Abstract
In yeast Saccharomyces cerevisiae the N-linked sugar chain is modified at different positions by the addition of mannosylphosphate. The mnn6 mutant is deficient in the mannosylphosphate transferase activity toward mannotetraose (Karson, E. M., and Ballou, C. E. (1978) J. Biol. Chem. 253, 6484-6492). We have cloned the MNN6 gene by complementation. It has encoded a 446-amino acid polypeptide with the characteristics of type II membrane protein. The deduced Mnn6p showed a significant similarity to Kre2p/Mnt1p, a Golgi alpha-1, 2-mannosyltransferase involved in O-glycosylation. The null mutant of MNN6 showed a normal cell growth, less binding to Alcian blue, hypersensitivity to Calcoflour White and hygromycin B, and diminished mannosylphosphate transferase activity toward the endoplasmic reticulum core oligosaccharide acceptors (Man8GlcNAc2-PA and Man5GlcNAc2-PA) in vitro, suggesting the involvement of the MNN6 gene in the endoplasmic reticulum core oligosaccharide phosphorylation. However, no differences were observed in N-linked mannoprotein oligosaccharides between Deltaoch1 Deltamnn1 cells and Deltaoch1Deltamnn1Deltamnn6 cells, indicating the existence of redundant genes required for the core oligosaccharide phosphorylation. Based on a dramatic decrease in polymannose outer chain phosphorylation by MNN6 gene disruption and a determination of the mannosylphosphorylation site in the acceptor, it is postulated that the MNN6 gene may be a structural gene encoding a mannosylphosphate transferase, which recognizes any oligosaccharides with at least one alpha-1,2-linked mannobiose unit.
Highlights
In yeast Saccharomyces cerevisiae the biosynthesis of Nlinked oligosaccharides has been studied in detail
Mannosylphosphate transferase activity was decreased in the mnn6 mutant, it was still uncertain whether the mnn6 mutation affected the Nlinked core oligosaccharide phosphorylation
Cloning of the MNN6 Gene—The original mnn6 mutant exhibited a reduced amount of phosphomannan on the cell wall and showed less binding to Alcian blue, a dye that binds to a phosphate moiety of cell surface mannoproteins [11]
Summary
Yeast Strains and Media—Strain TO3-6D, used for the cloning of MNN6, was a meiotic segregant from a cross of LB1425-1B, kindly provided by C. YS126-47D and its isogenic mnn disruptant strain XW43 were used for oligosaccharide analysis. The BglII site is located 333 base pairs upstream from the ATG, and the BclI site is found 558 base pairs upstream from the stop codon (see Fig. 4A) This digestion removed a 1116-base pair fragment encompassing 261 amino acids of the MNN6 sequence and further replaced it with a BglII fragment containing the complete ADE2 gene from pASZ11 [26]. Haploid yeast strains were used to transform with the linearized mnn6::ADE2 DNA fragments (Fig. 4A). Man5GlcNAc2-PA acceptor (25 pmol) prepared from strain YS133-1D mannoproteins was used for the enzyme assay. PAoligosaccharides were detected by fluorescence (excitation 5 320 nm; emission 5 400 nm)
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