Investigating the Roles of Anionic Phospholipids in Endocytic Trafficking in Mammalian and S. cerevisiae Cells

A fundamental feature of eukaryotic cells is their compartmentalization into organelles. This allows for the segregation of biochemical processes, such as protein translation, energy production, macromolecule catabolism, and the transcription of genetic materials. Organelles are delineated by membrane bilayers that are composed of transmembrane and peripheral proteins and lipids. For organelles to maintain their functionality, they must constantly refresh or receive new lipids and proteins. Additionally, organelles are not static structures found in the cytoplasm but are highly mobile and moved around the cell using microtubule-based motors. Two main mechanisms are responsible for the movement of material between organelles. This includes vesicular transport of proteins and lipids and non-vesicular movement of lipids. Non-vesicular transport is best characterized to occur at sites of contact between the ER and other organelles. However, other contacts have been described which do not rely on the ER. Importantly, not only are lipids essential components of the bilayer of organelles but they also contribute to the identity of the organelle, signal transduction, functionality, and vesicular transport. This thesis examines the roles of negatively charged lipids, specifically phosphatidylserine (PtdSer) and phosphorylated variants of phosphatidylinositol (PIPs) on organelle identity, vesicle budding and transport, and the relative roles for vesicular and non-vesicular transport in the subcellular distribution of PtdSer and cell polarity. Results from my studies reveal i) a redistribution of the phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) to endomembrane structures upon PIPKH (PIP5K homolog) overexpression that is accompanied by an accumulation of PIPs, which increases the negative surface charge of endomembrane that in turn leads to the relocalization of polycationic proteins. ii) PtdSer is not only a cargo of Snx4 and Retromer mediated retrograde transport pathways but is also essential to support the formation and recycling of cargo, and iii) Osh6 and Osh7, two proteins involved in the non-vesicular transport of PtdSer, are dispensable with regards to the polarization of PtdSer in the plasma membrane of yeast and not required to support robust cell polarity pathways. Together, my results highlight the importance of anionic phospholipids in organelle identity and endocytic trafficking.Ph.D.2021-11-15 00:00:0