Progress 10/01/12 to 01/17/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Deceased -08/31/16
Publications
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Scientific Community (graduate students, postdoctoral fellows, faculty members, and others): participants at scientific conferences - i) Western Photosynthesis Conference; ii) Gordon Research Conference on membrane protein folding; iii) American Society of Plant Biologist annual meeting. Attendees at Biology Academic Success Center Road To Research Seminar Series. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Four graduate students, one of which graduated and turned to be a postdoctral researcher, and several undergraduate students were involved in this project. All were trained by the PI at weekly laboratory meetings and individual meetings (at least once per week). In addition, the PI helped their professional development by instructing manuscript writing and preparation for oral presentations at a campus group meeting as well as poster presentation at a national meeting. How have the results been disseminated to communities of interest?The results were published in scientific journals (the Journal of Biological Chemistry, and Frontiers in Plant Science) and presented at National meetings. What do you plan to do during the next reporting period to accomplish the goals?For the essential chloroplast outer membrane proteins, we will use the established genetic system to test structure-function relationships, and also use biochemical assays to gain further insights into the mechanism of their targeting. For the peptidase, we will use a genetic complementation assay to define the importance of its redox status, and also use both genetic and biochemical assays to elucidate its intraorganellar targeting mechanism.
Impacts
What was accomplished under these goals?
In photosynthetic eukaryotes, oxygenic photosynthesis as well as the biosynthesis of amino acids, nucleotides, lipids, and various other molecules occurs in chloroplasts. Previously we had demonstrated that two homologous proteins located in the outer membrane of the chloroplast envelope are essential for viability of plants from the embryonic stage. During the reported period, we have been establishing a novel genetic system to test their roles in post-germination stage of plant development. We have also started defining the mechanisms of their targeting from the cytoplasm to the chloroplast, then to the chloroplast envelope. Many protein components of photosynthetic machinery are made with a signal peptide, which needs to be cleaved upon reaching the final destination and before assembly. Our previous results demonstrated that the peptidase responsible for the signal removal depends on redox status of its Cys residues for the activity in vitro, and that its targeting to the membrane requires ATP hydrolysis. During the reported period, we have obtained data to support that the redox status of this enzyme may be compensated by other factors in vivo, and that the targeting of this enzyme involves interaction with a molecular chaperone in the chloroplast stroma. The second finding is particularly interesting in light of the adaptation of membrane protein targeting mechanism, from co-translational to post-translational mechanism during evolution of chloroplasts.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Inoue K (2015) Emerging knowledge of the organelle outer membranes - research snapshots and an updated list of the chloroplast outer envelope proteins. Frontiers in Plant Science 6, 278.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Gou JY, Li K, Wu K, Wang X, Lin H, Cantu D, Uauy C, Dobon-Alonso A, Midorikawa T, Inoue K, S�nchez J, Fu D, Blechl A, Wallington E, Fahima T, Meeta M, Epstein L, Dubcovsky J (2015) Wheat stripe rust resistance protein WKS1 reduces the ability of the thylakoid-associated ascorbate peroxidase to detoxify reactive oxygen species. Plant Cell 27, 1755-1770
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Endow JK, Singhal R, Fernandez DE, Inoue K (2015) Chaperone-assisted post-translational transport of plastidic type I signal peptidase 1. Journal of Biological Chemistry 290, 28778-28791.
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2016
Citation:
Klasek L, Inoue K (2015) Dual protein localization to the envelope and thylakoid membranes within the chloroplast. International Rreview of Cell and Molecular Biology.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Day M, Inoue K. Targeting of an Essential ?-Barrel Protein to the Chloroplast Outer Membrane. Plant Biology 2015, American Society of Plant Biology Annual meeting.
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Scientific Community (graduate students, postdoctoral fellows, faculty members, and others): attendants at the seminar held at Dept. Biochemistry and Molecular Biology, University of Massachusetts (Amherst, MA); participants in American Society of Plant Biologist annual meeting; participants in Gordon Research Conference on photosynthesis Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Three graduate students and a postdoctral fellow were involved in this project. All were trained by the PI at weekly laboratory meetings and individual meetings (at least once per week). In addition, the PI helped their professional development by instructing manuscript writing and preparation for oral presentations at a campus group meeting as well as poster presentation at a national meeting. How have the results been disseminated to communities of interest? The results were published in scientific journals (the Plant Journal and Frontiers in Plant Science) and presented at National meetings as well as at Department of Biochemistry and Molecular Biology at University of Massachusetts. What do you plan to do during the next reporting period to accomplish the goals? We will define the targeting mechanism of the outer membrane proteins as well as the peptidase. We will also complete analysis of the mutant plants we have developed to answer questions about the function of the outer membrane protein as well as the significance of the redox-dependent activity of the peptidase.
Impacts
What was accomplished under these goals?
In photosynthetic eukaryotes, oxygenic photosynthesis as well as the biosynthesis of amino acids, nucleotides, lipids and various other molecules occurs in chloroplasts. Previously we had demonstrated that two homologous proteins located in the outer membrane of the chloroplast envelope are essential for viability of plants from the embryonic stage. During the reported period, we have defined their phylogenetic relationships, and also found that they use different mechanisms to be sorted to the chloroplast. Many components of photosynthetic machinery are made with a targeting signal, which needs to be cleaved upon reaching the final destination and before assembly. We have used biochemical assays to demonstrate that the activity of the peptidase responsible for the signal removal depends on redox status of Cys residues, and that the enzyme itself is targeted to the photosynthetic membrane via an energy-dependent pathway.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Inoue K, Glaser E (2014) Processing and degradation of chloroplast extension peptides. In Theg SM, Wollman F-A, eds, Plastid Biology (Springer, New York) Advances in Plant Biology 5, 305-323
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Midorikawa T, Endow, JK, Dufour J, Zhu J, Inoue K (2014) Plastidic type I signal peptidase 1 is a redox-depenent thylakoid processing peptidase. The Plant Journal, 80, 592-603
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Day PM, Potter D, Inoue K (2014) Evolution and targeting of Omp85 homologs in the chloroplast outer envelope membrane. Frontiers in Plant Science 5, 535
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Schnell D, Paila Y, Richardson L, Inoue E. Parks E. Integration of protein import with nuclear gene expression during chloroplast biogenesis. Plant Biology 2014, American Society of Plant Biology Annual meeting.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Endow JK, Inoue K. Unraveling the intraorganellar targeting mechanism of plastidic type I signal peptidase 1. lant Biology 2014, American Society of Plant Biology Annual meeting.
|
Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Scientific Community: participants in Western Photosynthesis Conference; participants in International Colloquium on Endocytobiology and Symbiosis; attendants at Plant Molecular & Cellular Biology Graduate Program Seminar in University of Florida. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? A graduate student and a postdoctral fellow were involved in this project. Both were trained by the PI at weekly laboratory meetings and individual meetings (at least once per week). In addition, the PI helped their professional development by instructing manuscript writing and prepartion for oral presentations at a campus group meeting. How have the results been disseminated to communities of interest? The results were published in scientific journals (the Plant Journal and FEBS Letters) and presented at the Plant Molecular & Cellular Biology Graduate Program Fall 2013 Seminar series in University of Florida. What do you plan to do during the next reporting period to accomplish the goals? For objective 1, we will attempt to identify and characterize components of the PsbO complex. For objective 2, we will finalize the analysis of Plsp1 targeting to the thylakoid in vitro and will test importance of conserved residues for Plsp1 targeting in vivo. For Objective-3, we will focus on characterizing the mechanism and significance of oxidative folding for activity of Plsp1 both in vitro and in vivo.
Impacts
What was accomplished under these goals?
This project is aimed to define the mechanisms of photosynthetic membrane development and protein sorting/assembly. The main subject of the research is removal (processing) of targeting signal sequences from the thylakoid lumenal proteins by an enzyme called Plsp1. There are three specific objectives. Objective-1 is to examine if/how lumenal proteins interact with the thylakoid membrane if the targeting sequences are not removed, and if/how accumulation of such unprocessed lumenal proteins affects thylakoid development. We used in vitro assay to examine the fates of three lumenal proteins. They are two oxygen-evolving complex subunits (PsbO and PsbP) and a soluble electron carrier plastocyanin (PetE). All the three proteins were found to associate tightly with the membrane, instead of being released free in the lumen, when removal of the targeting signal was inhibited by mutation around the processing site. Among the three proteins, PsbO was found to be incorporated into a large protein complex while PsbP remained as a monomer. Interestingly, formation of the PsbO complex was disrupted by addition of a reducing agent or by mutation of some but not all of Cys residues within PsbO. These results suggest that Cys thiol oxidation is required for the PsbO complex formation. By contrast to these two proteins, uncleaved PetE was quickly degraded under light. Further analyses showed that the PetE degradation activity was enhanced by light-driven proton motive force and ATP hydrolysis. Together, the obtained results indicate the presence of multiple mechanisms for controlling unprocessed lumenal proteins in the thylakoid. An implication of this result is a possibility that protein maturation may play a regulatory role in protein quality control. Objective-2 is to elucidate the mechanism by which Plsp1 is targeted to the chloroplast internal membranes. We have used in vitro assay to show that Plsp1 is targeted to the membrane via a stromal intermediate and that its membrane integration appears to require ATP hydrolysis. We have also generated Plsp1 variants with mutations in several conserved residues in order to understand their importance for membrane targeting. Objective-3 is to test if and how the signal peptide cleavage links two energy-related processes, proton gradient formation and protein translocation across the membrane. For this, we have demonstrated that Plsp1 forms a stable complex with PGRL1. PGRL1 is a known component of the cyclic electron flow around photosystem I and a recent result by another group suggested that it acts as an elusive ferredoxin-ubiquinone oxidoreductase. A genetic study indicated that complex formation with PGRL1 is not required for the functionality of Plsp1. Finally, during the
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Endow JK, Inoue K (2013) Stable complex formation of thylakoidal processing peptidase and PGRL1. FEBS Letters 587, 2226-2231
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Midorikawa T, Inoue K (2013) Multiple fates of nonmature lumenal proteins in thylakoids. The Plant Journal 76, 73-86
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Midorikawa T, Vu S, Inoue K (2013) The fate of immature thylakoid lumen proteins. Proceeding for 22nd Western Photosynthesis Conference.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Inoue K (2013) Chloroplast biogenesis. Proceeding for 22nd Western Photosynthesis Conference.
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The chloroplast is an organelle of photosynthetic eukaryotes. Its metabolic activity, such as oxygenic photosynthesis and the biosynthesis of nucleotides, amino acids, lipids, storage polysaccharides and various pigments, supports lives of most organisms on earth. The goal of the project is to advance our mechanistic understanding of the development of this important organelle by achieving two specific aims. The first aim is to elucidate molecular details of carotenoid metabolism. The second aim is to understand the mechanism and significance of signal peptide cleavage in the development of photosynthetic membranes (thylakoids). Signal peptide cleavage in thylakoids is catalyzed by thylakoidal processing peptidase (TPP). There are two TPP isoforms in higher plants - Plsp1 and Plsp2. Plsp1 acts not only as TPP, but also as envelope processing peptidase to cleave an envelope-sorting signal from Toc75. Toc75 is a protein import channel and co-exists with its paralog called OEP80 in the chloroplast outer envelope membrane. There two paralogous proteins originated from a protein in the ancestral cyanobacterium, and play distinct functions essential for plant development form the embryonic stage. However, the exact function of OEP80 remains elusive. Completed activities include: i) demonstration of Plsp1 forming a complex with a protein involved in photosynthetic electron flow; ii) examination of the significance of the Plsp1 complex using genetic tools; iii) discovery that disruption of the signal peptide removal results in multiple fates of the immature proteins by biochemical assays; iv) molecular genetic and biochemical studies of OEP80; v) review of available studies about nuclear-encoded plastid proteins essential for embryogenesis; vi) teaching and mentoring four undergraduate students, one graduate student and three postdoctoral fellows. Collaboration with confocal microscope facility on campus has been fostered. Some of the results were disseminated to scientific community by presentations at Western Photosynthesis Conference and Gordon Conference on Protein Transport Across Cell Membrane, as well as by an invited lecture at Nara Institute of Science and Technology (Nara, Japan). PARTICIPANTS: (1) Principal investigator: Kentaro Inoue, who oversaw the entire projects, designed and conducted experiments, analyzed and disseminated the results. (2) Other personnel (all are for aim 2): Garrett Shafer, Simon Vu, Andrew Zareie, Erin Oriley, undergraduate students; Joshua Endow, a graduate student; Takafumi Midorikawa, Mehdi Nafati, Agostinho Gomes-Rocha, postdoctoral fellows. TARGET AUDIENCES: Scientific Community: participants in Western Photosynthesis Conference and Gordon Research Conference on Protein Transport across Cell Membranes; graduate students and scientists at Nara Institute of Science and Technology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
By various genetic and biochemical assays, we showed that Plsp1 forms a complex with PGRL1. This complex is not detectable in the pgrl1-knockout mutant. Known substrates of Plsp1 accumulate as mature forms in the pgrl1-null mutant. In addition, only a few % of the entire PGRL1 seems to form a complex with Plsp1. Hence, we hypothesize that PGRL1-Plsp1 complex formation may negatively regulate the activity of Plsp1. The reaction catalyzed by Plsp1 is indirectly dependent on photosynthetic energy conversion (proton motive force and production of ATP). The reaction itself is hydrolysis, which does not require energy input. However, the presentation of the substrate protein to Plsp1 is made possible by protein transport, which uses either ATP in the stroma or proton motive force across the thylakoid membrane. Various works have suggested that PGRL1 plays a role in regulating proton gradient across the thylakoid membrane. Notably, a recent paper suggested that PGRL1 acts as ferredoxin:plastoquinone oxidoreductase, which is the key and elusive enzyme of the cyclic electron flow around photosystem I. Hence, our discovery about the Plsp1 may indicate a previously unknown link between the energy-producing process (photosynthetic electron flow) and energy-consuming process (protein transport). By biochemical assays, we showed that lumen-targeting proteins take several distinct fates if their signal removal is prevented. More specifically, inhibition of the signal removal by mutation around the processing site results in i) oligomeric formation of the 33-kD component of oxygen evolving complex (PsbO); ii) light-dependent degradation of the lumenal electron carrier, plastocyanin; iii) accumulation of a monomeric form of the 23-kD component of oxygen evolving complex (PsbP). These results suggest the presence of multiple protein quality systems in the photosynthetic membranes. Finally, we established a molecular genetic assay system to examine the structure-function relationships of OEP80. This system is useful for addressing questions about chloroplast biogenesis and evolution, which are very important problems in the organelle biology.
Publications
- Hsu S-C, Nafati M, Inoue K (2012) OEP80, an essential protein paralogous to the chloroplast protein translocation channel Toc75, exists as a 70-kD protein in the Arabidopsis thaliana chloroplast outer envelope. Plant Molecular Biology 78(1-2), 147-158
- Nafati M, Inoue K (2013) Indispensable roles of plastids in Arabidopsis thaliana embryogenesis update. Advances in Genome Science, accepted.
- Inoue K, Gasser E (2013) Processing and degradation of chloroplast extension peptides. In Plastid Biology (eds. Theg SM, Wollman F-A), accepted.
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The goal of the project is to advance our mechanistic understanding of chloroplast development by achieving two specific aims. Aim 1 is to elucidate molecular details of carotenoid metabolism. Aim 2 is to understand the mechanism and significance of signal peptide cleavage in the development of photosynthetic membranes (thyalkoids). Signal peptide cleavage is catalyzed by thylakoidal processing peptidase (TPP). Multiple TPP isoforms exist in higher plants. Plsp1 is one of TPP isoforms. Completed activities include: i) in vitro import assay of key enzymes of carotenoid biosynthesis in melon (via collaboration with Dr. Li Tian's group); ii) phylogenetic analysis of TPPs; iii) analysis of the TPP gene expression; iv) genetic complementation assay with seedling-lethal plsp1-null plants; v) biochemical assays for Plsp1 targeting; vi) analysis of formation of the Plsp1 complex by blue native polyacrylamide gel electrophoresis (BN-PAGE); vii) teaching and mentoring one undergraduate student, one graduate student, and two postdoctoral fellows. Collaborations with various facilities on campus (Proteomics Facility at Genome Center and Electron Microscopy Laboratory at Department of Pathology and Laboratory) have been fostered. Some of the results were disseminated to scientific community by presentations at Gordon Research Conference on Photosynthesis, FEBS workshop on Plant Organellar Signaling, and Department of Energy Basic Energy Science Photosynthetic Systems Meeting, as well as by invited lectures at Kyoto Prefectural University, Okayama University, Kyoto Sangyo University, Ludwig-Maximilians-Universitat, and Leibniz-Institute of Plant Biochemistry. The PI also visited Merritt College Biology Department (Oakland, CA). PARTICIPANTS: (1) Principal investigator: Kentaro Inoue, who oversaw the entire projects, designed and conducted experiments, analyzed and disseminated the results. (2) Other personnel (all are for aim 2): Garret Shafer, undergraduate student; Joshua Endow, a graduate student; Nicholas Ruppel and Takafumi Midorikawa, postdoctoral fellows, Brett S. Phinney, collaborator. TARGET AUDIENCES: Scientific Community: Department of Energy Basic Energy Science Photosynthetic Systems Principal Investigators; participants in Gordon Research Conference on Photosynthesis and FEBS workshop on Plant Organellar Signaling. and Department of Energy Basic Energy Science Photosynthetic Systems Meeting' scientists at Kyoto Prefectural University, Okayama University, Kyoto Sangyo University, Ludwig-Maximilians-Universitat, and Leibniz-Institute of Plant Biochemistry. A postsecondary minority institution: Biology Department, Merritt College. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
By in vitro protein import assay, we confirmed plastid localization of the key enzymes of carotenoid biosynthesis, namely phytoene synthases, from melon. By phylogenetic analyses, we demonstrated that TPPs originated before the endosymbiotic event, and that duplication leading to multiple TPPs occurred prior to the gymnosperm-angiosperm separation. Gene expression analysis and genetic complementation assay allowed us to demonstrate that the TPP isoforms have distinct functions. By biochemical assays, we found that targeting of Plsp1 to the chloroplast membrane depends on ATP hydrolysis and azide-sensitive component. Finally, combination of genetic assay and BN-PAGE analysis, we demonstrated that Plsp1 forms a tight complex that contains a protein that was previously shown to play a role in cyclic electron flow. This is an interesting finding because it may indicate a connection between protein targeting, which requires energy input, and photosynthetic electron flow. Besides the works on TPP, we used the biochemical assay to characterize a novel protein that is located in the chloroplast envelope and plays a role in plastid differentiation in embryo-derived tissues during seedling growth.
Publications
- Hsu S-C, Endow JK, Ruppel NJ, Roston RL, Baldwin AJ, Inoue K (2011) Functional diversificatio of thylakoidal processing peptidases in Arabidopsis thaliana. PLoS ONE 6(11), e27258
- Inoue K (2011) Emerging roles of the chloroplast outer envelope membrane. Trends in Plant Science 16(10), 550-557
- Ruppel NJ, Logsdon CA, Whippo CW, Inoue K, Hangarter RP (2011) A mutation in Arabidopsis seedling plastid development1 affects plastid differentiation in embryo-derived tissues during seedling growth. Plant Physiology 155(1), 342-353
- Qin X, Coku A, Inoue K, Tian L (2011) Expression, subcellular localization, and cis-regulatory structure of duplicated phytoene synthase genes in melon (Cucumis melo L.). Planta 234(4), 737-748
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The goal of the project is to further our mechanistic understanding of chloroplast development by achieving two specific aims. Outputs completed during the reporting period are related to aim 2, which is to understand the mechanism and significance of signal peptide cleavage in the development of thylakoids. Completed activities include i) biochemical analyses of a mutant plant that lacks Plsp1, the enzyme responsible for signal peptide cleavage; ii) genetic complementation assay: transformation of the plant that lacks Plsp1 with DNA constructs encoding various Plsp1 isoforms and their derivatives, and analysis of the resultant plants; iii) biochemical assays for Plsp1 targeting; iv) generation of a plant that accumulates fluorescent protein-tagged Plsp1; v) teaching and mentoring a postdoctoral fellow, a junior specialist, and undergraduate and graduate students for the experiments related to the project. Collaborations with various facilities on campus (Proteomics Facility at Genome Center, Electron Microscopy Laboratory at Department of Pathology and Laboratory Medicine, and Microscopy and Imaging Facility at Department of Molecular and Cellular Biology) have been fostered. Some of the results were disseminated to scientific community by presentations at American Society of Plant Biologists annual meeting and Microscopy Program Class at Merritt College. PARTICIPANTS: (1) Principal investigator: Kentaro Inoue, who oversaw the entire projects, designed and conducted experiments, analyzed and disseminated the results. (2) Other personnel: Rebecca Shipman-Roston and Joshua Endow, graduate students who worked on objectives 3 and 4; Shih-Chi Hsu, a junior specialist who was involved in objective 5; Nicholas Ruppel, a postdoctoral fellow who worked on objective 5; Corey Lee and Robert Shih, undergraduate students who were involved in objective 4 and 5, respectively; Catalina Damoc and Brett S. Phinney, collaborators who contributed to objective 3. TARGET AUDIENCES: Scientific community: American Society of Plant Physiologists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Biochemical analyses of the mutant plant that lacks Plsp1 have allowed us to obtain fundamental knowledge significant enough to be included in a publication. Furthermore, the obtained knowledge helped us to develop a more defined hypothesis about the mechanism of thylakoid development, which was included in another publication. Based on results of the genetic complementation assay, Plsp1 isoforms were proposed to have distinct functions. By the biochemical assay, Plsp1 was found to be isorted to thylakoids via a stromal intermediate, and its membrane targeting depends on a soluble ATPase, which is known to mediate protein targeting to thylakoid lumen, but not to thylakoid membrane as in the case of Plsp1. This is an interesting finding and may add to the field as a novel mechanism of thylakoid protein sorting. Finally, the plant that accumulates fluorescent protein-tagged Plsp1 will be used to conduct in vivo imaging of protein sorting.
Publications
- Endow, J.K., Ruppel, N.J., and Inoue, K. (2010). Keep the balloon deflated: The significance of protein maturation for thylakoid flattening. Plant Signaling & Behavior, 5(6): 721-723.
- Shipman-Roston, R.L., Ruppel, N.J., Damoc, C., Phinney, B.S., and Inoue, K. (2010). The significance of protein maturation by plastidic type I signal peptidase 1 for thylakoid development in Arabidopsis thaliana chloroplasts. Plant Physiology, 152(3): 1297-1308.
- Ruppel, N.J., Hsu, S-C., Shih, R., Chen, Y-T., and Inoue, K. (2010) The role of plastidic type I signal peptidase isoforms in Arabidopsis thaliana. American Society of Plant Biologists, Montreal, Canada. August, 2010.
|
Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The project consists of six objectives. For objectives 1 and 2, activities include collection of tomato plants transformed with a carotenogenic gene and data analyses. Relevant subjects were disseminated as a lecture for Postharvest Technology Short Course, and also as a report to California Citrus Research Board. For objectives 3 and 4, suborganellar localization of Plsp1 was examined under electron microscope, and its correlation with gene expression and enzyme activity was demonstrated. Furthermore, detailed biochemical, molecular, and morphological analyses of the mutant plants that lack Plsp1 were conducted. Several in vitro assay systems were also tested. Collaborations with Proteomics Facility at Genome Center and Electron Microscopy Laboratory at Department of Pathology and Laboratory Medicine were achieved. Obtained data were analyzed and disseminated at Western Photosynthesis Conference and Department of Energy Photosynthetic Systems Research Meeting. For objective 5, constructs necessary to knockdown expression of genes encoding certain type I signal peptidase isoforms were prepared, and introduced into Arabidopsis plants via an Agrobacterium-mediated method. In addition, a construct encoding Plsp2 was prepared and introduced into plsp1-null plants, and its consequence to the plant phenotype was analyzed. There is no notable outcome for objective 6. PARTICIPANTS: (1) Principal investigator: Kentaro Inoue. He oversaw the entire projects, conducted experiments, analyzed and disseminated the results. (2) Other personnel: Rebecca Shipman-Roston, Shih-Chi Hsu, and Joshua Endow: Graduate students who worked mainly on objectives 3 and 4. Nicholas Ruppel: A postdoctoral fellow who worked on objective 5. Robert Shih: An undergraduate student who worked on objective 5. TARGET AUDIENCES: Scientific community: American Society of Plant Physiologist and others. Local agricultural community: California Citrus Research Board. International postharvest community: Participants of Postharvest Short Course. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
There are two major impacts. The first impact is on the current understanding of the development of thylakoids, the essential photosynthetic membrane systems in chloroplasts. According to the well-accepted model, thylakoids are derived form the organelle envelope although molecular details of this process remain unknown. The presence of a number of proteins in both the envelope and thylakoids has been reported although its physiological significance was not explored. In the work related to objective 4, we showed that Plsp1 is located in both the envelope and thylakoids, and that this dual-localization depends on the development of chloroplasts and correlates with the accumulation of the substrates of Plsp1 in the two subcompartments. This work established the rationale for the multiple localizations of Plsp1, helping define testable questions that will further our understanding of thylakoid biogenesis. The second impact concerns the biological significance of protein maturation. Related to objective 3, we demonstrated that i) Plsp1 seems to be the main type I signal peptidase among three isoforms in Arabidopsis chloroplasts; ii) an unprocessed lumen protein exists in the stromal vesicles that are abundant in the mutants that lack Plsp1; and iii) incomplete maturation of the protein translocation channel Toc75 appears to be dispensable for normal chloroplast development. These findings reveal the new level of complexity of protein maturation and also pave a new path for investigations on this well known yet under-explored research subject.
Publications
- Hsu S-C, Inoue K (2009) Two evolutionary conserved essential β-barrel proteins in the chloroplast outer envelope membrane. BioScience Trends 3, 168-178
- Ikegami A, Akagi T, Potter D, Yamada M, Sato A, Yonemori K, Kitajima A, Inoue K (2009) Molecular identification of 1-Cys peroxiredoxin and anthocyanidin/flavonol 3-O-galactosyltransferase from proanthocyanidin-rich young fruits of persimmon (Diospyros kaki Thunb.). Planta 230, 841-855
- Shipman RL, Inoue K (2009) Suborganellar localization of plastidic type I signal peptidase 1 depends on chloroplast development. FEBS Letters 583, 938-942
|
Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: This project consists of six objectives. Objective 1 is to compare carotenoid metabolism in unripe green, degreening, ripe orange, and regreened ripe fruit peel of Valencia orange. We have found by HPLC analysis that regreened peel of ripe fruits accumulates chlorophylls a and b as well as the three major photosynthetic carotenoids (violaxanthin, lutein and β-carotene) in a roughly similar ratio as does the unripe green peel. We have also employed TaqMan reverse-transcriptase mediated quantitative polymerase chain reaction to estimate the amount of transcripts encoding nine proteins involved in carotenoid biosynthesis, seven for chlorophyll metabolism, and eight for proteins that play roles in plastid development. Among them, lycopene epsilon cyclase, carotene epsilon-hydroxylase, and chlorophyll synthase are involved in the biosynthesis of photosynthetic pigments (lutein for the first two and chlorophylls for the last one, respectively), which do not accumulate in the ripe orange peel. Interestingly, however, the level of transcripts encoding these three proteins was found to increase during degreening (ripening). This result might indicate that during the early stage of ripening, these fruits are already preparing for regreening by accumulating transcripts for the biosynthesis of photosynthetic pigments. Another interesting finding from the TaqMan analysis is that some genes appear to express higher in the regreened peel than in the green peel of unripe fruits. Among them is plastoglobulin, which is known to respond to various stresses. Thus, the regreened peel might be experiencing higher stress than is the green peel of unripe fruits. Objective 2 is to attempt to reconstitute and characterize carotenogenic complexes in chloroplasts. We are currently in preparing Arabidopsis and tomato plants that produce the tagged lycopene epsilon cyclase, which will be examined in the next year or so. Objectives 3-5 have been incorporated into the project funded by US Department of Energy (grant number DE-FG02-08ER15963) from July, 2008, which aims to i) elucidate the significance of gene duplications giving rise to multiple chloroplastic signal peptidases, and ii) define the mechanism by which Plsp1 is targeted to the chloroplast internal membranes. First, we have successfully prepared plastids from the plsp1-null mutant plants and found two proteins, plastocyanin and OE23, as new putative Plsp1 substrates. We also showed by immunoblotting assay that at least one of the Plsp1 orthologs is present in the mutant plastids. Finally, an electron microscopic immunolocalization study has revealed that Plsp1 is located evenly in the envelope and thylakoids of developing chloroplast, whereas it is mainly found in thylakoids of developed chloroplasts. There is no specific progress to report for the objective 6. Besides the proposed six objectives, we took a genetic approach to show that a gene encoding a protein paralogous to one of Plsp1 substrates is essential for development and/or maintenance of plastids from the embryonic stage using Arabidopsis thaliana. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
I was invited to present and share our findings from objective 1 with various people including those from industries at the Plant Growth Regulation Society of America Annual Meeting in August, 2008. Although there is no obvious impact yet, I received various feedback comments, some of which appear to promise that our current as well as future findings should help growers develop strategies to improve crop quality. In addition, the research activity has involved two graduate students, a postdoctoral scholar, and two undergraduate intern students. They have been actively participating in not only experiments, but also research discussion. Thus, the project has had an impact on education.
Publications
- Goudeau D, Uratsu SL, Inoue K, daSilva FG, Leslie A, Cook D, Reagan R, Dandekar AM (2008) Tuning the orchestra: selective gene regulation and orange fruit quality. Plant Science 174, 310-320
- Hsu S-C, Patel R, Bedard J, Jarvis P, Inoue K (2008) Two distinct Omp85 paralogs in the chloroplast outer envelope membrane are essential for embryogenesis in Arabidopsis thaliana. Plant Signaling & Behavior 3, 1134-1135
- Inoue K, Qin X, Ikegami A (2008) Green or orange, Towards understanding the molecular basis of citrus fruit peel color development. Proceedings for the 35th Annual Meeting of Plant Growth Regulation Society of America, submitted.
- Patel R, Hsu S-C, Bedard J, Inoue K, Jarvis P (2008) The Omp85-related chloroplast outer envelope protein, OEP80, is essential for viability in Arabidopsis. Plant Physiology 148, 235-245
|
Progress 01/01/07 to 12/31/07
Outputs
The first objective aims to address the mechanism by which proteins are targeted to the chloroplast envelope membrane. In addition to identification of the targeting sequence which appears to be conserved among several proteins in the plastid envelope membranes, we made a discovery that a signal peptidase is involved in the processing of the targeting sequence. Quite interestingly, our genetic study has indicated that this peptidase is responsible not only for processing of a protein located in the plastid envelope membrane, but also for maturation of several thylakoidal proteins which are involved in the photosynthetic electron transfer reaction. Furthermore, our immunocytochemical study has shown that this enzyme is located in both the envelope and thylakoids of developing chloroplasts, but is mainly present in thylakoids of developed chloroplasts. Thus, our research has demonstrated the new level of complexity in the biogenesis of chloroplasts. We started addressing
mechanistic details of the action of this signal peptidase. We were invited to present these results at Western Photosynthesis Conference (January) and at The 21st Century COE program International Symposium on New Frontiers in Biosciences - Molecular Mechanisms in Cellular Regulation (January). We also presented part of our findings at American Society of Biologists Annual Meeting (July). We have published a review article on the plastid outer membrane protein. We also contributed to a publication on the study of protein targeting and maturation at the inner membrane of plastid envelope. The second objective is to identify the function of the newly identified paralog to the chloroplast protein translocation channel. We provided genetic evidence that this protein is essential for viability of plants from the embryonic stage, and presented this finding at American Society of Biologists Annual Meeting (July). In addition, we have started preparing a manuscript about this part of the
research for publication in a scientific journal. The third objective is to analyze proteome of fruit plastids. As stated in previous reports, we have been taking molecular biological approaches to study proteins involved in carotenoid biosynthesis in citrus. We obtained funding from California Citrus Research Board and started examining expression patterns of several genes responsible for the biosynthesis of plastid pigments (chlorophylls and carotenoids) as well as those involved in plastid development in citrus fruit peel during its color change (from green to orange, and orange back to green). We also started extending our research and using a model plant Arabidopsis as well as Escherichia coli to elucidate mechanistic details of carotenogenesis. Part of these works was presented at American Society of Biologists Annual Meeting (July). There is no specific progress to be reported for the fourth objective (analysis of ascorbate homeostasis in plastids).
Impacts
The research activity has involved two graduate students, two postdoctoral scholars, and two undergraduate students. They have been actively participating in not only experiments, but also research discussion. Thus, the project has had an impact on education.
Publications
- Tripp J, Inoue K, Keegstra K, Froehlich JE (2007) A novel Serine/Proline-rich domain in combination with a transmembrane domain is required for the insertion of AtTic40 into the inner envelope membrane of chloroplasts. The Plant Journal 52, 824-838
- Inoue K (2007) The chloroplast outer envelope membrane: the edge of light and excitement. Journal of Integrative Plant Biology 49, 1100-1111
|
Progress 01/01/06 to 12/31/06
Outputs
The research related to the first objective which concerns the mechanism by which proteins are targeted to the chloroplast outer envelope membrane has been funded by the Growth and Development program of USDA-CSREES between September 2003 and August 2006. Its detailed progress can be found in another report CA-D*-PLS-7168-CG. Briefly, we got further insights into the amino acid sequences which play important roles in targeting proteins to the chloroplast envelope and published this finding to a scientific journal in April 2006. Furthermore, we presented the data derived from this research at one international meeting in June 2006, and one domestic meeting in August 2006. The second objective is to identify the function of the newly identified paralog to the chloroplast protein translocation channel. We have continued elucidating this problem using biochemical and genetic reagents we have prepared in previous years and also this year. There is no original scientific
article published related to this objective. The third objective is to analyze proteome of fruit plastids. As stated in the last year's report, we have been taking molecular biological approaches to study proteins involved in carotenoid biosynthesis in citrus. The results reported in the previous year regarding characterization of catalytic activities and suborganellar localization of carotenogenic proteins from citrus has been summarized and published in a scientific journal in August 2006. Furthermore, we have generated genetic and biochemical reagents to get further insights into the mechanistic details of carotenogenesis. There is no specific progress to be reported for the fourth objective (analysis of ascorbate homeostasis in plastids).
Impacts
The research activity has involved two postdoctoral scholars, one international visiting postdoctoral scholar, two undergraduate students, and two 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 impact derived from accomplishing the first objective can be found in another report CA-D*-PLS-7168-CG. There is no specific impact to be reported from the research on the second and third objectives.
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 Inoue K, Furbee KJ, Uratsu S, Kato M, Dandekar AM, Ikoma Y (2006) Catalytic activities and chloroplast import of carotenogenic enzymes from citrus. Physiologia Plantarum 127:561-570
|
Progress 01/01/05 to 12/31/05
Outputs
The first objective of the research project concerns the mechanism by which proteins are targeted to the chloroplast outer envelope membrane. The research related to this objective has been funded by the Growth and Development program of USDA-CSREES since September, 2003, and hence its detailed progress can be found in another report CA-D-PLS-7168-CG. Briefly, we were able to identify a peptidase responsible for maturation of the chloroplast protein translocation channel and presented the results at an international meeting in April, 2005. We also published the data in a scientific journal. The second objective is to identify the function of the newly identified paralog to the chloroplast protein translocation channel. We were able to prepare biochemical and genetic reagents to address these problems. There is no original scientific article published related to this objective. However, we were involved in writing a review article regarding evolutionary origin of the
chloroplast protein translocation machinery. The third objective is to analyze proteome of fruit plastids. As reported last year, we started taking molecular approach to study proteins involved in plastid metabolism, more specifically, citrus carotenoids. We have isolated full length cDNA clones for carotenogenic proteins from citrus fruit peels, and expressed them in bacteria to confirm their catalytic activities. Furthermore, we performed protein import assay using isolated chloroplasts to verify their plastid targeting and also examined their suborganellar localization. These data were summarized and submitted for publication in a scientific journal in November, 2005. There is no specific progress to be reported for the fourth objective (analysis of ascorbate homeostasis in plastids).
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 main accomplishment from the first objective was identification of a protein named Plsp1 (plastidic type I signal peptidase 1). This enzyme is responsible for maturation of the protein translocation channel at the chloroplast outer envelope membrane. Interestingly, Plsp1 was targeted both to the envelope and also to thylakoids of chloroplasts. Disruption of the Plsp1 gene in the model plant Arabidopsis resulted in reduction of the development of plastid internal membranes such as thylakoids. These results have 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. As for the second objective, the paper we published last year (Inoue and Potter, 2004) has impacted on the field of membrane protein biogenesis as is evidenced by the fact that it was cited more than 10 times in other scientific articles.
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
- Reumann S, Inoue K, Keegstra K (2005) Evolution of the general protein import pathway of plastids. Molecular Membrane Biology 22: 73-86
- Gomez SK, Cox MM, Bedel JC, Inoue K, Alborn HT, Tunlinson JH, Korth K (2005) Lepidopteran herbivory and oral factors induce transcripts encoding terpene synthases in Medicago truncatula. Archives of Insect Biochemistry and Physiology 58: 114-127
|
Progress 01/01/04 to 12/31/04
Outputs
The first objective of the research project concerns the mechanism by which proteins are targeted to the outer membrane of the chloroplast envelope. The research related to this objective has been funded by the Growth and Development program of USDA-CSREES since September, 2003, and hence its progress can be found in another report CA-D*-POM-7168-CG. For the second objective, which is to identify the function of the newly identified protein in the chloroplast outer envelope homologous to the protein translocation channel, the following progress has been made in the reporting period. First, via a collaboration with Dr. Daniel Potter (Department of Pomology, UC Davis), we finished initial characterization of this protein named OEP80 described in last year's report, and published a research paper. The data were also reported at the Gordon Research Conference on Mitochondria and Chloroplasts in July, 2004. Second, a new graduate student joined the laboratory and started
studying OEP80. She started analyzing the Arabidopsis plants with mutations in the gene for this protein. Furthermore, she has prepared a recombinant form of OEP80 in bacteria which will be utilized to characterize this protein more in detail. The third objective was to analyze proteome of fruit plastids. Despite of several attempts, we have not been able to isolate plastids from any fruits yet. Instead of isolating fruit plastids and analyze their proteome, we started obtaining molecular reagents to study metabolism in plastids, namely cDNAs for enzymes potentially responsible for carotenoid biosynthesis, from citrus peel, via collaboration with Dr. Abhaya Dandekar (Department of Pomology, UC Davis). We have characterized functions of some of the clones. Furthermore, I published a review article regarding a recent finding of a key enzyme responsible for carotenoid biosynthesis. There is no specific progress report for the fourth objective, which is to analyze ascorbate homeostasis in
plastids.
Impacts
The publication by Inoue and Potter is related to the second objective, and has corrected the misconcept that was previously reported by a German group regarding evolutionary relationships among proteins related to chloroplast protein translocation channel. Although there has been no evident impact so far, a couple of papers have been prepared to discuss this work's impact on the field of the organelle evolution.
Publications
- Inoue, K. and Potter, D. 2004. The chloroplastic protein translocation channel Toc75 and its paralog OEP80 represent two distinct protein families and are targeted to the chloroplastic outer envelope by different mechanisms. The Plant Journal 39:354-365.
- Inoue, K. 2004. Carotenoid hydroxylation - P450 finally! Trends in Plant Science 9:515-517.
|
Progress 01/01/03 to 12/31/03
Outputs
The first two objectives of the research project concern problems with a protein in the outer membrane of the chloroplastic envelope, Toc75. This protein is postulated to function as a protein translocation channel in the envelope membrane, and is essential not only for biogenesis of the chloroplast, but also for viability of plants. I have obtained a two-year funding from the Growth and Development program of USDA-CSREES for a proposal related to the objective 1 of the research project, to identify the sequence that is necessary and sufficient to target the protein to the plastid envelope membrane. I made a poster presentation of results related to this objective at the 22nd Symposium in Plant Biology in Riverside, CA, in January, 2003. Any other progress report for this part can be found elsewhere (CA-D-POM-7168-CG). For the objective 2 of the research project, to identify the function of the newly identified protein homologous to Toc75, the following progress has
been made in the reporting period. First, I obtained antisera against a portion of the protein. A rotation graduate student, Hsien Men Easlon, and myself performed immunoblot experiments using these antisera and protein extracts from Arabidopsis chloroplasts, and found that the molecular weight of the protein was 80 K. Based on this finding and the predicted amino acid sequence, we named the protein as AtOEP80 for Arabidopsis thaliana Outer Envelope membrane Protein of 80 K. The antisera will be further utilized to analyze the function of AtOEP80. Furthermore, in collaboration with Dr. Daniel Potter (Department of Pomology, UC Davis), we have established phylogenetic relationships of proteins homologous to Toc75. We collected and analyzed sequences of proteins not only from plants, but also from a wide range of Gram negative bacteria including cyanobacteria which are believed to share the common ancestor with the higher plant chloroplasts. We found that OEP80 orthologues belong to a
family of proteins that include those from cyanobacteria, which is distinct from that of Toc75 orthologues. This finding would suggest that OEP80 is more ancestral form than Toc75. I also found that the mechanism for targeting of AtOEP80 to the chloroplastic envelope is different from that of Toc75. Comparison of the targeting mechanism of two paralogous proteins should be relevant to the long term objective of the research project. I started preparing a manuscript describing these findings for publication. There is no specific progress report for other two objectives.
Impacts
There is no evident impact so far. The plastid is a plant-specific organelle in which numerous important biochemical reactions, such as photosynthesis and biosynthesis of amino acids, lipids, and vitamins, take place. Understanding the biogenesis of this important organelle could develop new strategies to solve practical agricultural problems, for example, by manipulating its metabolism, in the future.
Publications
- Inoue, K. 2003. Protein translocation across biological membranes: cost what it may. Trends in Plant Science 8:360-363.
- Inoue, K. and Keegstra, K. 2003. A polyglycine stretch is necessary for proper targeting of the protein translocation channel precursor to the outer envelope membrane of chloroplasts. The Plant Journal 34:661-669.
- Davila-Aponte, J.A., Inoue, K. and Keegstra, K. 2003. Two chloroplastic protein translocation components, Tic110 and Toc75, are conserved in different plastid types from multiple plant species. Plant Molecular Biology 51:175-181.
|
|