Investigating the roles of Arabidopsis STIC1 and STIC2 in chloroplast protein transport

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 over 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 responsible for the reactions of photosynthesis (the process that captures sunlight energy and uses it to make sugars). As 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, as they play essential roles in the synthesis of oils, proteins and starch.
Although chloroplasts do contain DNA (a relic from their evolutionary past as free-living photosynthetic bacteria), and so can make some of their own proteins, >90% of the 3000 proteins needed to build a fully-functional chloroplast are encoded on DNA in the cell nucleus. Thus, most chloroplast proteins are made outside of the organelle in the cellular matrix known as the cytosol. As 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 evolved a sophisticated protein import apparatus, which uses energy (in the form of ATP) to drive the import of proteins from the cytosol. This import apparatus comprises two molecular machines: one in the outer envelope membrane called TOC (an abbreviation of "Translocon at the outer envelope of chloroplasts"), and another in the inner envelope membrane called TIC. Each machine is made up of several proteins which cooperate to ensure the efficiency of import. One of the features of the TIC machine is that it recruits a special class of proteins from the chloroplast interior, or stroma, called "chaperones". These stromal chaperones act like a motor as they use the energy from ATP to drive protein 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, tic40, has a defect in a TIC gene such that chloroplast protein import does not work efficiently. Several years ago we identified other mutations called stic1 and stic2 (stic stands for "suppressor of tic40") which significantly improve protein import efficiency in tic40. Recently, we discovered which genes (and therefore which proteins) the stic mutations affect: STIC1 belongs to a family of well-known protein transport factors that were not previously thought to act in the chloroplast envelope, while STIC2 is related to a group of bacterial proteins of unknown function. Interestingly, we have shown that STIC2 can bind to STIC1, as well to stromal chaperones and the TIC machine. Thus, we believe that STIC2 may be a new component of the aforementioned import motor. It may also help to guide newly-imported proteins from the TIC apparatus to their final destination, which is perhaps where STIC1 plays its role. We will do experiments to test these theories.
As chloroplasts carry out essential functions, and because protein import is essential for chloroplast development, it is not surprising that plants without a functional chloroplast protein import machinery are unable to survive (in fact, they die as embryos). Similarly, as we are all ultimately dependent upon plant products for survival, it follows that chloroplast protein import is essential on a global scale. As chloroplasts play major roles in the synthesis of many economically important products (e.g., oils, starch), a better understanding of how these organelles develop will enable us to enhance the productivity of crop plants or otherwise manipulate their products.

Arabidopsis stic mutations suppress tic40, which disrupts chloroplast protein import motor function and inner envelope protein re-insertion. STIC1 belongs to a well-known family of protein transport factors while STIC2 is related to bacterial proteins of unknown function. STIC2 interacts with STIC1, stromal motor chaperones and Tic110; it may be a cochaperone in the motor and/or a stromal guidance factor linking the TIC to targets in destination membranes (e.g. STIC1). We will define the roles of STIC1/2.

1. Aforesaid STIC2 interactions were revealed by immunoprecipitation (IP) and BiFC. This work will be corroborated by in vitro pull-down and extended to include analyses of STIC1.

2. Further IP and TAP assays (and LC-MS/MS) will aim to identify new STIC1/2 partners.

3. Pilot YFP and subfractionation work showed that STIC2 is stromal, while STIC1 is mainly thylakoidal but possibly also in the envelope. We will test the hypothesis that some STIC1 is in the envelope in refined YFP/fractionation studies and by immunogold EM; if it is not, we will assess how loss of its thylakoid function might suppress tic40.

4. To assess possible STIC2 functions (import motor vs. guidance factor) we will test: its ability to bind importing preproteins and the timing/location of such interactions; its ability to act as a cochaperone toward its main chaperone partner.

5. In vitro assays showed that the tic40 protein import defect is alleviated by stic2. Such assays will also be done for stic1. We will also assess the tic40 re-insertion defect in both mutants.

6. Genetic analyses will assess: the STIC1/2 relationship (they seem to act in the same pathway); STIC2/chaperone interactions; suppression specificity of stic1/2; the relationship between STIC2 and its homologue STL; functional conservation between STIC2 and its E. coli homologue.

7. A shared motif exists in STIC1 and -2; we will test its importance in planta and whether it enables binding of a mutual partner.

Academic impact will be substantial due to the work's interdisciplinarity as detailed in the Academic Beneficiaries section. This will manifest itself in several ways: 1) The project will contribute significantly to scientific advancement providing new knowledge with relevance in several overlapping fields and disciplines. 2) The project will stimulate international collaboration, mainly through the collaboration with Topel/Oxelman at Gothenburg University, but also due to the involvement of Dr Wu as RA who will return to China after the project to pursue an independent research career and with whom we expect to maintain collaborative links. 3) The project will contribute significantly to the health of UK plant science due to publicity surrounding the project, the interactions it will enable, and by our hosting of visitors from schools as this will generate enthusiasm for plant biology. 4) The project will deliver highly-trained individuals who will also contribute to the health of UK plant science. Training will result not only from the direct involvement of the research staff but also from Prof Jarvis' supervision of PhDs and MSc project students (enrolled on the University's Molecular Genetics or Bioinformatics courses), who will work on projects closely related to the proposed work and have daily interaction with the research staff.

In the longer term, industry, agriculture and society generally also stand to benefit from the work, due to the fundamental importance of the area in which the project is focused. Chloroplasts are the site of photosynthesis in plants and so are responsible for much of the world's primary productivity. Plastids synthesize a diversity of products (e.g. starch, amino acids, fatty acids) and many of these are vital in mammalian diets. Knowledge on plastid biogenesis resulting from the project may enable improvements in the quantity or quality of these products, or in the productivity of crops generally. 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 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, better understanding of plastid biogenesis will aid development of this technology.
As chloroplasts can contain >50% of leaf protein, foreign proteins can be expressed to very high levels in plastids. Manipulating the TIC machinery was shown to induce massive proliferation of the inner envelope membrane, without affecting plant growth. This may provide an environment for accumulation of foreign membrane proteins, which are difficult to express in bacteria and commercially important (e.g. 50% of drugs target membrane proteins). But the success of such methods will depend on proper protein targeting and at present the protein transport to the inner envelope is poorly understood. Our work may yield insight in this area, facilitating use of chloroplasts as membrane protein bioreactors.

The general public and schools will benefit as we will engage with them in various ways. We will develop a schools engagement activity on Chloroplast Biology as part of a two-day event entitled Dynamic DNA organized by GENIE, a Centre for Excellence in Teaching and Learning at the University. Through the University's Botanic Garden (which receives 40k visitors annually) we will contribute to a publicly-circulated newsletter, deliver a public lecture on project-related topics, prepare a display board for placement at the gardens, and contribute to well-attended educational activities for local schools. Finally, we will continue to accept visitors into our lab via different schemes (e.g. sixth-formers funded by the Nuffield Foundation) and engage the media via the University Press Office.