The regulated degradation of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase reporter construct occurs in the endoplasmic reticulum.
The rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase, is regulated at a number of levels. One important mechanism is regulation of the half-life of the protein by a controlled proteolytic system. This comes about in response to downstream products of the sterol biosynthetic pathway. Little is known about this system, including where in the cell this regulated degradation occurs. HMG CoA reductase resides in the endoplasmic reticulum. To localize the site of regulated degradation of HMG CoA reductase, we used a construct that fuses the N-terminal membrane-anchoring domain of HMG CoA reductase in-frame with beta-galactosidase as a reporter domain (HM-Gal). HM-Gal has previously been shown to reproduce faithfully the degradative properties of native HMG CoA reductase (Chun et al. (1990) J. Biol. Chem. 265, 22004-22010). CHO cells transfected with DNA encoding HM-Gal were exposed to mevalonic acid, which enhances the rate of HMG CoA reductase degradation several fold, and leads to the reduction of the steady state levels of HM-Gal by 80-90%. To accumulate HMG CoA reductase at the site of degradation, cells were simultaneously treated with N-acetyl-leucyl-leucyl-norleucinal (ALLN), which inhibits the protease responsible for reductase degradation. HM-Gal was localized morphologically by immunofluorescence and biochemically by measuring beta-galactosidase activity in Percoll gradients of cellular homogenates. Using either technique HM-Gal localization was indistinguishable from that of ER markers in both control cells and in cells treated to accumulate HMG CoA reductase at the site of degradation.(ABSTRACT TRUNCATED AT 250 WORDS)
Lecureux
L W
LW
Cell Biology Unit, Upjohn Company, Kalamazoo, MI 49001.
Wattenberg
B W
BW
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006207:Half-Life; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D008562:Membrane Glycoproteins; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
C028391:G protein, vesicular stomatitis virus; D007976:Leupeptins; D008562:Membrane Glycoproteins; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C063261:acetylleucyl-leucyl-norleucinal; D006903:Hydroxymethylglutaryl CoA Reductases; D001616:beta-Galactosidase; D010450:Endopeptidases