The role of dolicholmonophosphate in glycoprotein biosynthesis in Saccharomyces cerevisiae.

Abstract

1. In a crude particulate fraction from yeast mannose is transferred from GDP‐mannose to an endogenous “lipid” fraction, the monophosphates of dolichol‐14 to dolichol‐18. The same membrane fraction also catalyzes the mannosylation of glycoproteins. More than 80% of the total radioactivity in the glycoprotein fraction obtained from GDP‐[14C]mannose is released by β‐elimination. The small‐sized radioactive products of β‐elimination are mannose, mannobiose and mannotriose.2. Ageing of the particulate fraction leads to a drastic loss of mannosyl transfer activity from GDP‐[14C]mannose to dolichol monophosphate. To the same extent the amount of [14C]mannose obtained after β‐elimination of the glycoproteins decreases. The amount of radioactive mannobiose and mannotriose is, however, much less affected.3. With decreasing GDP‐mannose concentrations the amount of [14C]mannose obtained after β‐elimination increases as compared to the amount of radioactive mannobiose and mannotriose. The Km‐value for the incorporation of mannosyl groups directly linked to serine and/or threonine, therefore, is lower than the Km‐value for the transfer of subsequent mannosyl residues.4. The mannosylation of dolichol monophosphate from GDP‐[14C]mannose proceeds in the presence of Mg2+ and Mn2+ ions as well. When solely Mg2+ is present in the incubation mixture, β‐elimination of the glycoprotein produced yields almost exclusively mannose, and it is only in the presence of Mn2+ that the normal β‐elimination pattern is observed.5. Incubation of the particulate fraction with dolichol‐monophosphate‐[14C]mannose results in the formation of radioactive glycoprotein; again most of the radioactivity is released by β‐elimination but only [14C]mannose is obtained as product. When dolichol‐monophosphate [14C]mannose is incubated together with non‐radioactive GDP‐mannose, β‐elimination yields radioactive mannose, mannobiose and mannotriose. Mannobiose is found to be labelled in the reducing end only.6. The results are consistent with the assumption that dolichol monophosphate is involved in the mannosylation of a specific position in yeast glycoproteins, i.e. in the formation of mannosyl linkages to serine and/or threonine. The subsequent mannosylation proceeds directly from GDP‐mannose in a reaction obligatorily requiring Mn2+.7. After β‐elimination of the methanol‐insoluble material mannosylated in the presence of dolichol‐monophosphate [14C] mannose the residual insoluble radioactivity is to a large extent transformed into dialyzable material by pronase. Mannose, therefore, is transferred from dolichol‐monophosphate mannose also to glycoprotein positions not involving OH‐groups of serine or threonine.