The cell walls of yeast cells possess a large mannan structure mainly comprising of a linear alpha 1,6-linked mannose oligomer on the N-linked glycans. The biosynthesis of the mannan is initiated by ScOch1p alpha 1,6-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 degrees 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 Spmnn9 Delta and Spanp1 Delta mutants suggested that the mannan structure in S. pombe is synthesized sequentially by the alpha-mannosyltransferases in the order of SpOch1p, SpMnn9p and SpAnp1p. Immuno-precipitation 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. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.