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

UNLABELLED

The fission yeast is a widely employed model organism for studying the eukaryotic cell cycle. Like plants and bacteria, 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 α-amylases that are putative members of this evolutionarily conserved family of cell wall-modifying proteins. We found that unlike rod-shaped wildtype cells, 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 cells revealed that 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 cells depended on β-glucan upregulation and the cell wall integrity pathway that mediates it. While 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.

SIGNIFICANCE STATEMENT

This study utilized a range of imaging techniques and high-resolution solid-state NMR spectroscopy of intact cells to refine our understanding of cell wall composition. This study also determined that two related GPI-anchored α-amylase family proteins, Aah1 and Aah3, likely act as transglycosylases non-redundantly with an α-glucan synthase in the synthesis of α-glucan chains of appropriate content and size to support polarized growth and cell division. Our results also highlight the anti-fungal therapeutic potential of GPI-anchored enzymes acting in concert with glucan synthases.