Biosynthèse de Mannosyl‐Phosphoryl‐Dolichol dans la Membrane Externe des Mitochondires

Biosynthesis of Dolichol Phosphate Mannose in the Mitochondrial Outer Membrane Previous studies have shown that mannose incorporation into mitochondrial glycoproteins is catalysed by a mannosyl‐transferase, which is located in the mitochondrial inner membrane. It seems important to study the presence of mannosyl‐transferases which are able to catalyse the transfer of mannose into other acceptors, such as polyprenic intermediates. Mouse liver mitochondria catalyse the incorporation of mannose from GDP‐[ 14 C]mannose into three products: most of the radioactivity is in a product (A) soluble in chloroform/methanol (2/1, by vol.); mannose is also incorporated into product (B) soluble in chloroform/methanol/water (10/10/3, by vol.), and into a product which is insoluble in organic solvents and trichloroacetic acid and which is a glycoprotein. We have tried to localize in mitochondria the mannosyl‐transferases implicated in these reactions. Mitochondrial outer membranes were prepared by swelling purified mitochondria in phosphate buffer, and were purified on a discontinuous sucrose gradient. The purified outer membranes catalyse the incorporation of mannose from GDP‐[ 14 C]mannose in very large quantities into product A and in small quantities into product B. Different procedures (purification on silicic acid and DEAE‐cellulose, hydrolytic procedures and chromatographic properties) demonstrated the identity of product A with authentic dolichol phosphate mannose. Product B is lipid‐linked oligosaccharides. The biosynthesis of dolichol phosphate mannose depends on Mn 2+ . When ethyleneglycol bis(2‐aminoethyl) N,N′ ‐tetracetic‐acid (EGTA) is added to the mixture, the reaction is inhibited. When EGTA is added to a mixture containing preformed endogenous dolichol phosphate [ 14 C]mannose, the biosynthesis of the product stops, and the product is hydrolysed, with a concomitant apparition of lipid‐linked‐oligosaccharides. These results and the kinetics of the reactions suggest that dolichol phosphate mannose might be the mannosyl donor for the lipid‐linked‐oligosaccharides. The synthesis of dolichol phosphate mannose is reversed by the addition of GDP. The role of these products as intermediates in mitochondrial glycoprotein biosynthesis is discussed.