Impact of Fungal MAPK Pathway Targets on the Cell Wall

Abstract

The fungal cell wall is an extracellular organelle that provides structure and protection to cells. The cell wall also influences the interactions of cells with each other and surfaces. The cell wall can be reorganized in response to changing environmental conditions and different types of stress. Signaling pathways control the remodeling of the cell wall through target proteins that are in many cases not well defined. The Mitogen Activated Protein Kinase pathway that controls filamentous growth in yeast (fMAPK) was required for normal growth in media containing the cell wall perturbing agent Calcofluor White (CFW). A mass spectrometry (MASS-SPEC) approach and analysis of expression profiling data identified cell wall proteins and modifying enzymes whose levels were influenced by the fMAPK pathway. These include Flo11p, Flo10p, Tip1p, Pry2p and the mannosyltransferase, Och1p. Cells lacking Flo11p or Och1p were sensitive to CFW. The identification of cell wall proteins controlled by a MAPK pathway may provide insights into how signaling pathways regulate the cell wall.

Keywords: MAPK; cell wall; cell wall stress; cell–cell interactions; mannoproteins.

Figure 1

MAPK pathways that can regulate the cell wall in yeast. Not all proteins are shown, adapted from [77].

Figure 2

Role of MAPK pathways in regulating cell wall stress in yeast. (A–C) 0.1 O.D₆₀₀ of wild type (PC538) and indicated strains (see Table 1 for strain numbers) were spotted in 10-fold serial dilutions onto YEPD media supplemented with or without CFW at a final concentration of 50 µg/mL. Plates were incubated at 30 °C or 37 °C and photographed after two days of growth (panels A and C) or three days (panel B).

Figure 3

Analysis of cell-wall gene expression and mutant phenotypes. qPCR analysis was used to measure differences in expression levels of targets of identified by MASS SPEC analysis and gene expression profiling. (A) FLO10; (B) TIR gene family expression analysis from cells grown on agar media versus liquid. All asterisks are p < 0.05 for each pair; (C) colonies of wild-type (WT, PC538) cells and the indicated deletion mutants of members of the TIR family; (D) bar graph showing number of ruffles per radii in the wild type and the indicated TIR deletion mutants (strain numbers of TIR deletion mutants can be found in Table 1). Asterisks are p < 0.05 relative to WT. Additional qPCR analysis of targets identified in a comparative RNAseq experiment (Chow et al., under review; GSE115657) (E) TIP1 and PRY2; (F) OCH1 (WT, PC538; ste12Δ, PC1079). All asterisks are p < 0.05 relative to wild type.

Figure 4

Analysis of the role of Och1p in cell wall organization. (A) wild type (PC513) and the och1Δ (PC514) mutant were stained with CFW (middle panel) and ConA (right panel), 100× magnification, scale bar 5 microns; (B) SEM of wild-type or och1Δ mutant showing different cell wall architecture, 20.0k× magnification; (C) plate-washing assay and of wild type (PC538), ste12Δ (PC1079) and och1Δ (PC7133). WT colony diameter, 0.75 cm; (D) single cell invasive growth assay, 100× magnification, scale bar 5 microns (E) cell wall sensitivity of the och1Δ mutant, WT colony diameter, 0.75 cm; (F) SEM of wild-type (PC538) cells and the ste12Δ (PC1079) mutant, 5.0k× magnification. Assays with the och1Δ mutant were shown at different days to account for the growth defect of the mutant.