<Research questions>
1. How would the endosymbiont lose its autonomy and become an organelle in eukaryotic cells?
>> Elucidating the functions and sorting/assembly mechanisms of Toc75 and OEP80*, which are homologous to a family of proteins essential for the viability of Gram-negative bacteria.
2. How do the intraorganellar membrane systems develop?
>>Elucidating the mechanism by which Plsp1** is sorted to the envelope and thylakoid within a chloroplast.
>>Characterizing the citrus fruit peel that changes its color from green to orange, then back to green (dis-assembly and re-assembly of thylakoids).
3. What is the biological significance of gene duplications?
>>Comparing properties and functions of two sets of homologous proteins in chloroplasts (Toc75/OEP80* and Plsps/TPPs**).
4. What are the roles of chloroplasts in “no-photosynthetic” tissues?
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*Toc75 and OEP80 (also known as Toc75-V) are paralogous proteins in the outer envelope membrane of chloroplasts in Viridiplantae (Eckart et al. 2002; Inoue and Potter 2004). They belong to the Omp85 family, which includes proteins in the outer membranes of Gram-negative bacteria (including cyanobacteria, which share the common ancestor with chloroplasts) and mitochondria of eukaryotic cells. Hence, it is believed that Toc75/OEP80 derived from a protein in the ancient cyanobacterium. The homologs in bacteria (BamA) and mitochondria (Sam50/Tob55) play essential roles in sorting and integration of β-barrel proteins to the outer membrane (Knowles et al. 2009). Whether this is the case in cyanobacteria and chloroplasts remains elusive. In chloroplasts, Toc75 is the major component of the protein import apparatus: it forms a conducting channel for proteins synthesized in the cytosol. Establishment of protein import machinery must have been a prerequisite for the successful conversion of the endosymbiont to the organelle, driving the endosymbiont to give up its own genes to the nucleus of the host cell (because if it can import proteins, it would not need to invest energy to make them). Both Toc75 and OEP80 are indispensable for viability of plants from the embryonic stage (summarized in Hsu and Inoue 2009). One hypothesis is that OEP80 retains the original function of BamA in the ancestral cyanobacterium, whereas Toc75 evolved to gain a new function that is essential for the organelle. But this idea does not go beyond a hypothesis.
**TPP (Thylakoidal Processing Peptidase) catalyzes maturation of a subset of proteins in the thylakoid lumen, such as Oxygen Evolving Complex (OEC) subunits and plastocyanin, as well as several membrane-associated lumenal proteins such as VAR1 (FtsH1/5), VAR2 (FtsH2/8), and the chloroplast-encoded cytochrome f. In the model plant Arabidopsis thaliana (Arabidopsis), there are three TPP homologs named Plsp1 (At3g24590), 2A (At1g06870), and 2B (At2g30440) (Plsp stands for Plastidic type I signal peptidase; Hsu et al. 2011). Plsp2B was the first TPP whose cDNA was identified (Chaal et al. 1998).
Phylogenetic analysis revealed that TPP may have originated before the endosymbiotic event, and that there are two groups of TPP in seed plants: one includes Plsp1 and another comprises Plsp2A and Plsp2B. The duplication leading to the two groups predates the gymnosperm-angiosperm divergence, and the separation of Plsp2A and 2B occurred after the Malvaceae-Brassicaceae diversification (Hsu et al. 2011). Genetic and biochemical studies have demonstrated that Plsp1 is the main TPP isoform in Arabidopsis (Inoue et al. 2005; Shipman-Roston et al. 2010; Midorikawa and Inoue 2013; Midorikawa et al. 2014). Interestingly, Plsp1 is located not only in thylakoids, but also in the envelope, where it appears to catalyze maturation of Toc75 (Inoue et al. 2005; Shipman and Inoue 2009). In addition, thylakoid-localied Plsp1 forms a stable complex with PGRL1 (Endow and Inoue 2013). PGRL1 is postulated to catalyze electron transfer from ferredoxin to plastoquinone during antimycin A-sensitive cyclic electron flow (CEF) around Photosystem I (PSI) (Hertle et al. 2013). CEF around PSI plays versatile roles in the photosynthesis of higher plants (Johnson 2011). Thus, it is tempting to speculate that the stable complex formation of Plsp1 and PGRL1 might represent a novel link that connects photosynthesis and energy-consuming protein transport (Endow and Inoue 2013).
Results of expression analysis and genetic complementation assay using the plsp1-null plants suggest that the two Plsp2 isoforms are evolved to play a role different from that of Plsp1 (Hsu et al. 2011). Whether the two Plsp2 isoforms are simply redundant or they have specific roles remains elusive. Also unknown is if Plsp2 isoforms are also located in both the envelope and thylakoids.
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Background: Plastids are organelles essential and specific to all photosynthetic and some non-photosynthetic eukaryotes. Plastids support lives of all organisms on earth via their various metabolic activities including oxygenic photosynthesis. A plastid is surrounded by a double-membrane envelope. In higher plants, the undifferentiated protoplastid in meristematic cells ensures the continuity of plastids between generations, and develops into more complex plastids, such as chloroplasts in photosynthetic tissues, chromoplasts in non-green fruits, roots, and flower petals, and amyloplasts in roots.
According to the endosymbiotic theory, the chloroplast used to be a free-living bacterium, which shares the common ancestor with modern cyanobacteria, more than billions years ago. More than 2000 different proteins are postulated to be present in a plastid of higher plants [see Inoue 2007 (corrections) and references therein]. However, the plastid genome of most higher plants encodes less than 100 proteins. A vast majority of proteins found in plastids are encoded in the nuclear genome and synthesized on cytosolic ribosomes. They have to be targeted to the plastid and/or traverse the envelope membranes posttranslationally. Thus, protein import is an essential process for the biogenesis of plastids in two distinct ways:
1. Establishment of the protein import machinery was required for the successful conversion of the bacterial endosymbiont to the organelle in eukaryotic cells.
2. Posttranslational protein import is a prerequisite for the development and maintenance of the existing plastids.
Extensive biochemical studies by various laboratories have resulted in identifying proteinaceous machineries at the outer and inner membranes of the chloroplast envelope that are responsible for protein import. These components are also encoded in the nuclear genome although the mechanisms by which they are targeted to and assembled properly in the lipid bilayers are not completely understood yet. Furthermore, molecular details of the processes by which these components were acquired during the endosymbiotic event remain elusive.
Research Tools:
Biochemical assays:
-Preparation and fractionation of chloroplasts from seedlings of pea and Arabidopsis.
-In vitro protein import assay.
-Protein separation by SDS-PAGE, blue native-PAGE, and gel filtration chromatography.
-Separation and analysis of photosynthetic pigments by HPLC.
-Affinity purification of proteins.
-Antibody purification
-Co-immunoprecipitation.
Generation and utilization of mutant plants:
-Identification and characterization of Arabidopsis mutants (reverse genetics).
-Genetic complementation assays using constitutive, native, and tissue-specific promoters as well as an inducible system.
-RNAi (under adaptation).
-Transient expression in Nicotiana benthamiana.
Gene expression and protein accumulation analysis:
-Real-time PCR.
-Proteomics (with UCDavis Proteomics Facility).
-Immunoblotting.
Others:
-Electron microscopic study (observation and immunolocalization: with UCDavis Department of Pathology and Laboratory Medicine, EM Lab).
-Fluorescent light microscopic study (localization of GFP-tagged proteins in living cells).
-Citrus fruit peel culture.
Grant Support:
US NSF Molecular and Cellular Biosciences Program (’11-’16)
US DOE Energy Biosciences Program (’08-’11; ’11-’14; ’14-’17)
California Citrus Research Board (’07-’09)
UC Discovery Grant (’03-’07)
USDA-CSREES Growth and Development program (’03-’06)