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 /?-elimination. The small-sized radioactive products of /?-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 /?-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 /&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. 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 /?-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 dolicholmonophosphate mannose also to glycoprotein positions not involving OH-groups of serine or threonine.Glycosylated lipids play a role in the biosynthesis of glycoproteins in animals [l -51, fungi [6,7] and plants [S]. The lipophilic component in all these cases is probably dolichol monophosphate [2,4,9,10].In Succhuvomyces cerevisiue the lipophilic mannosyl acceptor has been shown by mass spectroscopy to consist of the monophosphates of the whole family of dolichols with 14 to 18 isoprene units [lo]. From -~ his 60th birthday. mannosylated dolichol monophosphates the mannosyl group is transferred by yeast membranes to glycoproteins. Preliminary evidence has been presented [7] threonine, only the mannosyl residue directly linked to Dedicated to Professor Dr 0. Hoffmann-Ostenhof on Abbreviations. Dolichol-P, dolichol monophosphate; dolichol-P-Man, dolichol-monophosphate-mannose ; GDPthat within oligomannose chains linked to serine/ Man, guanosine-diphosphate-mannose.Eur. J. Biochem. 46 (1974)

Solubilization and Characterization of the Initial Enzymes of the Dolichol Pathway from Yeast

“…After thorough mixing phases were separated by centrifugation. The upper phase was discarded and the lower phase containing radioactive glycolipids was washed as described in [23].…”

“…The reaction mixture was incubated for 25 min and the reaction was stopped by the addition of 2 ml of chloroformlmethanol (3 : 2, by vol.). After removing the denatured protein by centrifugation, the lipid phase was partitioned, washed [23] and the radioactive glycolipids were separated by thin-layer chromatography. Radioactive bands were located and the bands corresponding to Dol-PPGlcNAc and to Dol-PP-(GlcNAc)2 (arising from newly synthesized Dol-PP-GlcNAc) were scraped off the plates and counted.…”

Formation of Dolichol Monophosphate 2‐Deoxy‐d‐glucose and Its Interference with the Glycosylation of Mannoproteins in Yeast

“…were incubated in a total volume of 0.05 ml at 23 "C and terminated at the indicated time by the addition of 1 nil chloroforni/methanol (3/2, v/v). The lipid and the polymer fraction were extracted, washed and counted as described previously [22].…”

“…Synthesis involves the transfer of mannosyl groups from guanosine diphosphate mannose (GDP-Man) to the lipid intermediate followed by transfer of the lipid-bound hexose to endogenous acceptors. Once the first mannose is attached to the protein, subsequent mannoses are incorporated directly from the sugar nucleotide without the participation of dolichol [22,23].Recently methods have been worked out for the preparation of labelled or non-labelled guanosine diphosphate 2-deoxy-~-glucose (GDP-dGlc) and uridine diphosphate 2-deoxy-~-glucose (UDP-dGlc) [24]. This made it possible to investigate in vitro the transfer of 2-deoxy-~-g!ucose from these sugar nucleotides to yeast mannoproteins.…”

“…Synthesis involves the transfer of mannosyl groups from guanosine diphosphate mannose (GDP-Man) to the lipid intermediate followed by transfer of the lipid-bound hexose to endogenous acceptors. Once the first mannose is attached to the protein, subsequent mannoses are incorporated directly from the sugar nucleotide without the participation of dolichol [22,23].…”