Supplemental Materials

This article contains the following supporting material:

• Supplemental Figure 1 - Sequence alignment of ScSsk2 and CaSsk2. The primary amino acid sequences of ScSsk2 (encoded by YNR031c) and CaSsk2, as determined in this study, were aligned using ClustalW (Thompson et al., 1994). Dashes indicate single-residue gaps introduced to maximize the alignment. Amino acids conserved between ScSsk2 and CaSsk2 are indicated; black boldface = identical, black type = highly similar, gray boldface = similar. Non-conserved amino acids are shown in gray type. The putative Ssk1 binding region in CaSsk2, aligned with the Ssk1 binding domain in ScSsk2, is underlined. * represents the amino acid in CaSsk2 which corresponds to Thr1460 in ScSsk2. ‡ designates the amino acid substitutions STOP430K and STOP1344K in the CaSSK2 allele of BWP17, compared to the annotated CaSSK2 sequence in the Candida genome database.
• Supplemental Figure 2 - ssk2Δ and hog1Δ strains display equivalent phenotypes in response to osmotic and oxidative stress. Approximately 10³ cells, and 10-fold dilutions thereof, of exponentially growing wild-type (Wt-BWP17), hog1Δ (JC47), ssk2Δ (JC482), and ssk2Δ +SSK2 ste11Δ strains were spotted onto YPD plates containing the indicated additives; NaCl (0.3, 0.6, 0.9 M), sorbitol (0.8, 1.0, 1.2 M), t-BOOH (1.75, 2.0, 2.25 mM), and menadione (0.15, 0.25 and 0.35 mM). Plates were incubated at 30°C for 24 hrs.
• Supplemental Figure 3 - Ssk2 is required for Hog1 phosphorylation at high levels of osmotic stress. Western blot analysis of whole cell extracts isolated from wild type (Wt), ssk2Δ and ste11Δ cells after treatment for the specified times with 1.0 M NaCl. Phosphorylated Hog1 and total levels of Hog1 were detected as described in Figure 2.