The proposed research is aimed at the elucidation of the mechanisms underlying the apportionment of cholesterol among various cellular pools in mammalian cells. Studies from this laboratory have shown that in human fibroblasts newly-synthesized sterols en route from the rough endoplasmic reticulum (RER) to the plasma membrane become associated with a distinctive cholesterol-enriched membrane fraction which we call the sterol-rich organelle (SRO). The SRO will be purified and its composition analyzed. The specific mechanism which determines whether sterols in the RER are exported to the plasma membrane or esterified for storage will be sought. At issue is the interplay between esterified cholesterol and various cell cholesterol pools: the plasma membrane, the RER, newly synthesized cholesterol, ingested cholesterol, and cholesterol released from ester stores. The following model will be tested: (1) A sensor protein in the plasma membrane is critically sensitive to the cholesterol content of that membrane. (2) As cholesterol content rises above a threshold level, the sensor is activated and a message is sent to ACAT leading to its activation. The esterification of the cholesterol circulating through the RER from all sources is thereby controlled by the plasma membrane. Tests of this model: (1) The effect of cell cholesterol content on the activity of acyl-CoA:cholesterol acyltransferase (ACAT) will be examined. Of particular interest is whether changes in this enzyme activity correlate with plasma membrane or intracellular cholesterol. (2) Whether the enzymes that synthesize cholesterol are in the same region of the RER as ACAT will be determined. (3) Bilayer modifying agents will be sought which affect the putative sensor regulating ACAT. Finally, ACAT will be identified and isolated by its phosphorylation. These studies should increase our understanding of the disposition of cellular cholesterol, its incorporation into ester droplets, and, hopefully, the basis of atherosclerosis.
