Synthesis of Dolichol in a Polyprenol Reductase Mutant Is Restored by Elevation ofcis-Prenyl Transferase Activity

Abstract

CHB11-1-3 is a glycosylation mutant of Chinese hamster ovary (CHO) cells, isolated by screening mutagenized cells for those with decreased intracellular lysosomal enzyme activity [C. W. Hallet al.(1986)Mol. Cell. Biochem.72, 35–45]. CHB11-1-3 synthesizes the lipid polyprenol, the metabolic precursor of dolichol, rather than dolichol, indicating a defect in polyprenol reductase. This defect was demonstrated previously in Lec9 CHO mutants, and cell fusion experiments confirmed that CHB11-1-3 is a member of this complementation group. A revertant of CHB11-1-3, CHBREV, isolated for its ability to grow at 39°C, synthesizes dolichol at near-normal levels. CHBREV is probably a second-site revertant, because it synthesizes three to four times as much polyprenol as CHB11-1-3 and exhibits a similar elevation in the specific activity ofcis-prenyl transferase. This higher activity appears to reflect an increase in enzyme molecules rather than the presence of an activator or absence of an inhibitor. These results suggest that CHB11-1-3 is a “Km” mutant, because synthesis of higher amounts of the substrate of polyprenol reductase obviates the defect.