Intracellular Localization of GDP-Fucose

Abstract

The intracellular site of synthesis of the fucose-rich polysaccharlde slHme secreted by corn roots was localzed by monitoring the distribution of tions of corn roots. Root tip sectons were chopped in the presence of 0.56 molar sucrose and 100 millmolar Tris (pH 7.0). After a brief centrifugation, the homogenate was appled to a Sepharose 4B column (1.5 x 30 cm). The turbid, particulate portion of the superatant fraction eluted at the void volume. Ninety per cent of the enzyme activity was found in the pooled particulate fractions. The particulate fraction was purified on linear sucrose gradients. Gradient fractions were characterized by buoyant density, 280 nanometer absorbance, electron microscope observation, and distributions 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 data suggest that the synthesis of corn root slime begins in the endoplasmic reticulum and is completed in the dictyosomes prior to secretion; however, the possibility that more than one fucosyl transferase exists in corn roots is discussed.The process of slinie secretion by the peripheral root cap cells of corn was first studied using the electron microscope by Mollenhauer et al. (14). They described large vesicles produced by hypertrophied portions of the dictyosome cisternae that contained electron-dense material that was eventually secreted to the outside of the cell via an exocytotic mechanism. Their observations were verified and expanded by subsequent workers. The slime from corn was characterized as a high mol wt heteropolysaccharide rich in fucose and galactose (9,17). Secretion and synthesis of slime were studied with high resolution autoradiography (16,19) and by localization of fucose-containing polymers in subcellular fractions from roots incubated in [14CJglucose (2)(3)(4) or [3Hlfucose (20).These studies indicate that dictyosomes are involved in the secretion and probably the synthesis of corn root slime.To determine intracellular sites of biosynthesis of the fucoserich slime, organelle fractions were assayed for GDP-fucose:polysaccharide fucosyl transferase activity. This paper describes the organelle fractionation procedure, the results offucosyl transferase localization, and the conclusions that can be drawn from these experiments. MATERIALS AND METHODSPlant Material. Corn seeds (Zea mays cv. SX-17A, a gift of