Control of lipid synthesis and distribution during membrane biogenesis.

摘要

Eukaryotic cell membranes are composed of proteins and diverse types glycerophospholipids, sphingolipids, and sterols. Membrane lipids are non-randomly distributed throughout the endocytic and secretory pathways and along with resident proteins determine organelle identity. The heterogeneous localization of lipids is actively maintained despite a constant flow of membrane traffic between organelles. The mechanisms which coordinate lipid dynamics remain poorly understood. In this thesis, we investigated two aspects of membrane lipid control: synthesis and distribution.; In chapter 1, we studied phagocytosis in human embryonic kidney (HEK) 293 cells as an experimental system to investigate how cells coordinate lipid biosynthesis during the assembly of new membranes. In response to phagocytosis, 293 cells synthesized cholesterol and phospholipids at amounts equivalent to the surface area of the internalized particles. Lipid synthesis was accompanied by increased transcription of several lipogenic proteins including the low-density lipoprotein (LDL) receptor, enzymes required for cholesterol synthesis (3-hydroxy-3-methylglutaryl coenzyme A synthase and 3-hydroxy-3-methylglutaryl coenzyme A reductase) and fatty acid synthase. Phagocytosis triggered the activation of two lipogenic transcription factors, sterol regulatory element binding protein-1a(SREBP-1a) and SREBP-2. Phagocytosis-induced transcription and lipid synthesis require SREBP activation. These results identify SREBPs as essential regulators of membrane biogenesis.; Phagocytosis-induced expression of the LDL receptor suggests a role for extracellular cholesterol in membrane biogenesis. The LDL receptor mediates the uptake of cholesteryl ester-containing LDL particles into the endosomal system. In endosomes/lysosomes, cholesteryl esters are hydrolyzed by acidic cholesteryl ester hydrolase (aCEH) to cholesterol and fatty acid. Cholesterol is then redistributed from endosomes to membrane pools, mainly plasma membrane, by poorly characterized mechanisms.; In chapter 2, we have studied endosomal/lysosomal cholesteryl ester metabolism in mouse macrophages and with cell-free extracts. We show that net hydrolysis of cholesteryl ester is coupled to the transfer of cholesterol to membranes. When membrane cholesterol levels are low, absorption of cholesterol effectively drives cholesteryl ester hydrolysis. When cholesterol levels in acceptor membranes approach saturation or when cholesterol export is blocked, cholesterol is re-esterified in endosomes. Through this process, cells are able to modulate the endosomal dispensation of cholesterol to the cellular requirement for cholesterol.