ANALYSIS OF A CHINESE-HAMSTER OVARY CELL MUTANT WITH DEFECTIVE MOBILIZATION OF CHOLESTEROL FROM THE PLASMA-MEMBRANE TO THE ENDOPLASMIC-RETICULUM

The factors involved in shuttling cholesterol among cellular membranes have not been defined. Using amphotericin B selection, we previously isolated Chinese hamster-ovary cell mutants expressing defects in intr acellular cholesterol transport. Complementation analysis among seven mutants identified one cell line, mutant 3-6, with a unique defect. Th e present analysis revealed three key features of mutant 3-6. First, t ile movement or cholesterol both from the endoplasmic reticulum and th rough lysosomes to the plasma membrane was normal. However, when intac t 3-6 cells were treated with sphingomyelinase, movement of plasma mem brane cholesterol to acyl CoA: cholesterol acyltransferase in the endo plasmic reticulum was defective. Cellular cholesterol was mobilized to this enzyme upon activation by 25-hydroxycholesterol. Second, mutant 3-6 did not utilize endogenously synthesized sterol or low density lip oprotein-derived cholesterol for growth as effectively as parental Chi nese hamster ovary cells. Finally, despite normal movement of choleste rol to the plasma membrane, mutant 3-6 was amphotericin B resistant. T he plasma membrane cholesterol content was normal as assessed by chole sterol oxidase treatment and Semliki Forest virus fusion, which sugges ts that the 3-6 mutation alters the organization of cholesterol in the plasma membrane. Our characterization of this mutant cell line should facilitate the identification of gene(s) required for this transport pathway.