{'fraction_non_alphanumeric': 0.018960244648318043, 'fraction_numerical': 0.010397553516819572, 'mean_word_length': 5.816666666666666, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Cholesterol Trafficking for Steroid Biosynthesis in MA‐10 Mouse Tumor Leydig 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.