FIGURE 3:

Ste11 interacts with the PH^Ste5 domain. (A) Schematic representation of the interaction of Ste11 with Ste50 through their respective SAM domains in the natural HOG pathway (left) and through the RBL and grafted PH^Ste5 domain in the altered HOG pathway. (B) The altered HOG pathway format in A was used to delineate the boundary of the PH^Ste5 domain in yeast strain YCW1476 (ste50Δ ssk2Δ ssk22Δ) by monitoring its ability to grow on hyperosmotic media (with 0.75 M NaCl). (C) The RBL^Ste11 domain specifically interacts with the PH^Ste5 domain. Other PH domains replacing the Ste50-SAM domain were unable to activate the altered HOG pathway, indicating that they do not interact with the RBL^Ste11 domain. (D) The RBL^Ste11 domain interacts with the PH^Ste5 domain in vitro. Bacterially expressed, His-tagged RBL^Ste11 was incubated with glutathione–Sepharose bead–immobilized Ste5 fragments of either aa 373–537 (PH^Ste5L) or 373–523 (PH^Ste5S) as GST fusion or GST alone as control. His-tagged BRL^Ste11 copurified with the glutathione–Sepharose beads was revealed by Western blotting analysis with anti-His antibody (top) and the GST fusion with anti-GST antibody (bottom).