FIG. 2.

Time-dependent accumulation of ouabain sites (A), α₁(TM4/TM5) loop protein (A), α₁ mRNA (B), and PAB1 mRNA (B). The Na,K-ATPase or the α₁(TM4/TM5) loop was expressed from galactose-inducible promoters in the S288C mutant strain, as described in Materials and Methods. Membranes, cytosolic fractions, and total RNA were isolated from cells harvested at the indicated time points. Data represent one of two or three independent growth experiments with each yeast strain. Ouabain binding to crude membranes was performed with 15 nM [³H]ouabain. The amount of α₁(TM4/TM5) was determined by quantitative Western blotting. PAB1 mRNA, α₁ mRNA, or actin mRNA was detected by Northern slot blot analysis. Blots were quantified on a STORM PhosphorImager. RNA data were normalized to actin mRNA levels. The maximum value for each experiment was defined as 100%.