Solid-State NMR Analysis of Schizosaccharomyces pombe Reveals Role of α -Amylase Family Enzymes in Cell Wall Structure and Function

The fission yeast Schizosaccharomyces pombe is a widely employed model organism for studying the eukaryotic cell cycle. Like plants and bacteria, S. pombe must build a cell wall in concert with its cell cycle, but how cell wall-synthesizing and remodeling enzymes mediate this process remains unclear. Here we characterize the functions of Aah1 and Aah3, two related S. pombe α-amylases that are putative members of this evolutionarily conserved family of cell wall-modifying proteins. We found that unlike rod-shaped wildtype S. pombe cells, aah1 Δ aah3 Δ cells are nearly spherical, grow slowly, have thickened cell walls, and have severe defects in cell separation following cytokinesis. Solid-state NMR spectroscopy analyses of intact wildtype and aah1 Δ aah3 Δ cells revealed that aah1 Δ aah3 Δ cell walls are rigidified with a significant reduction in the α-glucan matrix, characterized by reduced amounts of the major α-1,3-glucan and the minor α-1,4-glucan within the rigid and mobile phases; this reduction was compensated for by a two-fold increase in β-glucan content. Indeed, viability of aah1 Δ aah3 Δ cells depended on β-glucan upregulation and the cell wall integrity pathway that mediates it. While aah1 Δ aah3 Δ cells resemble cells with impaired function of the transglycosylation domain of α-glucan synthase 1 (Ags1), increased expression of Aah3 does not compensate for impaired Ags1 function or vice-versa. Overall, our data suggest that Aah1 and Aah3 are required in addition to Ags1, likely downstream, for the transglycosylation of α-glucan chains to generate fibers of appropriate dimensions to support proper cell morphology, growth, and division.