Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32

Abstract

Fine-tuning the plasma-membrane permeability to essential nutrients is fundamental to cell growth optimization. Nutritional signals including nitrogen availability are integrated by the TORC1 complex which notably regulates arrestin-mediated endocytosis of amino-acid transporters. Ammonium is a ubiquitous compound playing key physiological roles in many, if not all, organisms. In yeast, it is a preferred nitrogen source transported by three Mep proteins which are orthologues of the mammalian Rhesus factors. By combining genetic, kinetic, biochemical and cell microscopy analyses, the current study reveals a novel mechanism enabling TORC1 to regulate the inherent activity of ammonium transport proteins, independently of arrestin-mediated endocytosis, identifying the still functional orphan Amu1/Par32 as a selective regulator intermediate. We show that, under poor nitrogen supply, the TORC1 effector kinase' Npr1' promotes phosphorylation of Amu1/Par32 which appears mainly cytosolic while ammonium transport proteins are active. Upon preferred nitrogen supplementation, like glutamine or ammonium addition, TORC1 upregulation enables Npr1 inhibition and Amu1/Par32 dephosphorylation. In these conditions, as in Npr1-lacking cells, hypophosphorylated Amu1/Par32 accumulates at the cell surface and mediates the inhibition of specific ammonium transport proteins. We show that the integrity of a conserved repeated motif of Amu1/Par32 is required for the interaction with these transport proteins. This study underscores the diversity of strategies enabling TORC1-Npr1 to selectively monitor cell permeability to nutrients by discriminating between transporters to be degraded or transiently inactivated and kept stable at the plasma membrane. This study further identifies the function of Amu1/Par32 in acute control of ammonium transport in response to variations in nitrogen availability.

Fig 1. Npr1 is required for Mep1 and Mep3 inherent transport activity.

(a) Immunodetection of Mep1 and Mep3 from membrane-enriched cell extracts. Wild-type (23344c) and npr1Δ (30788a) cells were grown in the presence of proline (0.1%) as nitrogen source. The plasma membrane proton ATPase Pma1 was detected as a loading control. (b) Immunodetection of Mep1 and Mep3 from membrane-enriched extracts treated (+) or not (-) with alkaline phosphatase (ALP). Wild-type (23344c) and npr1Δ (30788a) cells were grown in the presence of proline (0.1%) as nitrogen source. (c) Mep1-GFP and Mep3-GFP localization was observed by fluorescence microscopy in triple-mepΔ (31019b) and triple-mepΔ npr1-1 (31052c) cells transformed with the pGAL1Mep1-GFP or pGAL1Mep3-GFP low-copy-number vectors and grown in the presence of proline (0.1%), galactose (3%) and glucose (0.3%). (d) [¹⁴C]-methylammonium (0.5 mM) accumulation in proline-grown triple-mepΔ (31019b,│,□) and triple-mepΔ npr1 ^ts (MB063,▲,Δ) cells transformed with YCpMep1 after transfer in a similar medium preheated at 29°C (│,▲) or 37°C (□,Δ). (e) Immunodetection of Mep1 from membrane-enriched extracts of cells collected after the temperature shift as described in Fig 1d.

Fig 2. Mep1 and Mep3 overproduction suppresses Npr1 requirement.

(a) Growth tests on solid medium containing, as the sole nitrogen source, ammonium 3 mM (Am3) or glutamate 0.1% (Glt, positive growth control). Wild-type (23344c), npr1Δ (30788a), mep2Δ mep3Δ (31018b), mep2Δ mep3Δ npr1-1 (31059b), mep1Δ mep3Δ (31022a), mep1Δ mep3Δ npr1-1 (31059d), mep1Δ mep2Δ (31021c) and mep1Δ mep2Δ npr1-1 (31045c) cells were transformed with the empty vector pFL38. Triple-mepΔ (31019b) and triple-mepΔ npr1-1 (31052c) cells were transformed with the empty vector pFL38 and with the high-copy-number plasmids: YEpMep1, YEpMep2 and YEpMep3. (b) Immunodetection of Mep1, Mep2 or Mep3 from membrane-enriched extracts. In the case of Mep2, the cellular extracts were treated with N-glycosidase F. Cells were grown in the presence of proline (0.1%) as nitrogen source. The plasma membrane proton ATPase Pma1 was detected as a loading control.

Fig 3. The amu1 suppressor mutation restores growth of Npr1-lacking cells on ammonium.

Growth tests on solid medium containing, as the sole nitrogen source, ammonium 1 mM (Am1) or glutamate 0.1% (Glt, positive growth control), supplemented or not with methylammonium 50 mM. Wild-type (23344c), npr1Δ (30788a), npr1-1 (21994b), amu1Δ (MB139) and amu1Δ npr1Δ (36314b) cells were transformed with the empty vector pFL38. amu1-1 npr1-1 (31034c) cells were transformed with the empty vector pFL38 and YCpAmu1.

Fig 4. Amu1 is only conserved in fungi and contains a four-fold repeated motif.

Sequences were retrieved from the UniProt database [38] primarily by a BlastP search [39] and secondly by a FuzzPro search [40] using the following searching pattern, G-R-G-G-X-[AG]-N-X(30,110)-G-R-G-G-X-[AG]-N. A selection of Amu1/Par32 orthologues is displayed in the alignment; the aligned sequences being denoted by their accession numbers: PAR32_YEAST, Saccharomyces cerevisiae, Q59N81_CANAL, Candida albicans, F5HGC0_CRYNB, Crytococcus neoformans, Q6CMJ9_KLULA, Kluyveromyces lactis, Q4PFY6_USTMA, Ustilago maydis and C8VIZ6_EMENI, Aspergillus nidulans. The multiple sequence alignment was automatically generated by ClustalW [41] and manually adjusted using BioEdit [42]. The repeated motif characterizing the Amu1 protein family is highlighted in yellow.

Fig 5. The amu1 mutation restores the function of Mep1 and Mep3 in the absence of the Npr1 kinase.

Growth tests on solid medium containing, as the sole nitrogen source, ammonium 1 mM (Am1) or glutamate 0.1% (Glt, positive growth control). Wild-type (23344c), npr1Δ (30788a), amu1Δ (MB139), amu1-1 npr1-1 (31034c), triple-mepΔ (31019b), triple-mepΔ npr1-1 (31052c), triple-mepΔ amu1Δ (AM114), triple-mepΔ amu1Δ npr1-1 (31087c), mep2Δ mep3Δ (31018b), mep2Δ mep3Δ npr1-1 (31059b), mep2Δ mep3Δ amu1Δ (PVV003), mep2Δ mep3Δ amu1Δ npr1-1 (PVV001), mep1Δ mep3Δ (31022a), mep1Δ mep3Δ npr1-1 (31059d), mep1Δ mep3Δ amu1Δ (PVV007), mep1Δ mep3Δ amu1Δ npr1-1 (PVV005), mep1Δ mep2Δ (31021c), mep1Δ mep2Δ npr1-1 (31045c), mep1Δ mep2Δ amu1Δ (PVV011) and mep1Δ mep2Δ amu1Δ npr1-1 (PVV009) cells.

Fig 6. Amu1 phosphorylation is controlled by nitrogen supply, the Npr1 kinase and TORC1.

Immunodetection of Amu1-3HA from total cellular extracts. (a-b) AMU1-3HA (MB142) and AMU1-3HA npr1Δ (36307b) cells were grown in the presence of proline (0.1%) as nitrogen source. (b) Total cell extracts were treated (+) or not (-) with alkaline phosphatase (ALP). (c) AMU1-3HA (MB142) and AMU1-3HA npr1Δ (36307b) cells were grown in the presence of proline (0.1%, Pro), urea (0.1%), ammonium (5 or 20 mM, Am), glutamate (0.1%, Glt) or glutamine (0.1%, Gln) as nitrogen sources. (d) AMU1-3HA (MB142) and AMU1-3HA npr1Δ (36307b) cells were grown in the presence of ammonium (20 mM) as nitrogen source. At time t = 0, rapamycin or the drug vehicle alone (-, control) was added to the cell culture. (e) AMU1-3HA (MB142) cells were grown in the presence of ammonium (20 mM) as nitrogen source. At time t = 0, cells were transferred in a proline-medium or in a similar ammonium-medium (control). (f) AMU1-3HA (MB142) cells were grown in the presence of proline (0.1%) as nitrogen source. At time t = 0, ammonium (20 mM) was added to the cell culture. (g) AMU1-3HA (MB142), AMU1-3HA npr1Δ (36307b), AMU1-3HA psr1Δ (PVV152), AMU1-3HA psr2Δ (PVV158), AMU1-3HA psr1Δ psr2Δ (PVV160) cells were grown in the presence of proline (0.1%) as nitrogen source. At time t = 0, ammonium (20 mM) was added to the cell culture.

Fig 7. Amu1 localization is controlled by TORC1 and Npr1.

(a-b) Amu1-GFP and Mep1-mCherry localizations were observed by fluorescence microscopy. MEP1-mCherry (MB321) or MEP1-mCherry npr1Δ (MB325) cells were transformed with YCpAmu1-GFP and grown in the presence of proline (0.1%). (b) MEP1-mCherry (MB321) cells transformed with YCpAmu1-GFP were grown in the presence of proline (0.1%) as nitrogen source. At time t = 0, ammonium (Am, 20mM) or glutamine (Gln, 0.1%) was added to the cell culture during 1h (2^nd and 3^rd columns). Rapamycin or the drug vehicle alone (control, -) was added to the proline-grown cells. 1h later, ammonium (Am, 20 mM) was added to the cell cultures during 1h (4^th and 5^th columns). (c) Immunodetection of Amu1-GFP from total cellular extracts of amu1Δ (MB139) cells transformed with YCpAmu1-GFP. Rapamycin or the drug vehicle alone (control, -) was added to proline-grown cells. 1h later, ammonium (Am, 20 mM) was added to the cell cultures during 1h.

Fig 8. The phosphorylation state of Amu1 governs its localization and the integrity of the conserved motif is required for appropriate localization and inhibition function of the protein.

(a) Immunodetection of Amu1-3HA from total extracts of proline (0,1%)-grown cells. amu1Δ (MB139) and amu1Δ npr1Δ (36314b) were transformed with YCpAmu1-3HA, YCpAmu1^4R-A-3HA, and YCpAmu1^phos-3HA. The plasma membrane proton ATPase Pma1 was detected as a loading control. (b) Amu1-GFP, Amu1^4R-A-GFP and Amu1^phos-GFP localization was observed by fluorescence microscopy. amu1Δ (MB139) or amu1 npr1 (31034c or 36314b) cells were transformed with the corresponding YCpAmu1-GFP plasmid and grown in the presence of proline (0.1%). (c) Growth tests on solid medium containing, as the sole nitrogen source, ammonium 1 mM (Am1) or glutamate 0.1% (Glt, positive growth control), supplemented or not with methylammonium 50 mM (Mea50). Wild-type (23344c) and npr1Δ (30788a) were transformed with the empty vector pFL38. amu1Δ (MB139) and amu1Δ npr1Δ (36314b) cells were transformed with the empty vector pFL38, with the low-copy-number plasmids: YCpAmu1-HA, YCpAmu1^4R-A-HA, YCpAmu1^phos-HA, or with the high-copy-number plasmids: YEpAmu1-HA, YEpAmu1^4R-A-HA, YEpAmu1^phos-HA.

Fig 9. Amu1 interacts with Mep1 and Mep3 in vitro.

After spheroplasting, the cells were lysed and Amu1-3HA was immunoprecipitated with anti-HA. Immunoblots of the lysates and immunoprecipitates (IP) with anti-HA and anti-GFP antibodies are shown. (a) Wt (23344c, negative control)) and AMU1-3HA (MB142) cells transformed with pGAL1Mep1-GFP, pGAL1Mep2^N4Q-GFP or pGAL1Mep3-GFP were grown in the presence of proline (0.1%). Glutamine (gln, 0.1%) was added to AMU1-3HA (MB142) cells transformed with one of the 3 pGAL1Mep-GFP vectors (30 min). (b) Triple-mepΔ (31019b) cells transformed with pGAL1Mep1-GFP (negative control) and, triple-mepΔ amu1Δ (MB310) cells transformed with both pGAL1Mep1-GFP (LEU2) and YEpAmu1-HA (URA3) or with both pGAL1Mep1-GFP (LEU2) and YEpAmu1^4R-A-HA (URA3) were grown in the presence of proline (0.1%).

Fig 10. Three different strategies enable the TORC1-pathway to regulate plasma-membrane transport proteins.

Under non-preferred nitrogen supply, TORC1 is poorly active and Npr1 is hypophosphorylated and able to mediate the phosphorylation of its different targets. Npr1 mediates the phosphorylation of Amu1 which remains cytosolic, while Mep1 and Mep3 are kept active. Npr1 enables C-terminal phosphorylation of the Mep2 ammonium transport protein, thereby silencing an autoinhibitory domain and allowing Mep2 activity. Npr1 mediates phosphorylation of arrestin-like adaptors thereby protecting their amino acid permease (AAP) targets from endocytosis and vacuolar degradation. Under preferred nitrogen supply, TORC1 is upregulated, Npr1 is hyperphosphorylated and inhibited. In these conditions, Amu1 is dephosphorylated, and accumulates at the cell surface, physically interacts with Mep1 and Mep3, and mediates inhibition of ammonium transport. The non-phosphorylated autoinhibitory domain of Mep2 prevents the enhancer domain to activate the transport protein. Dephosphorylated arrestin-like adaptors recruit the Rsp5 ubiquitin-ligase to their AAP targets, which are ubiquitylated, endocytosed and degraded in the vacuole.