Abstract
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 and incorporation of a cleavable signal sequence resulted in a truncated soluble bovine Gal T that was rapidly secreted (within 1 h) from transfected COS-1 cells. Replacement of the signal/anchor domain of bovine Gal T with the signal/anchor domain of the human transferrin receptor resulted in the transport of the hybrid molecule to the cell surface of transfected COS-1 cells. Furthermore, a hybrid construct containing the signal/anchor domain of Gal T with ovalbumin was efficiently retained in the Golgi complex, whereas ovalbumin anchored to the membrane by the transferrin receptor signal/anchor was expressed at the cell surface of transfected COS-1 cells. Overall, these studies show that the hydrophobic, signal/anchor domain of Gal T is both necessary and sufficient for Golgi localization.
Highlights
The expression and localization of bovine @1,4-gal- The terminal carbohydratesequences of glycoproteins peractosyltransferase (Gal T)has been studied in mam- form a diverse range of biological roles,for example molecular malian cells transfected with Gal T cDNA constructs, recognition events such as targeting of lysosomal hydrolases and therole of the amino-terminaldomains of Gal T in (Kornfeld, 1987) and cell adhesion
Bovine Gal T found in glycoproteins and glycolipids, by catalyzing the cDNA was expressed, as active enzyme, transiently in transfer of galactose, from UDP-Gal, to the terminal nonreCOS-1cells and stablyin murine L cells, and the prod- ducing GlcNAc residues of oligosaccharide chains (Beyer et uct was shown to be localized to theGolgi complexby al., 1981; Strous, 1986). immunofluorescenceusing the polypeptide-specifican- The cDNAs encoding Gal T have been isolated from human tibodies
Synthetic peptides corresponding to thecytoplasmic tail of bovine Gal T4and aninsoluble bacterial fusion protein encoding the 147 carboxyl-terminal amino acids from the luminal domain of bovine Gal T were used as immunogens
Summary
M , MluI; N, NaeI; P, PuuII; Ps,PstI; S, StuI. More than the one BsaJI site is present in the plasmid, and the site referred to in the text is indicated by the asterisk. 6-e, structure of plasmids containing full-length Gal T cDNA and deletion mutants of Gal T. Aliquots of 12 well slides, the medium overlaying the cells was replaced with cell extracts (all containing the same Gal T activity) were incubated fresh medium (50 pl) containing either 10 p M of nocodazole or 5 pg/ with either 40 p1 of affinity-purified antibody or preimmune serum, ml brefeldin A, and the monolayers were incubated for 45 min at diluted in PBS, for 60 min on ice. The immune complexes were . (2 X lo[6] cells) were washed twice with DMEM (serum-free) and labeling mix, Du Pont-New England Nuclear, Australia; specific preincubated with PBS containing 20 mg/ml bovine serum albumin activity of ~-[~'S]methionine-1, 100 Ci/mmol) and incubated at 37 "C (Fraction V, Sigma) for 30 min. Samples were diluted with SDS electrophoresis sample buffer, boiled for 2 min, and analyzed by SDS-PAGE andfluorography
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