Figure 3.
Processing of HAC1u to HAC1i is specifically inhibited in yeast expressing R193A/R235A during ER stress. -Fold change in total HAC1 RNA level (A) and HAC1i mRNA level (B) in yeast carrying VC, WT (mRTA), or mutant RTA expression plasmids was quantified by qRT-PCR using total RNA prepared from cells grown in dextrose or galactose in the absence (Gal) or presence of ER stress (Gal + DTT). The y axis shows the average -fold change in mRNA compared with the same cells grown in dextrose, with error bars representing the range of HAC1 mRNA from two biological replicates using three technical replicates for each. Statistical analysis was conducted separately for each. Means with different letters show significant differences (p < 0.001). C, GFP fluorescence from a UPRE-GFP reporter measured by flow cytometry is shown. The y axis shows the GFP signal normalized to yeast lacking the UPRE-GFP reporter from a minimum of three biological replicates along with the S.E. (n = 3). Means with different letters show significant differences (p < 0.01). D, protein prepared from yeast carrying VC, WT, or mutant RTA expression plasmids grown in dextrose or galactose in the absence (Gal) or presence of ER stress (Gal + DTT) was analyzed on SDS-PAGE followed by Western blot analysis with polyclonal antibodies against HAC1 (top), RTA (middle), and Dpm1 (bottom). Uncropped Western blots are shown in Fig. S5.