The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase.
A hybrid gene has been constructed consisting of coding sequence for the membrane domain of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase linked to the coding sequence for the soluble enzyme Escherichia coli beta-galactosidase. Expression of the hybrid gene in transfected Chinese hamster ovary cells results in the production of a fusion protein (HMGal) which is localized in the endoplasmic reticulum. The fusion protein contains the high-mannose oligosaccharides characteristic of HMG-CoA reductase. Importantly the beta-galactosidase activity of HMGal decreases when low density lipoprotein is added to the culture media. Therefore, the membrane domain of HMG-CoA reductase is sufficient to determine both correct intracellular localization and sterol-regulation of degradation. Mutant fusion proteins which lack 64, 85, or 98 amino acid residues from within the membrane domain of HMG-CoA reductase are found to be localized in the endoplasmic reticulum and to retain beta-galactosidase activity. However, sterol-regulation of degradation is abolished.
Skalnik
D G
DG
Department of Biological Sciences, Stanford University, California 94305.
Narita
H
H
Kent
C
C
Simoni
R D
RD
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005696:Galactosidases / Q000235:genetics*; D005796:Genes; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D010957:Plasmids; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D006903:Hydroxymethylglutaryl CoA Reductases; D004262:DNA Restriction Enzymes; D005696:Galactosidases; D001616:beta-Galactosidase