Figure 10.

Yeast complementation experiments. The strain BK203-15B carries a lethal deletion of the TRG1 gene but is rescued by the centromer plasmid pWBK-PDI, which contains a copy of the wild-type TRG1 gene and a URA3 selection marker. BK203-15B was transformed with different pDS2 constructs (1 = yPDI, 2 = yPDImc, 3 = vector alone, 4 = Dd-PDI, 5 = Dd-PDI-HDEL, 6 = Dd-PDIΔC, and 7 = Dd-PDIΔC-HDEL) under the control of the yeast galactose-inducible GAL1-promoter. Clones from each strain were then transferred onto a plate containing galactose and 5-FOA (SGC/FOA) to counterselect for the presence of the centromer plasmid (A). Of the seven strains, only the five with functional PDI complemented the TRG1 deletion. Finally, these five strains were streaked as indicated in the diagram on rich medium with galactose or glucose (B). All five strains grew at indistinguishable speed on galactose (YPG, 2 d). In contrast, on glucose (YPD, 6 d), only yPDI grew rapidly, followed in order by Dd-PDIΔC-HDEL and then by both Dd-PDI and Dd-PDI-HDEL. Dd-PDIΔC exhibited virtually no complementation in this period. None of the strains picked from the 5-FOA plate grew on plates lacking uracil (−Ura), demonstrating that growth on YPG and YPD plates was not due to the presence of the centromer plasmid. yPDI, wild-type yeast PDI; yPDImc, yeast PDI with both thioredoxin CGHC boxes mutated to SGHS.