Arias et al. BMC Genomics 2011, 12:390 http://www.biomedcentral.com/1471-2164/12/390 Page 5 of 17 Table 1 S. cerevisiae mutants with increased levels of dually phosphorylated MAP Kinase Slt2 (Continued) YJR152W YLR292C YCR090C YDL173W YDR290W YGR022C YLR250W YLR338W YMR119W-A YNL058C YNL105W YNR014W YPL158C DAL5 SEC72 SSP120 5.7 2.9 2.0 4.1 4.2 3.9 5.2 7.2 3.2 3.0 5.5 2.5 3.0 Transport Transport Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Member of the allantoate family of the major facilitator superfamily (MFS) Component of ER protein-translocation subcomplex. Unknown function Unknown function Unknown function Unknown function Unknown function Unknown function Unknown function Unknown function Unknown function Unknown function Unknown function Levels of Slt2 phosphorylation (P-Slt2) correspond to the fold-change in each mutant strain relative to the wild-type calculated as described in Methods. Representative data of at least replicated experiments are shown. Functional categories and descriptions were assigned based on the information provided by the BIOBASE Knowledge Library Proteome. Genes were grouped together on the basis of their functional category. Those gene deletions that have been reported to be synthetic lethal with slt2Δ are labeled with a black dot. highlighted. The first subgroup comprises seven genes (SLA1, ABP1, BUD6, VRP1, ARC18, TPM1, DMA2) related to actin cytoskeleton organization. Actin cytoske- leton disruption has been shown to activate the CWI pathway, probably due to the induction of cell wall stress, but the precise molecular mechanism by which Slt2 is sti- mulated has not been fully established [42]. Moreover, related to this group we found the Doa4 deubiquitinating enzyme (Figure 3), which has been associated with cell morphology and actin cytoskeleton defects. In fact, DOA4 had a synthetic genetic interaction with SLA1 [43]. A second subgroup encompasses six mutants, including structural cell wall-related proteins. Three of them are glycosylphosphatidylinositol-anchored proteins (GPI- APs) on the cell surface: Gas1, which is a b-1,3-glucano- syltransferase; Ecm33, which is linked to cell wall mainte- nance; and Yps7, an aspartyl protease. The corresponding deletant strains have severe cell wall defects that may explain their high basal Slt2 activation. In fact, the com- pensatory response elicited in a gas1Δ strain has pre- viously been characterized, and it involves a significant induction in MLP1 expression [12]. In agreement with this, gas1Δ and ecm33Δ mutants have previously been shown to have a constitutively high level of Slt2 phosphorylation [44,45]. Moreover, within this second subgroup we found Las21, an ER membrane protein involved in the synthesis of the GPI core structure. The absence of this protein leads to global cell wall defects. Functionally linked to Las21, the protein Arv1 was identi- fied (Figure 3). This protein is involved in sphingolipid metabolism and has recently been related to the process of GPI synthesis and anchoring [46]. Finally, two proteins related to chitin metabolism, Chs6, which is involved in chitin synthase Chs3 activity, and Chs1 (chitin synthase I), which is required for repairing the chitin septum dur- ing cytokinesis [7] were uncovered in our screening. The identification of these mutants is significant since, due to functional redundancy and the existence of gene families, the deletion of certain individual genes encoding cell wall-related proteins does not usually lead to observable phenotypes. A third subset includes pmt1Δ, hoc1Δ and alg6Δ strains. All of them encode mannosyltransferase activities, and their selection in the screening is consis- tent with the finding that protein glycosylation of cell surface proteins is important for cell wall assembly [7,47]. Interestingly, transcriptional responses to O- and N-glycosylation defects in yeast include the fingerprint of the cell wall damage transcriptional profile [48,49]. Figure 2 Representative examples of yeast mutants with increased levels of Slt2 phosphorylation. Exponential phase cultures of the indicated yeast strains were taken and processed for immunoblotting as described under “Methods”. Western blots detecting the phosphorylated form of Slt2 (P-Slt2) and actin as loading control are shown. Numbers correspond to the P-Slt2 fold-change obtained from densitometric quantification of the P-Slt2 bands from Western blots normalized with respect to the actin bands, using the values of the wild- type strain as reference (fold-change set to 1.0).