Figure 2.

Interaction of PAT1 prey with APP-BaSS and various baits. (a) β-Galactosidase color assay: Yeast diploid cells (L40A5xEGY48) containing prey vector (pJG4–5) expressing PAT1 and bait vectors (p1979A) containing either the wild-type APP-BaSS (Tyr) or the mutant APP-BaSS (Ala) were grown on a galactose + histidine plate in the presence of -bromo-4-chloro-3-indolyl β-d-galactoside. These and other data are obtained from a pooled population of at least 5 colonies. (b) Growth assay: Yeast diploid cells obtained as above were grown on galactose plates lacking histidine with or without 20 μM 3-amino-1, 2,4-triazole (3-AT). (c) Quantitative β-galactosidase assay: Yeast diploid cells obtained as above were grown in galactose-containing liquid culture for 12–18 h, and the β-galactosidase activity was determined and normalized for cell density. Data represent average results from five individual colonies. Error bars indicate the standard deviation. Yeast cells (L40A5) transformed with the wild-type APP-BaSS bait vector alone were used as a negative control. (d) YSV90 yeast cells were cotransformed with PAT1 prey vector (in pJG4–5) and indicated bait vectors (in p1979A), and the transformants were grown on galactose-lacking leucine for 2–3 days. The names of proteins and the signaling activity of the sequence (BaSS or endocytic) are given on the left. Signal sequences fused to the DNA-binding domain of LexA are given on the right, and the residues crucial for signaling activity are underlined.