SP1 and ubiquitin-mediated control of chloroplast protein import

Chloroplasts and mitochondria are normal components of many cells - they are sub-cellular structures called organelles. Interestingly, these two organelles evolved from bacteria that were engulfed by other cells more than a billion years ago, and in many ways they still resemble free-living bacteria. Chloroplasts are found in plant cells, contain the green pigment chlorophyll, and are exclusively responsible for the reactions of photosynthesis (the process that captures sunlight energy and uses it to power the activities of the cell). Since photosynthesis is the only significant mechanism of energy-input into the living world, chloroplasts are of inestimable importance, not just to plants but to all life on Earth. Chloroplasts are also important in many other ways, since they play essential roles in the biosynthesis of oils, proteins and starch. Although chloroplasts do contain DNA (a relic from their ancient, evolutionary past as free-living photosynthetic bacteria), and so are able to make some of their own proteins, over 90% of the 3000 proteins needed to build a fully functional chloroplast are encoded on DNA in the cell nucleus. Most chloroplast proteins are therefore made outside of the chloroplast, in the cellular matrix known as the cytosol. Since chloroplasts are each surrounded by a double membrane, or envelope, that is impervious to the passive movement of proteins, this presents a significant problem. To overcome the problem, chloroplasts have evolved a sophisticated protein import apparatus, which uses energy (in the form of ATP) to drive the import of proteins from the cytosol, across the envelope, and into the chloroplast interior. This protein import apparatus comprises two molecular machines: one in the outer envelope membrane called TOC (an abbreviation of 'Translocon at the outer envelope membrane of chloroplasts'), and another in the inner envelope membrane called TIC. Each machine is made up of several different proteins which cooperate to ensure the efficiency of import. We work on a model plant called Arabidopsis that has many advantages for research, such as an availability of numerous mutants (each one with a mutation in a specific gene). One such mutant plant, ppi1, has a defect in a TOC gene such that chloroplast protein import does not work efficiently. Several years ago, we identified another mutation called sp1 (this stands for 'suppressor of ppi1'), which significantly improves protein import efficiency in ppi1. Very recently, we discovered that the defective gene in sp1 (the SP1 gene) encodes a type of regulatory protein called a 'ubiquitin E3 ligase'. These usually work by labelling-up unwanted proteins and targeting them for degradation. Because this control mechanism was not previously known to operate in chloroplasts, we believe that we have made an important breakthrough. We think that SP1 controls protein import efficiency by regulating the amount of the TOC machinery. In the sp1 mutant, this control mechanism is disturbed somewhat, allowing certain TOC proteins to accumulate to a higher level, thereby improving protein import efficiency. We will do experiments to test these theories. Because chloroplasts carry out essential functions, and because protein import is essential for chloroplast development, it should come as no surprise to learn that plants without a functional chloroplast protein import machinery are unable to survive (in fact, they die at the embryo stage). Thus, chloroplast protein import is an essential process for plants. Similarly, since we are all ultimately dependent upon plant products for survival, it follows that chloroplast protein import is essential on a global scale. Because chloroplasts play major roles in the synthesis of many economically important products (such as oils and starch), a more complete understanding of how these organelles develop will enable us to enhance the productivity of crop plants, or otherwise manipulate their products.

We found that SUPPRESSOR OF PPI1 LOCUS 1 (SP1) encodes a RING finger (RNF) ubiquitin E3 ligase of the chloroplast envelope. We propose that SP1 controls chloroplast protein import by targeting the Toc75 translocon component for degradation. This is highly novel as no role for the ubiquitin proteasome system (UPS) at the chloroplast surface was reported before. We will elucidate the role of SP1 and UPS in chloroplast import. 1. To confirm that Toc75 is a substrate of SP1, we will test the Toc75-SP1 association by several methods: in vitro pull-down using recombinant and in vitro translated proteins; in vivo co-immunoprecipitation using either epitope-tagging or SP1 antibody; possibly also BiFC, to confirm the interaction in intact cells if necessary. 2. To confirm that SP1 regulates turnover of Toc75 via the UPS, we will do ubiquitination assays in vitro (using recombinant proteins) and in vivo (in WT and sp1 plants, to test the requirement for SP1). We will also assay Toc75 stability in WT and sp1 cells, via protoplast transfection. 3. We will do biochemical tests to confirm that SP1 is an integral protein of the outer envelope membrane and to elucidate its orientation. 4. We will analyse functions of three SP1 domains: two transmembrane domains and the RNF. We will mutate each one and then assess for effects on: localization in vivo; ubiquitination in vitro; functionality in planta (complementation). 5. We will assess whether SP1 and its homologues, SPL1 and SPL2, have redundant or divergent roles. To do this we will study double and triple mutants (in the ppi1 and PPI1 backgrounds), and overexpress each of the genes (in the ppi1 and sp1 ppi1 backgrounds). 6. We will use tandem affinity purification and proteomics to seek additional substrates of SP1. Collaborative arrangements have been established. 7. We will assess for involvement of SUMO in chloroplast import, and conduct a new screen for sp mutants (the original screen was not saturating).

We propose basic research on the Arabidopsis model plant in an area of profound biological importance with great potential to yield results of economical or societal significance. Non-academic impact of the research is discussed below. Chloroplasts are the site of photosynthesis in plants, algae and some protists, and so mediate much of the world's primary productivity. As photosynthesis is the only significant mechanism of energy-input into the biosphere, chloroplasts are essential for plants and animals alike; thus, agriculture is wholly dependent on chloroplast biogenesis. Chloroplasts or other plastids also mediate many biosyntheses (starch, amino acids, fatty acids). Many of these products are vital in mammalian diets, and knowledge on plastid biogenesis may enable improvements in their quantity or quality. Since plastids are so integral to cellular metabolism, plastid biogenesis defects can cause plants to die during (pre)embryonic development. Chloroplasts contain ~3000 proteins but only ~100 are encoded by the plastome. Thus >90% of plastid proteins are nucleus-encoded and cytosolically-synthesized, so that chloroplast biogenesis is dependent on efficient operation of the TOC/TIC import machinery. Because of chloroplasts' uniquely important role in the biosphere, we are all dependent upon proper chloroplast development (including protein import) for survival. This emphasizes the importance of knowledge on chloroplast protein import. Plastids offer many opportunities for agricultural or industrial exploitation. Depletion of fossil fuels and environmental effects of their use demand that renewable materials are used by the chemical and fuel industries. Biofuels have attracted much attention recently, and will likely become more significant as cost and efficiency issues are resolved. As raw materials for biofuel production are derived largely via chloroplast processes, improved understanding of plastid biogenesis will aid development of this important technology. As chloroplasts may contain >50% of total leaf protein, foreign proteins can be expressed to extremely high levels in plastids. Plastid manipulation may also enable accumulation of foreign proteins that would be harmful elsewhere in the cell. Plastids are inherited maternally and so (in relation to transplastomics) the possibility for transgene out-crossing is minimized. Knowledge on plastids may have medical or veterinary applications, as apicomplexan parasites (malaria, toxoplasmosis, coccidiosis) contain a relict plastid. Moreover, our work on SP1 may shed light on related processes in mitochondrial biogenesis; significantly, mitochondria and the ubiquitin proteasome system have both been implicated in ageing and neurodegenerative diseases. Short-term commercialization is not currently envisaged, but relevant mechanisms are nonetheless in place. For example, we will liaise with Leicester's Biobator which promotes commercial activities resulting from University research, and the Enterprise and Business Development Office which advises on intellectual property, its protection and commercialization. We will engage the public and broader community in various ways. We will continue to accept visitors into our lab via different schemes; e.g., sixth-formers funded by the Nuffield Foundation, undergraduates funded by the Genetics or Biochemical Societies, and ESF-funded trainee technicians. The Nuffield scheme gives school students an insight into scientific research and promotes outreach and science education. The Department is invited to present at local schools and colleges, and we will actively support this role. Further engagement will be through our lab web pages and our participation in undergraduate teaching. Our research directly influences undergraduate teaching, as research developments are presented in tutorials and lectures. We will engage the media via the University Press Office, with press releases on new publications and discoveries.