchanged between the two aqueous phases, the cytoplasm and the chloroplast stroma. The chloroplast envelope is also the location where the biosynthesis and accumulation of various lipids take place. By contrast to the inner membrane, which contains a number of specific transporters and acts as the permeability barrier, the chloroplast outer membrane has often been considered a passive compartment derived from the phagosomal membrane. However, the presence of galactoglycerolipids and β-barrel membrane proteins support the common origin of the outer membranes of the chloroplast envelope and extant cyanobacteria. Furthermore, recent progress in the field underlines that the chloroplast outer envelope plays important roles not only for translocation of various molecules, but also for regulation of metabolic activities and signaling processes. The chloroplast outer envelope membrane offers various interesting and challenging questions that are relevant to the understanding of organelle biogenesis, plant growth and development, and also membrane biology in general.
