Secretion in yeast: reconstitution of the translocation and glycosylation of alpha-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 alpha and invertase. The precursors were translated in a yeast lysate from mRNA obtained by in vitro transcription of the MF alpha 1 and SUC2 genes. Inclusion of yeast microsomes resulted in the glycosylation of the alpha-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.