Quantification of Atg18 localization and asymmetric localization of Atg18 on the daughter vacuole of budding yeast

Phospholipids play a key role in intracellular signaling. Membrane-bound phosphatidylinositols (PIs) are examples of those signaling lipids. They are phosphorylated from three different places, and these phosphorylations attract different proteins. In this thesis, phosphatidylinositol 3, 5 biphosphate PI(3,5)P2 was investigated. The autophagy protein Atg18 localize on vacuole membranes in a way that correlates with PI(3,5)P2 levels on the vacuoles. Therefore, if Atg18 can be utilized as a PI(3,5)P2 marker is inquired. To quantify Atg18 localization on the vacuoles, the EzColocalization plugin was used in ImageJ-FIJI. This plugin provided us with a Pearson Correlation Coefficient, which indicates the amplitude of Atg18 localization on the vacuole. The deletion of PI(3,5)P2 regulatory complex proteins, VAC7, VAC14, and FAB1, was investigated along with Atg18 mutants that either can only bind PI(3,5)P2, or cannot bind any PI at all. The quantified results showed that Atg18 localization on vacuoles, indeed, increases with elevated PI(3,5)P2 levels. Moreover, it was observed that Atg18 localizes only to the daughter vacuole after the vacuole segregates to the daughter cell. Using the EzColocalization plugin, I was able to show this phenomenon in numbers. Through several analyses, I found that daughter-specific Atg18 localization to the daughter vacuole mostly starts right after vacuole segregation ends, within 5 minutes around metaphase-anaphase transition, and ends 5 minutes after the spindle breakdown, persisting on daughter vacuole for 30 minutes on average. 49% of the mid-log phase population showed sole Atg18 daughter-specific localization from the end of vacuole segregation until cytokinesis, while more than 95% of the population showed daughter-specific Atg18 localization at some point. Whether other PI(3,5)P2 regulators show asymmetry was also investigated. Timelapse of Vac14 showed no asymmetry. Population analysis of cells by measuring fluorescence intensity of still images also showed that Vac7, Vac14, and Fab1 do not localize asymmetrically. On the other hand, two more proteins, Sch9 and Ivy1, were also analyzed since they are relevant to PI(3,5)P2 signaling. Sch9 was slightly enriched on the mother vacuole, whereas Ivy1 was strongly enriched on the daughter vacuole. Atg18-Sloop mutant that only binds PI(3,5)P2, was enriched on the daughter vacuole as well. Altogether, the findings of this thesis suggest that Atg18 vacuole localization correlates with the PI(3,5)P2 production and it is likely that it may reflect PI(3,5)P2 levels. This thesis also further presents evidence that the daughter and the mother vacuole exhibits compartment specific behavior in terms of some PI(3,5)P effectors (Atg18, Sch9) and regulators (Atg18 and Ivy1).