SpMnn9p and SpAnp1p form a protein complex involved in mannan synthesis in the fission yeast Schizosaccharomyces pombe

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The cell walls of yeast cells possess a large mannan structure mainly comprising of a linear α1,6-linked mannose oligomer on the N-linked glycans. The biosynthesis of the mannan is initiated by ScOch1p α1,6-mannosyltransfease, and elongated by the mannan polymerase complexes M-Pol I and II in the Golgi of Saccharomyces cerevisiae. Here, we functionally characterized SpMnn9 and SpAnp1 proteins in the fission yeast Schizosaccharomyces pombe; these proteins are homologs of S. cerevisiae M-Pol II complex proteins ScMnn9p and ScAnp1p. Cells harboring disruptions in Spmnn9+ and Spanp1+ genes showed slower growth at 37°C and an increased sensitivity to hygromycin B, characteristic of a glycosylation defect. Results obtained from the acid phosphatase assay and high-performance liquid chromatography analysis of N-linked glycans in Spmnn9Δ and Spanp1Δ mutants suggested that the mannan structure in S. pombe is synthesized sequentially by the α-mannosyltransferases in the order of SpOch1p, SpMnn9p and SpAnp1p. Immunoprecipitation and split YFP analyses demonstrated that SpMnn9p and SpAnp1p form the M-Pol-II like complex. Together, these results provided an improved understanding of the mechanism of mannan synthesis by SpMnn9p and SpAnp1p in S. pombe.

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Strains, media and genetic methods

Wild-type S. pombe ARC039 (h-ura4-C190T leu1-32) strain was provided by the National BioResource Project (NBRP). Standard rich medium (YES) and synthetic minimal medium (MM) were used for growing S. pombe as described (29). For transformation of S. pombe cells, the lithium acetate method was used (30,31). Standard methods used for genetic manipulation have been described previously (32). Escherichia coli XL1-Blue (Stratagene, La Jolla, CA, USA) was used for all cloning procedures.

Gene disruption

The Spmnn9+

Phenotypic characterization of Spmnn9Δ and Spanp1Δ cells

To investigate the effects of deletions of Spmnn9+ and Spanp1+ genes on the cell growth and glycosylation, Spmnn9Δ and Spanp1Δ cells were constructed. Both Spmnn9Δ and Spanp1Δ cells were aberrantly shaped and swollen, and appeared to be inherently aggregating in cultures with approximately 5–10 cells, similar to the Spoch1Δ cells (39) (Fig. 1A). EDTA treatment, which is used for disaggregating flocculation mutants (40), did not abolish the aggregations of the Spmnn9Δ and the Spanp1Δ cells (data

Discussion

The mechanism underlying the biosynthesis of mannan structure in S. cerevisiae has been extensively investigated, leading to the identification of the genes involved in mannan biosynthesis and elucidation of the coordinated structure of mannan. However, molecular details of the mechanism of mannan biosynthesis in S. pombe still remains largely elusive, even though the genes homologous to those involved in mannan biosynthesis in S. cerevisiae were predicted to be present in S. pombe. Here we

Acknowledgments

This work was partly supported by the Project for Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of New Industrial Science and Technology Frontiers by the Ministry of Economy, Trade and Industry (METI) of Japan, New Energy and Industrial Technology Development Organization (NEDO).

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  • §

    Present address: Department of Life Science, Faculty of Science and Engineering, Setsunan University, 17-8 Ikeda-Nakamachi, Neyagawa, Osaka 572–8508, Japan.

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