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Down-regulation of TORC2-Ypk1 signaling promotes MAPK-independent survival under hyperosmotic stress.

Muir A, Roelants FM, Timmons G, Leskoske KL, Thorner J - Elife (2015)

Bottom Line: Moreover, hyperosmotic conditions block TORC2-dependent Ypk1-mediated Fps1 phosphorylation, causing channel closure, glycerol accumulation, and enhanced survival under hyperosmotic stress.These events are all Hog1-independent.Our findings define the underlying molecular basis of a new mechanism for responding to hypertonic conditions.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
In eukaryotes, exposure to hypertonic conditions activates a MAPK (Hog1 in Saccharomyces cerevisiae and ortholog p38 in human cells). In yeast, intracellular glycerol accumulates to counterbalance the high external osmolarity. To prevent glycerol efflux, Hog1 action impedes the function of the aquaglyceroporin Fps1, in part, by displacing channel co-activators (Rgc1/2). However, Fps1 closes upon hyperosmotic shock even in hog1∆ cells, indicating another mechanism to prevent Fps1-mediated glycerol efflux. In our prior proteome-wide screen, Fps1 was identified as a target of TORC2-dependent protein kinase Ypk1 (Muir et al., 2014). We show here that Fps1 is an authentic Ypk1 substrate and that the open channel state of Fps1 requires phosphorylation by Ypk1. Moreover, hyperosmotic conditions block TORC2-dependent Ypk1-mediated Fps1 phosphorylation, causing channel closure, glycerol accumulation, and enhanced survival under hyperosmotic stress. These events are all Hog1-independent. Our findings define the underlying molecular basis of a new mechanism for responding to hypertonic conditions.

No MeSH data available.


Related in: MedlinePlus

TOR Complex 2 (TORC2)-dependent Ypk1-mediated regulation of Fps1 is independent of Hog1 and Rgc1 and Rgc2.(A) Cultures of Fps1-3xFLAG (yGT21), Fps1570A-3xFLAG (yGT24), Fps13A-3xFLAG (yGT22), Fps1-3xFLAG hog1Δ (yAM275), Fps1570A-3xFLAG hog1Δ (yAM291-A) and Fps13A-3xFLAG hog1Δ (yAM278) strains were adjusted to A600 nm = 1.0 and serial dilutions were then spotted onto YPD plates containing the indicated concentration of arsenite. Cells were allowed to grow for 2 days at 30°C prior to imaging. (B) As in (A), except Fps1IVAA-3xFLAG (yAM308-A), Fps1(3A)IVAA-3xFLAG (yAM309-A), Rgc27A-HA (yAM315) and Fps13A-3xFLAG Rgc27A-HA (yAM318) strains were tested. The Fps1IVAA mutation prevents Hog1 binding to and regulation of Fps1, and Rgc27A cannot be displaced from Fps1 because it cannot be phosphorylated by Hog1; both mutations render the channel constitutively open and make cells arsenite sensitive (Lee et al., 2013). (C) Fps1-3xFLAG (yAM271-A) or Fps13A-3xFLAG (yAM272-A) strains were co-transformed with PMET25-Rgc2-HA (p3151) and PMET25-Fps1-3xFLAG (pAX302) or PMET25-Fps13A -3xFLAG (pAX303) plasmids. After Rgc2-HA and Fps1-3xFLAG expression, Fps1 was immuno-purified with anti-FLAG antibody-coated beads (see ‘Materials and methods’). The bound proteins were resolved by SDS-PAGE and the amount of Rgc2-HA present determined by immunoblotting with anti-HA antibody. (D) Wild-type (BY4741), hog1Δ (YJP544) or Fps13A-3xFLAG hog1Δ (yAM278) strains were grown and serial dilutions of these cultures plated onto synthetic complete medium lacking tryptophan with 2% dextrose and the indicated concentration of sorbitol. Cells were grown for 3 days prior to imaging.DOI:http://dx.doi.org/10.7554/eLife.09336.009
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fig3: TOR Complex 2 (TORC2)-dependent Ypk1-mediated regulation of Fps1 is independent of Hog1 and Rgc1 and Rgc2.(A) Cultures of Fps1-3xFLAG (yGT21), Fps1570A-3xFLAG (yGT24), Fps13A-3xFLAG (yGT22), Fps1-3xFLAG hog1Δ (yAM275), Fps1570A-3xFLAG hog1Δ (yAM291-A) and Fps13A-3xFLAG hog1Δ (yAM278) strains were adjusted to A600 nm = 1.0 and serial dilutions were then spotted onto YPD plates containing the indicated concentration of arsenite. Cells were allowed to grow for 2 days at 30°C prior to imaging. (B) As in (A), except Fps1IVAA-3xFLAG (yAM308-A), Fps1(3A)IVAA-3xFLAG (yAM309-A), Rgc27A-HA (yAM315) and Fps13A-3xFLAG Rgc27A-HA (yAM318) strains were tested. The Fps1IVAA mutation prevents Hog1 binding to and regulation of Fps1, and Rgc27A cannot be displaced from Fps1 because it cannot be phosphorylated by Hog1; both mutations render the channel constitutively open and make cells arsenite sensitive (Lee et al., 2013). (C) Fps1-3xFLAG (yAM271-A) or Fps13A-3xFLAG (yAM272-A) strains were co-transformed with PMET25-Rgc2-HA (p3151) and PMET25-Fps1-3xFLAG (pAX302) or PMET25-Fps13A -3xFLAG (pAX303) plasmids. After Rgc2-HA and Fps1-3xFLAG expression, Fps1 was immuno-purified with anti-FLAG antibody-coated beads (see ‘Materials and methods’). The bound proteins were resolved by SDS-PAGE and the amount of Rgc2-HA present determined by immunoblotting with anti-HA antibody. (D) Wild-type (BY4741), hog1Δ (YJP544) or Fps13A-3xFLAG hog1Δ (yAM278) strains were grown and serial dilutions of these cultures plated onto synthetic complete medium lacking tryptophan with 2% dextrose and the indicated concentration of sorbitol. Cells were grown for 3 days prior to imaging.DOI:http://dx.doi.org/10.7554/eLife.09336.009

Mentions: Fps1 can be negatively regulated by Hog1 via two mechanisms: Hog1 phosphorylation of Fps1 stimulates its internalization and degradation (Thorsen et al., 2006; Mollapour and Piper, 2007); Hog1 phosphorylation closes the channel by displacing bound Fps1 activators (Rgc1 and Rgc2) (Beese et al., 2009; Lee et al., 2013). We found, however, that Fps13A was still in the closed state, as judged by arsenite resistance, in the total absence of Hog1 (hog1Δ) (Figure 3A), or in an Fps1 mutant (Fps1IVAA) that cannot bind Hog1 or where the activator cannot be displaced from Fps1 by Hog1 phosphorylation (Rgc27A) (Lee et al., 2013) (Figure 3B). Thus, closure of the Fps1 channel by lack of Ypk1 phosphorylation occurs independently of any effects requiring Hog1. Consistent with this conclusion, presence or absence of Ypk1-mediated Fps1 phosphorylation had no effect on Fps1-Rgc2 interaction (Figure 3C).10.7554/eLife.09336.009Figure 3.TOR Complex 2 (TORC2)-dependent Ypk1-mediated regulation of Fps1 is independent of Hog1 and Rgc1 and Rgc2.


Down-regulation of TORC2-Ypk1 signaling promotes MAPK-independent survival under hyperosmotic stress.

Muir A, Roelants FM, Timmons G, Leskoske KL, Thorner J - Elife (2015)

TOR Complex 2 (TORC2)-dependent Ypk1-mediated regulation of Fps1 is independent of Hog1 and Rgc1 and Rgc2.(A) Cultures of Fps1-3xFLAG (yGT21), Fps1570A-3xFLAG (yGT24), Fps13A-3xFLAG (yGT22), Fps1-3xFLAG hog1Δ (yAM275), Fps1570A-3xFLAG hog1Δ (yAM291-A) and Fps13A-3xFLAG hog1Δ (yAM278) strains were adjusted to A600 nm = 1.0 and serial dilutions were then spotted onto YPD plates containing the indicated concentration of arsenite. Cells were allowed to grow for 2 days at 30°C prior to imaging. (B) As in (A), except Fps1IVAA-3xFLAG (yAM308-A), Fps1(3A)IVAA-3xFLAG (yAM309-A), Rgc27A-HA (yAM315) and Fps13A-3xFLAG Rgc27A-HA (yAM318) strains were tested. The Fps1IVAA mutation prevents Hog1 binding to and regulation of Fps1, and Rgc27A cannot be displaced from Fps1 because it cannot be phosphorylated by Hog1; both mutations render the channel constitutively open and make cells arsenite sensitive (Lee et al., 2013). (C) Fps1-3xFLAG (yAM271-A) or Fps13A-3xFLAG (yAM272-A) strains were co-transformed with PMET25-Rgc2-HA (p3151) and PMET25-Fps1-3xFLAG (pAX302) or PMET25-Fps13A -3xFLAG (pAX303) plasmids. After Rgc2-HA and Fps1-3xFLAG expression, Fps1 was immuno-purified with anti-FLAG antibody-coated beads (see ‘Materials and methods’). The bound proteins were resolved by SDS-PAGE and the amount of Rgc2-HA present determined by immunoblotting with anti-HA antibody. (D) Wild-type (BY4741), hog1Δ (YJP544) or Fps13A-3xFLAG hog1Δ (yAM278) strains were grown and serial dilutions of these cultures plated onto synthetic complete medium lacking tryptophan with 2% dextrose and the indicated concentration of sorbitol. Cells were grown for 3 days prior to imaging.DOI:http://dx.doi.org/10.7554/eLife.09336.009
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fig3: TOR Complex 2 (TORC2)-dependent Ypk1-mediated regulation of Fps1 is independent of Hog1 and Rgc1 and Rgc2.(A) Cultures of Fps1-3xFLAG (yGT21), Fps1570A-3xFLAG (yGT24), Fps13A-3xFLAG (yGT22), Fps1-3xFLAG hog1Δ (yAM275), Fps1570A-3xFLAG hog1Δ (yAM291-A) and Fps13A-3xFLAG hog1Δ (yAM278) strains were adjusted to A600 nm = 1.0 and serial dilutions were then spotted onto YPD plates containing the indicated concentration of arsenite. Cells were allowed to grow for 2 days at 30°C prior to imaging. (B) As in (A), except Fps1IVAA-3xFLAG (yAM308-A), Fps1(3A)IVAA-3xFLAG (yAM309-A), Rgc27A-HA (yAM315) and Fps13A-3xFLAG Rgc27A-HA (yAM318) strains were tested. The Fps1IVAA mutation prevents Hog1 binding to and regulation of Fps1, and Rgc27A cannot be displaced from Fps1 because it cannot be phosphorylated by Hog1; both mutations render the channel constitutively open and make cells arsenite sensitive (Lee et al., 2013). (C) Fps1-3xFLAG (yAM271-A) or Fps13A-3xFLAG (yAM272-A) strains were co-transformed with PMET25-Rgc2-HA (p3151) and PMET25-Fps1-3xFLAG (pAX302) or PMET25-Fps13A -3xFLAG (pAX303) plasmids. After Rgc2-HA and Fps1-3xFLAG expression, Fps1 was immuno-purified with anti-FLAG antibody-coated beads (see ‘Materials and methods’). The bound proteins were resolved by SDS-PAGE and the amount of Rgc2-HA present determined by immunoblotting with anti-HA antibody. (D) Wild-type (BY4741), hog1Δ (YJP544) or Fps13A-3xFLAG hog1Δ (yAM278) strains were grown and serial dilutions of these cultures plated onto synthetic complete medium lacking tryptophan with 2% dextrose and the indicated concentration of sorbitol. Cells were grown for 3 days prior to imaging.DOI:http://dx.doi.org/10.7554/eLife.09336.009
Mentions: Fps1 can be negatively regulated by Hog1 via two mechanisms: Hog1 phosphorylation of Fps1 stimulates its internalization and degradation (Thorsen et al., 2006; Mollapour and Piper, 2007); Hog1 phosphorylation closes the channel by displacing bound Fps1 activators (Rgc1 and Rgc2) (Beese et al., 2009; Lee et al., 2013). We found, however, that Fps13A was still in the closed state, as judged by arsenite resistance, in the total absence of Hog1 (hog1Δ) (Figure 3A), or in an Fps1 mutant (Fps1IVAA) that cannot bind Hog1 or where the activator cannot be displaced from Fps1 by Hog1 phosphorylation (Rgc27A) (Lee et al., 2013) (Figure 3B). Thus, closure of the Fps1 channel by lack of Ypk1 phosphorylation occurs independently of any effects requiring Hog1. Consistent with this conclusion, presence or absence of Ypk1-mediated Fps1 phosphorylation had no effect on Fps1-Rgc2 interaction (Figure 3C).10.7554/eLife.09336.009Figure 3.TOR Complex 2 (TORC2)-dependent Ypk1-mediated regulation of Fps1 is independent of Hog1 and Rgc1 and Rgc2.

Bottom Line: Moreover, hyperosmotic conditions block TORC2-dependent Ypk1-mediated Fps1 phosphorylation, causing channel closure, glycerol accumulation, and enhanced survival under hyperosmotic stress.These events are all Hog1-independent.Our findings define the underlying molecular basis of a new mechanism for responding to hypertonic conditions.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
In eukaryotes, exposure to hypertonic conditions activates a MAPK (Hog1 in Saccharomyces cerevisiae and ortholog p38 in human cells). In yeast, intracellular glycerol accumulates to counterbalance the high external osmolarity. To prevent glycerol efflux, Hog1 action impedes the function of the aquaglyceroporin Fps1, in part, by displacing channel co-activators (Rgc1/2). However, Fps1 closes upon hyperosmotic shock even in hog1∆ cells, indicating another mechanism to prevent Fps1-mediated glycerol efflux. In our prior proteome-wide screen, Fps1 was identified as a target of TORC2-dependent protein kinase Ypk1 (Muir et al., 2014). We show here that Fps1 is an authentic Ypk1 substrate and that the open channel state of Fps1 requires phosphorylation by Ypk1. Moreover, hyperosmotic conditions block TORC2-dependent Ypk1-mediated Fps1 phosphorylation, causing channel closure, glycerol accumulation, and enhanced survival under hyperosmotic stress. These events are all Hog1-independent. Our findings define the underlying molecular basis of a new mechanism for responding to hypertonic conditions.

No MeSH data available.


Related in: MedlinePlus