title: Analysis of a Chinese hamster ovary cell mutant with defective mobilization of cholesterol from the plasma membrane to the endoplasmic reticulum.
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.
authors.affiliation: Department of Physiology, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: N L
authors.initials: NL
authors.lastname: Jacobs
authors.forename: B
authors.initials: B
authors.lastname: Andemariam
authors.forename: K W
authors.initials: KW
authors.lastname: Underwood
authors.forename: K
authors.initials: K
authors.lastname: Panchalingam
authors.forename: D
authors.initials: D
authors.lastname: Sternberg
authors.forename: M
authors.initials: M
authors.lastname: Kielian
authors.forename: L
authors.initials: L
authors.lastname: Liscum
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002455:Cell Division; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D002788:Cholesterol Esters / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000378:metabolism; D008078:Cholesterol, LDL / Q000378:metabolism; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000378:metabolism; D008247:Lysosomes / Q000737:chemistry; D008561:Membrane Fusion; D009154:Mutation; D010743:Phospholipids / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D002785:Sterol O-Acyltransferase / Q000378:metabolism
chemical_list: D002788:Cholesterol Esters; D008078:Cholesterol, LDL; D008077:Lipoproteins, LDL; D010743:Phospholipids; D000666:Amphotericin B; D002784:Cholesterol; D002789:Cholesterol Oxidase; D002785:Sterol O-Acyltransferase; D013108:Sphingomyelin Phosphodiesterase