Transport and topology of galactosyltransferase in endomembranes of HeLa cells.

Abstract

HeLa [human cervical carcinoma] cell membranes were studied for the distribution and orientation of the Golgi marker enzyme UDP-galactose:.beta.-D-N-acetylglucosamine .beta., 1-4 transferase (GT). Short pulse labeling in the presence of [35S]methionine resulted in 2 precursor species (MW = 44,000 and 47,000), present in a microsomal fraction with a density of 1.18 g/ml in sucrose, presumably derived from the rough endoplasmic reticulum. Processing of the N-linked oligosaccharide(s) occurred only after the precursor molecules migrated to lighter density fractions, presumably derived from the Golgi complex. The mature GT molecules (MW = 54,000) contain O-linked oligosaccharides as shown by .beta.-elimination of metabolically incorporated [3H]galactose. The O-glycosylation occurred mainly in the light density fractions. The topology of GT was studied on membrane fractions after labeling with [35S]methionine as well as immunocytochemically on ultrathin cryosections at the EM level. Both the antigenic determinants of GT and polypeptide chain are present intramembraneously and at the luminal side of the membranes of the Golgi complex and rough endoplasmic reticulum.