Relationship of DFG16 to the Rim101p pH response pathway in Saccharomyces cerevisiae and Candida albicans

Abstract

Many fungal pH responses depend upon conserved Rim101p/PacC transcription factors, which are activated by C-terminal proteolytic processing. The means by which environmental pH is sensed by this pathway are not known. Here, we report a screen of the Saccharomyces cerevisiae viable deletion mutant library that has yielded a new gene required for processed Rim101p accumulation, DFG16. An S. cerevisiae dfg16Delta mutant expresses Rim101p-repressed genes at elevated levels. In addition, Candida albicans dfg16Delta/dfg16Delta mutants are defective in alkaline pH-induced filamentation, and their defect is suppressed by expression of truncated Rim101-405p. Thus, Dfg16p is a functionally conserved Rim101p pathway member. Many proteins required for processed Rim101p accumulation are members of the ESCRT complex, which functions in the formation of multivesicular bodies (MVBs). Staining with the dye FM4-64 indicates that the S. cerevisiae dfg16Delta mutant does not have an MVB defect. We find that two transcripts, PRY1 and ASN1, respond to mutations that affect both the Rim101p and MVB pathways but not to mutations that affect only one pathway. The S. cerevisiae dfg16Delta mutation does not affect PRY1 and ASN1 expression, thus confirming that Dfg16p function is restricted to the Rim101p pathway. Dfg16p is homologous to Aspergillus nidulans PalH, a component of the well-characterized PacC processing pathway. We verify that the previously recognized PalH homolog, Rim21p, also functions in the S. cerevisiae Rim101p pathway. Dfg16p is predicted to have seven membrane-spanning segments and a long hydrophilic C-terminal region, as expected if Dfg16p were a G-protein-coupled receptor.

FIG. 1.

Processing of Ura3-V5-Rim101p and Rim101-HA2p. (A, B) Protein extracts from MATa deletion library S. cerevisiae strains containing a URA3-V5-RIM101 plasmid were analyzed on an anti-V5 immunoblot to visualize the unprocessed and processed forms of the protein. Protein amounts loaded were approximately equal, as determined by Ponceau-S staining, with the exception of panel A, lanes 10, 12, 15, 17, and 19, and panel B, lanes 1, 4, and 6. These lanes were intentionally loaded with two or three times as much protein, as indicated above the lane, in order to detect the epitope. (C) Protein extracts from yeast strains WXY169 (RIM101-HA2 DFG16) and YKB167 (RIM101-HA2 dfg16Δ) were analyzed on an anti-HA immunoblot to visualize the processing of the epitope-tagged Rim101p, expressed from the native RIM101 locus.

FIG. 2.

Comparison of gene expression changes in the rim101Δ strain to dfg16Δ, rim21Δ, and snf7Δ strains. Microarray signals were expressed as log₂ ratios of each S. cerevisiae mutant strain compared to the wild-type strain (see Fig. S1 in the supplemental material), and all 25 Rim101p-responsive transcripts (18) that differed by at least twofold in the comparison of rim101Δ to the wild type reported here were selected. The log₂ expression ratio in each comparison of mutant and wild type is plotted on the ordinate, and the log₂ expression ratio in the comparison of rim101Δ and the wild type is plotted on the abscissa. Mutants include the dfg16Δ strain (A), the rim21Δ strain (B), and the snf7Δ strain (C).

FIG. 3.

Northern blot analysis of Rim101p-repressed genes. Wild-type and mutant S. cerevisiae strains, as indicated above each lane, were grown in YPD medium and then shifted to YPD medium, pH 8, for approximately 4 h before RNA was isolated. Each lane contained 20 μg total RNA; lanes 1 to 10 and 11 to 20 show two different Northern blots prepared in parallel. The blots were probed for NRG1 or SMP1, as indicated on the left of each panel, and then stripped and probed for the loading control, ENO1.

FIG. 4.

Staining of vacuolar and prevacuolar compartments with FM4-64. Wild-type (WT) and mutant S. cerevisiae strains (A to J) and C. albicans strains (K to P), as indicated to the left of the micrographs, were stained with FM4-64. Cells were visualized with visible Nomarski optics (A, C, E, G, I, K, M, and O). FM4-64 fluorescence was visualized for the same fields (B, D, F, H, J, L, N, and P). All images are shown at the same magnification.

FIG. 5.

Northern blot analysis of Snf7p-responsive genes. (A) Wild-type and mutant S. cerevisiae strains, as indicated above each lane, were grown in YPD medium and then shifted to YPD medium, pH 8, for approximately 4 h before RNA was isolated. Each lane contained 20 μg of total RNA; lanes 1 to 10 and 11 to 20 show two different Northern blots prepared in parallel. The blots were probed for PRY1 or ASN1, as indicated on the left of each panel, and then stripped and probed for the loading control, ENO1. (B) Probe intensities relative to the wild type were normalized for loading against the ENO1 signal for the Northern blots in panel A.

FIG. 6.

Filamentation of C. albicans wild-type and mutant strains. Colonies were grown on M199 (pH 8) plates for 3 days at 37°C. The wild-type C. albicans reference strain DAY185 (A) was compared to C. albicans strains with mutations rim101/rim101Δ (D) and dfg16Δ/dfg16Δ (B) and to a dfg16Δ/dfg16Δ strain that had been complemented through integration of HIS1-DFG16 plasmid pKJB026 at the HIS1 locus (C). Both mutant C. albicans strains were transformed with plasmids pRIM101-405 (E, H) and pRIM101 (F, I) integrated into the RIM101 locus and empty vector controls (D, G). All strains in this comparison were prototrophic. All images are shown at the same magnification.