The extracellular domain is required for Sln1 kinase activation by wall perturbations. A, representative field showing localization of SLN1-GFP (pSS1881) or sln1ΔECD-GFP (pSS1904). BF, bright field. B, Western analysis showing reduced levels of sln1ΔECD compared with full-length SLN1. SLN1, JF2007 (sln1Δ) carrying full-length SLN1-myc (pCLM994); sln1ΔECD, JF2007 carrying sln1ΔECD-myc (pCLM1774). C, comparison of Hog1 phosphorylation kinetics in SLN1 (JF2008 (sln1Δ sho1Δ) carrying full-length (pCLM994)) and sln1ΔECD (JF2008 (sln1Δ sho1Δ) carrying sln1ΔECD (pCLM1774)) strains following addition of 0.4 M NaCl. D, Northern (RNA) hybridization analysis of RNA prepared from wild type
Cultures from the sin 1 (sln1Δ) strain JF2007, or sin 1 (sln1Δ) ccw 12 (sln1Δ), strain JF2368 transformed with the SLN 1 expression plasmids, pCLM994 (SLN1) or pCLM1774 (sln1 (sln1Δ) ECD), and treated with 1 unit/ml zymolyase (+) where indicated.
NCA3 expression was normalized to expression of the SLN1-SKN7-independent CDC33 gene. The extent of induction by zymolyase is shown relative to the untreated SLN1 or sln 1(sln1Δ)ECD strains