Abstract
The degradation of the microsomal enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is highly regulated and is dependent on both a sterol and non-sterol derivative of mevalonic acid (MVA). We recently proposed that the non-sterol component is derived from farnesyl diphosphate (FPP), presqualene pyrophosphate, or squalene (Correll, C. C. and Edwards, P. A. (1994) J. Biol. Chem. 269, 633-638). In the current study, we have used digitonin-permeabilized cells to further define this MVA-derived non-sterol component required for the regulated degradation of HMG-CoA reductase. The addition of either FPP or farnesol to digitonin-permeabilized cells resulted in a rapid and dose-dependent degradation of HMG-CoA reductase. The effect of FPP, but not farnesol, was blocked by the phosphatase inhibitor sodium fluoride. The enhanced degradation of HMG-CoA reductase in permeabilized cells specifically required farnesol, since the addition of any of the structurally related isoprenoids geraniol, geranyl diphosphate, geranylgeranyl diphosphate, nerolidol, or all-cis-farnesol, or of the non-sterol squalene to the permeabilized cells did not stimulate enzyme degradation. The present studies demonstrate for the first time that the accelerated degradation of HMG-CoA reductase can be initiated in vitro. Further, since farnesol is shown to be specifically required for the enhanced degradation of the enzyme in vitro, we propose that this isoprenoid alcohol is important in this process in intact cells.
Highlights
Met-18b-2Cells Permeabilized by DigitoniRnelease Cytosolic from 20 to 50%(data not shown)
The finding that eitherof the Enzymes but Retain Membrane-bound HMG-CoA Reductase- substrates of HMG-CoAreductase increases thestability of the Digitonin permeabilization of Met-18b-2 cells that stably ex- protein in permeabilized cells may indicate that protein conpress P-galactosidase resulted in a greater than90% decrease formation plays a role in the susceptibility of the enzyme to in thecell-associated activity of the cytosolicenzymes phospho- degradation
That hadnot beentreated withdigitonin (Fig. 1).Further, digi- In agreement with earlier studies (8, 9), incubation of pertonin permeabilization of cells radiolabeled with T~-an~~S-labemleabilized cells for 4 h in thepresence of rat liver cytosol (0.5 resulted in more than 50%loss of trichloroacetic acid-precipi- mg/ml) or an ATP-generating system did not significantly affect table protein (Fig. 1).In contrast, there wasno significant loss the amountof radiolabeled HMG-CoA reductase protein
Summary
The incorporation of T~-an~~S-label into tocteallular proteins was quantified by precipitation of proteins with trichloroacetiaccid followed scribed under “Experimental Procedures.” The amount of radiolabeled HMG-CoA reductase declined less than 25% when the permeabilized cells were incubated for 4 h under standard conditions that included either anNADPH-generating system The finding that eitherof the Enzymes but Retain Membrane-bound HMG-CoA Reductase- substrates of HMG-CoAreductase increases thestability of the Digitonin permeabilization of Met-18b-2 cells that stably ex- protein in permeabilized cells may indicate that protein conpress P-galactosidase resulted in a greater than90% decrease formation plays a role in the susceptibility of the enzyme to in thecell-associated activity of the cytosolicenzymes phospho- degradation.
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