Intracellular glycerol levels modulate the activity of Sln1p, a Saccharomyces cerevisiae two-component regulator

Abstract

The HOG mitogen-activated protein kinase pathway mediates the osmotic stress response in Saccharomyces cerevisiae, activating genes like GPD1 (glycerol phosphate dehydrogenase), required for survival under hyperosmotic conditions. Activity of this pathway is regulated by Sln1p, a homolog of the "two-component" histidine kinase family of signal transduction molecules prominent in bacteria. Sln1p also regulates the activity of a Hog1p-independent pathway whose transcriptional output can be monitored using an Mcm1p-dependent lacZ reporter gene. The relationship between the two Sln1p branches is unclear, however, the requirement for unphosphorylated pathway intermediates in Hog1p pathway activation and for phosphorylated intermediates in the activation of the Mcm1p reporter suggests that the two Sln1p branches are reciprocally regulated. To further investigate the signals and molecules involved in modulating Sln1p activity, we have screened for new mutations that elevate the activity of the Mcm1p-dependent lacZ reporter gene. We find that loss of function mutations in FPS1, a gene encoding the major glycerol transporter in yeast activates the reporter in a SLN1-dependent fashion. We propose that elevated intracellular glycerol levels in the fps1 mutant shift Sln1p to the phosphorylated state and trigger the Sln1-dependent activity of the Mcm1 reporter. These observations are consistent with a model in which Sln1p autophosphorylation is triggered by a hypo-osmotic stimulus and indicate that the Sln1p osmosensor is tied generally to osmotic balance, and may not specifically sense an external osmolyte.

Fig. 1. Identification of nrp0831 complementing sequences on chromosome XII

Diagram of the transcriptional orientation and boundaries of the open reading frames included on the genomic library plasmid complementing the nrp0831 mutant. Complementation by the original library clone and two subclones (shown below the map) is shown in the upper part of the table. FPS1 plasmid, pWT1037; SDH2 plus YLL042 plasmid, pWT1039; vector, pRS315. Recessiveness of the nrp0831 mutation is shown in the lower part of the table. β-Galactosidase values are given in Miller units and are the averages of n assays.

Fig. 2. Effect of mutation in the FPS1 gene on transcript levels from the P-lacZ reporter

An equal amount of RNA (5 μg, lanes 1 and 2; 10 μg, lanes 3 and 4; 20 μg, lanes 5 and 6) from wild type (JF1566, lanes 1, 3, and 5) and fps1Δ (JF1825, lanes 2, 4, and 6) strains was subjected to Northern (RNA) hybridization using lacZ and URA3 probes sequentially. Transcript levels were quantitated using PhosphorImage analysis (Molecular Dynamics). lacZ transcript levels were normalized to URA3 levels, to control for loading differences between lanes.

Fig. 3. Mcm1 protein levels are unaffected by mutations in FPS1

Protein extracts prepared from wild type (JF1567, lanes 1–3), nrp0831 (JF1709, lane 4), fps1Δ (JF1732, lane 5), sln1–22 (JF1567, lane 6), and sln1–22 fps1Δ (JF1733, lane 7) strains carrying pGY107, the 2-μm MCM1-Myc plasmid (lanes 1–7) and the wild type strain lacking pGY107 (JF1709, lane 8) were subjected to SDS-polyacrylamide gel electrophoresis in duplicate. One gel was treated with Coomassie Brilliant Blue (not shown) to examine loading and an identical gel was treated with anti-Myc antibody for Western analysis as described in detail under “Experimental Procedures.” A 2-s exposure following chemiluminescence detection is shown. Lanes 1, 2, and 3 were loaded with 0.5, 1, and 2 × protein, respectively; while lanes 4–8 each contained 1 × protein.

Fig. 4. Effect of sorbitol on P-lacZ transcript levels in the fps1 mutant

RNA was harvested from early log phase cells treated with 1.0 M sorbitol or untreated for the times (min) indicated and subjected to Northern hybridization analysis. Levels of lacZ hybridization were quantitated by PhosphorImage analysis (Molecular Dynamics) of the blot. Hybridization of the same blot to the DED1 probe was used for normalization. Values shown below the image were calculated as the ratio of lacZ/DED1 hybridization at a given time divided by the ratio of lacZ/DED1 hybridization at time 0 for a given growth condition.

Fig. 5. Model illustrating the effect of FPS1 deletion on the activities of Sln1 regulated pathways

In the absence of Fps1p, intracellular glycerol levels rise relative to extracellular levels. This differential in osmotic strength may be sensed by the cell as a hypo-osmotic environment. This activates Sln1p kinase activity and the Sln1p phosphorelay pathway and leads to the Ypd1p-dependent phosphorylation of the two receiver molecules, Ssk1p and Skn7p. Phosphorylation inactivates Ssk1, whereas phosphorylation of Skn7 leads to its activation. The phosphorylated form of Skn7 acts either directly or indirectly to activate transcription of the P-lacZ reporter.