Abstract
The in vivo turnover rate of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the mevalonate (MVA) pathway, is accelerated when excess MVA or sterols are added to the growth medium of cells. As we have shown recently (Roitelman, J., Bar-Nun, S., Inoue, S., and Simoni, R. D. (1991) J. Biol. Chem. 266, 16085-16091), perturbation of cellular Ca2+ homeostasis abrogates the MVA-accelerated degradation of HMG-CoA reductase and HMGal. Here we show that, in contrast, the sterol-accelerated degradation of HMG-CoA reductase is unaffected by Ca2+ perturbation achieved either by Ca2+ ionophore or by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. The differential effects of Ca2+ perturbation can be attributed neither to global alteration in protein synthesis nor to inhibition of MVA conversion to sterols. Yet, such manipulations markedly reduce the incorporation of MVA into cellular macromolecules, including prenylated proteins. Furthermore, we directly demonstrate that MVA gives rise to at least two distinct signals, one that is essential to support the effect of sterols and another that operates independently of sterols. Our results indicate that the cellular signals operating in the MVA-accelerated turnover of HMG-CoA reductase are distinct from those involved in the sterol-regulated degradation. A working model for the degradation pathway is proposed.
Highlights
The in vivo turnover rate of the endoplasmic reticu- ments [2,3,4] and, toa lesser extent, by the ER’/pre-Golgi lum protein 3-hydroxy-3-methylglutarylcoenzyme A degradation machinery of proteinsin the central vacuolar (HMG-CoA)reductase, the rate-limiting enzyme in thesystem [5, 6], very little is known about the signal(s) that mevalonate (MVA) pathway, is accelerated when ex- initiate the selective tagging of target proteins
Perturbation achieved either by Ca2+ionophore or by reductase is extended. When these demands are satisfied by inhibitors of the endoplasmic reticulum Ca2+-ATPase. abundant sterols or MVA, HMG-CoA reductase is rapidly
The lysate was Inthis article we report thatthe sterol and nonsterol "signals" that regulate the degradation of HMG-CoA reductase are distinct, as determinebdy their sensitivity to perturbation of cellular Ca2+homeostasis
Summary
Effects of Cellular ea2+Perturbationon the MVA- and ized, as described [10]. Cells-CHO-HMGal cells [13] were maintained in MEM containing 5% FCS and Geneticin a t 250 pg/ml. In pulse-labeled cells, chased in Ca2+-containing medium in the presence of excess MVA, the degradation of HMG-CoA reductase and HMGal is accelerated 2-3-fold relative to the basal rate InCa2+-free medium(Fig. containing medium, perturbation of cellular Ca2+by the addition of ionomycin extends thehalf-life of both proteins, and it totally abolishes the effect of MVA. This differential effect of thapsigargin on theMVA-. -i cedures" and chased in Ca2+-containing a (panel A ) or Ca2+-free(panel B ) MEM in the absence ( 0 )or presence of 20 mM MVA (0),1p M ionomycin (W), or both (0).A t the indicated time points, cells werleysed and HMG-CoA reductase.
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