Isolation of Chinese hamster cell mutants defective in the receptor-mediated endocytosis of low density lipoprotein

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Abstract

This paper describes a procedure for the isolation of mutant cells with defects in receptor-mediated endocytosis. The procedure takes advantage of the unique structure of low density lipoprotein, a plasma cholesterol transport protein that enters cells by receptor-mediated endocytosis. LDL contains a core of cholesteryl ester that can be extracted and reconstituted with hydrophobic molecules that convert the LDL into a toxic or fluorescent particle. Mutagenized Chinese hamster ovary cells were incubated with reconstituted LDL containing toxic 25-hydroxycholesteryl oleate. Wild-type cells take up this lipoprotein via the LDL receptor, liberate the 25-hydroxycholesterol in lysosomes, and die. To identify colonies of receptor-deficient cells from among the few survivors of the first selection step, we incubated the cells with LDL reconstituted with a fluorescent cholesteryl ester and picked colonies that failed to accumulate fluorescence. The two-step isolation procedure yielded receptor-deficient cells at a frequency of 1 in 105. The mutant cells grew in the presence of LDL reconstituted with 25-hydroxycholesteryl oleate at concentrations 100-fold higher than those that killed parental cells. The altered phenotypes have remained stable for more than 200 population doublings under non-selective conditions. Inasmuch as LDL can be coupled to ligands that bind to receptors other than the LDL receptor, the above method may have general utility in isolating cells with mutations affecting other receptor systems.

Original languageEnglish (US)
Pages (from-to)167-184
Number of pages18
JournalJournal of Molecular Biology
Volume150
Issue number2
DOIs
StatePublished - Aug 5 1981

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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