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 sln1Δ strain, JF2007, or sln1Δ ccw12Δ strain, JF2368, transformed with the SLN1 expression plasmids, pCLM994 (SLN1) or pCLM1774 (sln1ΔECD), 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 sln1ΔECD strains.