Secretion in yeast: Reconstitution of the translocation and glycosylation of α-factor and invertase in a homologous cell-free system

Abstract

A homologous cell-free system has been derived from the yeast Saccharomyces cerevisiae that allows the translation, translocation, and glycosylation of the precursors of yeast mating factor α and invertase. The precursors were translated in a yeast lysate from mRNA obtained by in vitro transcription of the MFα1 and SUC2 genes. Inclusion of yeast microsomes resulted in the glycosylation of the α-factor precursor, which was demonstrated to be sequestered within the membrane vesicles. Similar results, including signal sequence cleavage, were observed for invertase. Processing of secretory proteins translated in a yeast lysate could not be achieved using microsomes derived from canine pancreas, nor were yeast microsomes active in a wheat germ translation system.