Identification of HRD1 as an important actor in Z-1AT-mediated toxicity in a toxicity assay set up in <i>S</i>. <i>cerevisiae</i>.

<p>(<b>A</b>) Empty Vector, Wild-type (WT) 1AT and Z-1AT expressing cells were grown in inducible media and spotted onto nitrocellulose. After 48 hr of growth at 30°C, the cells were rinsed from the nitrocellulose with distilled water and then immunoblotted for 1AT. (<b>B</b>) Yeast screen methodology summary. The expression of Z-1AT is not toxic in yeast. As a consequence we proposed to identify genes whose presence is necessary for this absence of toxicity, the latter being revealed upon target gene deletion. The Euroscarf library of individual gene deletion mutants is transformed by expression plasmids for either Z-1AT, WT-1AT or the empty vector. Individual colonies are picked and plated in 96-well plates. The growth test is then initiated by inducing the expression of the recombinant 1AT proteins on specific media. Clones with defective growth are selected and the DNA barcode sequence for gene identification purposes. In brief, through this screen, we identified 171 KO strains in which Z-1AT toxicity was slightly changed compared to the empty vector. Then, we have tested these 171 KO strains for the toxicity induced by the WT-1AT expression and found 31 KO mutants in whom Z-1AT was more toxic than the WT-1AT. Finally, only 5 genes displayed a clear human ortholog. (<b>C</b>) Yeast cells transformed with empty vector, WT-1AT or Z-1AT were grown in liquid synthetic complete medium to log phase. Cells were spotted on plates containing SD medium (2% dextrose) to repress 1AT expression or SG medium (2% galactose) to induce 1AT expression, and were incubated at 30°C for 3 days. Shown is fourfold serial dilution starting with equal numbers of cells. Spotting assays for <i>HRD1</i> deletion strain (right panels) and the parental control strain (BY4247 –left panels) are shown.