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-meth ylglutaryl-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 respo nse 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 reti culum. To localize the site of regulated degradation of HMG CoA reduct ase, we used a construct that fuses the N-terminal membrane-anchoring domain of HMG CoA reductase in-frame with P-galactosidase as a reporte r domain (HM-Gal). HM-Gal has previously been shown to reproduce faith fully the degradative properties of native HMG CoA reductase (Chun et al. (1990) J. Biol. Chem. 265, 22004-22010). CHO cells transfected wit h DNA encoding HM-Gal were exposed to mevalonic acid, which enhances t he rate of HMG CoA reductase degradation several fold, and leads to th e reduction of the steady state levels of HM-Gal by 80-90%. To accumul ate HMG CoA reductase at the site of degradation, cells were simultane ously treated with N-acetyl-leucyl-leucyl-norleucinal (ALLN), which in hibits the protease responsible for reductase degradation. HM-Gal was localized morphologically by immunofluorescence and biochemically by m easuring beta-galactosidase activity in Percoll gradients of cellular homogenates. Using either technique HM-Gal localization was indistingu ishable from that of ER markers in both control cells and in cells tre ated to accumulate HMG CoA reductase at the site of degradation. We co nclude that the regulated degradation of HMG CoA reductase occurs in t he ER or an ER-like compartment.