Intracellular Localization of GDP-Fucose

Abstract

The intracellular site of synthesis of the fucose-rich polysaccharide slime secreted by corn roots was localized by monitoring the distribution of GDP-fucose:polysaccharide fucosyl transferase activity in subcellular fractions of corn roots. Root tip sections were chopped in the presence of 0.56 M sucrose and 100 mM Tris (pH 7.0). After a brief centrifugation, the homogenate was applied to a Sepharose 4B column (1.5 .times. 30 cm). The turbid, particulate portion of the supernatant fraction eluted at the void volume. Of the enzyme activity, 90% was found in the pooled particulate fractions. The particulate fraction was purified on linear sucrose gradients. Gradient fractions were characterized by buoyant density, 280 nm absorbance, EM observation, and distribution of NADH-cytochrome c oxidoreductase and fucosyl transferase activities. Two peaks of fucosyl transferase activity were observed: one in fractions rich in smooth endoplasmic reticulum, and another in fractions rich in dictyosome cisternae and dictyosome vesicles. The synthesis of corn root slime apparently begins in the endoplasmic reticulum and is completed in the dictyosomes prior to secretion; however, the possibility that more than 1 fucosyl transferase exists in corn roots is discussed.