Characterisation of peroxisomes metabolism and segregation in yeast

Abstract

Eukaryotic cells are distinguished by the presence of membrane-enveloped structures, called organelles. Organelles play a vital role in cellular organisation as they create unique intracellular microenvironments that sustain the complex metabolism of eukaryotic cells. One of these structures is entitled the peroxisome. Peroxisomes contributes to cell’s metabolism, particularly to fatty acid beta-oxidation. Although the role of peroxisomes in metabolism has been extensively studies in the model yeast Saccharomyces cerevisiae, and many of the proteins involved in peroxisome metabolism have been identified in this yeast. The peroxisome proteome of most other yeasts is still uncharacterised and identification of pivotal proteins that illustrate the biochemical reactions that take place within this organelle and therefore its metabolic functions is unexplored. Limited approaches have been used to identify peroxisomal proteome. In this study, I focused on the yeast Debaryomyces hansenii, an organism known for accumulating high amount of lipid and used in biotechnology research. Initially potential peroxisomal proteins were identified via bioinformatic research, particularly identifying Peroxisome Targeting Signals type 1 and type 2 containing proteins (PTS1 or PTS2, respectively). In addition, a relatively new technique entitled proximity-dependent labelling (BioID) was utilised by fusing a promiscuous E. coli biotin ligase mutant (BirA*) to green fluorescent protein containing a strong PTS1. The BioID enriched proteins were identified via mass spectrometry and analysed in terms of their peroxisome targeting signal, function, predicted metabolic function pathway and related information. Multiple proteins were identified containing a PTS1. Interestingly and surprisingly, two proteins involved in amino sugar metabolism were identified implying a new role for fungal peroxisomes in amino sugar metabolism. This shows the validity of the approach to identify new peroxisomal functions in less well studied organisms. In the second part of this thesis I report the structural analysis of Pex3-Pex19 interaction in S. cerevisiae. AlphaFold prediction was employed to analyse the interaction of the Pex3 C-terminal domain and with the Pex19 N-terminus. The predicted structure suggested a series of electrostatic interactions. The predicted interactions between Pex3 and Pex19 were tested by mutagenesis of Pex19 and analysis in vivo confirmed a loss of Pex19 function in peroxisome biogenesis. Additionally, a preliminary genetic screen was designed to identify potential regulators of the Inp1-Pex3 tethering complex, particularly kinases, phosphatases and ubiquitin ligases. Multiple candidates were identified and classified according to their effect, further investigation is required to understand how these candidates regulate the tether. In summary, this study furthers our understanding of the functions of fungal peroxisomes and their biogenesis.