Subcellular localization and topology of beta(1-->4)galactosyltransferase that elongates beta(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-[(14)C]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 180-2000 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.
Geshi
Naomi
N
Biotechnology Group, Danish Institute of Agricultural Sciences, Thorvaldsensvej 40. 8. 2., 1871, Frederiksberg C, Copenhagen, Denmark. n.geshi@dias.kvl.dk
Jørgensen
Bodil
B
Ulvskov
Peter
P
D015153:Blotting, Western; D003902:Detergents / Q000494:pharmacology; D019286:Endopeptidase K / Q000494:pharmacology; D005685:Galactans / Q000737:chemistry / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000201:enzymology*; D009237:N-Acetyllactosamine Synthase / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D010368:Pectins / Q000096:biosynthesis*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011755:Pyrophosphatases / Q000378:metabolism; D011198:Solanum tuberosum / Q000201:enzymology*; D013395:Sucrose / Q000378:metabolism
C411147:1,4-beta-galactan; D003902:Detergents; D005685:Galactans; C042491:rhamnogalacturonan I; D011092:Polyethylene Glycols; D013395:Sucrose; D010368:Pectins; D017830:Octoxynol; C010615:Nonidet P-40; D009237:N-Acetyllactosamine Synthase; D019286:Endopeptidase K; D011755:Pyrophosphatases; C023273:uridine diphosphatase