FIG. 9.

Effects of protein misfolding on GCN4 translation. S288C mutant yeast cells carrying either the translational GCN4-lacZ reporter fusion (A) or the UPR-lacZ fusion (B) and an expression plasmid for α₁β₁ Na,K-ATPase, α₁(N713A)β₁-Na,K-ATPase, or α₁(S715A)β₁-Na,K-ATPase or the empty vector were grown as described in Materials and Methods to an OD₄₅₀ of 1.0. One-fifth of each culture was transferred to 15, 20, 25, 30, or 35°C and induced after 30 min with 2% galactose dissolved in growth medium lacking glucose but containing amino acids. After 48 h, specific β-galactosidase activities were determined in purified cytoplasmic fractions, and ouabain binding capacities were determined in purified membrane fractions (C). The ouabain binding capacity was defined as 100% for membranes isolated from cells grown at 15°C. S288C mutant yeast cells carrying the GCN4-lacZ fusion or the UPR-lacZ reporter were separated into three portions (D). Each culture was supplemented with 1 μg/ml tunicamycin, 15 mM β-mercaptoethanol, or 50 mM 3-aminotriazole. β-Galactosidase activities were measured in cytosolic fractions from cells harvested after 18 h. The data shown are from two or three independent growth experiments with triple determinations of each data point.