Figure 9.

Biochemical characterization of the subcellular distribution of free [¹⁴C]cholesterol. (A) hem1Δ (YRS1707) and hem1Δ aus1 pdr11Δ (YRS1962) mutant cells were grown in the presence of [¹⁴C]cholesterol for 24 h at 24°C. Equal OD units of cells were harvested, and membranes were separated on an Accudenz density gradient. Fractions were collected, lipids were extracted and separated by TLC, and free sterols were quantified by radioscanning. (B) Distribution of marker proteins. Membranes from hem1Δ (YRS1707) mutant cells grown in cholesterol were fractionated on an Accudenz density gradient, and the distribution of marker proteins was examined by Western blotting with antibodies against the proteins indicated. Organelles are indicated as follows: PM, plasma membrane; LP, lipid particles; VAC, vacuole; and Mito, mitochondria. (C) Distribution of free cholesterol in anaerobic nonviable mutants. Strains of the indicated genotype were cultivated as in A, membranes were fractionated on an Accudenz gradient, lipids were extracted and separated by TLC, and the distribution of free [¹⁴C]cholesterol was determined by radioscanning.