Investigating the role of yeast Glutathione peroxidase 3 and related proteins in mitochondria and peroxisomes

In Saccharomyces cerevisiae, Gpx3 is a H2O2 sensor that resides in the cytosol, the mitochondrial intermembrane space (IMS) and the peroxisomal matrix. Upstream translation from a non-AUG codon after exposure to H2O2 results in production of an N- terminally extended form, N18Gpx3, that targets the protein to mitochondria in a more efficient manner. Whilst the major role of Gpx3 in the cytosol is known, notably its interaction with (and activation of) the transcription factor Yap1, the role/s of Gpx3 and N18Gpx3 in the mitochondrial IMS and peroxisomal matrix are yet to be fully determined. Previous work published by our lab and collaborators identified the oxidoreductase Mia40 as an interactor of Gpx3; however, others were anticipated. Here, we identified two new putative IMS interactors of Gpx3: Cytochrome c (Cytc) and Cytochrome b2 (Cytb2). Cytc is an abundant electron carrier in the IMS that plays a role in both respiration and the mitochondrial IMS assembly (MIA) oxidative folding pathway, and in mammals Cytc is also involved in triggering apoptosis. In contrast, little is known about Cytb2. We focused our attention on the potential Gpx3-Cytc interaction and found that by mutating Cys64 of Gpx3, a highly conserved cysteine residue with no known function, its interaction with Cytc was affected. As both Mia40 and Cytc are components of the MIA pathway, we hypothesise that Gpx3 plays a role in oxidative folding and may be a regulator of this pathway under oxidative stress. Next, we uncovered six small proteins with no previous mitochondrial associations and went on to investigate one in particular, Ypl107w, an uncharacterised protein containing a highly conserved oxidoreductase-like domain (OXLD) that, despite being present in thousands of proteins across hundreds of species, has an unknown function. We confirmed its targeting and localisation to the mitochondrial matrix and hypothesise, based on bioinformatics and BN-PAGE data, that Ypl107w has many interactors and may be involved in mitochondrial assembly. There may also be a link between Ypl107w, Gpx3 and Cytc as ypl107wD mitochondria have lower amounts of both Gpx3 and Cytc. Finally, we confirmed the presence of Gpx3 in peroxisomes and discovered a shorter version of Mia40 along with Trx1 and Trx2 also residing in peroxisomes. We hypothesise that the targeting of these proteins occurs by previously unrecognised peroxisomal targeting signals, and that the MIA pathway may operate in both mitochondria and peroxisomes. Altogether, the work presented in this thesis adds to our knowledge of yeast Gpx3 in regards to its compartment-specific roles