DISTINCT COMPARTMENTALIZATION OF TGN46 AND BETA-1,4-GALACTOSYLTRANSFERASE IN HELA-CELLS

The Golgi proteins, TGN46 and GalT, were characterized in human HeLa c ells using specific polyclonal and monoclonal antibodies, A bacteriall y expressed soluble recombinant TGN46 protein was used to raise rabbit polyclonal antibodies and used to probe HeLa cell extracts. Human TGN 46 had an apparent molecular mass of 100 to 120 kDa which reflects ext ensive glycosylation. Epifluorescence light microscopy indicated subst antial colocalization of TGN46 and GalT. However, confocal laser micro scopy and three-dimensional reconstruction of double-labeled HeLa cell s revealed large areas of colocalization but also specific differences in the distribution of these two proteins within the Golgi apparatus. Importantly, quantitative immunoelectron microscopy showed that there was little overlap between the distribution of GalT and TGN46. Approx imately 75% of GalT was in the Golgi stack, whereas 80% of TGN46 was d etected in tubules. Distinct GalT-positive regions within the Golgi ci sternal stack were not labeled for TGN46.