On Cholesterol Transport Between Membranes

The studies described in this dissertation focus on investigation of the pathways for transport of cholesterol from one organelle to another in animal cells. Cells have evolved elaborate transport mechanisms to assure an optimum cholesterol content within their membranes. Dysregulation of cholesterol transport causes common diseases, including atherosclerosis. The major source of cellular cholesterol comes from Low Density Lipoprotein (LDL). When plasma membranes are low in cholesterol, cells produce LDL receptors which bind LDL and mediate its uptake by endocytosis and its delivery to lysosomes. Within lysosomes the cholesteryl esters of LDL are hydrolyzed. The free cholesterol binds to a soluble lysosomal protein called Niemann Pick C2 (NPC2) which delivers it to a membrane-embedded protein called NPC1 which inserts the cholesterol into the lysosome membrane. From there the cholesterol moves to the plasma membrane (PM) through a pathway that is unknown. When the PM becomes saturated with cholesterol, any excess is transported to the endoplasmic reticulum (ER) to repress production of LDL receptors and to be stored in lipid droplets. The work described here 1) showed that triazole antifungal drugs inhibit lysosomal cholesterol export by binding to the membrane domain of NPC1 2) used itraconazole to solve the crystal structure of NPC1 at 3.3Å, 3) revealed that cholesterol is transported out of lysosomes through interactions between two or more NPC1 molecules, and 4) utilized CRISPR-Cas9 whole-genome knockout screens to identify all the genes involved in the transport and uptake of LDL cholesterol. From these screens in the latter study, we discovered that a specific phospholipid, phosphatidylserine (PS), is required for PM-to-ER cholesterol transport. These studies provide supporting evidence towards a vision of one-way directional transport of LDL-derived cholesterol from lysosomes to the PM to the ER.