Figure 3.

Depleting Tti2 below endogenous levels supports viability. (A) Yeast strains CY6971 and BY4742 were grown to saturation in YP medium containing galactose, then diluted 1:100 in the same medium, and grown for 16 hr to an optical density of 2.0 at 600 nm; 10⁸ cells were then harvested before blocking translation and 2, 4, 6, and 8 hr after adding 35 µg/ml of cycloheximide. For each time point, cell pellets were washed with lysis buffer then immediately stored at –80°. Total protein was extracted from each cell pellet using an optimized extraction protocol (von der Haar 2007) and in the presence of protease inhibitors. A 1.0-µl aliquot of lysate was loaded per time point and separated by SDS-PAGE. Tti2 was detected by Western blotting with anti-Myc antibody, and the bottom of the gel was stained with Coomassie Brilliant Blue for a loading control. (B) GAL10-TTI2 expression in glucose-containing medium. Yeast strain CY6070 (lanes 1, 2, and 3), CY6971 (lanes 5 and 8), and BY4742 (no tag control; lane 4) were grown to stationary phase in glucose-containing YP, diluted 1:20 in fresh medium and grown for 8 hr before harvesting. Protein was extracted with glass beads and the indicated amounts separated by SDS-PAGE and Western blotted with an anti-Myc antibody to detect tagged Tti2. Lane numbers are listed between the blot and the bottom of the gel, which was stained with Coomassie Brilliant Blue for a loading control (CBB). Lane 6 was left open to separate galactose control lanes. (C) CY7086 (genomically encoded C-terminal Myc⁹-tagged TTI2, WT-TTI2; lanes 1, 2, 3, and 7), CY6971 (GAL10-TTI2; lanes 4, 5, 6, and 8), and BY4742 (no tag; lane 9) were grown to saturation in YP media containing either raffinose or galactose, then diluted 1:20 in the same medium and grown for 8 hr. Protein was extracted by bead lysis and the indicated amounts separated by SDS-PAGE and Western blotted as described above. The bottom portion of the gel was stained with Coomassie Brilliant Blue. (D) CY6971, CY7172 (YCplac33-TTI2-TTI2), and BY4742 were grown to stationary in media depleted of leucine and containing either raffinose or galactose, diluted 1:20 in the same medium, and grown for 8 hr before harvesting. Protein extraction and Western blotting was performed as in (B).