FIG. 3.

CY028-producing S. cerevisiae cells under inducing conditions. Cells were cryofixed, freeze-substituted with 0.3% uranyl acetate-0.01% glutaraldehyde in methanol, and subsequently low-temperature embedded in Lowicryl HM20. After double immunogold labeling with anti-cutinase (1:250) and goat anti-rabbit with conjugated 6-nm gold (1:10) and with anti-BiP (1:200) and goat anti-rabbit with conjugated 10-nm gold (1:10), CY028 cutinase and BiP were located in aggregates, which were confined to the ER and the vacuoles. (A) Low-power magnification of a CY028-producing cell, showing the labeled area of aggregates (asterisk) and vacuoles (V and V′) containing cutinase and BiP. Bar = 250 nm. Nu, nucleus. (B) High-power magnification of a labeled aggregate (asterisk) and vacuole (V′) with 6-nm gold particles indicating the location of CY028 cutinase and with 10-nm gold particles indicating the location of BiP. Bar = 100 nm.