The goal of this research is the elucidation of the topographic organization of cholesterol biosynthesis within mammalian cells. The starting point is our recent demonstration in cultured human fibroblasts that several sterol intermediates are nonuniformly distributed among subcellular fractions. This has lead to the hypothesis that cholesterol biosynthesis is not taken to completion at a single locus in the smooth endoplasmic reticulum. The proposed approach will involve subcellular fractionation of cell homogenates by equilibrium sucrose density centrifugation, rate zonal sedimentation in sucrose gradients and two phase aqueous partition. Cultured fibroblasts will be incubated with 3H-acetate to label biosynthetically so as to induce the accumulation of the intermediates squalene, squalene oxide and lanosterol: (a) incubation at 10 degrees C; (b) treatment with 4,4,10 beta - Trimethyl-trans-decal-3 beta-ol, an inhibitor of 2,3-oxidosqualene cyclase: and (c) exposure to an unidentified inhibitor of the conversion of lanosterol to cholesterol which we discovered recently. The subcellular distribution of nascent cholesterol and its precursors will be compared with the distribution of markers for the smooth and rough endoplasmic reticulum, Golgi apparatus, nucleus and plasma membrane. Since digitonin forms a specific, stoichiometric complex with cholesterol in membranes and thereby increases their density, it will be used to distinguish at what point precursor sterols become associated with cholesterol-rich membranes. We propose to adapt digitonin as a probe for the identification and isolation of cholesterol- and lanosterol-rich membranes by making antibodies to digitonin and by preparing galactosylated derivatives of digitonin reactive with cognate lectins. Two other specific hypotheses also will be tested: (1) Are intracellular cholesterol precursors carried by acidic vesicles? (2) Is the pathway for the delivery of newly synthesized cholesterol to the plasma membrane related to those for newly synthesized phospholipids and proteins? In view of the central role of cholesterol in human cardiovascular disease, an understanding of the topography of cholesterol biosynthesis and the mechanism leading to the concentration of cholesterol in the cell surface membrane is of fundamental interest.
