The function and substrate of the ABC transporters of the mitochondria

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.

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.