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.