Chinese hamster cell mutant resistant to ML236B (Compactin) is defective in endocytosis of low-density lipo-protein.

A fungal metabolite, ML236B (Compactin), isolated from Penicillium citrinum, is a specific inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (EC 1.1.1.34). Three ML236B-resistant (ML236Br) mutants, MF-1, MF-2, and MF-3, were isolated from V79 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The fluctuation test showed 2.2 X 10(-6) mutants per cell per generation of a spontaneous mutation frequency of ML236Br clones. These ML236Br clones showed a four- to fivefold-higher resistance to the drug than did their parental V79. Radioactive acetate, but not mevalonate, incorporation into the sterol fraction increased about 10-fold in ML236Br clones in comparison with that in V79. The cellular level of HMG-coenzyme A reductase in three ML236Br mutants was found to be a few-fold higher than that of V79 when cultured in the presence of lipoproteins. The 125I-labeled low-density lipoprotein-binding assay showed binding activity in three ML236Br clones comparable to that of the parental V79 cells. By contrast, an internalization assay of 125I-labeled low-density lipoprotein into the cells showed significantly reduced activity in three ML236Br clones in comparison with V79.

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Sterols accelerate degradation of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase encoded by a constitutively expressed cDNA.

Molecular and Cellular Biology ◽

10.1128/mcb.5.4.634 ◽

1985 ◽

Vol 5 (4) ◽

pp. 634-641 ◽

Cited By ~ 99

Author(s):

D J Chin ◽

G Gil ◽

J R Faust ◽

J L Goldstein ◽

M S Brown ◽

...

Keyword(s):

Coenzyme A ◽

Reductase Activity ◽

Chinese Hamster Ovary Cells ◽

Low Density Lipoprotein ◽

Chinese Hamster ◽

Density Lipoprotein ◽

Simian Virus 40 ◽

Low Density ◽

Reductase Mrna ◽

Coenzyme A Reductase

A recombinant plasmid containing a full-length cDNA for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase was introduced by calcium phosphate-mediated transfection into UT-2 cells, a mutant line of Chinese hamster ovary cells that lack 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus require low density lipoprotein-cholesterol and mevalonate for growth. We selected a line of permanently transfected cells, designated TR-36 cells, that expressed high levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus grew in the absence of low density lipoprotein and mevalonate. Constitutive synthesis of reductase mRNA in TR-36 cells was driven by the simian virus 40 early promoter, and therefore the mRNA was not suppressed by sterols, such as 25-hydroxycholesterol or cholesterol derived from low density lipoprotein, which normally suppresses transcription of reductase mRNA when the reductase gene is driven by its own promoter. Although TR-36 cells continued to synthesize large amounts of reductase mRNA and protein in the presence of sterols, reductase activity declined by 50 to 60%. This decline was caused by a twofold increase in the rate of degradation of preformed enzyme molecules. The current data demonstrate that sterols accelerate the degradation of reductase protein independently of any inhibitory effect on the synthesis of the protein.

Download Full-text

Sterols accelerate degradation of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase encoded by a constitutively expressed cDNA

Molecular and Cellular Biology ◽

10.1128/mcb.5.4.634-641.1985 ◽

1985 ◽

Vol 5 (4) ◽

pp. 634-641

Author(s):

D J Chin ◽

G Gil ◽

J R Faust ◽

J L Goldstein ◽

M S Brown ◽

...

Keyword(s):

Coenzyme A ◽

Reductase Activity ◽

Chinese Hamster Ovary Cells ◽

Low Density Lipoprotein ◽

Chinese Hamster ◽

Density Lipoprotein ◽

Simian Virus 40 ◽

Low Density ◽

Reductase Mrna ◽

Coenzyme A Reductase

A recombinant plasmid containing a full-length cDNA for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase was introduced by calcium phosphate-mediated transfection into UT-2 cells, a mutant line of Chinese hamster ovary cells that lack 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus require low density lipoprotein-cholesterol and mevalonate for growth. We selected a line of permanently transfected cells, designated TR-36 cells, that expressed high levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus grew in the absence of low density lipoprotein and mevalonate. Constitutive synthesis of reductase mRNA in TR-36 cells was driven by the simian virus 40 early promoter, and therefore the mRNA was not suppressed by sterols, such as 25-hydroxycholesterol or cholesterol derived from low density lipoprotein, which normally suppresses transcription of reductase mRNA when the reductase gene is driven by its own promoter. Although TR-36 cells continued to synthesize large amounts of reductase mRNA and protein in the presence of sterols, reductase activity declined by 50 to 60%. This decline was caused by a twofold increase in the rate of degradation of preformed enzyme molecules. The current data demonstrate that sterols accelerate the degradation of reductase protein independently of any inhibitory effect on the synthesis of the protein.

Download Full-text