Figure 7.

Synergistic effect of zinc overload and AhPDF1.1b expression on UPRE activation and ERAD. (A) Expression of the UPRE-GFP reporter system in yeast cells expressing the pFL38H empty vector (EV) or the native AhPDF1.1b (AhPDF1.1b) or the mutated C15A-C36A AhPDF1.1b (AhPDF1.1b C15A-C36A). The GFP levels were acquired through a synchrone spectrum from 430 to 530 nm. Yeast cells were cultivated on agar plates with increasing zinc concentrations and picked up on their exponential growth phase (see Fig.4A.2). The fluorescence was normalized by the number of cells given by the A_600nm. At least three biological repetitions were done for each experiment. Error bars correspond to standard errors, n = 3. (B) Drop-test performed with yeast cells expressing the pFL38H empty vector (EV) or the native AhPDF1.1b (AhPDF1.1b) or the mutated C15A-C36A AhPDF1.1b (AhPDF1.1b C15A-C36A). An overnight yeast culture was diluted to the A_600nm levels indicated and 10 μL were spotted on YNB medium supplemented or not with 30 mmol/L ZnSO₄. (C) HAC1 mRNA splicing in yeast cells expressing or not the native AhPDF1.1b and grown on agar plates supplemented or not with 20 mmol/L zinc. After total RNAs extraction, HAC1 mRNA splicing was monitored by RT-PCR. PCR products were separated by electrophoresis on a 1% agarose gel. The positions of the bands corresponding to the precursor (p) or the spliced (s) HAC1 mRNA are indicated. (D) The expressions of KAR2 and HRD1 were monitored by Real-time RT-PCR. mRNA were isolated from yeast expressing the pYX212 empty vector (EV) or expressing the native AhPDF1.1b (AhPDF1.1b) and grown on control conditions or in the presence of 20 mmol/L ZnSO₄ (Zn). Three biological replicates were analyzed and transcript quantifications were expressed relatively to the expression of two housekeeping genes: ACTIN and TUBULIN. Error bars correspond to standard errors, n = 3. The asterisk (*) indicated Student's t-test P ≤ 0.05 when compared AhPDF1.1b-Zn with the other conditions.