Fig 1.
The agp2Δ mutant confers resistance to CHX.
A, spot test analysis to determine CHX sensitivity of yeast strains. BY4741 and agp2Δ strains of OD 600 ~0.6 were serially diluted and spotted onto YPD agar containing increasing concentrations of CHX 0, 0.177, 0.355, 0.71 and 1.77 μM. The plates were grown at 30°C and imaged after 48 hrs. B, Growth rate of cells in the absence and presence of CHX. The results are representative of three independent experiments.
Fig 2.
One-dimensional PAGE analysis of crude plasma membrane extracts showing proteins with differences in expression level between the WT and the agp2Δ mutant.
One-dimensional 8% SDS-PAGE gel analysis of crude plasma membrane extract from the BY4741 (WT) and the agp2Δ strains. Lane 1 WT and lane 2 agp2Δ were extracts prepared from cells without CHX treatment, and lane 3 WT and lane 4 agp2Δ were extracts prepared from cells treated with 3.5 μM of CHX for 15 minutes. An equal amount of protein was loaded on the gel and electrophoresed at 110 V until the dye front exited the gel. The analysis is representative of three independent experiments. Arrows indicated by the following colours red, purple, blue, green, and brown show the difference in protein levels between the WT and the agp2Δ.
Fig 3.
The significant differentially expressed proteins with log2 fold change > = 2 and alpha < = 0.05 are visually represented as k-means clustered, protein-wise data-centered heatmap. Similar samples are clustered into column-based clusters while proteins with similar expression patterns are clustered into row-based clusters. The parameter k = 6 was used and the proteins were enriched into 6 repertoires.
Fig 4.
Clustered bar chart of functional categories.
The 80 significant differentially expressed proteins were broadly categorized based on their functional annotations specified in the Saccharomyces Genome Database (SGD). The proteins highly expressed in WT and agp2Δ mutant are represented as blue and orange bars, respectively. The functional categories of these proteins represent the y-axis with the x-axis being the protein count in each category.
Fig 5.
Volcano-plot of agp2Δ untreated (-) vs. WT untreated (-).
The gene names of significant differentially expressed proteins with log2 fold change > 0.5 and -log10 P-value > 2 are labeled.
Fig 6.
Visual representation of the results of differential testing (agp2Δ mutant versus the WT) for gene expression (RNAseq derived gene dataset: 5744 genes). The data points are colored as described in the legend. The gene symbols of the data points upregulated/downregulated in agp2Δ mutant strain with log2 fold change >2 and scoring top 15 Padj values are labeled.
Fig 7.
Heatmap representing the expression levels of the top 60 significant genes along with their log2 fold changes.
Fig 8.
PDR5 gene expression is controlled by Agp2.
A, RT-PCR analysis showing the upregulation of the PDR5 gene in the agp2Δ mutant. The ACT1 gene was used as a control. B, Quantification of the fold induction of the PDR5 gene in the agp2Δ mutant. C, Promoter region of PDR5 showing the location of the primer pairs for qRT-PCR used in the ChIP analysis. PDRE, Pleiotropic Drug Resistance Element PDRE1 5ʹ-TCCGCGGA-3ʹ and the variant PDRE2 5ʹ-TCCGTGGA-3ʹ. D, Results of the ChIP analysis indicating the enrichment of Agp2 onto the 5’-upstream region, spanning– 440 bp to– 173 bp, of the PDR5 gene. The analysis was repeated twice.
Fig 9.
Cumulative log2 fold change profile of functional categories.
The functional categories of DEGs are represented on the y-axis and the cumulative log2 fold change of all genes categorized into a particular functional category is shown on the x-axis.
Fig 10.
The hypothetical models illustrate the unique roles of the permease Agp2 in response to stress caused by the fungicide CHX.
A, Agp2 is a plasma membrane-bound protein and a fraction is localized to the nucleus in association with the promoter of the PDR5 gene. B, Agp2 can sense CHX, becomes rapidly ubiquitinylated, and triggers its degradation. This scenario would promote the expression of the PDR5 gene. C, In the absence of Agp2, the PDR5 expression is depressed and the cells are resistant to CHX.