A genome-wide gene overexpression screen for mitophagy mutants in the yeast Saccharomyces cerevisiae

2017-03-03T01:19:21Z (GMT) by Lucarelli, Giuseppe Andrea
Autophagy is a fundamental pathway conserved amongst all eukaryotic cells that is necessary for the degradation of intracellular structures, including organelles, to maintain cellular homeostasis. Being a fundamental process, autophagy is linked with many other intracellular pathways, and its dysfunction is associated with many human diseases. Mitochondria can be degraded by autophagy after delivery to the vacuole (in yeast) or lysosome (in mammalian cells) in a selective process termed mitophagy. Significant contribution to the understanding of mitophagy has come from research on the model organism Saccharomyces cerevisiae. Two non-essential gene deletion screens have identified two genes, ATG32 and ATG33, encoding components of the mitophagy pathway both localised at the outer mitochondrial membrane (OMM). Atg32p acts as the essential receptor activated by phosphorylation allowing interaction with core autophagy proteins resulting in mitophagy. However, our knowledge of mitophagy and its regulation in yeast is incomplete requiring further investigation. To this end, I developed a high-throughput imaging assay based on the use of a mitochondrially targeted fluorescent protein biosensor called mt-Rosella. The assay is performed using a 96-well format in a semi-automated fashion and allows the screening of different strain libraries under a range of different conditions. Using this assay I investigated the effects of gene overexpression on mitophagy using a library comprised of 1588 high-copy number plasmids that cover more than 95% of the yeast genome including essential genes (previously excluded from the deletion library). Each plasmid contains on average 5 contiguous genes each under the expression control of their endogenous promoter. The screen revealed that increased expression of a number of genes can affect mitophagy. Among such genes, I found that OM14 encoding the OMM protein Om14p confers a strong decrease in mitophagy when overexpressed in cells. Subsequently a similar phenotype was observed in cells in which the OM14 gene was deleted. Om14p is required for co-translational import of proteins into mitochondria and also forms a complex with the OMM proteins Por1p and Om45p. Preliminary results indicate that lack of Om14p leads to a significant reduction in amounts of Atg32p in cells suggesting the involvement of Om14p in the co-translational import of Atg32p in mitochondria. Another line of evidence indicates that the absence of Om14p causes a perturbation in the ultrastructure of mitochondria suggesting a role for Om14p in mitochondrial architecture, the alteration of which could indirectly affect proteins essential for mitophagy, including Atg32p. Further investigation is required to fully understand the role of Om14p in mitophagy.