BETA-1,4 N-ACETYLGALACTOSAMINYLTRANSFERASE (GM2 GD2/GA2 SYNTHASE) FORMS HOMODIMERS IN THE ENDOPLASMIC-RETICULUM - A STRATEGY TO TEST FOR DIMERIZATION OF GOLGI MEMBRANE-PROTEINS/

Many Golgi membrane-bound glycosyltransferases exist as intermolecular disulfide bonded species, some of which have been demonstrated to be homodimers, Evidence for homodimer formation has come primarily from r adiation inactivation experiments, We utilized an alternative strategy to test for homodimer formation of the cloned beta 1,4 N-acetylgalact osaminyltransferase (GalNAcT) responsible for synthesis of the glycosp hingolipids GM2, GD2, and GA2, We stably transfected CHO cells with my c epitope-tagged GalNAcT, which localizes primarily to the Golgi, and a hemagglutinin (HA) epitope-tagged GalNAcT fusion protein in which th e cytoplasmic domain of GalNAcT was replaced by an ER retention signal , We then sought evidence for dimer formation between the two forms of GalNAcT, Immunoprecipitation with anti-myc or anti-HA co-immunoprecip itated the HA-tagged form or the myc-tagged form, respectively, provid ing evidence for the physical association of the two forms of GalNAcT, As a result of this association, GalNAcT/myc increased in the ER as d emonstrated by Western blots and immunofluorescence, The rapid formati on of dimers provided further evidence for dimer formation occurring i n the ER. In summary, these results demonstrate that GalNAcT forms hom odimers as a result of intermolecular disulfide bond formation in the ER, Furthermore, this ER motif strategy is potentially useful for demo nstrating homodimer formation of other Golgi enzymes.