FIG. 3.

Analysis of eIF6 depletion in yeast cells and its effect on cell growth. (A) Schematic representation of the plasmid pUB-TIF6. The plasmid was constructed as described under Materials and Methods. Abbreviations: UAS, the upstream activation sequence of the GAL10 promoter; Ub, ubiquitin gene; X, the codon for arginine; lacI, a restriction fragment that encodes amino acid residues 318 to 346 of the lac repressor; HA, an epitope from the influenza virus hemagglutinin protein. (B) Exponentially growing cultures of W303α (○) and KSY603 (•) were diluted to an A₆₀₀ of about 0.03 in either YPGal (galactose) medium or YPD (glucose) medium. Cell growth was monitored by measuring the A₆₀₀. To keep cultures in exponential growth, they were diluted in fresh medium whenever the A₆₀₀ reached 0.8 U. (C) At the indicated times following the shift from YPGal to either YPGal (left) or YPD (right), cell lysates were prepared from KSY603 as described in Materials and Methods. Approximately 50 μg of protein from each cell lysate was electrophoresed through an SDS–15% polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane. The blot was then probed with peroxidase-coupled anti-HA monoclonal antibodies to detect eIF6 fusion protein. The decrease in the amount of eIF6 observed at the 6-h time point in YPGal is due to a gel loading error.