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INVESTIGATION INTO THE MOLECULAR MECHANISMS OF IMPORT OF MITOCHONDRIAL SMALL TIM PROTEINS
[Thesis]. Manchester, UK: The University of Manchester; 2012.
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Abstract
Protein import is essential for the biogenesis of mitochondria, as the majority (99%) of mitochondrial proteins are synthesised in the cytosol and thus, have to be imported into mitochondria for their function. The biogenesis of many cysteine-containing proteins of the mitochondrial intermembrane space (IMS), such as members of the small TIM and Cox17 families, is regulated by their thiol-disulphide redox state. Only the Cys-reduced precursors can be imported into mitochondria, whereas oxidised forms cannot. Their import and oxidative folding in the IMS is driven by the IMS disulphide relay system, known as mitochondrial import and assembly (MIA) pathway, whose central components are the oxidoreductase Mia40 and the sulphydryl oxidase Erv1. Currently, little is known about how the MIA precursors are maintained in the cytosol in an import-competent form, and whether they interact with the translocase of the outer membrane (TOM complex) to enter the IMS. In addition, the MIA-mediated protein folding events occurring in the IMS that lead to the generation of fully oxidised substrates are still under investigation. Using Tim9 and yeast as models, studies presented in this thesis showed that Tim9 binding to the mitochondrial outer membrane (OM) does not depend on the receptors of the TOM complex, and occurs without regard to the redox state of the precursor proteins. In addition, it is shown that the oxidised and reduced precursors share the same binding site on the OM, and that this binding site is not important for the translocation process across the OM (Chapter 3). Studies in this thesis investigated the role of the cytosolic thioredoxin and glutaredoxin systems in the biogenesis of mitochondria. Firstly, in vivo studies provided the evidence that the cytosolic thioredoxin system but not the glutaredoxin system is required for growth of yeast cells under respiratory conditions. Secondly, in vivo studies provided the first proof that the Trx system is required for the biogenesis of small Tim proteins. In vitro studies confirmed that the Trx1 system facilitates import of small Tim proteins into isolated mitochondria by maintaining the precursors in a reduced and therefore competent form (Chapter 5). Finally, in vitro studies showed that Mia40 is able to promote the full oxidation of Tim9. Efficient release of Tim9 from Mia40 required the presence of all cysteine residues of Tim9, as effective oxidation and concomitant release from Mia40 failed upon mutation of single cysteine residues. Finally, the study showed that reduced glutathione resolved rapidly the Mia40-Tim9 mixed-disulphide complexes, probably accelerating and/or promoting the Tim9 oxidative (Chapter 4).