Expression of soluble active human beta 1,4 galactosyltransferase in Saccharomyces cerevisiae.

Sequences coding for the cytoplasmic and transmembrane domains were removed from the cDNA of the human Golgi resident membrane protein beta 1,4 galactosyltransferase (gal-T). The remaining sequences coding for the stem and catalytical domains of this glycosyltransferase were fused to sequences coding for the yeast invertase signal sequence. The hybrid was inserted together with a constitutive yeast promoter and a terminator into a E. coli/yeast shuttle vector. Saccharomyces cerevisiae strain BT150 transformed with this new expression vector expressed enzymically active soluble enzyme, whereas no activity was detectable in mock-transformed yeasts. The enzyme product was identified by HPLC analysis and shown to correspond to the expected product N-acetyllactosamine.

Kin recognition between medial Golgi enzymes in HeLa cells.

The medial Golgi enzymes, N-acetylglucosaminyltransferase I (NAGT I) and mannosidase II (Mann II), and the trans Golgi enzyme, beta-1,4-galactosyltransferase (GalT) were each retained in the endoplasmic reticulum (ER) by grafting on the cytoplasmic tail of the p33 invariant chain. Transient and stable expression of p33/NAGT I in HeLa cells caused relocation of endogenous Mann II to the ER and transient expression of p33/Mann II had a similar effect on endogenous NAGT I. Neither of these endogenous medial enzymes were affected by transient expression of p33/GalT. These data provide strong evidence for kin recognition between medial Golgi enzymes and suggest a role for them in the organization of the Golgi stack.

Purification and characterization of recombinant human beta 1-4 galactosyltransferase expressed in Saccharomyces cerevisiae.

A protease-defective strain of Saccharomyces cerevisiae (BT 150) was used to express full-length cDNA of HeLa cell beta-D-N-acetylglucosaminide-beta-1,4-galactosyltransferase (gal-T). To ascertain import of the recombinant gal-T into the secretory pathway of yeast cells, metabolically labeled enzyme was immunoprecipitated from extracts of yeast transformants, analysed by SDS/PAGE/fluorography and tested for sensitivity to treatment with endoglycosidase-H. Untreated recombinant gal-T had an apparent molecular mass of 48 kDa, which was reduced to 47 kDa after treatment, indicating that the recombinant enzyme was N-glycosylated and, therefore, competent for translocation across the membranes of the endoplasmic reticulum. Using specific gal-T assays employing N-acetylglucosamine or glucose in combination with alpha-lactalbumin as exogenous acceptor substrates, recombinant gal-T enzyme activity could readily be detected in crude homogenates. Analysis of the disaccharide products by 1H-NMR spectroscopy demonstrated that only beta-1-4 linkages were formed by the recombinant gal-T. The recombinant gal-T was detergent solubilized and subsequently purified by affinity chromatography on N-acetylglucosamine-derivatized Sepharose followed by alpha-lactalbumin-Sepharose. The purified enzyme preparation had a specific activity comparable to that of the soluble gal-T isolated from human milk. Furthermore, kinetic parameters determined for both acceptor and donor substrates of both enzymes differed only slightly. This work shows that yeast provides an appropriate host system for the heterologous expression of mammalian glycosyltransferases.

Saccharomyces cerevisiae a- and alpha-agglutinin: characterization of their molecular interaction.

An O-glycosylated protein of approximately 18 kDa responsible for mating type specific agglutination has been isolated from Saccharomyces cerevisiae a cells, purified to homogeneity and via peptide sequences the gene was cloned by PCR. An open reading frame codes for a protein of 69 amino acids. A minimum of five serine and five threonine residues of the mature protein are glycosylated. alpha-Agglutinin is a highly N-glycosylated protein of approximately 250 kDa. Both purified agglutinins form a specific 1:1 complex in vitro. Pretreatment of alpha-agglutinin, but not of alpha-agglutinin, with diethylpyrocarbonate (DEPC) prevents formation of the complex; treatment of alpha-agglutinin in the presence of alpha-agglutinin protects the former from DEPC inactivation. By carboxy terminal shortening of the alpha-agglutinin gene and by replacing three of its eight histidyl residues by arginine, the active region of alpha-agglutinin for interaction with alpha-agglutinin has been defined. Neither the N- nor the O-linked saccharides of the two agglutinins seem to be essential for their interaction.

Immunocytochemical localization of the Golgi apparatus using protein-specific antibodies to galactosyltransferase.

Polyclonal antisera to human milk galactosyltransferase have been widely used as immunocytochemical reagent to visualize the Golgi apparatus. Immunochemical analysis revealed partial specificity of these antisera to glycan epitopes expressed on galactosyltransferase and other gene products (R. A. Childs et al., Biochem. J. 238, 605-611 (1986)). Since glycan-specific reagents such as lectins are known to label the Golgi apparatus by virtue of their exclusive glycan specificity, Golgi localization of galactosyltransferase by use of these polyclonal antisera remained elusive. In order to demonstrate authentic Golgi localization of the galactosyltransferase peptide, we expressed the enzyme in Escherichia coli as non-glycosylated beta-galactosidase-fusion proteins and used them as affinity matrix to protein-epitope purified polyclonal antibodies from antisera to the milk enzyme, and to induce protein-specific antisera by injecting them into rabbits, respectively. A short fusion protein comprising most of the substrate binding sites and a protein which included the stem region of galactosyltransferase were expressed. The short fusion protein was poorly immunogenic whereas the long fusion protein induced a high-titer antiserum. Protein-epitope purified antibodies derived from polyclonal antisera to the milk enzyme as well as antibodies to the long fusion protein cross-reacted with milk galactosyltransferase and with fusion protein as demonstrated by immunoblotting and enzyme-linked immunosorbent assay (ELISA) and produced typical Golgi morphologies in both HeLa and CaCo2 cells when used for immunocytochemical localization of galactosyltransferase. We conclude that previous immunolocalization studies have correctly been interpreted as Golgi localization of the galactosyltransferase protein and that antigenicity to the protein moiety is confirmed to the N-terminal third of the enzyme.

Cloning of the glutamine:fructose-6-phosphate amidotransferase gene from yeast. Pheromonal regulation of its transcription.

The activity of the amino sugar-synthesizing enzyme L-glutamine:D-fructose-6-phosphate aminotransferase (EC 2.6.1.16) in haploid a cells of Saccharomyces cerevisiae increases 1.7-fold after alpha factor addition. The gene (the gene should be called GFA1 for glutamine:fructose-6-phosphate amidotransferase) for the enzyme has been cloned by complementing the gcn1 mutation (Whelan, W. L., and Ballou, C. E. (1975) J. Bacteriol. 125, 1545-1557). Its expression is increased 2-3 times within 15 min when the mating pheromone is added. The gene codes for a protein of 716 amino acids in length. It is highly homologous (64%) to the corresponding gene of Escherichia coli, except for a sequence coding for 83 amino acids (numbers 204-286), which is lacking in E. coli. The amino-terminal region of the coding sequence also shows a high degree of homology to the corresponding sequence of the E. coli and S. cerevisiae L-glutamine:phosphoribosylpyrophosphate amidotransferase. In the promotor region of the S. cerevisiae L-glutamine:D-fructose-6-phosphate amidotransferase gene the heptanucleotide "TGAAACA," shown to be required for pheromone control of transcription (Kronstad, J. W., Holly, J. A., and MacKay, V. L. (1987) Cell 50, 369-377), is present six times.