Characterization of the TOC complexes which define distinct client-specific chloroplast protein import pathways in Arabidopsis

How chloroplasts efficiently import thousands of different proteins. 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 TOC machine, which performs two essential functions. Firstly, it recognizes those proteins that need to be imported as they arrive at the chloroplast surface. Secondly, it forms a channel through the outer membrane so that the proteins can pass across, once recognized. We will study the first of these functions: recognition. To carry out this function, the TOC machine uses special molecules called receptors, which bind to the proteins as they arrive at the chloroplast envelope. Quite recently, we found that there are actually several different types of receptor, and we think that they probably exist so that chloroplasts can efficiently recognize all of the many different proteins they need to import. In other words, we think that each of the receptors has a degree of specificity for a particular subset of the proteins that must be imported. This project will test these ideas, since we think that the different types of receptor are very important, helping to ensure the formation of fully functional chloroplasts. 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.

The aim is to characterize the TOC complexes that define distinct, client-specific (or, preprotein-specific) chloroplast protein import pathways in Arabidopsis. Different Arabidopsis TOC complexes will be purified using tandem affinity purification (TAP). To do this, we will use well-characterized Arabidopsis transgenics expressing TAP-tagged versions of the receptors, atToc33 and atToc34, which are thought to act in different import pathways. Proteins in the purified TOC complexes will be studied by SDS-PAGE, immunoblotting and mass spectrometry (the latter in collaboration with K. Lilley, Cambridge). More detailed studies will include assessments of complex size by BN-PAGE and size exclusion chromatography. We will also assess for dynamic changes in the complexes in response to TOC receptor knockout mutations. If novel factors are identified in association with the complexes, their detailed characterization will be the subject of a future project proposal. To corroborate the results of the TAP experiments, we will do in vivo studies using bimolecular fluorescence complementation (BiFC). We will test for interaction specificity between different members of the two Arabidopsis receptor families (atToc33, atToc34 vs. atToc159, atToc132, atToc120, atToc90). We expect to observe distinct association preferences amongst the receptor isoforms, leading to the formation of distinct TOC complexes. Experiments will be done in transiently transformed Arabidopsis protoplasts, using methods well established in the laboratory. Finally, we will conduct a preliminary study on preprotein-TOC receptor binding using nuclear magnetic resonance (NMR) spectroscopy (in collaboration with P. Crowley, Dublin), since there is a lack of structural information on these interactions. We will focus on two receptors (atToc33, atToc34) and two, functionally-different preproteins (plastocyanin, thioredoxin). We expect the data to reveal determinants of binding and specificity.