MUTANTS IN DOLICHOL SYNTHESIS - CONVERSION OF POLYPRENOL TO DOLICHOL APPEARS TO BE A RATE-LIMITING STEP IN DOLICHOL SYNTHESIS

Chinese hamster ovary (CHO) cells of the Lec9 recessive complementatio n group display a distinctive profile of resistance to a variety of to xic lectins. In addition, they accumulate cis-alpha-unsaturated polypr enol and use mainly polyprenol rather than dolichol to synthesize the glycosylated lipids used in asparagine-linked glycosylation of protein s. The primary defect in these cells is thought to result from a defic iency in polyprenol reductase activity. Three new mutants were isolate d and determined to have qualitatively, although not quantitatively, s imilar lectin resistance profiles to Lec9 cells. Two of these mutants (Abr(R) and Ric(R)) also contained polyprenol rather than dolichol. Th e lectin resistance profile of an independent mutant which accumulates polyprenol, F2A8, was also found to be qualitatively similar to the L ec9 pattern. The relationship among these mutants was analysed in more detail by construction of cell-cell hybrids. Lectin resistance profil es of the hybrids demonstrated that Abr(R), Ric(R) and F2A8 fell into the Lec9 complementation group. Analysis of prenols in the hybrids als o showed that F2A8 was a member of the Lec9 group. Surprisingly, a sig nificant fraction of the prenols found in Lec9 X Parent hybrids was po lyprenol (up to 30% of the neutral fraction), whereas the prenols foun d in Parent X Parent hybrids were nearly exclusively dolichol (97% of the neutral lipid fraction). Therefore, reduction of polyprenol to dol ichol appears to be a rate-limiting step in the synthesis of dolichol since hybrids with differing numbers of wild-type alleles can be bioch emically distinguished.