The role of the membrane domain in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

We have constructed a series of mutations in the membrane and linker domains of Syrian hamster 3-hydroxy-3-methylglutaryl-(HMG) CoA reductase in order to determine the regions critical for the regulated degradation of the enzyme. In transfected Chinese hamster ovary cells, we have expressed a fusion protein, HMGal, which consists of the membrane and linker domains of the Syrian hamster HMG-CoA reductase fused to beta-galactosidase. Using this fusion protein, we have determined that a deletion of 64 amino acids from the central region of the membrane domain causes the protein to be degraded extremely rapidly. In addition, deletion of PEST sequences has little effect on degradation, but deletion of the linker domain makes the protein's degradation insensitive to sterols and mevalonate. In addition to deletion mutations, we have systematically replaced each hydrophobic, putative membrane spanning region of the membrane domain with the first transmembrane sequence from bacteriorhodopsin. Replacement of span 4 has no effect on degradation. Replacements of spans 5 or 6 result in a protein which has a normal basal rate of degradation, but this rate of degradation is not accelerated by mevalonate, low density lipoprotein, or 25-hydroxycholesterol. Replacement of span 3 results in a protein whose degradation is similarly not accelerated by sterols or mevalonate, but since this protein might be mislocalized, these results are inconclusive. Replacement of span 7 yields a short-lived protein which is degraded more rapidly in response to mevalonate but not in response to exogenous sterols. Replacement of span 8 extends both the basal and mevalonate-accelerated half-life about 5-fold. This work begins to define the critical regions for regulated degradation within the membrane domain of HMG-CoA reductase.