Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking.

Abstract

This paper reports the isolation and characterization of Chinese hamster ovary cell mutants defective in low density lipoprotein (LDL)- cholesterol trafficking. The parental cell line was 25-RA, which possesses LDL receptors and various cholesterogenic enzyme activities that are partially resistant to down regulation by exogenous sterols (Chang, T. Y., and J. S. Limanek. 1980. J. Biol. Chem. 255:7787-7795). Because these cells accumulate a large amount of intracellular cholesteryl ester when grown in medium containing 10% fetal calf serum, mutagenized populations of 25-RA cells were grown in the presence of a specific inhibitor of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which depleted their cholesteryl ester stores. Without this cholesterol ester storage, 99% of 25-RA cells die after 5-d growth in cholesterol starvation medium, while the mutant cells, which accumulate free cholesterol intracellularly, survived. In two mutant clones chosen for characterization, activation of cholesteryl ester synthesis by LDL was markedly reduced in the mutant cells compared with 25-RA cells. This lack of activation of cholesterol ester synthesis in the mutant cells could not be explained by defective uptake and/or processing of LDL or by a decreased amount of ACAT, as determined by in vitro enzyme activity. Mutant cells grown in the presence of LDL contain numerous cytosolic particles that stain intensely with the fluorescent compound acridine orange, suggesting that they are acidic. The particles are also stained with filipin, a cholesterol-specific fluorescent dye. Indirect immunofluorescence with a monoclonal antibody specific for a lysosomal/endosomal fraction revealed a staining pattern that colocalized with the filipin signal. The mutant phenotype was recessive. The available evidence indicates that the mutant cells can take up and process LDL normally, but the hydrolyzed cholesterol accumulates in an acidic compartment, probably the lysosomes, where it can not be transported to its normal intracellular destinations.

Transport of LDL-derived cholesterol from the NPC1 compartment to the ER involves the trans-Golgi network and the SNARE protein complex

“…S1 A). The labeled lipids were extracted and analyzed on silver nitrate impregnated TLC plates (47); the percentage hydrolysis of 3 H-CL and the percentage re-esterification of 3 H-LDL-CHOL were calculated as described (2). To conduct the cholesterol efflux experiments, cells were incubated with 4% 2-hydroxypropyl-␤-CD in Medium D at 37°C during the last 10 min of the chase period (2).…”

Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

“…The CT mutant cell line is isolated as one of the cholesterol-trafficking mutants from mutagenized RA cells (9). It contains the same gain-of-function mutation in SCAP.…”

“…8). Chinese hamster ovary (CHO) mutants that are defective in the NPC1 protein have been isolated and characterized (9)(10)(11). We compared the cholesterol trafficking activities between the NPC1 mutants [CT43 (CT) and CT60] and their parental cell line 25RA (RA) (12,13), and showed that NPC1 also participates in the transport of PM-derived cholesterol to the ER.…”

Niemann–Pick Type C Mutations Cause Lipid Traffic Jam

“…For these studies, NPC1 was transiently expressed in CT60 cells, a Chinese hamster ovary cell mutant in the same complementation class as NPC1 fibroblasts, which store LDL-cholesterol in perinuclear lysosomes (47,48). A plasmid expressing enhanced green fluorescent protein was co-transfected to identify transfected cells (34,40).…”