SYNTHESIS OF DOLICHOL IN A POLYPRENOL REDUCTASE MUTANT IS RESTORED BYELEVATION OF CIS-PRENYL TRANSFERASE-ACTIVITY

CHB11-1-3 is a glycosylation mutant of Chinese hamster ovary (CHO) cel ls, isolated by screening mutagenized cells for those with decreased i ntracellular lysosomal enzyme activity [C. W. Hall et 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 previo usly in Lec9 CHO mutants, and cell fusion experiments confirmed that C HB11-1-3 is a member of this complementation group, A revertant of CHB 11-1-3, CHBREV, isolated for its ability to grow at 39 degrees C, synt hesizes dolichol at near-normal levels. CHBREV is probably a second-si te revertant, because it synthesizes three to four times as much polyp renol as CHB11-1-3 and exhibits a similar elevation in the specific ac tivity of cis-prenyl transferase. This higher activity appears to refl ect an increase in enzyme molecules rather than the presence of an act ivator or absence of an inhibitor. These results suggest that CHB11-1- 3 is a ''K-m'' mutant, because synthesis of higher amounts of the subs trate of polyprenol reductase obviates the defect. (C) 1997 Academic P ress