The catalytic site of the pectin biosynthetic enzyme alpha-1,4-galacturonosyltransferase is located in the lumen of the Golgi.
Alpha-1,4-galacturonosyltransferase (GalAT) is an enzyme required for the biosynthesis of the plant cell wall pectic polysaccharide homogalacturonan (HGA). GalAT activity in homogenates from pea (Pisum sativum L. var. Alaska) stem internodes co-localized in linear and discontinuous sucrose gradients with latent UDPase activity, an enzyme marker specific for Golgi membranes. GalAT activity was separated from antimycin A-insensitive NADH:cytochrome c reductase and cytochrome c oxidase activities, enzyme markers for the endoplasmic reticulum and the mitochondria, respectively. GalAT and latent UDPase activities were separated from the majority (80%) of callose synthase activity, a marker for the plasma membrane, suggesting that little or no GalAT is present in the plasma membrane. GalAT activities in proteinase K-treated and untreated Golgi vesicles were similar, whereas no GalAT activity was detected after treating Golgi vesicles with proteinase K in the presence of Triton X-100. These results demonstrate that the catalytic site of GalAT resides within the lumen of the Golgi. The products generated by Golgi-localized GalAT were converted by endopolygalacturonase treatment to mono- and di-galacturonic acid, thereby showing that GalAT synthesizes 1-->4-linked alpha-D-galacturonan. Our data provide the first enzymatic evidence that a glycosyltransferase involved in HGA synthesis is present in the Golgi apparatus. Together with prior results of in vivo labeling and immunocytochemical studies, these results show that pectin biosynthesis occurs in the Golgi. A model for the biosynthesis of the pectic polysaccharide HGA is proposed.
Sterling
J D
JD
Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, 220 Riverbend Road, Athens, Georgia 30602-4712, USA.
Quigley
H F
HF
Orellana
A
A
Mohnen
D
D
D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology; D014453:Glucuronosyltransferase; D016695:Glycosyltransferases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology; D008958:Models, Molecular; D018532:Pisum sativum / Q000166:cytology / Q000201:enzymology*; D010368:Pectins / Q000096:biosynthesis* / Q000378:metabolism; D010940:Plant Proteins; D011755:Pyrophosphatases / Q000378:metabolism; D013347:Subcellular Fractions; D013395:Sucrose; D014574:Uronic Acids / Q000378:metabolism
D010940:Plant Proteins; D014574:Uronic Acids; D013395:Sucrose; D010368:Pectins; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C438373:alpha-1,4-galacturonosyltransferase; D011755:Pyrophosphatases; C023273:uridine diphosphatase; C003181:polygalacturonic acid