Yeast cell wall, membrane, and soluble marker polypeptides identified by comparative two-dimensional electrophoresis.

Abstract

Yeast cell wall, plasma membrane, total spheroplast, and total soluble protein fractions were isolated from exponentially growing Saccharomyces cerevisiae batch cultures. The cell wall, plasma membrane, and soluble protein fractions were obtained by mechanical disruption of intact yeast cells under identical osmotic conditions. Electron micrographs of purified wall fractions appeared free of vesicular membrane contamination and micrographs of plasma membrane vesicles were free of cell wall contamination. Various stages of cell wall purification were monitored by electron microscopy and comparative two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This resulted in the identification of a glycopeptide designated 16w in the cell wall fraction, with an apparent isoelectric point of 5.0 and an apparent molecular weight of 25 000. Protein analyses of soluble and plasma membrane protein fractions failed to detect component 16w. Two-dimensional protein analyses of total cellular homogenates were capable of resolving the cell wall glycopeptide 16w. However, protein separations of spheroplasts formed by glusulase degradation of the cell wall complex did not detect 16w. These observations suggest that component 16w is unique to the cell wall fraction. In addition, comparison of two-dimensional gels of soluble and plasma membrane proteins, with a total cellular homogenate, tentatively identified several polypeptides unique to each of the soluble and plasma membrane fractions.