title: Structure, function and mitochondrial targeting of sideroflexin-3 (SFXN3)
Creators: name: Elder, Amy
Publisher: name: University of Glasgow
Year: 2023

description: Mitochondria possess a double membraned structure, consisting of a semipermeable outer membrane (OM) and an impermeable inner membrane (IM). IMlocalised mitochondrial carriers (MCs) are required to allow passage of small molecules across the IM, enabling communication of matrix and cytosol environments. Sideroflexins (SFXN) are a family of MCs comprised of five (SFXN1-5) members in mammals which are each nuclear-encoded and imported into the mitochondria via TIM22 and the carrier import pathway. Mammalian SFXN3 has been the subject of several recent studies, largely owing to the belief that SFXN3 may provide neuroprotection in Parkinson’s Disease. Despite the surge in interest, the experimental structure, range of functions and mitochondrial targeting information of SFXN3 remain unknown. The first part of this thesis aimed to predict the transmembrane domain (TM) structure and orientation of SFXN3 at the IM. Through use of topology and AlphaFold structure predictions, we found that SFXN3 is likely a largely helical protein, with 4 TMs, a short Cterminal domain, large N-terminal domain, and beta-strand region conserved across all SFXNs. Topology predictions could not reliably deduce orientation of the N- and C-termini of SFXN3 at the IM. The second part of this thesis aimed to identify the mitochondrial targeting information of SFXN3. Import of 35S-radiolabelled truncations of SFXN3 into isolated mitochondria revealed that the N-terminal domain of SFXN3 may contain mitochondrial targeting information, and three predicted TMs of the protein may enhance efficiency of import. However, reproducibility of said result was not assessed and must be confirmed in future. Further study involved purification of Tim9/10, an IMS-localised chaperone complex which interacts with carrier pathway substrates. Incubation of purified Tim9/10 with an SFXN3 peptide spot array suggests that the Nterminal domain, fourth transmembrane region and preceding loop domain can interact with Tim9/10 complex. This could imply that these regions of SFXN3 are essential for the chaperone interaction and thus mitochondrial import. The final part of this thesis initially aimed to characterise the function of SFXN3. Due to problems in obtaining an SFXN3-knockdown cell line, the aims shifted to characterising function of budding yeast SFXN homolog, fungal sideroflexin-1 (FSF1). Analysis of DFSF1 yeast growth revealed that FSF1 was not essential for mitochondrial respiration or iron transport. However, deletion of FSF1 decreased steady state levels of Fe-S cluster protein, mitochondrial aconitase. This preliminary finding may suggest that FSF1 could play a role in Fe-S cluster biogenesis. However, further study is required to confirm this. Overall, this thesis provides a detailed analysis of SFXN3 structure, narrows down the sequence regions involved in mitochondrial targeting, and provides evidence which disputes certain proposed functions of FSF1. This provides a starting point upon which further study can build to ultimately determine if SFXN3 is a feasible therapeutic target for neurodegenerative disorders.