Hog1 molecules truncated up to D315 are activated by Pbs2, but molecules shorter than Y337 are not autoactivated.

<p><b>A... </b><i>hog1</i>Δ cells harboring either an empty vector (negative control) or vectors expressing either wild type Hog1 (Hog1^WT), or the truncated proteins Hog1^F343, Hog1^Y337, Hog1^F322, Hog1^D315 and Hog1^V313 were plated in five dilutions (starting from 200,000 cells) on plates containing YPD supplemented with 1 M NaCl. All strains were also plated on medium with no NaCl and all grew equally well on these plates (not shown). <b>B.</b> Phosphorylation levels of Hog1^WT, intrinsically active Hog1 variant (Hog1^D170A) and truncated Hog1 proteins Hog1^F343, Hog1^Y337 Hog1^F322, Hog1^D315 and Hog1^V313 were tested when expressed in <i>hog1</i>Δ cells, before (−) and 10 minutes after (+) exposure to 1 M NaCl. 20 μg of protein lysates were loaded on each lane. <b>C... </b><i>hog1</i>Δ cells expressing the indicated truncated proteins, carrying either Thr174, or Ala174, were plated in five dilutions (starting from 10,000 cells) on plates containing YPD supplemented with 1 M NaCl. All strains were also plated on medium with no NaCl and all grew equally well on these plates (not shown). <b>D.</b> Phosphorylation levels of the indicated Hog1 proteins, expressed in <i>hog1</i>Δ cells were tested as described in panel B. 30 μg of protein lysates were loaded on each lane. <b>E... </b><i>hog1</i>Δ<i>pbs2</i>Δ cells harboring same plasmids described in panel A were plated in five dilutions, starting from 100,000 on plates containing YPD supplemented with 0.8 M NaCl. Growth of cells expressing the intrinsically active Hog1 variant (Hog1^D170A) served as a positive growth control. All strains were also plated on medium with no NaCl and all grew equally well on these plates (not shown). <b>F.</b> Phosphorylation levels of the indicated Hog1 proteins were tested as described in panel B, but when expressed in <i>hog1</i>Δ<i>pbs2</i>Δ cells. 20 μg of protein lysates were loaded on each lane.