Abstract
The cell wall is important for maintenance of the structural integrity and morphology of fungal cells. Besides beta-glucan and chitin, alpha-glucan is a major polysaccharide in the cell wall of many fungi. In the fission yeast Schizosaccharomyces pombe, cell wall alpha-glucan is an essential component, consisting mainly of (1,3)-alpha-glucan with approximately 10% (1,4)-linked alpha-glucose residues. The multidomain protein Ags1p is required for alpha-glucan biosynthesis and is conserved among cell wall alpha-glucan-containing fungi. One of its domains shares amino acid sequence motifs with (1,4)-alpha-glucan synthases such as bacterial glycogen synthases and plant starch synthases. Whether Ags1p is involved in the synthesis of the (1,4)-alpha-glucan constituent of cell wall alpha-glucan had remained unclear. Here, we show that overexpression of Ags1p in S. pombe cells results in accumulation of (1,4)-alpha-glucan. To determine whether the synthase domain of Ags1p is responsible for this activity, we overexpressed Ags1p-E1526A, which carries a mutation in a putative catalytic residue of the synthase domain, but observed no accumulation of (1,4)-alpha-glucan. Compared with wild-type Ags1p, this mutant Ags1p showed a markedly reduced ability to complement the cell lysis phenotype of the temperature-sensitive ags1-1 mutant. Therefore, we conclude that, in S. pombe, the production of (1,4)-alpha-glucan by the synthase domain of Ags1p is important for the biosynthesis of cell wall alpha-glucan.
Highlights
The ␣-glucan synthase Ags1p was identified in the fission yeast Schizosaccharomyces pombe using a temperature-sensitive mutant strain, ags1-1ts, the cells of which lyse at the restrictive temperature due to a weakened cell wall unable to withstand internal osmotic pressure (7)
Our recent observation that cell wall ␣-glucan from S. pombe consists of ϳ10% (1,4)-linked ␣-glucose residues (19) prompted us to investigate whether the SYN domain is involved in the synthesis of this cell wall ␣-glucan constituent
The previously described putative catalytic residues of E. coli GlgA and human muscle glycogen synthase are highly conserved in the Ags SYN domains (Fig. 2)
Summary
Ags, ␣-glucan synthase; SYN, synthase; ORF, open reading frame; TGL, transglycosylase. Ags1p SYN Domain Synthesizes (1,4)-␣-Glucan in Fission Yeast glycogen synthase GlgA from the bacterium Agrobacterium tumefaciens (15) and the Archaea Pyrococcus abyssi (16) provide a basis for our understanding of the catalytic mechanism of these synthases. Both the N- and C-terminal halves fold into a subdomain with a Rossmann-type fold, a classical structural motif characterized by a central -sheet flanked by several ␣-helices. Our recent observation that cell wall ␣-glucan from S. pombe consists of ϳ10% (1,4)-linked ␣-glucose residues (19) prompted us to investigate whether the SYN domain is involved in the synthesis of this cell wall ␣-glucan constituent. Our data demonstrate that the S. pombe Ags1p SYN domain is involved in cell wall ␣-glucan biosynthesis by producing (1,4)-␣-glucan
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