Appearance of crystalloid endoplasmic reticulum in compactin-resistant Chinese hamster cells with a 500-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Abstract

We have developed a line of Chinese hamster ovary cells with a 500-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the membrane-bound enzyme that controls cholesterol synthesis. This line, designated (UT-1, was obtained by stepwise adaptation of cells to growth in increasing concentrations of compactin, a competitive inhibitor of reductase. Reductase accounts for approximately 2% of total cell protein in UT-1 cells, as calculated from enzyme specific activity and by immunoprecipitation of reductase after growth of cells in [35S]methionine. After solubilization in the presence of the protease inhibitor leupeptin and electrophoresis in NaDodSO4/polyacrylamide gels, reductase subunits from UT-1 cells were visualized by immunoblotting as a single band (Mr = 62,000). To accommodate the increased amounts of reductase, UT-1 cells developed marked proliferation of tubular smooth endoplasmic reticulum (ER) membranes, as revealed by immunofluorescence and electron microscopy. The ER tubules were packed in crystalloid hexagonal arrays. When UT-1 cells were incubated with low density lipoprotein, reductase activity was suppressed by 90% in 12 hr and the crystalloid ER disappeared. UT-1 cells should be useful for studies of the regulation of reductase and also for studies of the synthesis and degradation of smooth ER.