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. 2002 Aug;13(8):2651–2663. doi: 10.1091/mbc.02-03-0043

Figure 8.

Figure 8

Cytoplasmic localization of Wis1 MAPKK is essential for nuclear translocation of Spc1 MAPK in response to stress. (A) NES of wis1:GFP was substituted with the wild-type and mutant NLS from the simian virus 40 large-T antigen to construct wis1NLS:GFP and wis1NLS*:GFP alleles, respectively. The wis1:GFP spc1:HA6H (CA1174), wis1NLS:GFP spc1:HA6H (CA1225), and wis1NLS*:GFP spc1:HA6H (CA1207) 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. Spc1 phosphorylation in the wis1NLS strain was partially compromised, whereas the wis1NLS* strain showed normal Spc1 activation. (B and C) wis1NLS:GFP spc1:myc (CA1212) (B) and wis1NLS*: GFP spc1:myc (CA1296) (C) strains were exposed to 0.6 M KCl for the indicated times. Cells were fixed and subjected to immunofluorescence microscopy with anti-myc antibodies. The wis1NLS strain was completely defective in nuclear accumulation of Spc1 after osmostress, which was only partially restored by the NLS* mutation.