Figure 7.
NES of Wis1 MAPKK is not essential for Spc1 MAPK activation and localization. (A) wis1:GFP spc1:HA6H (CA1174) and wis1LA:GFP spc1:HA6H (CA1175) strains were grown to early-log phase at 30°C in YES medium and exposed to 0.6 M KCl for the indicated times. Spc1HA6H was purified by Ni2+-nitrilotriacetic acid chromatography, followed by immunoblotting with anti-phospho-p38 and anti-HA antibodies. (B) wis1LA:GFP spc1:myc strain (CA1170) was cultured and stressed as in A. Cells were fixed and subjected to immunofluorescence microscopy with anti-myc antibodies. Like in wild-type cells, Spc1 transiently accumulates in the nucleus upon stress in the wis1LA mutant strain.