Stress conditions promote yeast Gap1 permease ubiquitylation and down-regulation via the arrestin-like Bul and Aly proteins

Abstract

Gap1, the yeast general amino acid permease, is a convenient model for studying how the intracellular traffic of membrane transporters is regulated. Present at the plasma membrane under poor nitrogen supply conditions, it undergoes ubiquitylation, endocytosis, and degradation upon activation of the TORC1 kinase complex in response to an increase in internal amino acids. This down-regulation is stimulated by TORC1-dependent phosphoinhibition of the Npr1 kinase, resulting in activation by dephosphorylation of the arrestin-like Bul1 and Bul2 adaptors recruiting the Rsp5 ubiquitin ligase to Gap1. We report here that Gap1 is also down-regulated when cells are treated with the TORC1 inhibitor rapamycin or subjected to various stresses and that a lack of the Tco89 subunit of TORC1 causes constitutive Gap1 down-regulation. Both the Bul1 and Bul2 and the Aly1 and Aly2 arrestin-like adaptors of Rsp5 promote this down-regulation without undergoing dephosphorylation. Furthermore, they act via the C-terminal regions of Gap1 not involved in ubiquitylation in response to internal amino acids, whereas a Gap1 mutant altered in the N-terminal tail and resistant to ubiquitylation by internal amino acids is efficiently down-regulated under stress via the Bul and Aly adaptors. Although the Bul proteins mediate Gap1 ubiquitylation of two possible lysines, Lys-9 and Lys-16, the Aly proteins promote ubiquitylation of the Lys-16 residue only. This stress-induced pathway of Gap1 down-regulation targets other permeases as well, and it likely allows cells facing adverse conditions to retrieve amino acids from permease degradation.

Keywords: Amino Acid Transport; Arrestin; Endocytosis; Stress; TOR Complex (TORC); Ubiquitin; Yeast.

FIGURE 1.

Model for ammonium-induced ubiquitylation of the Gap1 permease. Left, on a poor nitrogen source, the Npr1 kinase is active and phosphorylates the Bul adaptors. These bind to 14-3-3 proteins and are thus inhibited. Hence, Gap1 is not ubiquitylated. Right, upon uptake of ammonium (or amino acids) into the cells, the internal concentration of amino acids increases. This leads to stimulation of the TORC1 kinase complex. Active TORC1 inhibits by phosphorylation the Npr1 kinase and somehow stimulates the Sit4 phosphatase. The Bul adaptors thus undergo dephosphorylation, dissociate from the 14-3-3 proteins, and recruit the Rsp5 Ub ligase to the Gap1 permease, which is ubiquitylated. Npr1 inactivation also induces Rsp5-dependent monoubiquitylation of the Bul proteins, but the role of this modification remains unknown.

FIGURE 2.

Rapamycin triggers ubiquitylation and endocytosis of Gap1. A, strain EK008 (gap1Δ ura3) transformed with the pJOD10 (YCpGAL-GAP1-GFP) plasmid was grown on galactose/proline medium. Glucose was added for 2 h to repress Gap1 synthesis, and when indicated, Am (20 mm) and/or Rap (200 ng/ml) was/were added for 2 h. Gap1-GFP localization was examined by fluorescence microscopy. Cells were labeled with FM4-64 to stain the vacuolar membrane. B, strain EK008 (gap1Δ ura3) transformed with pJOD10 (YCpGAL-GAP1-GFP) or pCJ038 (YCpGAL-GAP1^K9R,K16R-GFP) was grown on galactose/proline medium. Cells were collected before and 15 and 30 min after Rap (200 ng/ml) addition. Crude cell extracts were immunoblotted with anti-GFP antibodies. C, strains EK008 (gap1Δ ura3) and CJ005 (gap1Δ rsp5(npi1) ura3) were transformed with pJOD10 (YCpGAL-GAP1-GFP) or pCJ038 (YCpGAL-GAP1^K9R,K16R-GFP). Growth conditions were as in A. Gap1-GFP localization was examined by fluorescence microscopy. DIC, differential interference contrast.

FIGURE 3.

Bul1, Bul2, Aly1, and Aly2 arrestin-like adaptors contribute to rapamycin-induced ubiquitylation and down-regulation of Gap1. A, strain JA493 (gap1Δ bul1Δ bul2Δ ura3) transformed with pJOD10 (YCpGAL-GAP1-GFP) was grown on galactose/proline medium. Glucose was added for 2 h to repress Gap1 synthesis, and when indicated, Am (20 mm) or Rap (200 ng/ml) was added for 2 h. Gap1-GFP localization was examined by fluorescence microscopy. B, strains MA056 (gap1Δ aly1Δ ura3), MA059 (gap1Δ aly2Δ ura3), 35101a (gap1Δ aly1Δ aly2Δ ura3), and MA062 (gap1Δ bul1Δ bul2Δ aly1Δ aly2Δ ura3) transformed with pJOD10 (YCpGAL-GAP1-GFP) were grown on galactose/proline medium. Glucose was added for 2 h to repress Gap1 synthesis, and when indicated, Rap (200 ng/ml) was added for 2 h. Gap1-GFP localization was examined by fluorescence microscopy. C, strains EK008 (gap1Δ ura3), JA493 (gap1Δ bul1Δ bul2Δ ura3), 35101a (gap1Δ aly1Δ aly2Δ ura3), and MA062 (gap1Δ bul1Δ bul2Δ aly1Δ aly2Δ ura3) transformed with the pJOD10 (YCpGAL-GAP1-GFP) plasmid were grown on galactose/proline medium. Cells were collected before and 10 min after Rap (200 ng/ml) addition. Crude cell extracts were immunoblotted with anti-GFP antibodies. DIC, differential interference contrast.

FIGURE 4.

Bul1, Bul2, Aly1, and Aly2 remain phosphorylated and Bul2 still binds to the 14-3-3 protein Bmh2 after rapamycin addition. A, strains MA025 (gap1Δ BUL1-FLAG bul2Δ ura3) and MA032 (gap1Δ BUL2-HA ura3) transformed with pJOD10 (YCpGAL-GAP1-GFP) were grown on galactose/proline medium. Cells were collected before and 30 min after Am (50 mm) or Rap (200 ng/ml) addition. Crude cell extracts were immunoblotted with anti-FLAG or anti-HA antibodies. B, strain 23344c (ura3) transformed with pFL38 (URA3) was grown on glucose/proline medium. Cells were collected before and 30, 60, and 120 min after Rap (200 ng/ml) addition. Crude cell extracts were immunoblotted with anti-Bmh antibodies. C, strain MA032 (gap1Δ BUL2-HA ura3) transformed with pRS426-GST or pRS426-GST-BMH2 was grown on glucose/proline medium. Cells were collected before and 30 or 60 min after Am (50 mm) or Rap (200 ng/ml) addition, respectively. The cells were lysed, and GST was pulled down as described under “Experimental Procedures.” Lysates and pulldown fractions were immunoblotted with anti-GST or anti-HA antibodies. Two exposure times (1 and 2 min) are shown. D, strains JA986 (ALY1-FLAG ura3) and JA997 (ALY2-HA ura3) transformed with pFL38 (URA3) were grown on glucose/proline medium. Cells were collected before and 30 and 60 min after Rap (200 ng/ml) addition. Crude cell extracts were immunoblotted with anti-FLAG or anti-HA antibodies. E, strains and growth conditions were the same as those in D. Crude cell extracts were treated or not with alkaline phosphatase and immunoblotted with anti-FLAG or anti-HA antibodies.

FIGURE 5.

Schematic topology model of the Gap1 protein. Residues shown in gray in the N-terminal tail (NT), C-terminal tail (CT), and intracellular loops (L2, L4, L6, L8, and L10) are those which, when replaced with alanines, cause retention of Gap1 in the ER. Each number close to colored residues corresponds to the Gap1 mutant where these residues have been replaced with alanines. Residues labeled in green in the NT correspond to the ubiquitin-acceptor lysines (Lys-9 and Lys-16). The NT region between brackets and including the four amino acids replaced in the Gap1-112 mutant (red) is the region required for down-regulation mediated by the dephosphorylated Bul proteins in response to internal amino acids. Residues shown in blue identify the Gap1-132 and Gap1-134 mutants resistant to down-regulation mediated by the phosphorylated Bul proteins under stress or in the presence of rapamycin. Residues in brown (Gap1-108) and orange (Gap1-103); Gap1-134; Gap1-136; Gap1-157; Gap1-162; Gap1-164; Gap1-165, and Gap1-170 identify the mutants resistant to down-regulation mediated by the phosphorylated Aly proteins under the same conditions.

FIGURE 6.

Several Gap1 C-terminal mutants resist rapamycin-induced down-regulation via the Bul or Aly adaptors. A, strains EK008 (gap1Δ ura3), JA493 (gap1Δ bul1Δ bul2Δ ura3), 35101a (gap1Δ aly1Δ aly2Δ ura3), and MA062 (gap1Δ bul1Δ bul2Δ aly1Δ aly2Δ ura3) transformed with pMA74 (YCpGAL-GAP1-112-GFP) were grown on galactose/proline medium. Glucose was added for 30 min to repress Gap1 synthesis, and when indicated, Rap (200 ng/ml) was added for 3 h. Gap1-GFP localization was examined by fluorescence microscopy. B, strains JA493 (gap1Δ bul1Δ bul2Δ ura3) and 35101a (gap1Δ aly1Δ aly2Δ ura3) were transformed with pJOD10 (YCpGAL-GAP1-GFP) or pCJ038 (YCpGAL-GAP1^K9R,K16R-GFP) or with a plasmid encoding one of the indicated Gap1 mutants. Growth conditions were the same as in A. Gap1-GFP localization was examined by fluorescence microscopy. C, same strains as in B were transformed with pJOD10 (YCpGAL-GAP1-GFP) or pCJ038 (YCpGAL-GAP1^K9R,K16R-GFP) or with a plasmid encoding one of the indicated Gap1 mutants. Growth conditions were as in A. Gap1-GFP localization was examined by fluorescence microscopy. D, strain EK008 (gap1Δ ura3) was transformed with pJOD10 (YCpGAL-GAP1-GFP) or a plasmid encoding one of the indicated Gap1 mutants. Growth conditions were as in A. Gap1-GFP localization was examined by fluorescence microscopy. Cells were labeled with FM4-64 to stain the vacuolar membrane. E, same strains as in D (precluding pJOD10) were grown on galactose/proline medium. Glucose was added for 2 h to repress Gap1 synthesis, and when indicated, Am (20 mm) was added for 2 h. Gap1-GFP localization was examined by fluorescence microscopy. DIC, differential interference contrast.

FIGURE 7.

Aly1 and Aly2 adaptors promote Gap1 ubiquitylation on lysine 16 only. A, strains EK008 (gap1Δ ura3), JA493 (gap1Δ bul1Δ bul2Δ ura3), 35101a (gap1Δ aly1Δ aly2Δ ura3), and MA062 (gap1Δ bul1Δ bul2Δ aly1Δ aly2Δ ura3) transformed with pMA51 (YCpGAL-GAP1-108-GFP) or pMA47 (YCpGAL-GAP1-106-GFP) were grown on galactose/proline medium. Glucose was added for 30 min to repress Gap1 synthesis, and when indicated, Rap (200 ng/ml) was added for 3 h. Gap1-GFP localization was examined by fluorescence microscopy. B, strains EK008 (gap1Δ ura3), JA493 (gap1Δ bul1Δ bul2Δ ura3), and 35101a (gap1Δ aly1Δ aly2Δ ura3) transformed with pEL003 (YCp-GAP1-GFP), pEL005 (YCp-GAP1^K9R,K16R-GFP), pEL018 (YCp-GAP1^K9R-GFP), or pEL021 (YCp-GAP1^K16R-GFP) were grown on glucose/proline medium, and when indicated, Rap (200 ng/ml) was added for 3 h. Gap1-GFP localization was examined by fluorescence microscopy. C, strain JA493 (gap1Δ bul1Δ bul2Δ ura3) transformed with pEL003 (YCp-GAP1-GFP), pEL018 (YCp-GAP1^K9R-GFP), or pEL021 (YCp-GAP1^K16R-GFP) was grown on glucose/proline medium. Cells were collected before and 10 min after Rap (200 ng/ml) addition. Crude cell extracts were immunoblotted with anti-GFP antibodies. DIC, differential interference contrast.

FIGURE 8.

Stress conditions induce Gap1 down-regulation via the phosphorylated Bul and Aly adaptors. A, strains EK008 (gap1Δ ura3), JA493 (gap1Δ bul1Δ bul2Δ ura3), 35101a (gap1Δ aly1Δ aly2Δ ura3), and MA062 (gap1Δ bul1Δ bul2Δ aly1Δ aly2Δ ura3) transformed with pJOD10 (YCpGAL-GAP1-GFP) were grown on galactose/proline medium. Glucose was added for 2 h to repress Gap1 synthesis, and when indicated, H₂O₂ (0.88 mm) or ethanol (EtOH) (10%) was added for 2 h or the cells were transferred from 29 to 37 °C for 1 h. Gap1-GFP localization was examined by fluorescence microscopy. B, strain EK008 (gap1Δ ura3) was transformed with pJOD10 (YCpGAL-GAP1-GFP) or pMA74 (YCpGAL-GAP1-112-GFP). Growth and stress conditions were as in A. Gap1-GFP localization was examined by fluorescence microscopy. C, strain EK008 (gap1Δ ura3) transformed with pJOD10 (YCpGAL-GAP1-GFP) or pCJ038 (YCpGAL-GAP1^K9R,K16R-GFP), strain 35101a (gap1Δ aly1Δ aly2Δ ura3) transformed with pMA31 (YCpGAL-GAP1-134-GFP), and strain JA493 (gap1Δ bul1Δ bul2Δ ura3) transformed with pMA51 (YCpGAL-GAP1-108-GFP) or pMA33 (YCpGAL-GAP-135-GFP) were grown on galactose/proline medium. Stress conditions were as in A. Gap1-GFP localization was examined by fluorescence microscopy. D, strains MA025 (gap1Δ BUL1-FLAG bul2Δ ura3) and MA032 (gap1Δ BUL2-HA ura3) transformed with pJOD10 (YCpGAL-GAP1-GFP) were grown on galactose/proline medium. Cells were collected before and 30 min after Am (50 mm), Rap (200 ng/ml), H₂O₂ (0.88 mm), or ethanol (10%) addition or after transfer from 29 to 37 °C. Crude cell extracts were immunoblotted with anti-FLAG or anti-HA antibodies. DIC, differential interference contrast.

FIGURE 9.

Lack of Tco89 causes constitutive Gap1 down-regulation via a mechanism involving the Bul and Aly adaptors and the C-terminal tail of Gap1. A, strains 23344c (ura3) and FA198 (tco89Δ ura3) transformed with pFL38 (URA3) were tested for growth on solid medium containing the indicated compound as sole nitrogen source. For each growth condition, both strains were grown on the same plate. B, strains EK008 (gap1Δ ura3) and MYC003 (gap1Δ tco89Δ ura3) transformed with pCJ004 (YCpGAL-GAP1) or pCJ034 (YCpGAL-GAP1^K9R,K16R) were grown on galactose/proline medium. Glucose was added for 2 h to repress Gap1 synthesis. The initial uptake rate of ¹⁴C-labeled citrulline (75 μm), reflecting Gap1 activity, was then measured. C, strains MYC003 (gap1Δ tco89Δ ura3), MYC004 (gap1Δ tco89Δ bul1Δ bul2Δ ura3), and MYC009 (gap1Δ tco89Δ aly1Δ aly2Δ ura3) transformed with pJOD10 (YCpGAL-GAP1-GFP), pCJ038 (YCpGAL-GAP1^K9R,K16R-GFP), or pEL021 (YCpGAP1^K16R-GFP) were grown on galactose/urea medium. Glucose was added for 2 h to repress Gap1 synthesis. Gap1-GFP localization was examined by fluorescence microscopy. D, strain MYC003 (gap1Δ tco89Δ ura3) transformed with pJOD10 (YCpGAL-GAP1-GFP), pMA74 (YCpGAL-GAP1-112-GFP), or pMYC016 (YCpGAL-GAP1-134–135-GFP) was grown on raffinose/urea medium. Galactose was added, and the culture was incubated overnight to induce Gap1 synthesis, and then glucose was added for 1.5 h to repress Gap1 synthesis. Gap1-GFP localization was then examined by fluorescence microscopy. DIC, differential interference contrast.

FIGURE 10.

Can1, Lyp1, and Fur4 permeases are down-regulated by Tco89 depletion, rapamycin addition, or stress. A, strains EK008 (gap1Δ ura3), MYC003 (gap1Δ tco89Δ ura3), and 41453c (tco89Δ rsp5(npi1) ura3) transformed with pJOD10 (YCpGAL-GAP1-GFP), pMA185 (YCpGAL1-CAN1-GFP), pNAM001 (YCp-LYP1-GFP), or pFL38-gF-GFP (YCpGAL1-FUR4-GFP) were grown on galactose/urea medium, except for strain 41453c, which was grown on galactose/proline and strains harboring the Fur4 plasmid, which were grown on galactose/ammonium. Glucose was added for 2 h to repress permease synthesis, and cells were visualized by fluorescence microscopy. B, strain EK008 (gap1Δ ura3) transformed with pJOD10 (YCpGAL-GAP1-GFP), pMA185 (YCpGAL1-CAN1-GFP), pNAM001 (YCp-LYP1-GFP), or pFL38-gF-GFP (YCpGAL1-FUR4-GFP) was grown on galactose/proline medium except for strains with the Fur4 plasmid, which were grown on galactose/ammonium. Glucose was added for 2 h to repress permease synthesis, and when indicated, Rap (200 ng/ml), H₂O₂ (0.88 mm), or ethanol (10%) was added for 2 h or cells were transferred from 29 to 37 °C for 1 h. The cells were then visualized by fluorescence microscopy. C, strains EK008 (gap1Δ ura3) and JA983 (gap1Δ art1Δ ura3) transformed with pMA185 (YCpGAL1-CAN1-GFP) were grown on galactose/ammonium medium. Glucose was added for 30 min to repress Can1 synthesis, and when indicated, Rap (200 ng/ml) was added for 3 h. Can1-GFP localization was then examined by fluorescence microscopy. D, strains MYC003 (gap1Δ tco89Δ ura3) and 35246a (gap1Δ tco89Δ art1Δ ura3) transformed with pMA185 (YCpGAL1-CAN1-GFP) plasmid were grown on galactose/urea medium. Glucose was added for 2 h to repress Can1 synthesis. Can1-GFP localization was then examined by fluorescence microscopy. DIC, differential interference contrast.

FIGURE 11.

Model for amino acid- and stress-induced ubiquitylation of Gap1 permease. In cells growing on a poor nitrogen source, the TORC1 kinase complex is active for its ability to somehow prevent the phosphorylated Bul and Aly proteins (bound to 14-3-3 proteins) from promoting Gap1 ubiquitylation. When TORC1 is inhibited by rapamycin, stress conditions, or lack of its Tco89 subunit, this negative control is thus relieved, and the Bul and Aly proteins (remaining phosphorylated and bound to 14-3-3 proteins) act through C-terminal regions of Gap1 to induce its Rsp5-dependent ubiquitylation on Lys-9 or Lys-16 residues (Bul proteins) or only the Lys-16 residue (Aly proteins) in the permease's N-terminal tail. When the internal concentration of amino acids increases, active TORC1 further gains the ability to promote the dephosphorylation of the Bul proteins (which also undergo monoubiquitylation). These dephosphorylated Bul proteins dissociate from the 14-3-3 proteins, thereby gaining the ability to induce Gap1 ubiquitylation by acting through an N-terminal region of the permease close to the Lys-9 and Lys-16 residues.