Figure 6. Interaction of PhLOP1 with bSUG1.

AD-bSUG1 was cotransformed with the indicated BD-Phd, BD-PhLOP1, or truncated BD-PhLOP1 fusion constructs into yeast CG-1945. The transformants were divided equally and grown in the presence of histidine (-Leu-Trp) or absence of histidine (-Leu-Trp-His) and incubated at 30 °C. (A) Schematic alignment representing the BD constructs of Phd (1-246 aa), PhLOP1 (53-246 aa) and truncated PhLOP1s (carboxy terminal deletions: C delta (d) 20 aa, 40 aa, 60 aa, 100 aa or amino terminal delection: N d 166 aa) and their interaction with bSUG1 by estimating the growth on -Leu-Trp-His plates. (-): Small (<1 mm), pale colonies (background growth because of HIS3 protein "leaky" expression in CG-1945). (+): Small (<1 mm), pink colonies. (++): Medium sized (1-2 mm), pink colonies. (+++): Big (2-3 mm), pink colonies. (++++): Robust (>3 mm), pink colonies. (B) The yeast transformants grown on histidine supplemented synthetic (-Leu-Trp) plates were collected after 4 days incubation at 30 °C and a pool of colonies was seeded at 1,000 cells/ml into selective medium with histidine (-Leu-Trp) or without histidine (-Leu-Trp-His). After incubating the cultures for 2 days at 30 °C with shaking, growth was measured at OD₆₀₀. Data represent the mean ± SD of triplicate assays of a representative experiment and is presented as a percentage of the OD₆₀₀ of yeast grown in selective medium supplemented with histidine (-Leu-Trp). (C) A pool of colonies was collected from samples grown on -Leu-Trp plates and prepared for b-galactosidase liquid assay as described in methods. Data represent the mean ± SD of two independent experiments done in triplicate. The b-galactosidase activity is expressed in standard units multiplied by 1,000. The b-galactosidase standard units = 1,000 x OD₄₂₀ / time (min) x OD₆₀₀.