Plasma membrane cholesterol: removal and insertion into the membrane and utilization as substrate for steroidogenesis.

The plasma membrane cholesterol content of MA-10 Leydig tumor cells is depleted by trophic hormone stimulation and repleted by incubating the cells with low density lipoprotein. The present studies used subcellular fractionation to investigate the membranes involved in steroid hormone synthesis. The results showed that the plasma membrane was the major source of cholesterol substrate and that the cholesterol content changed independently of any mass changes in membrane protein or phospholipid. Membrane phospholipid composition also did not change as membrane cholesterol content decreased or increased, a finding inconsistent with the proposal that phospholipid composition dictates the amount of cholesterol contained in a membrane. The mitochondria of the MA-10 cells were cholesterol rich, containing more cholesterol per unit protein or phospholipid than the plasma membrane. This cholesterol was presumably in the outer mitochondrial membrane, since virtually all of the cholesterol of intact mitochondria was accessible to cholesterol oxidase. Although there was a high concentration of mitochondrial cholesterol, this cholesterol was largely inert as a substrate for steroidogenesis, and plasma membrane cholesterol was incorporated into steroid hormones without ever equilibrating with the mitochondrial cholesterol pool.