Investigating the function of a ClpC/Hsp100-type chaperone in chloroplast preprotein 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 (which is a relic from their ancient, evolutionary past as free-living photosynthetic bacteria), and are therefore able to make some of their own proteins, over 90% of the 3000 or so proteins required to build a fully functional chloroplast are encoded on DNA within the cell nucleus. The majority of 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.
This project is focused on the TIC machine, and in particular on a protein called Hsp93 which is associated with the TIC complex. This Hsp93 protein is an ATPase (i.e. it hydrolyses ATP to release energy), and is a member of a family of proteins called the "molecular chaperones". Such chaperone proteins are able to bind to other proteins, particularly when they are in an unfolded state. In doing this, some chaperones can exert a "pulling force" on the target protein, to facilitate its passage from one location to another. Based on several lines of evidence, Hsp93 is thought to provide the driving force for chloroplast protein import, and to act by pulling on those proteins that need to be imported (i.e. it is believed to be a core part of the so-called "chloroplast protein import motor"). Thus, much of the ATP consumption that occurs during the import mechanism is tentatively attributed to Hsp93. However, direct proof of these hypotheses is still lacking. We propose to test these ideas directly, by manipulating the activities of the Hsp93 protein in intact plants, and assessing the consequences of such manipulations on chloroplast protein import efficiency.
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. What is more, since chloroplasts play a major role in the synthesis of many economically important products (such as oils and starch), a more complete understanding of how these organelles develop may enable us to enhance the productivity of crop plants, or otherwise manipulate their products.

Hsp93 is a chloroplast ClpC/Hsp100-type chaperone that partitions between the envelope and the stroma, and it is thought to have two different roles: 1) at the envelope TIC complex it is thought to drive chloroplast protein import; 2) with stromal ClpP it is thought to target proteins for degradation. We will study the first of these roles as it is not yet supported by conclusive evidence.
Studying Arabidopsis hsp93 knockouts supported a role for Hsp93 in import, but could not causally isolate the mutant's import defects from proteolytic defects. To separate these functions, we mutated critical residues in Hsp93 that affect just one of the protein's roles, and then tested for complementation of hsp93 knockout plants. In collaboration with A. Clarke (Gothenburg; who is interested in proteolysis), we will study these transgenics in detail to gauge the relative importance of Hsp93's two roles.
Chloroplast protein import consumes stromal ATP, but this usage has not yet been assigned to a particular component. To test the importance of Hsp93 ATPase activity for import, we will interfere with the protein's ability to bind or hydrolyse nucleotides, and then assess the consequences in relation to protein import in plants.
Unpublished data imply that Hsp93 is recruited to the envelope by a well-known TIC protein. We will study this critical Hsp93-TIC interaction in detail, in vitro and in vivo. Use of deletion series will map the interaction, while its regulation will be assessed by applying different nucleotides, clients or partners. We will also elucidate complex size and stoichiometry.
Hsp93 forms a hexameric ring in the stroma, but its oligomeric state at the envelope is unknown. If envelope Hsp93 is hexameric, it likely acts by threading preproteins through the ring's axial channel; if it is not, another mechanism must operate. To address this issue, and to identify possible Hsp93 interactors at the envelope, we will characterize envelope-bound Hsp93 complexes.

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 outlined formal collaboration with A. Clarke at Gothenburg University, but also due to the involvement of Dr Flores-Perez as RA who maintains strong scientific links with Barcelona University where she obtained her PhD. 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 are commercially important (e.g. 50% of drugs target membrane proteins). But the success of such methods will depend on proper protein targeting, of which we presently have only superficial understanding. Our work on Hsp93 will significantly advance our knowledge in this area, thereby facilitating the use of chloroplasts as 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.