A galactosyltransferase from the fission yeast Schizosaccharomyces pombe.

Abstract

Abstract. A membrane-associated galactosyltransferase has been purified to homogeneity from the fission yeast, Schizosaccharomyces pombe. The enzyme has a molecular weight of 61,000 and is capable of transferring galactose from UDP-galactose (UDP-Gal) to a variety of mannose-based acceptors to form an or-l,2 galactosyl mannoside linkage. Immunofluorescence localization of the protein is consistent with the presence of the enzyme in the Golgi apparatus of S. pombe. This, together with the presence of terminal, or-linked galactose on the N-linked oligosaccharides of S.pombe secretory proteins, suggests that the galactosyltransferase is an enzyme involved in the processing of glycoproteins transported through the Golgi apparatus in fission yeast. Is recent years, two complementary approaches have been taken to identify components involved in protein transport from the endoplasmic reticulum, through the Golgi apparatus, to the cell surface. The genetic approach, exemplified by Schekman and his co-workers, has identified several classes of gene products involved at different stages of protein secretion in Saccharomyces cerevisiae, from initial translocation and glycosylation to final delivery to the cell surface (for review, see Schekman, 1985). The use of cell-free systems, initiated by Rothman, has increased our understanding of the biochemical mechanisms involved in vesicular traffic, primarily through the Golgi apparatus of mammalian cells (for review see Pfeffer and Rothman, 1987). The demonstration that yeast contains all the cytosolic components required for mammalian intra-Golgi transport (Dunphy et al., 1986) and the recent finding that the sec18 gene product of S. cerevisiae is functionally equivalent to one of the purified components of the Golgi transport system (N-ethylmaleimide-sensitive factor) (Wilson et al., 1989) demonstrate the efficacy of a combined genetic and biochemical approach to the questions of vesicular trafficking.The primary method for tracing the progress of a protein through the transport pathway is to follow the processing of N-linked oligosaccharides. In the endoplasmic reticulum of mammalian cells, N-linked Glc3MangGlcNAc2 oligosaccharides are first trimmed to a MansGlcNAc2 core, and then, as the glycoprotein is transported through the Golgi apparatus, processed by a series of mannosidases and sugar transferases to a final complex structure containing mannose, additional GIcNAc, galactose (Gal) t and sialic acid (for review, see Kornfeld and Kornfeld, 1985). Elucidation of the pathway of N-linked oligosaccharide processing and immunolocalization of some of the enzymes involved have demonstrated 1. Abbreviations used in this paper: Gal, galactose; Glc, glucose; GIcNAc, N-acetyl-glucosarnine; Man, mannose. that the Golgi apparatus in mammalian cells consists of distinct biochemical compartments through which secretory, lysosomal, and plasma membrane proteins pass in a vectorial fashion (for review, see Dunphy and Rothman, 1985). The relatively simple processing of N-linked oligosac...

S. pombe btn1, the orthologue of the Batten disease geneCLN3, is required for vacuole protein sorting of Cpy1p and Golgi exit of Vps10p

“…The morphology of S. pombe Golgi resemble those of mammalian cells (Chappell and Warren, 1989). In wild-type cells, the multiple Golgi were easily recognisable as electron-dense ribbons or flattened cisternae of length ~0.5-0.8 μm, often with a globular (or beaded) appearance (Fig.…”

Identification of Novel α1,3-Galactosyltransferase and Elimination of α-Galactose-containing Glycans by Disruption of Multiple α-Galactosyltransferase Genes in Schizosaccharomyces pombe

“…Preparation of Solubilized Microsomal Proteins-Solubilized enzymes from various strains were prepared as described (39). Briefly, cells were grown in minimal medium at 30°C, harvested in early stationary phase, washed with ice-cold water, and suspended in 50 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 0.25 M sucrose, and Complete EDTA-free protease inhibitor mixture (Roche Applied Science).…”

“…Galactosyltransferase Assay-The galactosyltransferase assay was carried out as described (39). The reaction mixture in a total volume of 30 l contained 60 g of solubilized enzyme, 100 mM HEPES-NaOH, pH 7.0, 0.1 M sucrose, 1 mM MnCl 2 , 1 mM UDP-Gal, 10 pmol PA-oligosaccharides, and was incubated at 37°C for 12 h. The reaction was terminated by boiling for 5 min.…”

Galactose-Specific Recognition System in the Fission Yeast <i>Schizosaccharomyces pombe</i>

“…Galactosylation consists of three steps including biosynthesis of UDP-Gal in the cytosol (45,46), transport of UDP-Gal from the cytosol into the lumen of the Golgi (47)(48)(49), and transfer of a Gal residue from UDP-Gal by Golgi-localized galactosyltransferases (28)(29)(30). In S. pombe, seven α1,2-galactosyltransferase-related genes have been identified.…”

“…In S. pombe, seven α1,2-galactosyltransferase-related genes have been identified. Gma12p was purified first as a major α1,2-galactosyltransferase prior to cloning of the gene (28,29). The gmh1 + -gmh6 + genes were identified as homologs of gma12 + (30,50).…”