Radioactive Labeling and Fractionation of Fission Yeast Walls

Abstract

Fungal cells contain an essential structure external to the cell, made of polysaccharides and proteins, termed the cell wall. Polysaccharides represent ∼96% of the cell wall on a dry-weight basis. They are complex insoluble polymers connected to each other by covalent linkages and hydrogen bonds with specific localizations in the cell wall and septum. Fission yeast contains three β-glucans (a major branched β(1,3)-glucan, a minor linear β(1,3)-glucan, and a minor branched β(1,6)-glucan), two α-glucans (a major α(1,3)-glucan and a minor α(1,4)-glucan), and a minor amount of galactomannan-linked glycoproteins. We provide here a simple protocol to label uniformly the cell wall using d-[U-¹⁴C]-glucose as a carbon source and to fractionate the cell wall into the three or four main cell wall components: galactomannoproteins, α-glucan, and β-glucan, which can be subdivided into β(1,3)-glucan and β(1,6)-glucan. This simple protocol uses enzymatic and chemical fractionations of the different cell wall components that permit the quantification of each polysaccharide in the cell wall and in the cell. This protocol is very useful for the analysis of the many morphological alterations caused by a variety of cellular processes that ultimately affect the cell wall and thus cell morphogenesis.