Stone, Scot2023-03-1520232023-022023-03-15February 2https://hdl.handle.net/10388/14523The mitochondrion is now recognized as a critical hub in cellular homeostasis and the last 25 years have seen rapid growth in studies that investigate the mechanistic basis of organelle signalling. Copper is an essential metal ion integral to the activity of several important metabolic enzymes and there is a growing appreciation of the role mitochondria fulfil in regulating cellular copper homeostasis. Yet it is unclear how copper handling pathways within the organelle influence cellular copper regulation. Localized to the inner mitochondrial membrane, the cuproprotein SCO2 plays a critical role in cytochrome c oxidase biogenesis and copper homeostasis. Classically, pathogenic mutations in the SCO2 gene such as the E140K substitution result in a lethal cardioencephalomyopathy owing to a severe deficiency in both processes. More recently, SCO2 mutations like the P169T variant have been described that cause a non-lethal neurological disorder providing further evidence of the strong genotype-phenotype correlation associated with mitochondrial disease presentation. To fully understand SCO2 function, we fused a promiscuous biotin ligase, BirA*, to wild-type SCO2 and the SCO2 E140K and P169T variants to allow us to identify interacting partners by the proximity-dependent ligation technique called, BioID. We found that in Flp-In T-REx HEK293 cells, the E140K-BirA* and P169T-BirA* SCO2 baits showed a notable reduction in biotinylation despite their expression being comparable to that of the wild-type-BirA* SCO2 bait. In contrast, these same baits were variably expressed when transduced into SCO2 patient and control fibroblasts, which resulted in different biotinylation intensities. Overexpression of untagged or BirA* tagged, wild-type SCO2 rescued the COX deficiency in SCO2 patient fibroblasts, while E140K-BirA* SCO2 did not. Optimization parameters relevant to streptavidin bead enrichment for the isolation of biotinylated proteins revealed that incubation time with biotin and protein quantity greatly affected yield. Proteins isolated via BioID in SCO2-BirA* expressing patient fibroblasts identified 220 high-confidence interactors, eight of which were considered novel among the SCO2 interactome. Sideroflexin 3, a protein that is highly expressed in neurons, was the top interactor. Future studies will investigate how the SCO2 interactors influence cellular copper homeostasis and, in turn, modulate disease phenotype.application/pdfencopperBioIDSCO2signallingmitochondriaredoxThesis2023-03-15