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This paper describes a rapid and efficient two-step procedure for the isolation of mutant cells with defects in receptor-mediated endocytosis. The procedure takes advantage of two fungal metabolites, compactin (ML236B), a potent inhibitor of cholesterol biosynthesis, and amphotericin B, a polyene antibiotic that forms toxic complexes with sterols in membranes. Mutagen-treated Chinese hamster ovary cells were preincubated overnight in a medium containing mevalonate, low density lipoprotein (LDL), and compactin (Mev/LDL/Com). At the end of the preincubation period, wild-type cells were cholesterol replete while mutant cells that could not utilize the cholesterol in LDL were cholesterol deficient. Subsequent incubation with amphotericin B for 6 hr killed most of the wild-type cells. After a second round of Mev/LDL/Com-amphotericin B selection, endocytosis-defective clones appeared at a frequency of approximately equal to 2.6 X 10(-5). Some of these clones expressed LDL receptor-defective phenotypes and fell into one of two previously defined classes of mutation. Sensitivity of the mutants to infection by vesicular stomatitis virus suggested that the mutations do not disrupt the coated pit-coated vesicle pathway of endocytosis. Minor modifications in the Mev/LDL/Com-amphotericin B selection permit the isolation of cholesterol auxotrophs and might allow the isolation of conditional-lethal mutations. Because LDL can be coupled to ligands that bind to receptors other than the LDL receptor, Mev/LDL/Com-amphotericin B selection may permit the isolation of mutant cells with defects that specifically disrupt other endocytic pathways.

proceedings of the national academy of sciences of the united states of america

Amphotericin B selection of mutant Chinese hamster cells with defects in the receptor-mediated endocytosis of low density lipoprotein and cholesterol biosynthesis.