The function and substrate of the ABC transporters of the mitochondria

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

Abstract

In buildings, walls separate rooms and doors regulate entry and exit. By analogy, membranes separate parts of living cells, and transporters control which substances move across. Knowing what each transporter does is important, as this will increase our understanding of the processes separated by the membrane, and allow us to manipulate the transport. Mitochondria are like rooms in which energy is generated and where many delicate chemical reactions take place (for instance for making vitamins or metal cofactors). The mitochondrial membranes contain many transporters, to supply the furnaces with fuel and to provide raw materials for chemical reactions. Energy, products and waste then need to be exported. We are interested in a special type of transporter, the ABC transporters of the mitochondria (ATMs). This is because ATMs export vital, but yet unidentified materials out from the mitochondria to the rest of the cell, catalyzing key biological reactions. ABC stands for ATP binding cassette, which is a feature of the transporter protein that defines the group it belongs to. The ATMs are present in all organisms, and defects lead to disease in humans and low yield in plants. The aim of this research proposal is to find out what is exactly the substance transported by these mitochondrial transporters. Earlier studies of ATMs have used yeast, because this is a simple organism to use as an experimental 'model'. These studies revealed that damage to the ATM transport system disrupts the production of small iron-sulphur structures outside the mitochondria. These iron-sulphur structures are essential for cell metabolism, growth and survival. Recently, our group and collaborators have found another chemical process that requires ATM, which is the synthesis of molybdenum-organic compounds. This was discovered in plants, because yeast does not have this compound (but humans do). So now we have two processes that are dependent on ATMs, and we can better speculate what could be transported. One of the possibilities is a small sulphur compound, which is part of the iron-sulphur structures as well as the molybdenum compound. It is also possible that the transporter can carry quite different precursor molecules for each of the metal structures. This is a hypothesis that we can test relatively easily, especially with the technical progress made recently to study pure preparations of ABC transporters. This also allows us to look at the ATM transporters that are damaged in different ways, and find out why they do not work properly. Again, plants have been very useful for this, because several plant lines with a damaged ATM3 transporter were found. In the end, we will learn how the transporter works and what it can transport, while increasing our understanding of the processes in the cell depending on it and their significance for human health and plant growth.

Technical Summary

The ABC transporters of the mitochondria (ATM) have an important function in metal homeostasis in all eukaryotes. Mutation of ATM3 results in heavy metal accumulation in (crop) plants, and mutations in the orthologous ABCB7 cause ataxia with anaemia in human patients. However, the substate(s) of these transporters has not been identified. Recently we found that ATM3 is the principal mitochondrial ABC transporter in the model plant Arabidopsis and that it is involved in molybdenum cofactor (Moco) biosynthesis, in addition to the previously established role in iron-sulphur (Fe-S) cofactor assembly. These data point to two possibilities, namely either ATM3 has a broad substrate specificity including both Fe-S cluster and Moco precursors, or ATM3 transports a sulphide compound, since sulphur is found in both Moco and Fe-S clusters. Which possibility is correct will be determined by a combination of in vivo and in vitro approaches: i) identification of precursor molecules or thiol compounds accumulating in mitochondria in plant and yeast atm mutants ii) establish the role of glutathione in transport; iii) test candidate substrates in ATPase activity assays of reconstituted Arabidopsis ATM3, yeast Atm1 and mitochondrial membranes; iv) molecular characterization and homology modelling of atm3 mutants. Together, these experiments will identify the substrate(s) of Arabidopsis ATM3 and its functional orthologues in yeast and human.

Publications

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Balk J (2014) Iron cofactor assembly in plants. in Annual review of plant biology

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Schaedler TA (2015) Structures and functions of mitochondrial ABC transporters. in Biochemical Society transactions

 
Description ABC transporters of the mitochondria (ATM) are essential for cytosolic Fe-S cluster assembly. Previous studies suggested that a compound containing sulphur (but not iron) in an unknown chemical form is exported from the mitochondria into the cytosol and that glutathione (GSH) plays a role in the export process. Therefore, the aim of this study is to characterize the substrate specificity of the plant AtATM3 and its orthologue yeast ScAtm1.



We are currently characterizing sulphur and thiol containing compounds in isolated mitochondria using High Pressure Liquid Chromatography and mass spectrometry as analytical tools. These measurements will allow quantification of the compounds of interest (e.g. GSH, cysteine, _-glutamyl cysteine). Analysis of mitochondria of selected atm mutants might contribute to the identification of compounds that accumulate in the mutant mitochondria and thus to the characterization of the substrate specificity of ATMs. Analysis of seedlings of atm3 mutants also showed that atm3 mutants are sensitive to BSO, an inhibitor of the first step of glutathione synthesis, and as a result display reduced growth. The connection between the BSO-induced growth phenotype and atm3 mutations is not understood and is the focus of our current investigations.
Exploitation Route Biotechnology - there is a growing interest in expressing Fe-S proteins in eukaryotes, for example hydrogenase in yeast, or nitrogenase in crop plants. A knowledge of proteins required for the assembly of Fe-S cofactors on these proteins, including ATMs, is therefore critical. Crop improvement - the biosynthesis of two plant hormones, ABA and auxin, depends on the function of ATM3 (Bernard et al 2009; Teschner et al 2010). These hormones are important for seed germination and shoot growth, respectively.



Health and disease - mutations in ABCB7, the human homologue of yeast ATM1 / plant ATM3, results in ataxia with anaemia. Although a rare genetic disease, treatment of these patients with the substrate of ABCB7 may improve their quality of life.
Sectors Healthcare

 
Description The main paper describing our findings was recently (August 2014) published as a Paper of the Week in the Journal of Biological Chemistry. This means that the editorial board members and associate editors of JBC determined that our paper was in the top 2 percent of manuscripts they will review in a year in significance and overall importance. About 50 to 100 papers are selected from the more than 6,600 we publish each year. We anticipate that our findings will be used by others.
First Year Of Impact 2013
Sector Education,Healthcare
Impact Types Societal,Economic

 
Description Collaboration with Dr Florian Bittner and Professor Ralf Mendel, TU Braunschweig, Germany. 
Organisation Braunschweig University of Technology
Country Germany 
Sector Academic/University 
PI Contribution A PhD student from the Bittner/Mendel group will visit to test Precursor Z (Moco biosynthesis) in ATM transport assays, May 2012.
Start Year 2012
 
Description Collaboration with Dr Markus Wirtz and Prof Rudiger Hell, University of Heidelberg, Germany. 
Organisation Heidelberg University
Country Germany 
Sector Academic/University 
PI Contribution Measurement of thiol compounds in isolated mitochondria.
Start Year 2011
 
Description Collaboration with dr Helene Puccio, IGBMC in Illkirch, Strasbourg, France 
Organisation Institute of Genetics and Molecular and Cellular Biology (IGBMC)
Country France 
Sector Academic/University 
PI Contribution Aim is to obtain data from mouse models of ABCB7 that would support the hypothesis that ATMs/ABCB7 transport a thiol compound. Dr Puccio has provided antibodies against mouse ABCB7 to monitor expression in L. lactis. Data from the transport assays may be combined with the unpublished mouse model data.
Start Year 2012
 
Description Collaboration with dr Markus Schwarzländer, University of Oxford and prof Andreas Meyer, University of Bonn. 
Organisation University of Bonn
Country Germany 
Sector Academic/University 
PI Contribution Donation of Grx-roGFP reporter construct, and help with confocal analysis for GSH/GSSG analysis
Start Year 2012
 
Description Collaboration with dr Markus Schwarzländer, University of Oxford and prof Andreas Meyer, University of Bonn. 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Donation of Grx-roGFP reporter construct, and help with confocal analysis for GSH/GSSG analysis
Start Year 2012
 
Description Collaboration with dr Tatjana Hildebrandt, University of Hannover, and prof Massimo Zeviani, Milan 
Organisation Carlo Besta Neurological Institute
Country Italy 
Sector Public 
PI Contribution Provision of human ETHE1 expression construct for enzymatic detection of glutathione-persulphide
Start Year 2011
 
Description Collaboration with dr Tatjana Hildebrandt, University of Hannover, and prof Massimo Zeviani, Milan 
Organisation Gottfried Wilhelm Leibniz Universit├Ąt Hannover
Country Germany 
Sector Academic/University 
PI Contribution Provision of human ETHE1 expression construct for enzymatic detection of glutathione-persulphide
Start Year 2011
 
Description Invited talk at Biochemical Society Conference (Chester, UK) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact Invited talk
Year(s) Of Engagement Activity 2015
 
Description Organiser of visit by >60 participants from Tokyo Institute of Technology (Japan) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Organiser of visit by >60 members of the Tokyo Institute of Technology during 2 days to Cambridge and the Department of Pharmacology
Year(s) Of Engagement Activity 2013