Fig. 4. Requirement for the Sho1 cytoplasmic linker region and the SH3 domain for Sho1 function in the osmotic stress response. (A) Schematic representation of the Sho1 constructs used in this experiment. The pSte2–Sho1 chimeric construct is described in Figure 2. Its derivatives, pSte2–Sho1-A and pSte2–Sho1-B, contain deletions within the Sho1 linker region. In pSte2–Sho1-C, the Sho1 linker region is replaced with an unrelated sequence of similar length (150 amino acids) derived from the SLN1 gene. pSte2–Sho1-D contains a truncation of Sho1 in which the SH3 domain is replaced by the PBS2 coding region. (B) Sho1–Ste2 chimeric constructs were transformed into the yeast strain MY007 (ssk2Δ ssk22Δ sho1Δ), and the osmosensitivity of the transformed cells was tested on YPD plates containing 1.5 M sorbitol. (C) Tyrosine phosphorylation of Hog1 at various times after the addition of 0.4 M NaCl to the medium. The same cells used in (A) were subjected to immunoblot analysis using anti-phosphotyrosine antibody 4G10.