Beta 1,4-galactosyltransferase: a short NH2-terminal fragment that includes the cytoplasmic and transmembrane domain is sufficient for Golgi retention.

Abstract

Beta 1,4-galactosyltransferase (beta 1,4-GT) is a Golgi-resident, type II membrane-bound glycoprotein that functions in the coordinate biosynthesis of complex oligosaccharides. Additionally, beta 1,4-GT has been localized to the cell surface of a variety of cell types and tissues where it is proposed to function in intercellular recognition and/or adhesion. Thus beta 1,4-GT is an appropriate molecule to be used in analyzing the molecular basis for retention of a membrane-bound enzyme in the Golgi complex and its subsequent or alternative transport to the cell surface. Previously we have shown that the gene for bovine and murine beta 1,4-GT is unusual in that it specifies a short (SGT) and long (LGT) form of the enzyme (Russo, R. N., Shaper, N. L., and Shaper, J. H. (1990) J. Biol. Chem. 265, 3324-3331). The only difference between the two related forms is in the primary structure of the cytoplasmic domains, where LGT has an NH2-terminal extension of 13 amino acids. In this study, we have tested the hypothesis that LGT and SGT are differentially retained in the Golgi or directed to the cell surface. LGT, SGT or chimeric proteins, containing the NH2-terminal cytoplasmic and transmembrane domain of SGT and LGT fused to the cytoplasmic protein pyruvate kinase, were each stably expressed in Chinese hamster ovary cells. Proteins expressed from each construct were localized by immunofluorescence staining exclusively to a perinuclear region, identified as the Golgi by co-localization with wheat germ agglutinin. Furthermore, the subcellular distribution of both SGT and LGT was restricted to the trans-Golgi compartment as assessed by EM immunoelectron microscopy. These data suggest that both forms of beta 1,4-GT are resident trans-Golgi proteins and that an NH2-terminal segment containing the cytoplasmic and transmembrane domains of SGT (39 amino acids) or LGT (52 amino acids) is sufficient for Golgi retention.