The yeast H+-ATPase Pma1 promotes Rag/Gtr-dependent TORC1 activation in response to H+-coupled nutrient uptake
- Université Libre de Bruxelles, Biopark, Belgium
- Institut de Recherches Microbiologiques J.-M. Wiame, Belgium
Figures
Figure 1 with 2 supplements
Uptake of β-alanine via the Gap1 permease causes Rag/Gtr-dependent TORC1 activation without increasing the internal pools of amino acids.
(A) Top. Wild-type (w–t) and gap1Δ cells were grown on Gluc Pro medium and [14C]-β-ala (0.5 mM) was added to the medium before measurement of the incorporated radioactivity at various times. Bottom. …
Figure 1—figure supplement 1
Uptake of β-alanine promotes ubiquitylation and downregulation of the inactive Gap1-126 permease via TORC1-dependent stimulation of the Bul arrestins.
(A) gap1Δ BUL2-HA cells expressing Gap1 from a plasmid were grown on Gluc Pro medium and NH4+ (20 mM) or β-ala (0.5 mM) was added to the culture for 4 min.
Rap was added to half of the culture for 30 min before addition of NH4+ or β-ala. Crude cell extracts were immunoblotted with anti-HA and anti-Pgk antibodies. The results show that the Rap-sensitive change in Bul2-HA migration upon addition of NH4+, previously shown to reflect its dephosphorylation and monoubiquitylation (Merhi and AndreAndré, 2012), also occurs upon addition of β-ala. Addition of β-ala thus activates TORC1. (B) w-t, gap1Δ, npi1-1/rsp5, and bul1Δ bul2Δ cells expressing the inactive Gap1-126-GFP from a plasmid, and w-t cells expressing Gap1-126(K9R-K16R)-GFP also from a plasmid, were grown on Gal Pro medium. Glucose was added for 30 min to stop Gap1-126 or Gap1-126(K9R,K16R) neosynthesis prior to β-ala addition (0.5 mM) for 4 min. Crude cell extracts were prepared and immunoblotted with anti-GFP and anti-Pma1 antibodies. The results show that addition of β-ala induces the appearance of the typical two upper bands corresponding to ubiquitylated forms of Gap1. As expected, this ubiquitylation is impaired if the two Ub acceptor lysines (K9 and K16) of the permease are mutated. Ubiquitylation of Gap1-126 is impaired in the gap1Δ, bul1Δ bul2Δ and hypomorphic npi1/rsp5 mutants. Uptake of β-ala via the endogenous Gap1 permease thus promotes Ub-dependent downregulation of the inactive Gap1-126 protein (C) Left. w-t and gap1Δ cells expressing Gap1-126-GFP from a plasmid were grown on Gal Pro medium. Glucose was supplied for 30 min to stop Gap1 neosynthesis. [14C]-β-ala (0.5 mM) was added to the medium before measuring the incorporated radioactivity at various times. The result shows that [14C]-β-ala is incorporated into cells expressing the endogenous Gap1 permease. Right. Fluorescence microscopy analysis of cells in panel C. Cells were grown on Gal Pro medium. Glucose was added for 1.5 hr to stop Gap1 neosynthesis, and β-ala (0.5 mM) was added for 30 min or 1 hr before analysis by fluorescence microscopy. CMAC staining (blue) was used to highlight the vacuole. The result shows that uptake of β-ala by Gap1 elicits endocytosis of the inactive Gap1-126-GFP protein.
Figure 1—figure supplement 2
An anti-(P) T737-Sch9 antibody recognizes phosphorylated Sch9 after NH4+ addition.
w-t cells transformed with pFL38 (URA3) and +Δ cells (JW00035) co-transformed with pCJ366 (TRP1-LEU2-HIS3) and pFL38 (URA3) were grown on Gluc Pro medium (for JW00035 cells, the growth medium was …
Figure 2
Uptake of β-ala via the endogenous Put4 or the heterologous HcGap1 permease also promotes TORC1 activation.
(A) w-t, gap1Δ, and gap1Δ put4Δ cells were grown on Gluc urea medium. [14C]-β-ala (0.1 or 2 mM) was added to the medium before measuring the incorporated radioactivity at various times. (B) Top. …
Figure 3
Uptake of arginine via an endogenous or a heterologous permease stimulates TORC1 even if arginine catabolism is impaired.
(A) Left. w-t, gap1Δ, can1Δ, and gap1Δ can1Δ cells were grown on Gluc Pro medium. [14C]-L-Arg (0.5 mM) was added to the medium before measuring the incorporated radioactivity at various times. …
Figure 4
H+influx coupled to transport promotes Rag/Gtr-dependent stimulation of TORC1.
(A) The β-ala uptake activities of Gap1 (in w-t cells grown on Gluc Pro medium), Put4 (in gap1Δ cells grown on Gluc urea medium), and HcGap1 (in gap1Δ cells expressing HcGap1 and grown on Gluc Pro …
Figure 5 with 1 supplement
H+diffusion via a protonophore promotes Rag/Gtr-dependent stimulation of TORC1.
(A) w-t cells expressing pHluorin (strain ES075) were grown on Gluc Pro medium. The cytosolic pH was monitored for 30 min at regular intervals. FCCP (20 µM) or its solvent (0.2% EtOH) was added at 1 …
Figure 5—figure supplement 1
Addition of the FCCP uncoupler stimulates trehalase activity in N-deprived cells.
w-t cells were incubated for 16 hr in Gluc medium (pH 6.1) devoid of any N source. FCCP (20 μM) or its solvent (0.2% EtOH, control) was added to the cells and trehalase activity was measured in cell …
Figure 6
Inhibition of the vacuolar V-ATPase activates TORC1.
(A) w-t cells expressing pHluorin (strain ES075) were grown on Gluc Pro medium. The cytosolic pH was monitored for 30 min at regular intervals. Concanamycin A (CMA) (1 µM) or its solvent (0.2% EtOH) …
Figure 7 with 2 supplements
TORC1 activation in response to increased cytosolic H+involves the Pma1 H+-ATPase.
(A) GAL1p-PMA1 pma2Δ cells transformed with the YCp(Sc)PMA1, YEp(Np)PMA4882Ochre, or pFL36 (empty) plasmid (-) were grown for 3 days on solid medium with Pro as sole N source and Gal or Gluc as …
Figure 7—figure supplement 1
Pma4882Ochre from tobacco fails to compensate for the lack of Pma1 as regards TORC1 activation in a pma1Δ pma2Δ mutant
(A) Left. The pma1Δ pma2Δ strain co-transformed with the YCp(Sc)PMA1 or YEp(Np)PMA4882Ochre plasmid and a LEU2-HIS3-LYS2 (pCJ315) plasmid were grown on Gluc Pro medium (pH = 6.5) containing 0.00225% …
Figure 7—figure supplement 2
TORC1 is not stimulated by Gln, Leu, or Arg, transported into Pma4882Ochre expressing cells
(A) GAL1p-PMA1 pma2Δ cells co-expressing (Sc)Pma1 or (Np)Pma4882Ochre and HA-Npr1 from plasmids were grown on Gluc NH4+ medium. After a shift to Gluc Pro medium for 3 hr, [14C]-L-Gln (0.012 mM to (Sc…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information | |
|---|---|---|---|---|---|
| Antibody | anti-GFP (mouse monoclonal) | Roche (Belgium) | 11814460001 ; RRID : AB_390913 | (1:10000) | |
| Antibody | anti-Pma1 (rabbit polyclonal) | (De Craene et al., 2001) | RRID : AB_2722567 | (1:5000) | |
| Antibody | anti-HA (12CA5) (mouse monoclonal) | Roche (Belgium) | 11583816001 ; RRID : AB_514506 | (1:5000) | |
| Antibody | anti-Pgk (mouse monoclonal) | Invitrogen/Life technologies (Belgium) | 459250 ; RRID : AB_221541 | (1:10000) | |
| Antibody | anti-(P)T737-Sch9 (rabbit purified polyclonal antibody) | This paper | RRID : AB_2722566 | GeneCust compagny; rabbit purified polyclonal antibody; against CKFAGF(pT)FVDESAIDE; (1:2500) | |
| Antibody | anti-Sch9Total (rabbit polyclonal) | (Prouteau et al., 2017) | (1:20000) | ||
| Antibody | anti-mouse IgG (whole Ab), HRP conjugate (polyclonal) | GE Healthcare/Fisher Scientific (Belgium) | NA931 ; RRID : AB_772210 | (1:10000) | |
| Antibody | anti-rabbit IgG (whole Ab), HRP conjugate (polyclonal) | GE Healthcare/Fisher Scientific (Belgium) | NA934 ; RRID : AB_772206 | (1:10000) | |
| Commercial assay or kit | Glucose Assay Kit | Sigma-Aldrich (Germany) | GAGO20 | Manufacture instructions | |
| Chemical compound, drug | R-5000 Rapamycin | LC Laboratories (U.S.A.) | 53123-88-9 | 200 ng/ml | |
| Chemical compound, drug | CellTracker Blue CMAC Dye | Life technologies (Belgium) | C2110 | ||
| Chemical compound, drug | Lumi-LightPlus Western blotting substrate | Roche (Belgium) | 12015196001 | Manufacture instructions | |
| Chemical compound, drug | Digitonin | Sigma-Aldrich (Germany) | D141 | ||
| Chemical compound, drug | Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) | Sigma-Aldrich (Germany) | C2920 | 20 µM | |
| Chemical compound, drug | Concanamycin A (Folimycin) | Abcam (U.K.) | ab144227 | 1 µM | |
| Chemical compound, drug | Bafilomycin A1 | Cell Signaling Technology (France) | 54645S | 1 µM | |
| Chemical compound, drug | Alanine, β-[1–14C] | Hartmann analytic (Germany) | ARC0183 | ||
| Chemical compound, drug | Arginine, L-[14C(U)] | Perkin-Elmer (Belgium) | NEC267E2 | ||
| Chemical compound, drug | Leucine, L-[14C(U)] | Perkin-Elmer (Belgium) | NEC279E0 | ||
| Chemical compound, drug | Glutamine, L-[14C(U)] | Perkin-Elmer (Belgium) | NEC4510 | ||
| Chemical compound, drug | Fructose, D-[14C(U)] | Hartmann analytic (Germany) | ARC0116 | ||
| Software, algorithm | GraphPad Prism 5 | RRID:SCR_015807 | Statistical analysis and graphs representation | ||
Table 1
Yeast strains used in this study
https://doi.org/10.7554/eLife.31981.015| Strain | Genotype | Reference or source |
|---|---|---|
| 23344 c | ura3 | Laboratory collection |
| EK008 | gap1Δ ura3 | Lab collection |
| ES032 | can1Δ ura3 | (Gournas et al., 2017) |
| ES029 | gap1Δ can1Δ ura3 | (Gournas et al., 2017) |
| MA032 | gap1Δ BUL2-HA ura3 | (Merhi and AndreAndré, 2012) |
| 27038a | npi1-1rsp5 ura3 | (Hein et al., 1995) |
| OS27-1 | bul1Δ bul2Δ ura3 | Lab collection |
| 34210 c | gap1Δ put4Δ ura3 | Lab collection |
| 33007 c | gap1Δ ura3 leu2 | Lab collection |
| 30911b | car1 ura3 | Lab collection |
| CG059 | gap1Δ can1Δ ura3 leu2 | (Gournas et al., 2017) |
| OS26-1 | gtr1Δ gtr2Δ ura3 | Lab collection |
| CEN.PK2-1c | leu2-3,112 ura3-52 trp1-289 his3-Δ1 MAL2-8c SUC2 hxt17Δ | (Wieczorke et al., 1999) |
| EBY.VW4000 | hxt1-17Δ gal2Δ stl1Δ agt1Δ mph2Δ mph3Δ leu2-3,112 ura3-52 trp1-289 his3-Δ1 MAL2-8c SUC2 | (Wieczorke et al., 1999) |
| I3 | EBY.VW4000 URA3::FSY1 | (Rodrigues de Sousa et al., 2004) |
| CG146 | seh1Δ ura3 | This study |
| CG148 | pib2Δ ura3 | This study |
| CG150 | iml1Δ ura3 | This study |
| 35652d | fcy1 ura3 | This study |
| ES075 | uga1::loxP-kanMX-loxP-GPD1p-pHluorin ura3 | This study |
| YPS14-4 | ade 2–101, leu2Δ1, his3-Δ200, ura3–52, trp1Δ63, lys2–801 pma1Δ::HIS3, pma2Δ::TRP1 + YCp-(Sc)PMA1 (LEU2) | (Supply et al., 1993) |
| PMA4–882Oc hre | ade 2–101, leu2Δ1, his3-Δ200, ura3–52, trp1Δ63, lys2–801 pma1Δ::HIS3, pma2Δ::TRP1 + YEp-PMA1p-(Np)PMA4882ochre (LEU2) | (Luo et al., 1999) |
| JW00035 | leu2-3-112 ura3-1 trp1-1 his3-11-15 ade2-1 can1-100 sch9Δ::TRP1 | (Wilms et al., 2017) |
| JX023 | GAL1p-PMA1 pma2Δ ura3 leu2 | This study |
Table 2
Plasmids used in this study
https://doi.org/10.7554/eLife.31981.016| Plasmid | Description | Reference or source |
|---|---|---|
| pFL38 | CEN-ARS (URA3) | (Bonneaud et al., 1991) |
| pFL36 | CEN-ARS (LEU2) | (Bonneaud et al., 1991) |
| p416 GAL1 | CEN-ARS GAL1p (URA3) | (Mumberg et al., 1994) |
| pJOD10 | p416 GAL1p-GAP1-GFP (URA3) | (Nikko et al., 2003) |
| pCJ038 | p416 GAL1p -GAP1(K9R,K16R)-GFP (URA3) | (Lauwers and André, 2006) |
| pMA065 | p416 GAL1p -GAP1-126-GFP (URA3) | (Merhi et al., 2011) |
| pMA091 | p416 GAL1p -GAP1-126-K9R,K16R-GFP (URA3) | (Merhi et al., 2011) |
| pCH500 | CEN-ARS-GAP1 (URA3) | (Hein and André, 1997) |
| pHcGAP1 | YEp-HcGAP1 (URA3) | (Wipf et al., 2002) |
| pES103 | YEp-HA-NPR1 (LEU2) | This study |
| pAS103 | YEp-HA-NPR1 (URA3) | (Schmidt et al., 1998) |
| pCJ315 | CEN-ARS (LEU2-HIS3-LYS2) | Lab collection |
| pMYC008 | YCp-AGP1p-LACZ (HIS3 TRP1 LEU2) | Lab collection |
| pES154 | YCp-AGP1p-LACZ (HIS3 TRP1) | This study |
| pHl-U | YEp-ADH1p-pHluorin (URA3) | (Orij et al., 2009; Zimmermannova et al., 2015) |
| pHl-I | YCp-loxP-kanMX-loxP-GPD1p-pHluorin (URA3) | (Orij et al., 2009; Zimmermannova et al., 2015) |
| pCJ366 | YEp (TRP1-LEU2-HIS3) | Lab collection |
| pPS15-P1 | YCp-(Sc)PMA1 (LEU2) | (Supply et al., 1993) |
| pPMA4882ochre | YEp-PMA1p-(Np)PMA4882ochre (LEU2) | (Luo et al., 1999) |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.31981.017
