Abstract:

HMG-CoA reductase (HMGR) is a 97kDa glycoprotein of the eddoplasmic reticulum (ER) which catalyzes the formation of mevalonate (MVA), the rate limiting step in the cholesterol biosynthetic pathway. The turnover of HMGR is regulated by the cellular levels of sterol and nonsterol MVA-derived isoprenoids; when cells are starved for sterols/MVA, the enzyme is degraded with a halflife (tl/2) of 7-15h. The rate of HMGR degradation is accelerated 3-5 fold (tl/2 2-3h) in sterols/MVA replete cells. This regulation is imparted to the enzyme by its elaborated membrane spanning domain. We have selected for a stable Chinese hamster ovary-derived cell-line, designated L-90, that grows in the presence of 90M lovastatin, a potent inhibitor of HMGR. Similar to other statin-resistant cell lines (e.g., UT-1), L-90 cells overexpress tiMGR as a result of gene amplification. The cells also have crystaUoid ER. Surprisingly, L90 cells do not accelerate the rate of HMGR degradation in response to sterols or MVA. This abnormal phenotype appears to b due to a defect in tran,s-acting factor(s) because L-90 cells also do not regdlate the turnover of transfected HMGai, a 150kDa fusion protein between the membrane domain of tIMGR and bacterial t3-galactosidase. Therefore, L-90 cells should prove useful in identifying cellular factor(s) that operate in the regulated degradation of HMGR. One such factor may be a 97kDa protein, which is not HMGR, that co-immunoprecipitates with HMGal in transfected L-90 cells.