Progress 01/01/06 to 12/31/06
Outputs
The overall goal of the research project was to understand the mechanism by which the protein translocation channel Toc75 is directed to, and assembled in the chloroplast outer envelope. Since Toc75 catalyzes a reaction essential for the viability of plants, achieving this goal is expected to have high impacts on future research relevant to growth and development of plants. There were three objectives in this project. The first objective was to identify sequences in the transit peptide that are essential for targeting Toc75 to the chloroplast outer envelope. The results described in the last year's report have been summarized and published in a scientific journal in April, 2006. The second objective was to test a hypothesis that a signal peptidase is responsible for maturation of Toc75. As reported in last year, we have provided several pieces of evidence to support this hypothesis which were summarized and published in a scientific journal in November 2005. We have
started elucidating more mechanistic details of the function and biological significance of the signal peptide cleavage in the chloroplast biogenesis by biochemical, genetic, and cytological approaches. These results have been presented at one international symposium in June, 2006, and at one domestic meeting in August, 2006. The third objective was to test if the Toc75 transit peptide targets various passenger proteins to the outer envelope membrane. We have shown that a soluble passenger protein was directed to the chloroplast envelope, both in the soluble and membrane fractions in vitro. We also identified several chloroplast proteins other than Toc75 that contain the feature similar to that of the Toc75 transit peptide, the polyglycine stretch, and showed that they are targeted to the chloroplast envelope membranes.
Impacts
The research activity has involved a postdoctoral scholar, undergraduate and graduate students. They have been actively participating in not only experiments, but also research discussion. Thus, the project has had an impact on education. Our findings with the involvement of the type I signal peptidase in the biogenesis of chloroplasts have drawn some attention in the research field, and the principal investigator has been invited to give talks at several domestic and international meetings.
Publications
- Baldwin AJ, Inoue K (2006) The most C-terminal tri-glycine segment within the polyglycine stretch of the pea Toc75 transit peptide plays a critical role for targeting the protein to the chloroplast outer envelope membrane. FEBS Journal 273:1547-1555
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Progress 01/01/05 to 12/31/05
Outputs
The overall goal of the research project is to understand the mechanism by which the protein translocation channel Toc75 is targeted to, and assembled in the chloroplast outer envelope. These processes are essential for the biogenesis of plastids and thus for viability of plants. Three objectives were proposed to accomplish. The first objective was to identify features in the transit peptide that are essential for directing Toc75 to the chloroplast outer envelope. We dissected a part of the transit peptide that was previously shown to be important for envelope targeting into several segments, and found that a specific region is important. This part of work was reported at American Society of Plant Biologists annual meeting (July, 2005). Data have also been summarized and prepared for submission to a scientific journal. The second objective was to examine whether or not a signal peptidase is responsible for maturation of Toc75. We were able to make the larger progress
with this objective among three. First, we proved that Toc75 is a substrate of a signal peptidase by showing that it was processed by a bacterial enzyme in vitro. Second, we identified Arabidopsis plants that lack one of plastidic signal peptidase (named Plsp1) and found that they accumulate immature forms of Toc75. Interestingly, these plants contain immature forms of not only Toc75, but also a thylakoidal lumen protein OE33. The mutant plants also show a reduction of plastid internal membrane biogenesis. We showed that Plsp1 was targeted both to the chloroplast envelope and thylakoids by in vitro import assay. These data suggest that Plsp1 may have multiple functions: i.e., one is to process Toc75 at the envelope, and another is to mediate maturation of thylakoidal proteins. We reported these results at American Society of Biochemistry and Molecular Biology annual meeting (April, 2005). There are two publications related to this part of the project. The one published in
"Photosynthesis: Fundamental Aspects to Global Perspectives" describes analysis to establish the hypothesis. Another paper published in Journal of Cell Biology provides biochemical and genetic data to support the idea that a signal peptidase is responsible for maturation of Toc75 and the biogenesis of plastid internal membranes. The third objective was to investigate whether or not the Toc75 transit peptide targets various passenger proteins to the outer envelope membrane. We have tried only one passenger protein, an enhanced green fluorescent protein (eGFP), for targeting activity of the Toc75 transit peptide both in vitro and in vivo. We also tested targeting activity of different versions of the Toc75 transit peptide using eGFP. In addition to the above three objectives, we investigated the targeting mechanism of a newly identified truncated form of Toc75 called atToc75-IV to the chloroplast outer envelope membrane. This work was included in a paper published in Plant Physiology.
Impacts
The research activity has involved a postdoctoral scholar, undergraduate and graduate students. They have been actively participating in not only experiments, but also research discussion. Thus, the project has had an impact on education. The discovery of Plsp1, the main accomplishment from the second objective, has generated a couple of new questions. i) Does the development of thylakoids depend on the maturation of the protein translocation channel at the chloroplast envelope membrane? ii) How is Plsp1 targeted to two locations, chloroplast envelope and thylakoids? Currently, there is no obvious impact from this accomplishment. However, addressing these new questions should have a high impact in the filed of the organelle biogenesis.
Publications
- Inoue K, Baldwin AJ, Shipman RL, Matsui K, Theg SM, Ohme-Takagi M (2005) Complete maturation of the plastid protein translocation channel requires a type I signal peptidase. The Journal of Cell Biology 171: 425-430
- Baldwin A, Wardle A, Patel R, Dudley P, Park SK, Twell D, Inoue K, Jarvis P (2005) A molecular-genetic study of the Arabidopsis toc75 gene family. Plant Physiology 138: 715-733
- Inoue K, Potter D, Shipman RL, Perea JV, Theg SM (2005) Involvement of a type I signal peptidase in biogenesis of chloroplasts - Towards identification of the enzyme for maturation of the chloroplast protein translocation channel. In Photosynthesis: Fundamental Aspects to Global Perspectives, eds. van der Est A and Bruce D. (Allen Press, Lawrelnce), pp. 933-935
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Progress 01/01/04 to 12/31/04
Outputs
The goal of the research project is to understand the mechanism by which the protein translocation channel Toc75 is targeted to, and assembled in the chloroplast outer envelope. These processes are essential for biogenesis of plastids and thus for viability of plants. A postdoctoral fellow funded by this grant joined the laboratory in March, 2004, and has been in charge of this project. The first objective of the project is to identify features in the transit peptide that are essential for directing Toc75 to the chloroplast outer envelope. The postdoctoral fellow has conducted a more detailed site-directed mutagenesis to refine the amino acid core sequence apparently responsible for envelope targeting in vitro. Furthermore, she has started testing the activity of the targeting sequences using transgenic plants in vivo. The second objective is to examine whether or not a signal peptidase is responsible for maturation of Toc75. As reported last year, we have found that
bacterial type I signal peptidase (SPase I) can catalyze this process. We identified three Arabidopsis SPases I which appeared to be localized in plastids. We conducted a reverse genetic screening and found that knockout of a gene for one of plastid-localized SPases I results in accumulation of unprocessed forms of Toc75. Currently, one graduate student is attempting to characterize the gene product biochemically. The third objective of the research project is to investigate whether or not the Toc75 transit peptide targets various passenger proteins to the outer envelope membrane. We found that the Toc75 transit peptide can target a soluble protein to the inter membrane space, but not to the outer envelope, in vitro. We are going to test if this is also the case in vivo using transgenic plants. Finally, some data regarding the SPase I were reported at the International Congress of Photosynthesis in September, 2004.
Impacts
No significant impact has been made during the reporting period.
Publications
- There is no publication for this research project during the reporting period. The presentation at the International Congress of Photosynthesis will be published in the proceedings in 2005.
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Progress 01/01/03 to 12/31/03
Outputs
The goal of the research project is to understand the mechanism by which the protein translocation channel Toc75 is targeted to, and assembled in the chloroplastic outer envelope. These processes are essential for biogenesis of plastids and ultimately, for viability of plants. During the reporting period, there is no significant progress for the first objective of the project, which is to identify features in the transit peptide that are essential for directing Toc75 to the chloroplastic outer envelope. For the second objective, which is to examine whether or not a signal peptidase is responsible for maturation of Toc75, I made the following progresses during the reporting period. First, by collaborating with Dr. Steve Theg's group in UC Davis, we found that a bacterial signal peptidase was able to catalyze the maturation of Toc75. Currently, we are conducting more detailed analysis of the activity of the bacterial enzyme against Toc75 and its mutated versions.
Second, we obtained seeds of Arabidopsis of which genes for putative signal peptidases are disrupted by T-DNA insertions, grew them, and started screening them for homozygous or heterozygous insertion lines. The goal of this genetic approach is to identify gene(s) encoding a signal peptidase responsible for the maturation of Toc75 in Arabidopsis. There is no significant progress for the third objective of the research project, which is to investigate whether or not the Toc75 transit peptide targets various passenger proteins to the outer envelope membrane. Finally, I identified a person for the postdoctoral position funded by this grant, who should start working on this project in March, 2004.
Impacts
The outcome of the research should help us design approaches to improve quality of crop plants by manipulating metabolism in the chloroplast, such as lipid biosynthesis, by targeting various enzymes to the envelope membrane.
Publications
- No publications reported this period
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