Localization of an alpha 1,2 galactosyltransferase activity to the Golgi apparatus of Schizosaccharomyces pombe.

Abstract

We have cloned a gene encoding an alpha 1,2 galactosyltransferase activity from Schizosaccharomyces pombe. The open reading frame of the gene (gma12 for galactomannan, alpha 1,2), combined with the previous protein purification (Chappell and Warren, 1989), predicts an O-linked glycoprotein with type II transmembrane topology. By homologous gene disruption, we have demonstrated that the gma12 gene product (gma12p) is nonessential. The deletion strain (gma12-D10::ura4) has a significantly reduced level of galactosyltransferase activity relative to the parental strain, but both in situ lectin binding and in vitro biochemical assays demonstrate the presence of further galactosyltransferase activity in addition to gma12p. Although gma12p is not the only galactosyltransferase in S. pombe, it produces a unique carbohydrate structure on the surface of the yeast cells. We have generated a polyclonal antiserum against this carbohydrate epitope and shown that gma12p is capable of synthesizing the epitope both in vitro and in vivo. Electron microscopic localization of the gma12+ specific epitope in gma12+ cells revealed that gma12p synthesizes the carbohydrate structure in the Golgi apparatus, and subsequent intracellular transport distributes the epitope to later stages of the secretory pathway. The immunolocalization studies confirm the presence of one or more galactosyltransferase activities in the Golgi apparatus in fission yeast.

BED1, a gene encoding a galactosyltransferase homologue, is required for polarized growth and efficient bud emergence in Saccharomyces cerevisiae.

“…The homology between Bedl and gmal2p/cdsl 1/cdsl3 was particularly significant over 4 regions in the luminal portion of these proteins (~30% identity and 65 % similarity). The gmal2p protein has been shown to be an a-l,2-galactosyltransferase (Chappell et al, 1994). Several galactosyltransferase activities have been detected in S. pombe (Chappell et al, 1994;Ballou and Ballou, 1995) and the grna12 ÷ gene belongs to a large family of related genes in S. pombe (Chappell, T., personal communication).…”

“…The gmal2p protein has been shown to be an a-l,2-galactosyltransferase (Chappell et al, 1994). Several galactosyltransferase activities have been detected in S. pombe (Chappell et al, 1994;Ballou and Ballou, 1995) and the grna12 ÷ gene belongs to a large family of related genes in S. pombe (Chappell, T., personal communication). Although the structures of the two proteins are clearly similar, Bedl has a larger cytoplasmic domain.…”

The Golgi apparatus: insights from filamentous fungi

“…1C). The Golgi apparatus among yeasts has similar diversification in its organization (Glick 1996): dispersed cisternae are observed in Saccharomyces cerevisiae (Preuss et al 1992), while stacks of cisternae are present in Schizosaccharomyces pombe (Chappell et al 1994) and Pichia pastoris (Gould et al 1992). It has been proposed that existence of either one or the other Golgi organization reflects the underlying differences in the organization of the endoplasmic reticulum (ER) exit sites: In P. pastoris, where the Golgi forms stacks, ER exit sites are discrete, confined at specific ER locales, while in S. cerevisiae, where the Golgi is dispersed, vesicles bud throughout the ER network (Rossanese et al 1999).…”

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

“…In contrast, S. pombe O-linked oligosaccharides have one or two galactose residues that can be attached to the α1,2-linked mannose backbone, giving rise to a branched Gal 0−2 Man 1−3 -Ser (or -Thr) structure (27). A major galactosyltransferase in S. pombe has been purified (28) and the corresponding gene was cloned and identified as gma12 + (29). The fission-yeast genome contains seven galactosyltransferase gene homologues, including gma12 + and gmh1 + -gmh6 + (29,30).…”

“…A major galactosyltransferase in S. pombe has been purified (28) and the corresponding gene was cloned and identified as gma12 + (29). The fission-yeast genome contains seven galactosyltransferase gene homologues, including gma12 + and gmh1 + -gmh6 + (29,30). Gma12p has been shown to be responsible for the galactosylation of O-linked oligosaccharides (30).…”