glycerol

Rgc1 and Rgc2 serve a redundant role in the regulation of glycerol efflux through the Fps1 glycerol channel

To understand the cause of the cell wall stress in the rgc1/2Δ mutant, we conducted a dosage suppressor screen for high-copy number plasmids that conferred growth at 37°C. A single class of strong suppressor was identified as the FPS1 gene (). FPS1 encodes an aquaglyceroporin that is the major facilitator of glycerol uptake and efflux in yeast ,. This plasma membrane channel protein also mediates uptake of toxic metalloids, such as arsenite and antimonite ,. One interpretation of the suppression result is that the rgc1/2Δ mutant experiences abnormally high turgor pressure from accumulation of glycerol, which yeast cells use as a compatible solute for osmoregulation. Measurement of intracellular glycerol concentration confirmed that the rgc1/2Δ mutant has a 5.9-fold higher glycerol level than wild-type cells under normal growth conditions, a value that is approximately half that of an fps1Δ mutant and approximately equal to that of wild-type cells exposed to hyper-osmotic shock (). To determine if excess intracellular glycerol is responsible for the phenotypic defects of this mutant, we blocked glycerol biosynthesis at the first committed and rate limiting step, glycerol-3-phosphate dehydrogenase (GPD) –. GPD is encoded by the paralogous genes GPD1 and GPD2. Deletion of either GPD1 or GPD2 alone did not suppress the lysis defect of the rgc1/2Δ mutant, but blocking glycerol biosynthesis completely by deletion of both GPD1 and GPD2 allowed growth at 37°C (), confirming that glycerol accumulation is responsible for the cell lysis defect. This also provides an explanation for the fortified cell wall of the rgc1/2Δ mutant as a response to the stress of abnormally high turgor pressure. Consistent with this interpretation, the gpd1/2Δ mutations relieved the cell wall stress signaling observed in the rgc1/2Δ mutant (). Finally, the gpd1/2Δ mutations relieved the caspofungin sensitivity of the rgc1/2Δ mutant ().

Rgc1 and Rgc2 serve a redundant role in the regulation of glycerol efflux through the Fps1 glycerol channel

We considered two possible explanations for the hyper-accumulation of glycerol in the rgc1/2Δ mutant – the mutant either produces excess glycerol, or it is impaired for glycerol efflux through Fps1. These hypotheses generate different predictions for the impact of the rgc1/2Δ mutations on the phenotype of an fps1Δ mutant. If the rgc1/2Δ mutant produces excess glycerol, this should exacerbate the lysis defect of an fps1Δ mutant, which is blocked for glycerol efflux. By contrast, if the rgc1/2Δ mutant is blocked for glycerol efflux through Fps1, loss of the glycerol channel should not result in an additive defect. shows that an fps1Δ mutant displays a temperature-sensitive growth defect that is slightly more severe than that of the rgc1/2Δ mutant, with a semi-permissive growth temperature of 34.5°C. The fps1Δ mutant growth defect at elevated temperature is also the result of cell lysis (data not shown). Significantly, the rgc1/2Δ fps1Δ triple mutant behaves identically to the fps1Δ mutant (), supporting the hypothesis that the rgc1/2Δ mutant is impaired for glycerol efflux through Fps1. To test this directly, we measured export of glycerol from cells exposed to a hypo-osmotic shock, a condition that would induce glycerol efflux through Fps1. We found that glycerol was released from wild-type cells, but not from the rgc1/2Δ mutant or the fps1Δ mutant (), supporting the conclusion that Rgc1 and Rgc2 regulate glycerol efflux through Fps1. The varied initial content of 14C-labeled glycerol among these mutants is a consequence of differential glycerol loading, reflecting the importance of Fps1 for glycerol influx as well as efflux. Finally, strong overexpression of RGC2 failed to suppress the temperature-sensitivity of the fps1Δ mutant (data not shown), thus establishing an epistatic relationship that places RGC1 and RGC2 above FPS1 in a common pathway for glycerol efflux.

Rgc1 and Rgc2 serve a redundant role in the regulation of glycerol efflux through the Fps1 glycerol channel

To determine the cause of the increased steady-state level of Fps1 in the rgc1/2Δ mutant, we conducted a test of Fps1 stability. Fps1 levels were followed in cells in which FPS1 transcription was shut down with the simultaneous inhibition of protein synthesis. We found that Fps1 was stabilized in the rgc1/2Δ mutant relative to wild-type cells (). Therefore, we conclude that increased intracellular glycerol in the rgc1/2Δ mutant, which is caused by a deficiency in Fps1 function, induces an increase in the level of weakly functional Fps1 through protein stabilization.