Identification of a cholesterol-regulated 53,000-dalton cytosolic protein in UT-1 cells and cloning of its cDNA.

Abstract

UT-1 cells, a clone of Chinese hamster ovary (CHO) cells, have a 100-1000-fold elevation in the amount of 3-hydroxy-3-methylglutaryl CoA reductase and therefore grow in the presence of compactin, an inhibitor of reductase. UT-1 cells also have a markedly increased amount of another protein with a MW of 53,000 and an isoelectric point of .simeq. 6. Whereas the reductase is an enzyme of the endoplasmic reticulum, the 53,000-dalton protein (termed the 53k protein) is in the cytosol. It is not precipitated by an antireductase antibody. Synthesis of the 53k protein, like that of the reductase, is suppressed when UT-1 cells are incubated with plasma low density lipoprotein (LDL). A library of recombinant plasmids containing double-stranded c[complementary]DNA from UT-1 cells was prepared. Using differential colony hybridization, recombinant plasmids were identified containing double-stranded cDNA inserts encoding mRNA expressed at high levels in UT-1 cells as compared with CHO cells. One of the plasmids, designated p53k-3, contained a 0.97-kilobase double-stranded cDNA that hybridized to a 3.8-kilobase mRNA. When translated in vitro, this 3.8-kilobase mRNA directed the synthesis of a protein identical to the cellular 53k protein as determined by 2-dimensional gel electrophoresis. The mRNA for the 53k protein was present in much larger amounts in UT-1 cells than in parental CHO cells. In both cell types, the content of this mRNA decreased markedly when the cells were incubated with LDL. Although the function of the 53k protein is not known, circumstantial evidence suggests that it may represent cytosolic 3-hydroxy-3-methylglutaryl CoA synthase, the enzyme preceding the reductase in the cholesterol biosynthetic pathway. Apparently, the synthesis of at least 2 proteins, the reductase and the 53k protein, are induced to high levels in compactin-resistant UT-1 cells and the synthesis of both is suppressed coordinately by LDL.