Subcellular localization and topology of β(1→4)galactosyltransferase that elongates β(1→4)galactan side chains in rhamnogalacturonan I in potato

The subcellular localization and topology of rhamnogalacturonan I (RG-I) beta(1-->4)galactosyltransferase(s) (beta[1-->4]GalTs) from potato (Solanum tuberosum L.) were investigated. Using two-step discontinuous sucrose step gradients, galactosyltransferase (GalT) activity that synthesized 70%-methanol-insoluble products from UDP-[C-14]Gal was detected in both the 0.5 M sucrose fraction and the 0.25/1.1 M sucrose interface. The former fraction contained mainly soluble proteins and the latter was enriched in Golgi vesicles that contained most of the UDPase activity, a Golgi marker. By gel-filtration analysis, products of 180-2,000 Da were found in the soluble fraction, whereas in the Golgi-enriched fraction the products were larger than 80 kDa and could be digested with rhamnogalacturonan lyase and beta(1,4)endogalactanase to yield smaller rhamnogalacturonan oligomers, galactobiose and galactose. The endogalactanase requires beta(1-->4)galactans with at least three galactosyl residues for cleavage, indicating that the enzyme(s) present in the 0.25/1.1 M Suc interface transferred one or more galactosyl residues to pre-existing beta(1-->4)galactans producing RG-I side chains in total longer than a trimer. Thus, the beta(1-->4)GalT activity that elongates beta(1-->4)-linked galactan on RG-I was located in the Golgi apparatus. This beta(1-->4)GalT activity was not reduced after treatment of the Golgi vesicles with proteinase, but approximately 75% of the activity was lost after treatment with proteinase in the presence of Triton X-100. In addition, the beta(1-->4)GalT activity was recovered in the detergent phase after treatment of Golgi vesicles with Triton X-114. Taken together, these observations supported the view that the RG-I beta(1-->4)GalT that elongates beta(1-->4)galactan was mainly located in the Golgi apparatus and integrated into the membrane with its catalytic site facing the lumen.