CLONED BETA-1,4N-ACETYLGALACTOSAMINYLTRANSFERASE - SUBCELLULAR-LOCALIZATION AND FORMATION OF DISULFIDE-BONDED SPECIES

Cloned human beta 1,4N-acetylgalactosaminyltransferase (GalNAcT) catal yses the synthesis of the glycosphingolipids GM2, GD2, and gangliotrio sylceramide. To determine the subcellular location of this enzyme and whether it exists in intermolecular disulfide bonded species, we stabl y transfected Chinese hamster ovary (CHO) cells with three myc epitope -tagged forms of the GalNAcT gene: the native enzyme; the lumenal doma in of GalNAcT fused to the cytoplasmic and transmembrane domains of N- acetylglucosaminyltransferase I (GNT); and the transmembrane and lumen al domains of GalNAcT fused to the cytoplasmic domain of the Iip33 for m of human invariant chain in order to retain the enzyme in the endopl asmic reticulum (ER). Immunoelectron microscopic analysis with anti-my c revealed that GalNAcT/myc was present throughout the Golgi stack, th e GNT/GalNAcT/myc form was restricted primarily to the medial Golgi ci sternae, and the Iip33/GalNAcT/myc form was restricted to the ER. Cell s transfected with each of the three constructs contained high levels of GM2 synthase activity in vitro, but only the GalNAcT/myc form and t he GNT/GalNAcT/myc forms were able to synthesize the GM2 product in vi vo. The enzyme produced by all three constructs was present in the tra nsfected cells in a disulfide bonded form having a molecular size cons istent with that of a homodimer or higher aggregate.