The role of tubulin in the steroidogenic response of murine adrenal and rat Leydig cells.

The Y-1 murine adrenal and CCL43 rat Leydig tumor cell lines were used as model systems for studying the role of tubulin in steroidogenesis. Prior to the stimulation of steroidogenesis it was observed that most of the tubulin present in these cells, as determined by indirect immunofluorescence, was in a 0.2-0.6 micrometers dia. granular form. When these cells were treated with ACTH and cAMP, respectively, it was observed that the granular form of tubulin was replaced by many organized microtubules. These granules were identified in the electron microscope using tubulin antibody/ferritin localization and appeared to be membrane-bound and identical to structures previously described as containing cholesterol. We have isolated these structures using cell homogenization and sucrose gradient centrifugation and analyzed the steroid composition by thin layer chromatography (94% cholesterol, 6% cholesterol ester). These granules also contained tubulin as determined by gel electrophoresis. In addition, they contained acid phosphatase as determined by their ability to hydrolize beta-glycerolphosphate. We suggest that tubulin may be involved in the sequestering of cholesterol by preventing its transport to the mitochondria where conversion to pregnenolone takes place, and that steroidogenesis is increased when tubulin is dissociated from the cholesterol granules.