Acute cholesterol depletion inhibits clathrin-coated pit budding

Many biologically important macromolecules are internalized into cells by c lathrin-coated pit endocytosis, The mechanism of clathrin-coated pit buddin g has been investigated intensively, and considerable progress has been mad e in characterizing the proteins involved in internalization, Membrane lipi d composition and the lateral organization of lipids and proteins within me mbranes are believed to play an important role in the regulation of membran e-trafficking processes. Here we report that membrane cholesterol plays a c ritical role in clathrin-coated pit internalization, We show that acute cho lesterol depletion, using beta-methyl-cydodextrin, specifically reduces the rate of internalization of transferrin receptor by more than 85%, without affecting intracellular receptor trafficking back to the cell surface. The effect on endocytosis is attributable to a failure of coated pits to detach from the plasma membrane, as visualized by using a green fluorescent prote in-clathrin conjugate in living cells. Ultrastructural studies indicate tha t acute cholesterol depletion causes accumulation of flat-coated membranes and a corresponding decrease in deep-coated pits, consistent with the possi bility that flat clathrin lattices are direct precursors of indented pits a nd endocytic vesicles in intact cells, We conclude that clathrin is unable to induce curvature in the membrane depleted of cholesterol.