Analysis of a Chinese hamster ovary cell mutant with defective mobilization of cholesterol from the plasma membrane to the endoplasmic reticulum

Natalie L. Jacobs, Biree Andemariam, Kathryn W. Underwood, Krishnanchali Panchalingam, David Sternberg, Margaret Kielian, Laura Liscum

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

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 intracellular cholesterol transport. Complementation analysis among seven mutants identified one cell line, mutant 3-6, with a unique defect. The present analysis revealed three key features of mutant 3-6. First, the movement of cholesterol both from the endoplasmic reticulum and through lysosomes to the plasma membrane was normal. However, when intact 3-6 cells were treated with sphingomyelinase, movement of plasma membrane cholesterol to acyl CoA: cholesterol acyltransferase in the endoplasmic 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 lipoprotein-derived cholesterol for growth as effectively as parental Chinese hamster ovary cells. Finally, despite normal movement of cholesterol to the plasma membrane, mutant 3-6 was amphotericin B resistant. The plasma membrane cholesterol content was normal as assessed by cholesterol oxidase treatment and Semliki Forest virus fusion, which suggests 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.

Original languageEnglish (US)
Pages (from-to)1973-1987
Number of pages15
JournalJournal of Lipid Research
Volume38
Issue number10
StatePublished - Oct 1 1997

Keywords

  • Low density lipoprotein
  • Somatic cell mutant
  • Sphingomyelinase

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology

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    Jacobs, N. L., Andemariam, B., Underwood, K. W., Panchalingam, K., Sternberg, D., Kielian, M., & Liscum, L. (1997). Analysis of a Chinese hamster ovary cell mutant with defective mobilization of cholesterol from the plasma membrane to the endoplasmic reticulum. Journal of Lipid Research, 38(10), 1973-1987.