Cytochemical evaluation of localization and secretion of a heterologous enzyme displayed on yeast cell surface

A starch-utilizing Saccharomyces cerevisiae strain was constructed by cell surface engineering. Distribution of the heterologous glucoamylase-alpha -a gglutinin fusion protein on the yeast cell was analyzed by indirect fluores cence microscopy using an anti-glucoamylase antibody. Most of the intense f luorescence was first localized in the small bud, then observed on the enti re cell wall of the daughter and mother cells. Fluorescence also accumulate d at the neck region. These observations suggest that the display of the he terologous protein on the cell surface is carried with other cell wall comp onents to the areas in which the cell wall is newly synthesized; the distri bution is controlled by the cell cycle. Then, the heterologous protein-enco ding gene was expressed in a sed mutant, in which secretory vesicles accumu late under restrictive temperature, and the produced protein was detected b y immunoelectron microscopy. Most of the gold particles that reacted with t he Fusion protein were not localized in vesicles but in expanding endoplasm ic reticulum. This phenomenon may be due to overproduction of the heterolog ous protein which was designed to be displayed on the cell wall. Artificial production of heterologous protein may have caused a relative shortage of glycosyl phosphatidylinositol anchors. (C) 2000 Federation of European Micr obiological Societies. Published by Elsevier Science B.V. All rights reserv ed.