Date of Award

Spring 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell Biology

First Advisor

Reinisch, Karin

Abstract

In eukaryotic cells, organelles are surrounded by membranes, which act as barriers to the cytosolic environment. Each subcellular membrane has a distinct lipid composition that is required for its unique organellar function, and is therefore is fundamental for cellular physiology. The unique distributions of organellar lipids result from highly regulated lipid transport networks and the activity of lipid metabolizing enzymes. Most phospholipids are initially synthesized in the ER and transferred to different organelles via vesicular or non-vesicular lipid transport pathways. Lipid transfer proteins (LTPs) localized at membrane contact sites mediate non-vesicular lipid transport. They contain a hydrophobic cavity to solubilize the hydrophobic “tail” of lipids. They either function as “shuttles” that typically ferry a single lipid at a time between membranes, or “bridges” that harbor hydrophobic channels along which more than one lipid can move between membranes at a time. For the first part of my thesis, I investigated the structure and function of VPS13, a novel lipid transfer “bridge” protein, and showed that the protein accommodates a 16nm long hydrophobic lipid transfer channel that mediates bulk lipid transfer. My work marked the identification of the first lipid transfer bridge in eukaryotes and raised several still open questions regarding the molecular mechanism of bridge-like LTPs. I further investigated VPS13’s WD40 domain to provide insights into how VPS13 interacts with membranes at membrane contact sites.The second part of my thesis focused on the modification of phosphatidylinositol (PI), which is essential in signalling. Phosphorylation on different positions of the head group of PI generates several phosphoinositide (PIP) species. Each of them has a unique subcellular localization. PI(3,5)P2 is one of the signature phosphoinositides in endolysosomal membranes, whose level is tightly upregulated in response to stimuli. PI(3,5)P2 is solely synthesized by the PIKfyve lipid kinase and its turnover is catalyzed by the Fig4 lipid phosphatase. Intriguingly, the two proteins, although catalyzing antagonistic reactions, are in the same complex together with a third protein, the scaffold Vac14. Little is known about how the activities of PIKfyve and Fig4 are regulated to prevent futile consumption of ATP. Combining structural and biochemistry studies, I gained insights into the overall architecture of the PIKfyve complex and into the regulatory mechanisms that govern PIKfyve and Fig4 activities.

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