Figure 7.

Stability of Hac1p of 238, 220, and 230 aa. (A) Transformants of the hac1Δ strain in which each of the mutant versions of the HAC1 gene described in Figure 6A had been introduced were grown at 30°C in SC(−Ura, Leu) medium to midlogarithmic phase, and aliquots were treated with cycloheximide (20 μg/ml). Immediately after the times indicated, the cultures were poured over ice and the cells were collected by centrifugation. Total proteins were extracted and analyzed by immunoblotting using anti-Hac1p and anti-Kar2p antibodies. For Hac1p of 220 aa, a shorter exposure of the film is also shown below. * denotes a nonspecific band. (B) The hac1Δ strain (KMY1045) was cotransformed with a plasmid carrying the mutant (230-type) HAC1 gene and the LEU2-selectable marker and a plasmid carrying the intron-less (238-type) HAC1 gene and the URA3-selectable marker. The resulting transformant was grown, pulse-labeled for 5 min with [³⁵S]methionine and [³⁵S]cysteine, and then chased for the times indicated as described in MATERIALS AND METHODS. The hac1Δ strain carrying either the 230-type or 238-type mutant HAC1 gene was pulse-labeled for 5 min and shown for comparison. Hac1p and Pdi1p (internal control) were immunoprecipitated and subjected to SDS-PAGE (12% gel). The positions of molecular mass markers, Pdi1p, and Hac1p of 238 aa and 230 aa are indicated. Radioactivities of each band were determined by using a BioImaging Analyzer BAS-2000 and corrected for Pdi1p values. Data from two separate experiments (indicated by circles and triangles) are plotted after normalization to the 0 time value of 230aa-Hac1p.