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

Lead Research Organisation: University of Oxford
Department Name: Plant Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/J009369/1 01/08/2012 30/09/2013 £402,473
BB/J009369/2 Transfer BB/J009369/1 01/10/2013 31/01/2016 £249,886
 
Description This project focused on the functional characterization of two chloroplast proteins, STIC1 and STIC2, identified using a genetic screening approach (for suppressors of the chloroplast import mutation tic40), and showed that the two proteins cooperate physically and functionally in a protein transport pathway that directs proteins to the chloroplast's thylakoid membranes.
Exploitation Route We and other may wish to further dissect the roles of the STIC1 and STIC2 proteins in the sorting of chloroplast thylakoid proteins.
Sectors Agriculture, Food and Drink,Energy

 
Description The outputs of this project hold considerable promise in relation to the manipulation and optimization of plastid protein biogenesis in crops, but it is yet too early to determine whether such potential will translate into real-world applications.
Sector Agriculture, Food and Drink,Energy
Impact Types Societal

 
Title Antibody against the STIC1 protein 
Description Antibody against the STIC1 protein 
Type Of Material Antibody 
Provided To Others? No  
Impact Furtherance of our BBSRC-funded research 
 
Title Antibody against the STIC2 protein 
Description Antibody against the STIC2 protein 
Type Of Material Antibody 
Provided To Others? No  
Impact Furtherance of our BBSRC-funded research 
 
Title Modification of pH2GW7 vector to enable expression of FLAG-tagged protein in transgenic plants 
Description Modification of pH2GW7 vector to enable expression of FLAG-tagged protein in transgenic plants 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Furtherance of our BBSRC-funded research 
 
Title Various double-mutant plant (Arabidopsis) genotypes to test interactions between stic1/2 and known thylakoid protein transport mutations 
Description Various double-mutant plant (Arabidopsis) genotypes to test interactions between stic1/2 and known thylakoid protein transport mutations 
Type Of Material Biological samples 
Provided To Others? No  
Impact Furtherance of our BBSRC-funded research 
 
Title Vectors and resulting transgenic plants (Arabidopsis) that overexpress STIC1/2 with FLAG or TAP tags, or the bacterial homologue of STIC2 
Description Vectors and resulting transgenic plants (Arabidopsis) that overexpress STIC1/2 with FLAG or TAP tags, or the bacterial homologue of STIC2 
Type Of Material Biological samples 
Provided To Others? No  
Impact Furtherance of our BBSRC-funded research 
 
Title Vectors for bacterial expression of STIC1 and STIC2 proteins 
Description Vectors for bacterial expression of STIC1 and STIC2 proteins 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Furtherance of our BBSRC-funded research 
 
Title Vectors for expression of STIC1 and STIC2 YFP fusion proteins in protoplasts 
Description Vectors for expression of STIC1 and STIC2 YFP fusion proteins in protoplasts 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Furtherance of our BBSRC-funded research 
 
Title Vectors for testing STIC1 and STIC2 interactions by bimolecular fluorescence complementation (BiFC) 
Description Vectors for testing STIC1 and STIC2 interactions by bimolecular fluorescence complementation (BiFC) 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Furtherance of our BBSRC-funded research 
 
Description Dr Enrique Lopez-Juez, Royal Holloway, University of London 
Organisation Royal Holloway, University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration on the import characteristics of newly identified mutant affecting the TIC machinery of chloroplasts
Collaborator Contribution Identification of the gene affected by the mutation
Impact Identification and characterization of a significant new mutant affecting the TIC machinery of chloroplasts; manuscript in preparation
Start Year 2012
 
Description Dr Mats Töpel, Department of Marine Sciences, University of Gothenburg, Sweden 
Organisation University of Gothenburg
Department Department of Marine Sciences
Country Sweden 
Sector Academic/University 
PI Contribution We have collaborated on the phylogenetic analysis of STIC2-related proteins, and other chloroplast proteins, as well as on the analysis of whole genome sequence data.
Collaborator Contribution Advanced expertise in bioinformatics and phylogenetic analysis.
Impact Ling, Q., Broad, W., Trösch, R., Töpel, M., Demiral Sert, T., Lymperopoulos, P., Baldwin, A. and Jarvis, R.P. (2019) Ubiquitin-dependent chloroplast-associated protein degradation in plants. Science 363: eaav4467. Bédard, J., Trösch, R., Wu, F., Ling, Q., Flores-Pérez, Ú., Töpel, M., Nawaz, F. and Jarvis P. (2017) Suppressors of the chloroplast protein import mutant tic40 reveal a genetic link between protein import and thylakoid biogenesis. Plant Cell 29: 1726-1747. Trösch, R., Töpel, M., Flores-Pérez, Ú. and Jarvis, P. (2015) Genetic and physical interaction studies reveal functional similarities between ALBINO3 and ALBINO4 in Arabidopsis. Plant Physiol. 169: 1292-1306.
Start Year 2015
 
Description Gordon Research Conference on Mitochondria and Chloroplasts, Barga, Italy (July 6-11, 2014) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Other audiences
Results and Impact Presentation delivered at conference.

Promoted awareness and outcomes of my BBSRC-funded work, and made contact with researchers in related fields leading possible future collaboration.
Year(s) Of Engagement Activity 2014
URL http://www.grc.org/programs.aspx?year=2014&program=mitochon
 
Description Plant Biology Europe FESPB/EPSO Congress, Dublin, Ireland (June 22-26, 2014) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Other audiences
Results and Impact Presentation as keynote speaker delivered at congress.

Promoted awareness and outcomes of my BBSRC-funded work, and made contact with researchers in related fields leading possible future collaboration.
Year(s) Of Engagement Activity 2014
URL http://europlantbiology.org/
 
Description Grenoble Alliance for Integrated Structural Cell Biology (GRAL) Workshop, Autrans, France (April 7-8, 2014) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Other audiences
Results and Impact Keynote presentation delivered at scientific workshop.

Promoted awareness and outcomes of my BBSRC-funded work, and made contact with researchers in related fields leading possible future collaboration.
Year(s) Of Engagement Activity 2014
URL http://www.labex-gral.fr/events
 
Description Hosting A-level student on Nuffield Research Placement (Adam Bills, King Edward VII College, Coalville) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact The student, Adam Bills from King Edward VII College, Coalville, participated in scientific research for a one month period, working alongside Dr Jocelyn Bedard in the laboratory.

The student developed better understanding of scientific research and biology during the course of his placement, and this will be likely to positively inform his future career choices.
Year(s) Of Engagement Activity 2013
URL http://www.nuffieldfoundation.org/nuffield-research-placements
 
Description Hosting of A-level student for work experience placement (Tom Bacon, Oakham School) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact The student, Tom Bacon from Oakham School, participated in scientific research during a one week placement, working alongside other researchers in the laboratory.

The student developed better understanding of scientific research and biology during the course of his placement, and this will be likely to positively inform his future career choices.
Year(s) Of Engagement Activity 2012
 
Description International Symposium on the Regulation of Photosynthetic Function, Guilin, China (August 16-20, 2014) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Other audiences
Results and Impact Presentation delivered at congress.

Promoted awareness and outcomes of my BBSRC-funded work, and made contact with researchers in related fields leading possible future collaboration.
Year(s) Of Engagement Activity 2014
URL http://rpf2014.csp.escience.cn/dct/page/1
 
Description Invited speaker at GRC Protein Transport Across Cell Membranes Meeting entitled "Protein Transport Across Cell Membranes: Mechanism, Structure, and Regulation" (Texas, USA, 2016) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was an invited speaker at this prestigious international meeting, which took place during 6-11 March 2016, at Hotel Galvez, Galveston, TX, USA.
Year(s) Of Engagement Activity 2016
URL https://www.grc.org/protein-transport-across-cell-membranes-conference/2016/
 
Description Keynote Speaker at Society of Experimental Biology (SEB) Annual Meeting (Gothenburg, Sweden, 2017) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was a keynote speaker at this prestigious international meeting, which took place during 3-6 July 2017, at Gothenburg, Sweden.
Year(s) Of Engagement Activity 2017
URL http://www.sebiology.org/events/event/seb-gothenburg
 
Description Plenary Speaker and Session Chair at the 27th International Conference on Arabidopsis Research (ICAR) (Gyeong Ju, Korea, 2016) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was a plenary speaker and session at the is prestigious international meeting, which took place during 29 June-3 July 2016, at Gyeong Ju, South Korea.
Year(s) Of Engagement Activity 2016
URL http://www.arabidopsisresearch.org/images/ICAR/ICAR2016_programofICAR2016_160418.pdf
 
Description Talk given to A-level students as part of Study Day at Oxford's Museum of Natural History 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact This talk was part of an engagement event (a Study Day) organized by Oxford's Museum of Natural History and Botanic Garden. It was a special day for A-level biologists and it focused on cells; my contribution was a presentation covering the evolution of the plant cell with a particular focus on chloroplast evolution. Approximately 300 students attended from a mixture of local schools. All students were between 16 and 18 years old and had chosen to study biology.
Year(s) Of Engagement Activity 2014
URL http://www.oum.ox.ac.uk/educate/index.htm