Figure 3. Identification of critical residues within Helix 8 repression domain.

(A) Sequence and structure of Helix 8 (5NQS). Helix 8 is colored green, and amino acids chosen for mutation are highlighted in light green in both the sequence and the structure. (B) Repression activity of indicated single- and double-alanine mutations. (C) Time-course flow cytometry of selected mutations of Helix 8 following auxin addition. TPLH3-8-IAA3 fusion proteins (black) were compared to indicated single mutations to alanine (red). Controls: Helix 1 (H1 – blue) and TPLN188 (dark green). (D) A series of alanine mutations (V145A, E146A, K148A, K149A, and the quadruple mutant Quad^AAAA chosen from A–C) were introduced into the TPLN188 bait construct and tested for interaction with wild-type TPLN188, AtMED21, and controls. Each single-alanine mutation reduces TPL interaction with AtMED21, while the quad mutation abrogated interaction. (E) The Helix 8 Quad^AAAA mutation was introduced into the TPLN188-IAA3 and TPLH3-8-IAA3 fusion proteins and compared to wild-type N188 in time-course flow cytometry. For all cytometry experiments, the indicated TPL construct is fused to IAA3. Every point represents the average fluorescence of 5–10,000 individually measured yeast cells (a.u.: arbitrary units). Auxin (IAA-10 µM) was added at the indicated time (gray bar, +Aux). At least two independent experiments are shown for each construct.