Article Figures & Data
Figures
- FIG. 1.
Overexpression of eng1+ or agn1+ suppresses the rho4Δ multiseptation phenotype. (A) Differential interference contrast images of rho4Δ cells transformed with pREP3X, pREP3Xeng1+, or pREP42Xagn1+ (pND02) and grown at 37°C for 22 h in the presence (EMM + T) or absence (EMM) of thiamine. Bar, 10 μm. (B) Quantification of the septation phenotype (n = 300). Calcofluor white was used to stain the septa. wt, wild type.
- FIG. 2.
Simultaneous overexpression of rho4+ and eng1+ or agn1+ causes dramatic cell lysis. (A) Wild-type cells transformed with plasmids pREP3Xeng1+ and pREP4X, pREP3X and pREP4Xrho4+, or pREP4Xrho4+ and pREP3Xeng1+ were grown at 32°C in EMM with 15 μM thiamine (nmt promoter off, top panel) or in EMM without thiamine (nmt promoter on, bottom panel) to induce rho4+ and eng1+ overexpression. (B) Images of wild-type cells transformed with plasmids pREP3X and pREP4X, pREP3Xrho4+ and pREP4X, pREP3X and pREP42Xagn1+, and pREP3Xrho4+ and pREP42Xagn1+. Cells were grown at 32°C in EMM without thiamine for 22 h and stained with methylene blue. Lysed cells appear dark. Bar, 10 μm.
- FIG. 3.
Phenotype of the eng1Δ rho4Δ double mutant. (A) Differential interference contrast images of eng1Δ, rho4Δ, and eng1Δ rho4Δ cells grown at 28°C or 37°C in rich medium. Bar, 10 μm. (B) Cell wall composition of wild-type, eng1Δ, rho4Δ, and eng1Δ rho4Δ cells grown in rich medium at 28°C or 37°C. [14C]glucose was added 4 h before harvesting the cells. The [14C]glucose radioactivity incorporated into each cell wall polysaccharide is shown. Values are the means from three independent experiments; the standard deviations for total carbohydrate values are shown.
- FIG. 4.
Eng1p glucanase activity is not dependent on rho4+ levels. β-1,3-Glucanase activity was assayed in extracts from wild-type (wt), eng1Δ, rho4Δ, and rho4+-overexpressing cells (A); in extracts from wild-type, rho4Δ, and rho4+-overexpressing cells containing pREP3Xeng1+ in the presence (hatched bars) or absence (black bars) of thiamine (B); and in culture supernatants of wild-type, eng1Δ, and rho4Δ cells grown at 37°C (C). Values are the means from at least three independent experiments. Error bars represent the standard deviations.
- FIG. 5.
Eng1p and Agn1p localizations are defective in rho4Δ cells grown at high temperature. (A and B) Wild-type and rho4Δ cells expressing eng1-GFP (A) or agn1-GFP (B) from their own promoter were grown at 36°C and observed by fluorescence microscopy. Arrows point to rho4Δ cells lacking Eng1-GFP or Agn1-GFP at the septa. DIC, differential interference contrast. (C) Localization of Bgs1-GFP and Bgs4-GFP in rho4Δ cells grown at 37°C. Bar, 10 μm.
- FIG. 6.
Agn1p and Eng1p levels are reduced in the cell walls and culture medium of rho4Δ cells grown at 37°C. Wild-type (wt) and rho4Δ cells containing agn1-GFP (A) or eng1-GFP (B) were analyzed by Western blotting. Proteins from cytoplasm, cell walls, and concentrated culture medium were separated in polyacrylamide gels containing sodium dodecyl sulfate. Gels were blotted and probed with monoclonal anti-GFP antibodies. Cells lacking the GFP tag were included as a negative control (c−). A 80-kDa specific band corresponding to Agn1-GFP and a 130-kDa specific band corresponding to Eng1-GFP are shown. Lower panels represent the loading control of the cytoplasm fraction, using tubulin in panel A or a nonspecific band in panel B.
Tables
- TABLE 1.
Strains
Strain Genotype PPG102 h − leu1-32 PPG371 h − leu1-32 ura4-D18 PPG1541 h − leu1-32 ura4-D18 rho4::kanMX6 YAB14 h − ura4-D18 eng1::kanMX6 PPG1568 h − ura4-D18 eng1::kanMX6 rho4::kanMX6 YAB18 h − leu1-32 eng1-GFP PPG1766 h − leu1-32 eng1-GFP rho4::kanMX6 KGY4398a h − ura4-D18 leu1-32 ade6-M210 agn1-GFP PPG3809 h + ura4-D18 leu1-32 ade6 agn1-GFP rho4::kanMX6 PPG4154 h + bgs1-GFP rho4::kanMX6 ade6-M210 his3-D1 PPG4156 h + bgs4-GFP rho4::kanMX6 ade6-M210 his3-D1 ↵ a Strain KGY4398 is from K. Gould's laboratory.
