FIG. 8.

Influence of expression level on GCN4 mRNA translation. S288C mutant cells carrying the translational GCN4-lacZ reporter fusion and the expression plasmid for α₁β₁ Na,K-ATPase or the empty vector were grown in amino acid-supplemented minimal medium, 0.5% glucose, and 3% glycerol at 30°C in the absence of leucine, in the presence of leucine, or with the expression plasmid integrated into the yeast chromosome. At an OD₄₅₀ of 1.0, cultures were induced with 2% galactose dissolved in growth medium lacking glucose but containing amino acids (as described in Materials and Methods). After 0, 4, 24, 48, and 72 h, specific β-galactosidase activities were determined in cytoplasmic fractions, and [³H]ouabain binding was determined in crude membranes with a [³H]ouabain concentration of 14.7 nM. Specific ouabain binding was corrected for nonspecific binding by subtracting the values obtained for the empty vector.