The signal for Golgi retention of bovine beta 1,4-galactosyltransferase is in the transmembrane domain.

作者：Rohan David Teasdale, G D'Agostaro, Paul A. Gleeson · 发表于：Journal of Biological Chemistry · 年份：1992 · DOI：10.1016/s0021-9258(19)50633-0 · 被引用次数：151 · 研究领域：Glycosylation and Glycoproteins Research、RNA and protein synthesis mechanisms、T-cell and B-cell Immunology

The expression and localization of bovine beta 1,4-galactosyltransferase (Gal T) has been studied in mammalian cells transfected with Gal T cDNA constructs, and the role of the amino-terminal domains of Gal T in Golgi localization examined. Here we demonstrate that the transmembrane (signal/anchor) domain of bovine Gal T contains a positive Golgi retention signal. Bovine Gal T was characterized in transfected cells with anti-bovine Gal T antibodies, affinity-purified from a rabbit antiserum using a bacterial recombinant fusion protein. These affinity-purified antibodies recognized native bovine Gal T and showed minimum cross-reactivity with Gal T from non-bovine sources. Bovine Gal T cDNA was expressed, as active enzyme, transiently in COS-1 cells and stably in murine L cells, and the product was shown to be localized to the Golgi complex by immunofluorescence using the polypeptide-specific antibodies. A low level of surface bovine Gal T was also detected in the transfected L cells by flow cytometry. The removal of 18 of the 24 amino acids from the cytoplasmic domain of bovine Gal T did not alter the Golgi localization of the product transiently expressed in COS-1 cells or stably expressed in L cells. Both the full-length bovine Gal T and the cytoplasmic domain deletion mutant were N-glycosylated in the transfected L cells, indicating both proteins have the correct N(in)/C(out) membrane orientation. Deletion of both the cytoplasmic and signal/anchor domains of bovine Gal T an...