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

Lead Research Organisation: University of Leicester
Department Name: Biology

Abstract

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.

Technical Summary

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.

Publications

10 25 50
 
Description Previous work from our laboratory and others suggested that different isoforms of the TOC receptors function in different import pathways with specificity for different preprotein clients. More specifically, the major receptor isoforms (atToc33 and atToc159) are thought to mediate the import of highly-abundant photosynthesis-related preproteins, while the minor receptor isoforms (atToc34 and atToc132/120) mediate the import of less abundant, non-photosynthetic or housekeeping preproteins. The assumption previously has been that the different receptor isoforms assemble differentially to form physically distinct translocon complexes. Our work challenges this assumption, as the results have shown that any given TOC receptor has the capacity to associate with any one of the other receptors in planta. The results obtained using TAP tagging and BiFC are consistent in this regard, as they both lead to the same conclusion. Given that the evidence supporting differing roles for the different receptors (in relation to photosynthetic and non-photosynthetic clients, as mentioned above) is strongly supported by many independent lines of evidence, one possible interpretation of our results is that multiple, different, client-specific import pathways operate within large multimeric TOC assemblies.
Exploitation Route The existence of distinct, client-specific preprotein import pathways may be important for the differentiation of different plastid types (for example, chloroplasts differentiate from etioplasts following the emergence of germinating seedlings into the light, leading to the establishment of photoautotrophic growth). Commercially important plastid developmental transitions (chloroplast to chromoplast) occur during fruit ripening in crops such as tomato, red bell pepper, and citrus. We are exploring the possibility that the regulation of client-specific import pathways may be utilized to manipulate this and other aspects of crop plant development. The existence of different, client-specific preprotein import pathways may allow for the manipulation of plastid biogenesis in crops, e.g. using a regulator of the import system identified in a different BBSRC project (BB/H008039/1).
Sectors Agriculture, Food and Drink,Energy

 
Description This work led to the realization that multiple, client-specific protein import pathways operate in plastids, and that these pathways are enabled by the existence of a diversity of client-specific protein import pathways, and by the operation of a regulator of the import machinery called SP1. We believe that manipulation of these import pathways will find beneficial applications in crops, by enabling manipulation of plastid functions and plastid development in a variety of different ways. This technology is covered by patent applications and is being promoted commercially.
First Year Of Impact 2012
Sector Agriculture, Food and Drink,Energy
Impact Types Societal

 
Title Antibody against the atToc120 protein 
Description A domain of atToc120 was expressed in bacteria, purified to homogeneity, and used to immunize rabbits (by Harlan Laboratories, Loughborough). The antiserum was affinity-purified and proven to be effective by ELISA and immunoblot analysis. This antibody was needed to analyse the translocon complexes purified during the course of the project. 
Type Of Material Antibody 
Provided To Others? No  
Impact The resource supports many on-going projects in the laboratory, and has enabled several publications. 
 
Title Antibody against the atToc132 protein 
Description A domain of atToc132 was expressed in bacteria, purified to homogeneity, and used to immunize rabbits (by Harlan Laboratories, Loughborough). The antiserum was affinity-purified and proven to be effective by ELISA and immunoblot analysis. This antibody was needed to analyse the translocon complexes purified during the course of the project. 
Type Of Material Antibody 
Year Produced 2012 
Provided To Others? Yes  
Impact The resource supports many on-going projects in the laboratory, and has enabled several publications. 
 
Title Antibody against the atToc34 protein 
Description A domain of atToc34 was expressed in bacteria, purified to homogeneity, and used to immunize rabbits (by Harlan Laboratories, Loughborough). The antiserum was affinity-purified and proven to be effective by ELISA and immunoblot analysis. This antibody was needed to analyse the translocon complexes purified during the course of the project. 
Type Of Material Antibody 
Provided To Others? No  
Impact The resource supports many on-going projects in the laboratory, and has enabled several publications. 
 
Title Antibody against the atToc75-III protein 
Description A domain of atToc75-III was expressed in bacteria, purified to homogeneity, and used to immunize rabbits (by Harlan Laboratories, Loughborough). The antiserum was affinity-purified and proven to be effective by ELISA and immunoblot analysis. This antibody was needed to analyse the translocon complexes purified during the course of the project. 
Type Of Material Antibody 
Year Produced 2012 
Provided To Others? Yes  
Impact This resource is supporting most of the research projects in our group, and is used in a large number of publications from the group. For example: Ling, Q., Huang, W., Baldwin, A. and Jarvis, P. (2012) Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasome system. Science 338: 655-659. 
 
Title Constructs for the analysis of TOC receptor interactions by BiFC 
Description Sequences encoding the various different TOC receptors were cloned into pSAT-based bimolecular fluorescence complementation (BiFC) vectors. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact This work is still in progress. 
 
Title Improved methodologies for TAP-based purification of translocon complexes, including preparation of IgG beads 
Description The methods for purification of chloroplast translocon complexes by the TAP approach were optimized and improved, in association with our collaborators led by Professor Felix Kessler. We also optimized a method for the preparation of human-IgG Sepharose beads, in order to reduce leaching of the IgG during the TAP procedure. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This resources several lines of ongoing research in the laboratory. 
 
Title Modification of the pNTAPi vector to include native promoters of translocon component genes 
Description The pNTAPi vector (Plant J., 2004, 38:172-181), for the generation of stably-transformed plants expressing a TAP-tagged form of the protein of interest, was modified to include the native atTOC33 and atTOC34 promoters. The standard version carries the constitutive CaMV 35S promoter. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact This work is still in progress. 
 
Description Professor Felix Kessler, University of Neuchâtel, Switzerland 
Organisation University of Neuchatel
Country Switzerland 
Sector Academic/University 
PI Contribution A collaboration was established to share knowledge and optimize methods for the native purification of chloroplast translocon complexes using the tandem affinity purification (TAP) technique. As part of this collaboration, a researcher visited the Kessler laboratory for 10 days in 2010 (18th-29th January, 2010). Since then, the collaboration has continued to evolve and take new directions, for example related to the involvement of post-translation modification in the regulation of chloroplast protein import. As a result of the collaboration, Professor was invited visit our department in Oxford on 22 November 2018, and to give a seminar here.
Collaborator Contribution A collaboration was established to share knowledge and optimize methods for the native purification of chloroplast translocon complexes using the tandem affinity purification (TAP) technique. As part of this collaboration, a researcher visited the Kessler laboratory for 10 days in 2010 (18th-29th January, 2010). Since then, the collaboration has continued to evolve and take new directions, for example related to the involvement of post-translation modification in the regulation of chloroplast protein import. As a result of the collaboration, Professor was invited visit our department in Oxford on 22 November 2018, and to give a seminar here.
Impact Jarvis, P. and Kessler, F. (2014) Mechanisms of chloroplast protein import in plants. In: Advances in Plant Biology: Plastid Biology (S.M. Theg and F.-A. Wollman, eds.) Springer, New York, pp. 241-270. Aronsson, H., Combe, J., Patel, R., Agne, B., Martin, M., Kessler, F. and Jarvis, P. (2010) Nucleotide binding and dimerization at the chloroplast pre-protein import receptor, atToc33, are not essential in vivo but do increase import efficiency. Plant J. 63: 297-311.
Start Year 2010
 
Title ATSP1, AN E3 UBIQUITIN LIGASE, AND ITS USE 
Description The invention relates to plants with improved phenotypes and related methods. These improved phenotypes are conferred by altering the expression of the SP1 gene which is involved in plastid development or altering the activity of the SP1 protein. 
IP Reference 16/643507 
Protection Patent application published
Year Protection Granted 2015
Licensed Yes
Impact A BBSRC Follow-on Funding Pathfinder grant was awarded in 2013 (BB/FOF/PF/15/12), which related to earlier patent filings of the same technology. A subsequent BBSRC Follow-on Funding Standard grant was applied for in 2017 (BB/R005591/1; application pending).
 
Title Transgenic Plants. AtSP1, An E3 Ubiquitin Ligase, and Its Use 
Description The invention relates to plants with improved phenotypes and related methods. These improved phenotypes are conferred by altering the expression of the SP1 gene which is involved in plastid development or altering the activity of the SP1 protein. 
IP Reference WO2014037735 
Protection Patent application published
Year Protection Granted 2014
Licensed Yes
Impact The technology is currently in development.
 
Title Improved methodologies for TAP-based purification of translocon complexes, including preparation of IgG beads 
Description The methods for purification of chloroplast translocon complexes by the TAP approach were optimized and improved, in association with our collaborators led by Professor Felix Kessler. We also optimized a method for the preparation of human-IgG Sepharose beads, in order to reduce leaching of the IgG during the TAP procedure. 
Type Of Technology New/Improved Technique/Technology 
Impact No actual Impacts realised to date 
 
Description 10th International Congress on Plant Molecular Biology (IPMB), 2012, Jeju South Korea 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited speaker International networking; inspiration for research, esteem

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description 2nd International Photosynthesis Workshop, 2009, Munich, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited speaker International networking; inspiration for research; esteem

no actual impacts realised to date
Year(s) Of Engagement Activity 2009
URL http://www.hfsp.org/frontier-science/awardees-articles/new-theories-origin-cerebral-cortical-convolu...
 
Description Chloroplast Biogenesis Session, Society for Experimental Biology (SEB) Annual Main Meeting, 2012, Salzburg, Austria 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other audiences
Results and Impact Session co-organizer International networking; inspiration for research; esteem

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Gordon Research Conference (GRC), Protein Transport Across Cell Membranes, 2010, Texas, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited speaker International networking; inspiration for research; esteem

no actual impacts realised to date
Year(s) Of Engagement Activity 2010
 
Description Gregor Mendel Institute, 2009, Vienna, Austria 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited seminar; guest International networking; inspiration for research; esteem

no actual impacts realised to date
Year(s) Of Engagement Activity 2009
 
Description Molecular and Cellular Biosciences Theme Research Day, 2012, University of Leicester 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Invited speaker Local networking; inspiration for research; esteem

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Plant Signaling and Behavior Meeting, 2009, Florence, Italy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited speaker International networking; inspiration for research; esteem

no actual impacts realised to date
Year(s) Of Engagement Activity 2009
 
Description University of Neuchatel, 2010, Neuchatel, Switzerland. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Seminar speaker (22 January 2010); guest. International collaboration with the laboratory of Professor Felix Kessler, University of Neuchatel

no actual impacts realised to date
Year(s) Of Engagement Activity 2010