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-agglutinin fusion protein on the yeast cell was analyzed by indirect fluorescence microscopy using an anti-glucoamylase antibody. Most of the intense fluorescence was first localized in the small bud, then observed on the entire cell wall of the daughter and mother cells. Fluorescence also accumulated at the neck region. These observations suggest that the display of the heterologous protein on the cell surface is carried with other cell wall components to the areas in which the cell wall is newly synthesized; the distribution is controlled by the cell cycle. Then, the heterologous protein-encoding gene was expressed in a sec1 mutant, in which secretory vesicles accumulate under restrictive temperature, and the produced protein was detected by immunoelectron microscopy. Most of the gold particles that reacted with the fusion protein were not localized in vesicles but in expanding endoplasmic reticulum. This phenomenon may be due to overproduction of the heterologous 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.
Shibasaki
Y
Y
Division of Material and Biological Sciences, Graduate School of Science, Japan Women's University, Bunkyo-ku, Tokyo, Japan.
Kamasawa
N
N
Shibasaki
S
S
Zou
W
W
Murai
T
T
Ueda
M
M
Tanaka
A
A
Osumi
M
M
D000373:Agglutinins / Q000235:genetics; D002453:Cell Cycle; D002473:Cell Wall / Q000201:enzymology* / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics; D005087:Glucan 1,4-alpha-Glucosidase / Q000032:analysis* / Q000235:genetics; D016253:Microscopy, Immunoelectron; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013213:Starch / Q000378:metabolism
D000373:Agglutinins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase