Identification of positive regulators of the yeast fps1 glycerol channel

Abstract

The yeast Fps1 protein is an aquaglyceroporin that functions as the major facilitator of glycerol transport in response to changes in extracellular osmolarity. Although the High Osmolarity Glycerol pathway is thought to have a function in at least basal control of Fps1 activity, its mode of regulation is not understood. We describe the identification of a pair of positive regulators of the Fps1 glycerol channel, Rgc1 (Ypr115w) and Rgc2 (Ask10). An rgc1/2Delta mutant experiences cell wall stress that results from osmotic pressure associated with hyper-accumulation of glycerol. Accumulation of glycerol in the rgc1/2Delta mutant results from a defect in Fps1 activity as evidenced by suppression of the defect through Fps1 overexpression, failure to release glycerol upon hypo-osmotic shock, and resistance to arsenite, a toxic metalloid that enters the cell through Fps1. Regulation of Fps1 by Rgc1/2 appears to be indirect; however, evidence is presented supporting the view that Rgc1/2 regulate Fps1 channel activity, rather than its expression, folding, or localization. Rgc2 was phosphorylated in response to stresses that lead to regulation of Fps1. This stress-induced phosphorylation was partially dependent on the Hog1 MAPK. Hog1 was also required for basal phosphorylation of Rgc2, suggesting a mechanism by which Hog1 may regulate Fps1 indirectly.

Figure 1. The rgc1/2Δ mutant displays an osmotic-remedial cell lysis defect.

(A) Temperature sensitive cell lysis defect of the rgc1/2Δ mutant. Diploid yeast strains were streaked onto YPD plates with or without 10% sorbitol for osmotic support and incubated at 37°C for 3 days. Strains were wild-type (DL3193), rgc2Δ (DL3181), rgc1Δ (DL3203), and rgc1/2Δ (DL3209). (B) The PH domain of Rgc2 is important for its function. Yeast strain DL3209 was transformed with pUT36, pUT36 MET25-rgc2 (1–420)-His₆ (p2808), or pUT36 MET25-rgc2 (1–720)-His₆ (p2809). Transformants were streaked onto YPD plates and incubated at 37°C for 3 days. The schematic shows the PH domain relative to the truncations tested.

Figure 2. The rgc1/2Δ mutant experiences cell wall stress.

(A) The rgc1/2Δ mutant is Zymolyase resistant. The strains from Figure 1A were grown to mid-log phase in YPD at 30°C, washed and resuspended in water to an initial density of A₆₀₀ ∼ 0.7 prior to treatment with Zymolyase 20T (150 µg/ml). Cell lysis was assessed by A₆₀₀ measurements at the indicated times. (B,C). The rgc1/2Δ mutant is under constitutive cell wall stress. (B) Wild-type yeast (DL3193), a rgc1/2Δ mutant (DL3209), and a rgc1/2Δ gpd1/2Δ mutant (DL3251) were transformed with a PRM5-lacZ (p1366) reporter plasmid to measure cell wall stress signaling. Transformants were grown to mid-log phase in selective medium at 23°C, followed by cell lysis and measurement of β-galactosidase activity. Each value represents the mean and standard deviation from three independent transformants. (C) Protein extracts from the strains in (B) were separated by SDS-PAGE and processed for immunoblot detection of activated Mpk1 (phospho-Mpk1) and total Mpk1 as a measure of cell wall stress signaling. (D) The rgc1/2Δ mutant is sensitized to caspofungin. The strains in (B) were grown to mid-log phase in YPD and 10-fold serial dilutions were spotted onto YPD plates with or without caspofungin (5 ng/ml) prior to incubation at 30°C for 3 days.

Figure 3. The rgc1/2Δ mutant is defective for glycerol efflux through Fps1.

(A) Suppression of the cell lysis defect of the rgc1/2Δ mutant by overexpressed FPS1. A rgc1/2Δ mutant (DL3209) was transformed with centromeric, or high-copy plasmids bearing FPS1 (pRS316-FPS1 or pRS202-FPS1, respectively), or vector (pRS316). Transformants were streaked onto a YPD plate and incubated for 3 days at 39°C. (B) Intracellular glycerol concentrations in wild-type (DL3193), rgc1/2Δ (DL3209), and fps1Δ (DL3234) strains. Cultures were grown to mid-log phase in YPD, diluted into YPD with or without sorbitol (to 1.8M) to induce hyper-osmotic shock (15 minutes). Each value represents the mean and standard deviation from three independent experiments. (C) The cell lysis defect of the rgc1/2Δ mutant is suppressed by blocking glycerol biosynthesis. Diploid yeast strains were streaked onto a YPD plate and incubated at 37°C for 3 days. Strains were: rgc1/2Δ (DL3209), rgc1/2Δ gpd1Δ (DL3237), rgc1/2Δ gpd2Δ (DL3254) and rgc1/2Δ gpd1/2Δ (DL3251). (D) The cell lysis defect of the rgc1/2Δ mutant is not additive with that of an fps1Δ mutant. Diploid yeast strains were streaked onto YPD plates and incubated for 3 days at the indicated temperatures. Strains were: wild-type (DL3193), rgc1/2Δ (DL3209), fps1Δ (DL3234), and rgc1/2Δ fps1Δ (DL3245). (E) The rgc1/2Δ mutant is blocked for glycerol efflux. Cells were pre-loaded with ¹⁴C-labelled glycerol (MES buffer with 300 mM glycerol), followed by hypo-osmotic shock (into MES buffer) for the indicated times. Strains were wild-type (DL3193), rgc1/2Δ (DL3209), and fps1Δ (DL3234). Each value represents the mean and standard deviation from three independent experiments.

Figure 4. The rgc1/2Δ mutant retains elevated levels of the Fps1 protein resulting from excess intracellular glycerol.

(A) Yeast strains were transformed with a plasmid expressing Fps1-FLAG under the control of the MET25 promoter (p2492), or empty vector. Extracts were prepared from cultures grown in selective medium, protein was separated by SDS-PAGE, and Fps1-FLAG was detected by immunoblot analysis using a mouse monoclonal α-FLAG antibody. Strains were: wild-type (DL3193), rgc1/2Δ (DL3209), and rgc1/2Δ gpd1/2Δ (DL3251). (B) FPS1-lacZ transcription is not altered in the rgc1/2Δ mutant. An FPS1-lacZ reporter plasmid (p2213) was transformed into a wild-type strain (DL3193) and a rgc1/2Δ mutant (DL3209). Transformants were grown to mid-log phase in selective medium at 30°C, followed by cell lysis and measurement of β-galactosidase activity. Each value represents the mean and standard deviation from three independent transformants. (C) A greater increase in Fps1 protein levels is seen when FPS1 is expressed from its native promoter. Yeast strains were transformed with a 2-micron plasmid expressing Fps1-Myc under the control of its own promoter (p2184), or empty vector. Extracts were treated as above, and Fps1-Myc was detected by immunoblot analysis using a mouse monoclonal α-Myc antibody. Strains were: wild-type (DL3193) and rgc1/2Δ (DL3209).

Figure 5. Localization of Rgc2.

(A) Rgc2-GFP₂ re-localizes from uniform cytoplasmic distribution to punctate spots near the cell periphery in response to hypo-osmotic shock. Wild-type diploid yeast cells (DL3193), transformed with a plasmid expressing Rgc2-GFP₂ (p2481), were grown to mid-log phase in SD medium, centrifuged briefly, and resuspended in distilled water to induce hypo-osmotic shock. Shocked cells were mounted for fluorescence microscopy and photographed within 10 seconds of shock. (B) Dissipation of Rgc2-GFP₂ spots after hypo-osmotic shock. Cells were treated as in (A) and photographed at the indicated times after shock. (C) The punctate spots of Rgc2-GFP₂ do not co-localize with those of Fps1-tdTomato. Wild-type cells (DL3193), co-transformed with p2481 and a plasmid expressing Fps1-tdTomato (p2489), were subjected to hypo-osmotic shock for 10 seconds and examined for co-localization of Rgc2 with Fps1.

Figure 6. Growth in the presence of 1M xylitol as a test of open channel behavior of fps1 alleles.

An rgc1/2Δ mutant that was also blocked for glycerol production (gpd1/2Δ; DL3246) was co-transformed with multi-copy plasmids bearing wild-type or the indicated open channel alleles of FPS1 and a centromeric plasmid bearing RGC1 (p2627), or a vector control (pRS313). Transformants were grown to mid-log phase in selective medium and 10-fold serial dilutions were spotted onto YPD plates with or without 1M xylitol prior to incubation at 30°C for 3 days or 2 days, respectively.

Figure 7. Mutants blocked for Fps1 function are resistant to arsenite toxicity.

(A) Diploid yeast strains were streaked onto YPD plates with the indicated concentration of arsenite and incubated at 30°C for 3 days. Diploid yeast strains were wild-type (DL3193), rgc1Δ (DL3203), rgc2Δ (DL3181), and rgc1/2Δ (DL3209). (B) The rgc1/2Δ mutations suppress the arsenite hyper-sensitivity of a hog1Δ mutant. Haploid yeast strains were wild-type (DL3187), fps1Δ (DL3226), rgc1/2Δ (DL3207), hog1Δ (DL3158), and rgc1/2Δ hog1Δ (DL3219).

Figure 8. Stress-induced phosphorylation of Rgc2.

(A) Stresses induce a band-shift in Rgc2 that is only partially dependent on Hog1. Wild-type (DL3187) or hog1Δ (DL3158) cells, transformed either with a plasmid that expresses Rgc2-His₆ (p2501), or vector control (V, pUT36), were treated with stresses that cause Fps1 opening (hypo-osmotic shock; H₂O), or closure (hyper-osmotic shock, or arsenite). Hypo-osmotic shock and hyper-osmotic (1.8M sorbitol) shock were for 1 minute, and arsenite (As) treatment was for 1 hour. Protein extracts were prepared and separated by SDS-PAGE for immunoblot detection of Rgc2-His₆. (B) The unstressed samples from (A) were run side-by-side to illustrate the Hog1-dependent band-shift of Rgc2-His₆. (C) Rgc2 band-shifts are caused by phosphorylation. Rgc2-His₆ was immuneprecipitated from extracts of wild-type (DL3187) cells treated as above, and subjected to calf intestinal phosphatase (CIP) treatment in the presence or absence of phosphatase inhibitor (Na₃VO₄). Immuneprecipitates were processed for immunoblot detection of Rgc2-His₆.