Filipin-cholesterol complexes form in uncoated vesicle membrane derived from coated vesicles during receptor-mediated endocytosis of low density lipoprotein.

Abstract

Filipin was widely used as an EM probe to detect 3-.beta.-hydroxysterols, principally cholesterol, in [human] cellular membranes. When it complexes with sterol, it forms globular deposits that disrupt the planar organization of the membrane. Previous studies showed that coated pits and coated vesicles, specialized membranes involved in receptor-mediated endocytosis, do not appear to bind filipin. These membranes may be low in cholesterol compared with the remainder of the plasma membrane. Since coated endocytic vesicles become uncoated vesicles during the transport of internalized ligands to the lysosome, studies were carried out to determine whether or not the membranes that surround these transport vesicles are unable to bind filipin and therefore, are also low in cholesterol. Cells were incubated with ferritin-conjugated ligands that bind to low density lipoprotein (LDL) receptors in coated pits. After allowing internalization of the conjugates, the cells were fixed in either the presence or absence of filipin. This permitted the identification of all of the vesicles involved in the transport of LDL to the lysosome and the determination as to whether the membranes of these vesicles were able to bind filipin. Coordinate with the dissociation of the clathrin coat from the endocytic vesicles, the membranes became sensitive to the formation of filipin-sterol complexes. All of the uncoated endocytic vesicle membranes, as well as the lysosomal membranes, bound filipin. Coated membrane may contain normal cholesterol levels, which is not easily detected with filipin, or that cholesterol rapidly moves into endocytic vesicles after the clathrin coat dissociates from the membrane.