FIG. 2.

CY028-producing S. cerevisiae cells under inducing conditions. Cells were cryofixed, freeze-substituted with 0.3% uranyl acetate-0.01% glutaraldehyde in methanol (A and B) and with 0.3% uranyl acetate-0.01% glutaraldehyde-0.3% osmium tetroxide-0.1% aminotriazole in methanol (C), and subsequently low-temperature embedded in Lowicryl HM20. After immunogold labeling with anti-cutinase (1:250) and goat anti-rabbit with conjugated 10-nm gold (1:10), CY028 cutinase was confined in aggregates of the ER. (A) Low-power magnification of a CY028-producing cell, showing the labeled area of an aggregate (asterisk) that had a total area that was about half the area of the nucleus (Nu). Bar = 250 nm. (B) High-power magnification of the labeled aggregate (asterisk) with 10-nm gold particles indicating the location of CY028 cutinase. Bar = 100 nm. (C) High-power magnification of triazole-stained membranes of the ER and 10-nm gold particles indicating the location of CY028 cutinase in the aggregates. Bar = 100 nm.