A Split-Ubiquitin Yeast Two-Hybrid Screen to Examine the Substrate Specificity of atToc159 and atToc132, Two Arabidopsis Chloroplast Preprotein Import Receptors
Figure 5
Comparative strengths of protein–protein interactions as determined by a quantitative β-galactosidase assay for additional prey proteins isolated from the screen using atToc132AG as the bait.
(A–D) Relative enzymatic activity of β-Gal in extracts from S. cerevisiae strain NMY51 that expressed atToc159G, atToc132G or atToc132AG-domain bait and also carried NubG-Prey construct as indicated in Table 1 and 2. Yeast cells were grown to A546 of ∼0.8 in SD (−Leu, −Trp) medium at 30°C, followed by measurements as described in materials and methods, and quantified according to the following formula: Activity = 1,000×OD615/V×t×OD546, were V is the volume of assay and t is the time of incubation. For β-Gal activity in cell extracts, the measured activity was normalised to that of mock co-transformed strains containing atToc159G, atToc132G or atToc132AG bait and empty vector, pR3-N for respective interactions. A relative β-Gal activity of 100% was arbitrarily assigned to the atToc159G bait containing (A–B) and atToc132G bait containing (C–D) pairwise interactions. The experiments were performed in triplicate and repeated at least twice. Error bars indicate ±SD. Values marked with asterisks are significantly different (Student’s t-test; P≤0.05). (A) Interaction in yeast co-expressing atToc159G or atToc132G bait and additional non-photosynthetic prey interactors identified in the screen using atToc132AG as the bait (Table 1). (B) Interaction in yeast co-expressing atToc159G or atToc132G bait and additional photosynthetic prey interactors identified in the screen using atToc132AG screening (Table 2). (C) Interaction in yeast co-expressing atToc132G or atToc132AG bait and additional non-photosynthetic prey interactors identified in the screen using atToc132AG screening (Table 1). (D) Interaction in yeast co-expressing atToc132G or atToc132AG bait and additional non-photosynthetic prey interactors identified in the screen using atToc132AG screening (Table 2).