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Title: Investigation of the Glutaredoxin system with the biogenesis of mitochondrial intermembrane space proteins
Author: Tran, Peter
ISNI:       0000 0004 5922 7824
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2016
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Mitochondrial protein biogenesis depends on the import of nucleus-encoded precursors from the cytosol. Import is highly regulated and specific for different subcompartments, with intermembrane space (IMS) import driven by an oxidative mechanism on conserved cysteine residues. Oxidative folding in the IMS is facilitated by the mitochondria import and assembly (MIA) pathway. Proteins can only be imported into the IMS in Cys-reduced unfolded forms, as oxidation prevents translocation into the IMS. How the import-competent forms are maintained in the cytoplasm is lesser characterised compared to the MIA pathway. Two recent studies suggest that the cytosolic Thioredoxin (Trx) and Glutaredoxin (Grx) reductase systems play a role in facilitating IMS protein import, with previous evidence identifying a role for yeast Trxs in small Tim protein biogenesis. In this study, the redox properties of the yeast Trx and Grx systems were investigated, as well as whether the yeast Grx system play a role in the biogenesis of two typical types of IMS precursor proteins. First, in vitro studies were carried out to determine the standard redox potentials (E°’) of the Trx and Grx enzymes. This was a quantifiable parameter of reducing activity and the results were described in Chapter 3. This study determined the E°’Trx1 value, which was shown to be a more effective reductant compared to other orthologs. Experimental limitations prevented the Grx system E°’ values being determined. Next, whether the Grx plays a role in the biogenesis of the CX3C motif-containing small Tim proteins were investigated using yeast genetic in vivo and biochemical analysis methods. The results were described in Chapter 4. There, Grxs were observed to not affect cell growth, but in using overexpressed Tim9 as an import model, significant differences were observed for the Grx system as GRX deletion significantly decreased overexpressed Tim9 levels. Study on the isolated mitochondria and cytosol with overexpressed Tim9 was unclear however. This study also observed a genetic interaction between GRX andYME1 that recovered cell functioning under respiratory conditions. Finally, whether the Grx system plays a role in the biogenesis of CX9C motif-containing proteins (Mia40, Mia40C and Cox17) was studied in Chapter 5. Whilst Mia40C (C-domain of Mia40) and Cox17 are imported into mitochondria via the MIA pathway, the full-length Mia40 is a substrate of the presequence-targeted TIM23 pathway. The results indicated that import of the full-length Mia40 was unaffected by GRX deletion. However, studies of an overexpressed Mia40C as a substrate of the MIA pathway, showed strong mitochondrial protein level decreases caused by deletion of the Grx proteins. This decrease was also accompanied by an accumulation of unimported Mia40C in the cytosol. Cox17 as an alternative MIA pathway substrate also showed decreased mitochondrial levels in the GRX deletion mutants. These results altogether suggest that the cytosolic Grx system can function in the biogenesis of CX9C motif-containing IMS proteins imported through the MIA pathway, as well as the CX3C small Tim proteins. The topic of how IMS proteins are degraded in the cell was also raised by the study of Yme1.
Supervisor: Not available Sponsor: BBSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Mitochondria ; Protein ; Oxidative folding ; Redox ; Protein import ; Protein degradation ; Intermembrane space ; Glutaredoxin ; Yeast