Edited by George CarmanHormone-sensitive acute steroid biosynthesis requires trafficking of cholesterol from intracellular sources to the inner mitochondrial membrane. The precise location of the intracellular cholesterol and its transport mechanism are uncertain. Perfringolysin O, produced by Clostridium perfringens, binds cholesterol. Its fourth domain (D4) retains cholesterol-binding properties but not cytotoxicity. We transfected steroidogenic MA-10 cells of mouse Leydig cell tumors with the mCherry-D4 plasmid. Tagged D4 with fluorescent proteins enabled us to track cholesterol. The staining was primarily localized to the inner leaflet of the plasma membrane and was partially released upon treatment with dibutyryl-cAMP (Bt 2 cAMP), a cAMP analog. Inhibitors of cholesterol import into mitochondria blocked steroidogenesis and prevented release of D4 (and presumably cholesterol) from the plasma membrane. We conclude that the bulk of the steroidogenic pool of cholesterol, mobilized by Bt 2 cAMP for acute steroidogenesis, originates from the plasma membrane. Treatment of the cells with steroid metabolites, 22(R)-hydroxycholesterol and pregnenolone, also reduced D4 release from the plasma membrane, perhaps evidence for a feedback effect of elevated steroid formation on cholesterol release. Interestingly, D4 staining was localized to endosomes during Bt 2 cAMP stimulation suggesting that these organelles are on the route of cholesterol trafficking from the plasma membrane to mitochondria. Finally, D4 was expressed in primary rat Leydig cells with a lentivirus and was released from the plasma membrane following Bt 2 cAMP treatment. We conclude that the plasma membrane is the source of cholesterol for steroidogenesis in these cells as well as in MA-10 cells.

The hormone‐sensitive and rate‐limiting step in steroid biosynthesis is the movement of cholesterol from intracellular sources to the inner mitochondrial membrane (IMM). Despite the numerous studies on cholesterol trafficking in steroidogenesis, the exact source and mechanism by which cholesterol is transported to IMM remains to be elucidated. D4 is the fourth domain of perfringolysin O protein, which has the ability to bind membranes containing greater than 30 mol% cholesterol of total lipid concentration with high affinity. mCherry‐tagged D4 was used to visualize cholesterol trafficking in MA‐10 cells. Confocal microscopy showed D4‐mCherry localized at the plasma membrane, but upon 45 minutes treatment with dibutyryl‐cAMP (dbcAMP) a significant reduction in plasma membrane labeling was observed. Functional inhibitors of proteins involved in cholesterol import into mitochondria and metabolism, blocked steroid formation and slowed down the movement of D4‐mCherry from the plasma membrane. Recombinant D4‐GFP protein readily bound the outer leaflet of the plasma membrane even after 120 minutes of dbcAMP stimulation, indicating that cholesterol was trafficked from the inner leaflet of the plasma membrane. D4‐mCherry also localized the late endosomes upon dbcAMP stimulation suggesting a route for the cholesterol from plasma membrane to mitochondria. These data suggest that the bulk of the steroidogenic pool of cholesterol, mobilized by cAMP for acute steroidogenesis, likely originates from the inner leaflet of the plasma membrane.Source: CIHR