The regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase requires a short-lived protein and occurs in the endoplasmic reticulum

Kristin Chun, Shoshana Bar-Nun, Robert D. Simoni

Research output: Contribution to journalArticle

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Abstract

A chimeric gene consisting of the coding sequence for the membrane domain of the endoplasmic reticulum protein, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, fused to the coding sequence for the soluble enzyme, β-galactosidase of Escherichia coli, has been previously constructed. This fusion protein, HMGal, has been localized to the membrane of the endoplasmic reticulum of Chinese hamster ovary cells transfected with this chimeric gene, and its β-galactosidase activity has declined in the presence of low density lipoprotein (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). In this report, we demonstrate that the loss of β-galactosidase activity results from the accelerated degradation of the HMGal protein. Taking advantage of a fluorescence-activated cell sorter technique, we have selected transfected cells which express sufficient levels of HMGal to improve its immunodetection. Based on pulse-chase experiments, the half-life of HMGal is 6.0 h, and, in the presence of 20 mM mevalonate, the half-life declines 1.7-fold. Under these conditions, mevalonate accelerates the degradation of HMG-CoA reductase in these cells 1.6-fold, from 8.4 h to 5.3 h, most probably by the same mechanism. This mevalonate-regulated degradation of HMGal is not due to a heteromeric association of HMGal with reductase, since the same effect has been observed in cells lacking the reductase protein. In addition, we demonstrate that inhibition of protein synthesis with cycloheximide abolishes the mevalonate-dependent accelerated degradation of HMGal, in agreement with previous studies which have presented indirect evidence that a short-lived protein is essential for mediating the loss of HMG-CoA reductase activity. Finally, using brefeldin A, we show that the mevalonate-dependent accelerated degradation of HMGal may occur in the endoplasmic reticulum.

Original languageEnglish (US)
Pages (from-to)22004-22010
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number35
StatePublished - Dec 15 1990
Externally publishedYes

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Hydroxymethylglutaryl CoA Reductases
Mevalonic Acid
Endoplasmic Reticulum
Galactosidases
Oxidoreductases
Degradation
Coenzyme A
Proteins
Half-Life
Brefeldin A
Genes
Membranes
Cycloheximide
Cricetulus
LDL Lipoproteins
Proteolysis
Ovary
Escherichia coli
Fluorescence
Fusion reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

The regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase requires a short-lived protein and occurs in the endoplasmic reticulum. / Chun, Kristin; Bar-Nun, Shoshana; Simoni, Robert D.

In: Journal of Biological Chemistry, Vol. 265, No. 35, 15.12.1990, p. 22004-22010.

Research output: Contribution to journalArticle

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