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Ubiquitylation of many plasma membrane proteins promotes their endocytosis followed by degradation in the lysosome. The yeast general amino acid permease, Gap1, is ubiquitylated and downregulated when a good nitrogen source like ammonium is provided to cells growing on a poor nitrogen source. This ubiquitylation requires the Rsp5 ubiquitin ligase and the redundant arrestin-like Bul1 and Bul2 adaptors. Previous studies have shown that Gap1 ubiquitylation involves the TORC1 kinase complex, which inhibits the Sit4 phosphatase. This causes inactivation of the protein kinase Npr1, which protects Gap1 against ubiquitylation. However, the mechanisms inducing Gap1 ubiquitylation after Npr1 inactivation remain unknown. We here show that on a poor nitrogen source, the Bul adaptors are phosphorylated in an Npr1-dependent manner and bound to 14-3-3 proteins that protect Gap1 against downregulation. After ammonium is added and converted to amino acids, the Bul proteins are dephosphorylated, dissociate from the 14-3-3 proteins, and undergo ubiquitylation. Furthermore, dephosphorylation of Bul requires the Sit4 phosphatase, which is essential to Gap1 downregulation. The data support the emerging concept that permease ubiquitylation results from activation of the arrestin-like adaptors of the Rsp5 ubiquitin ligase, this coinciding with their dephosphorylation, dissociation from the inhibitory 14-3-3 proteins, and ubiquitylation.
Studies conducted on yeast in the mid-1990s revealed that linkage of ubiquitin (Ub) to plasma membrane receptors or transporters is essential for their endocytosis followed by degradation in the vacuole (26, 29, 38). This ubiquitylation requires a Ub ligase enzyme, Rsp5 (Npi1) (18, 26, 30), which is also involved in several other cellular functions (31). This Ub-dependent downregulation mechanism is conserved in more complex cells (15) and often involves Rsp5-like Ub ligases, e.g., Nedd4 or AIP4/ITCH (62). The Rsp5 enzyme and mammalian homologs share the same global structure consisting of a C2 domain, two to four WW domains, and a C-terminal catalytic domain (HECT) (62). The Ub attached to permeases or receptors also acts as a signal for their sorting into vesicles budding into the lumen of the endosome via the multivesicular body (MVB) pathway, an essential step for subsequent delivery into the vacuolar/lysosomal lumen (36, 39, 59, 60, 67, 68).
In Saccharomyces cerevisiae, Rsp5 does not bind directly to permeases and is instead recruited onto them thanks to adaptor proteins (43). It binds to these adaptors via its WW domains, which recognize PPXY sequences (the PY motif). Among the PY-containing adaptors of Rsp5, some (e.g., Bsd2, Tre1/2, Ear1, and Ssh4) act mainly at the level of endosomes and/or the Golgi complex and generally include one or several transmembrane domains. Others are globally hydrophilic and promote ubiquitylation of plasma membrane proteins (39, 43). The latter category includes a family of proteins similar to arrestins (44, 56). Arrestins were first described as proteins binding to G-protein-coupled receptors and thereby arresting signaling (42). Yeast arrestin-related trafficking adaptors (ARTs), also termed alpha-arrestins, target various proteins according to various stimuli (39, 43, 56). For instance, Art1 promotes ubiquitylation of the Can1 arginine permease in response to cycloheximide and that of other amino acid permeases (AAPs) (Lyp1, Mup1, and Tat2) in the presence of excess substrate (44, 56), Aly2/Art3 promotes endocytosis of the Dip5 glutamate permease in response to excess substrate (25), and Art4/Rod1 mediates ubiquitylation of the Jen1 lactate permease in the presence of glucose (3). Recently, two studies have revealed that signal-induced ubiquitylation of specific permeases is triggered by direct regulation of arrestin-like adaptors (3, 44, 46).
The general amino acid permease Gap1 (34) is one of the yeast proteins used as a model to study Ub-dependent trafficking of plasma membrane proteins (39). The rate of Gap1 endocytosis is controlled by nitrogen (N). When cells grow on a poor N source like urea or proline, the GAP1 gene is highly transcribed and newly synthesized Gap1 accumulates at the plasma membrane in a highly active and stable form (12). When a good N source like ammonium (Am) at high concentration is provided to cells, Rsp5 catalyzes linkage of a short K63-based Ub chain to the K9 or K16 residue in the permease's N-terminal tail (39, 40, 64–66). The ubiquitylated permease is then internalized by endocytosis, sorted into the MVB pathway, and degraded in the vacuole (26, 40, 55, 65). While monoubiquitylation of Gap1, even on a single lysine, is sufficient for endocytic internalization of the permease, modification with a short K63-based Ub chain is essential for its subsequent sorting into the MVB pathway (40). In addition to Rsp5, ubiquitylation of Gap1 requires at least one of two redundant proteins, Bul1 and Bul2 (27, 64). These proteins harbor PY motifs enabling them to bind to the WW domains of Rsp5 (71), and they are distant members of the arrestin family (56), suggesting that they might act as adaptors to allow binding of Rsp5 to Gap1.
Signals and pathways controlling Gap1 ubiquitylation have been only partially investigated. A key factor is the Npr1 (nitrogen permease reactivator 1) kinase, first described as being required for full activity of Gap1 (22, 69) and later shown to counteract Gap1 ubiquitylation in cells growing on a poor N source (12). A key finding was that Npr1 is under the control of the TORC1 kinase complex (63). On a poor N source or in the presence of rapamycin, conditions of low TORC1 activity, Npr1 is weakly phosphorylated and active, whereas in the presence of amino acids, TORC1 is stimulated and promotes Npr1 hyperphosphorylation, causing its inactivation (19, 63). Hyperphosphorylation of Npr1 is also observed in mutant cells lacking the TORC1-regulated protein phosphatase 2A (PP2A)-related phosphatase Sit4 (32) or the Ptc1 PP2C phosphatase controlling Sit4 (20). The current model (Fig. 1A) proposes that activation of TORC1 by internal amino acids leads to reduced Sit4 activity, causing hyperphosphorylation and inactivation of the Npr1 kinase (11, 13, 45). TORC1 might also directly control Npr1 (8). Yet how Npr1 controls Gap1 ubiquitylation remains unknown.
In this work, we show that the control by nitrogen of Gap1 ubiquitylation proceeds via Npr1-mediated regulation of the Bul arrestin-like adaptors. On a poor N source, the Bul proteins are phosphorylated in an Npr1-dependent manner. Under these conditions, the Bul adaptors bind to 14-3-3 proteins, which inhibit the capacity of the Bul proteins to induce Gap1 downregulation. When ammonium is provided to the cells and converted to internal amino acids, or when Npr1 is inactivated in a thermosensitive mutant, the Bul proteins are dephosphorylated. This dephosphorylation depends on the Sit4 phosphatase and is coupled to dissociation of the Bul adaptors from 14-3-3 proteins. Furthermore, the Bul proteins undergo ubiquitylation. Our results, together with those of two recent studies (3, 46), suggest a widespread mechanism controlling permease ubiquitylation, which is discussed.
All S. cerevisiae strains used in this study (Table 1) derive from the Σ1278b wild type (1). Cells were grown at 29°C in minimal buffered medium, pH 6.1 (33). The main carbon source was glucose (3%) or galactose (3%) plus a low concentration of glucose (0.3%) to more readily initiate growth. In all experiments, proline (10 mM) was used as the sole N source of the medium. The sit4Δ and bmh1Δ bmh2Δ strains grew 2 to 2.5 times more slowly than did the wild type. When indicated, ammonium (20 or 50 mM) was added. Growth tests shown in Fig. 3A and Fig. 4B, obtained by spreading cell suspensions on solid media, were also carried out using spot dilutions and gave the same results (not shown). Strains MA001, MA003, and MA005 were isolated by insertion of a GAP1-deletion kanMX2 Geneticin resistance gene which was amplified by PCR using pUG06 plasmid as a template. Strains MA025 and MA032 were isolated by insertion of a DNA fragment amplified by PCR using as a template the pFA6a-6×GLY-3×FLAG-kanMX6 and pFA6a-6GLY-3×HA-kanMX6 plasmids, respectively (17). Strains MA013 and MA014 were isolated from EK008 by replacing the BUL1 and BUL2 genes, respectively, with a kanMX2 gene. Strain 35064c was isolated by replacing the SIT4 gene with kanMX2 in an ura3/ura3 diploid cell followed by sporulation. All oligonucleotides used to isolate inserted PCR DNA fragments are available upon request.
The plasmids used in this work are listed in Table 2. Plasmid pMA121 was constructed by recombination in yeast between the linearized pFL36 plasmid and PCR DNA fragments carrying the BUL1-FLAG gene. These DNA fragments (flanked by appropriate 40-bp recombination sequences) were obtained by PCR using total DNA of strain MA025 as the template. The pMA126, pMA130, and pMA136 plasmids were constructed by in vivo recombination in yeast between the linearized pMA121 plasmid and two PCR fragments carrying the BUL1 mutated sequences. The indicated mutations were introduced into the oligonucleotides used for the PCR. The pMA114 plasmid was constructed from pJOD10 by replacement of URA3 with LEU2. The pMA155 and pMA160 plasmids were obtained by recombination in yeast. The DNA fragments encoding PPSY and AASY regions were obtained by PCR using pMA121 and pMA136 as the templates, respectively. The accuracy of each newly isolated mutagenized plasmid was checked by sequencing.
For Western blot analysis, crude cell extracts were prepared and immunoblotted as previously described (26). After transfer to a nitrocellulose membrane (Schleicher and Schuell), mouse antihemagglutinin (anti-HA) (12CA5; Roche), anti-FLAG (F1804; Sigma), anti-Ub (p4D1; Santa Cruz), or anti-yeast 3-phosphoglycerate kinase (anti-PGK) (Invitrogen) antibodies were used at 1:10,000, 1:2,000, 1:500, and 1:5,000 dilutions, respectively. Primary antibodies were detected with horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G secondary antibody (GE Healthcare). For the glutathione S-transferase (GST) pulldown experiment, exponentially growing cells were first suspended in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40) supplemented with Complete EDTA-free protease inhibitor cocktail tablets (Roche), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM N-ethylmaleimide, and 50 μM protease inhibitor MG-132 (Sigma). The cells were lysed by vortexing with glass beads, and cell extracts were incubated for 20 min on ice and centrifuged for 30 min at 12,000 × g. GST-tagged Bmh2 was immunoprecipitated from the lysates with glutathione (GSH)-Sepharose beads (GE Healthcare). Proteins were analyzed by immunoblotting with anti-GST (Invitrogen) or anti-FLAG antibodies.
Exponentially growing cells were lysed with glass beads in 10% trichloroacetic acid (TCA). Precipitates were resuspended in 200 μl buffer 1 and boiled for 5 min at 95°C. Undissolved material was removed by centrifugation at 13,000 × g for 5 min. Eight hundred microliters of immunoprecipitation (IP) buffer was added, and lysates were incubated with EZview Red anti-FLAG M2 affinity gel beads (F2426; Sigma) with gentle rotation at 4°C for 2 h. The beads were washed once with 1 ml of each of the following buffers: B2, B3, B4, and B5. IP buffer contained 50 mM Tris (pH 7.5), 2 mM EDTA, 100 mM NaCl, 1.2% Triton X-100, 0.5% bovine serum albumin (BSA), protease inhibitor cocktail (Roche Applied Science), and 20 mM N-ethylmaleimide. B1 buffer contained 2% SDS containing bromophenol blue, protease inhibitor cocktail, N-ethylmaleimide (20 mM), and 100 μM protease inhibitor MG-132 (Sigma). B2 buffer contained 50 mM Tris (pH 7.5), 2 mM EDTA, 100 mM NaCl, 1% Triton X-100, and 0.2% SDS. B3 buffer contained 50 mM Tris (pH 7.5), 2 mM EDTA, 100 mM NaCl, 0.1% Triton X-100, 0.5% SDS, and 0.5% sodium deoxycholate (DOC). B4 buffer contained 50 mM Tris (pH 7.5), 2 mM EDTA, 500 mM NaCl, and 0.1% Triton X-100. B5 contained 50 mM Tris (pH 7.5), 2 mM EDTA, and 100 mM NaCl.
Crude cell extracts were prepared as described above, and proteins were suspended in SDS buffer (4% SDS, 0.1 M Tris-HCl [pH 6.8], 20% glycerol, 2% 2-mercaptoethanol). Dephosphorylation buffer, 1 M Tris (pH 8.0), and phenylmethylsulfonyl fluoride (PMSF) (1 mM) were added. Then, alkaline phosphatase (ALP; Roche Diagnostics) was added or not and the mixture was incubated for 2 h at 37°C. Proteins were then precipitated with 10% TCA, resuspended in sample buffer, and analyzed by immunoblotting.
Subcellular localization of Gap1-green fluorescent proteins (GFPs) was performed in cells growing exponentially in liquid glucose-proline or galactose-proline medium. When galactose was used as a carbon source, glucose was added (final concentration, 3%) for 2 h before visualizing cells so as to arrest Gap1-GFP neosynthesis. Labeling of the vacuolar membrane with FM4-64 was performed as described previously (55). Cells were laid down on a thin layer of 1% agarose and viewed at room temperature with a fluorescence microscope (Eclipse E600; Nikon) equipped with a 100× differential interference contrast numerical aperture (NA) 1.40 Plan-Apochromat objective (Nikon) and appropriate fluorescence light filter sets. Images were captured with a digital camera (DXM1200; Nikon) and ACT-1 acquisition software (Nikon) and processed with Photoshop CS (Adobe Systems).
The Gap1 permease is ubiquitylated and degraded when ammonium (Am) is provided to cells growing on proline or urea as sole N source (65). Uptake of Am is mediated mainly by three similar transport proteins, Mep1, -2, and -3 (48, 49). To determine if the Mep permeases are necessary to promote Am-induced Gap1 ubiquitylation and downregulation, we immunodetected Gap1 in cell extracts (Fig. 1B) and investigated the location of a functional Gap1-GFP fusion (Fig. 1C) in a gap1Δ mep1Δ mep2Δ mep3Δ mutant before and after addition of Am (20 mM). The same experiments were carried out in a gap1Δ mutant used as a control. On a medium with proline as sole N source (Pro medium), the plasmid-encoded Gap1 protein was not ubiquitylated and was present at the plasma membrane. Addition of Am to gap1Δ cells triggered internalization and delivery of Gap1 to the vacuolar lumen, as confirmed by colabeling with the FM4-64 fluorescent dye (Fig. 1C). This downregulation coincided with Gap1 ubiquitylation, as shown by the appearance of upper bands above the immunodetected Gap1 signal which were not visible when using the nonubiquitylable Gap1K9R,K16R mutant form (Fig. 1B). In the triple mep mutant, however, Gap1 was not ubiquitylated and remained largely stable at the plasma membrane (Fig. 1B and andC),C), indicating that Am must enter cells via the Mep permeases to induce Gap1 downregulation.
Once inside cells, Am condenses with α-ketoglutarate to yield glutamate, a reaction catalyzed by NADP+-dependent glutamate dehydrogenase I (Gdh1) (23, 47). Glutamate dehydrogenase 2 (Gdh2) is NAD+ dependent and involved mainly in glutamate catabolism (53). Yet, Gdh2 is also potentially capable of catalyzing glutamate synthesis when it is derepressed and when ammonium and α-ketoglutarate are abundant (70). We thus expressed Gap1-GFP in a gap1Δ gdh1Δ gdh2Δ mutant and found Gap1 to remain nonubiquitylated and stable at the plasma membrane after Am addition (Fig. 1B and andC).C). Hence, conversion of Am to glutamate is essential to normal Am-induced downregulation of Gap1.
As glutamate is the major N donor in reactions of amino acid biosynthesis, uptake of Am and its conversion to glutamate lead to an increased internal amino acid concentration (52). According to the current model of Gap1 control (Fig. 1A), this increase in internal amino acids activates TORC1 via the EGO complex, and TORC1 then counteracts the action of the Sit4 phosphatase via the Tap42 and Tip41 proteins (11, 13, 16, 45). As Sit4 promotes dephosphorylation of Npr1, its inactivation following TORC1 activation leads to hyperphosphorylaton of Npr1, which is inactivated (19, 63). Furthermore, Npr1 was found in a recent global analysis to interact physically with the Tor kinases (8), suggesting that it may be directly phosphorylated by TORC1. Using the conditions applied throughout this work (strains of the Σ1278b background growing on Pro or Pro+Am medium), we obtained data in full support of this model: in cells grown on Pro medium, an Npr1-HA form migrated through a gel as two close bands (Fig. 1D and andF)F) sensitive to treatment by alkaline phosphatase (ALP), indicating that both Npr1 forms were phosphorylated (Fig. 1D). After addition of Am, these bands were converted to three to four upper bands which were likewise ALP sensitive, indicating that Am induced hyperphosphorylation of Npr1 (Fig. 1D) (19, 63). This hyperphosphorylation was largely inhibited if the TORC1 inhibitor rapamycin was also present (Fig. 1E) or if the experiment was carried out in the triple mep mutant strain (Fig. 1F). Hence, addition of Am to proline-grown cells appears to induce Gap1 ubiquitylation via the following cascade: uptake of Am via the Mep permeases, conversion of Am to glutamate by Gdh enzymes, a probable increase in the internal concentration of amino acids, activation of the TORC1 complex, hyperphosphorylation and inactivation of Npr1, and ubiquitylation and endocytosis of the Gap1 permease.
We have previously reported that Gap1 is probably not a substrate of the Npr1 kinase, as it remains phosphorylated in an npr1Δ mutant (12). Npr1 thus more likely targets a protein promoting Gap1 ubiquitylation. Interestingly, Npr1 was recently reported to control phosphorylation of Art1, an Rsp5 adaptor controlling other AAPs (46). Another arrestin-like protein, Aly2/Art3, which localizes to endosomes, was found to be phosphorylated by Npr1 (58). Furthermore, a recent systematic search for proteins interacting with kinases and phosphatases revealed that Npr1 binds to the arrestin-like Bul1 and Bul2 proteins (8). We thus decided to further study the role of the Bul proteins in Gap1 downregulation. We first introduced several amino acid substitutions into Bul1 (Fig. 2). The mutant Bul1 proteins were analyzed in a growth assay monitoring Am-induced Gap1 endocytosis (Fig. 3A). The d isomer of histidine (d-His) is toxic and incorporated into cells via Gap1. On Pro medium, cells expressing Gap1 are thus poisoned by d-His, whereas on Am medium, cells resist d-His unless Gap1 ubiquitylation is prevented by the K9R and K16R substitutions in the permease. Sensitivity to d-His on Am plates was also observed for the bul1Δ bul2Δ mutant strain but not for the single bul1Δ and bul2Δ mutants, confirming the redundancy of the Bul proteins (Fig. 3A). Expression of native Bul1 in the bul1Δ bul2Δ strain restored resistance to d-His on Am medium, as expected. However, mutant forms of Bul1 altered in the PY motif (PPSY replaced with AASY) failed to restore resistance to d-His (Fig. 3A), indicating that the PY motif of Bul1 is essential to normal Gap1 downregulation. This was confirmed by examining the ubiquitylation and localization of Gap1-GFP before and after addition of Am (Fig. 3B and andC).C). In bul1Δ bul2Δ mutant cells expressing the native Bul1 protein, Gap1-GFP present at the plasma membrane was normally ubiquitylated and targeted to the vacuole after Am addition. In cells expressing no Bul factors, in contrast, Gap1-GFP failed to be ubiquitylated and remained stable at the cell surface, as expected. The latter phenotypes were also observed in cells expressing the Bul1 proteins altered in the PY motif (Fig. 3B and andC).C). Furthermore, the Gap1 permease was degraded after Am addition in cells expressing Bul1 but not in cells expressing no Bul1 or the mutant Bul1 altered in its PY motif (Fig. 3D). These results clearly show that the PY motif of Bul1 is required for normal ubiquitylation and downregulation of Gap1, thus suggesting that interaction between Rsp5 and Bul1 plays a crucial role in this process. We also isolated two Bul1 mutants bearing several substitutions in two highly conserved amino acid regions of the arrestin motif (Fig. 2). The results of Fig. 3 show that these two Bul1 mutants (which are expressed at a normal level [see below]) also failed to promote Am-induced Gap1 ubiquitylation and endocytosis. This suggests that the arrestin motif of Bul1 is crucial to its function, although the apparent loss of function of these two Bul1 mutants could be due to their eventual improper folding.
Addition of Am might induce interaction between the Bul adaptors and Gap1, subsequently causing Rsp5-dependent ubiquitylation of the permease. If this model is true, forcing the recruitment of Rsp5 to Gap1 should induce its ubiquitylation and downregulation, even on Pro medium. We found that a 38-amino-acid region including the PY motif is highly conserved between Bul1 and Bul2 (Fig. 2). We thus decided to fuse this sequence to the extreme N terminus of Gap1. Interestingly, the resulting PPSY-Gap1 chimera (Fig. 4A) failed to complement a gap1Δ mutation in d-His sensitivity tests, in contrast to the same protein in which the PPSY motif was replaced with AASY (Fig. 4B). We located the PPSY-Gap1-GFP chimera and found it to be delivered to the vacuolar lumen of cells growing on Pro medium (Fig. 4C). This constitutive targeting to the vacuole was also observed in bul1Δ bul2Δ mutant cells, showing that it does not require the Bul proteins (Fig. 4C). In contrast, the AASY-Gap1-GFP mutant protein was targeted to the plasma membrane (Fig. 4C). We next expressed the PPSY-Gap1 chimera in an end3Δ mutant strain defective in endocytosis and found the protein still to be largely present in the vacuolar lumen (Fig. 4D). These results show that the PPSY-Gap1 chimera is largely deviated from the secretory pathway to the vacuole without passing through the plasma membrane, i.e., it behaves like Gap1 newly synthesized in an npr1Δ mutant or in the wild type grown on Am (12, 64). The fact that the AASY-Gap1 protein is instead targeted to the plasma membrane (where it is functional) suggests that the PPSY-Gap1 protein is missorted to the vacuole because it recruits the Rsp5 Ub ligase in an unregulated manner. We next located the PPSY-Gap1-GFP chimera in the rsp5 (npi1) mutant, where the level of Rsp5 is markedly reduced (65). In this strain, where Gap1-GFP is not downregulated by Am, the PPSY-Gap1 chimera was found mainly at the plasma membrane (Fig. 4E). In the rsp5 (npi1) strain, consistently, PPSY-Gap1 conferred sensitivity to d-His (Fig. 4B).
Taken together, these data show that fusion to Gap1 of a highly conserved 38-amino-acid sequence of Bul1 including the PY motif is sufficient to induce its direct sorting from the secretory pathway to the vacuole. That this sorting depends on Rsp5 and the PY motif but not on the Bul proteins strongly suggests that the short PY-containing sequence of Bul1 is capable of recruiting Rsp5 to Gap1.
We next sought to determine whether Am-induced downregulation of Gap1 is accompanied by detectable changes at the level of the Bul adaptors. We thus isolated Bul1-FLAG and Bul2-HA C-terminal fusions that proved functional, as judged by their capacity to promote normal Am-induced Gap1-GFP downregulation (data not shown). Bul1-FLAG and Bul2-HA from cells grown on Pro medium displayed apparent molecular masses of 130 and 125 kDa, respectively (Fig. 5A and andB).B). In most experiments, it was in fact possible to distinguish two very close bands, of different intensities. Upon Am addition, the Bul1-FLAG and Bul2-HA bands changed into two more separate bands of similar intensities, one migrating slower and the other faster than the two close bands detected on Pro medium (Fig. 5A and andB).B). We show below that the upper band of the Bul1-FLAG signal corresponds to a ubiquitylated form of the protein. With respect to the lower bands, appearing after Am addition, we interpreted them as resulting from dephosphorylation. To test this interpretation, extracts of cells collected before or after Am addition were treated with alkaline phosphatase (ALP) (Fig. 5C). The Bul1-FLAG and Bul2-HA signals detected in ALP-treated extracts of Pro-grown cells were found to migrate faster than the signals detected in the corresponding untreated extracts, indicating that the Bul proteins are indeed phosphorylated under these growth conditions. Furthermore, the fast-migrating Bul1-FLAG and Bul2-HA signals detected after Am addition, with or without ALP treatment, seemed to comigrate with the signal detected in extracts of Pro-grown cells after ALP treatment (Fig. 5C), indicating that they correspond to dephosphorylated forms. In conclusion, the Bul adaptors seem to exist as two phosphorylated forms in cells grown on Pro medium and they undergo dephosphorylation when Am is provided to the cells.
As mentioned above, addition of Am also led to the appearance of an upper band for both Bul1 and Bul2, migrating above the dephosphorylated Bul1-FLAG or Bul2-HA signal. Migration of this upper band was not altered by treatment of cell extracts with ALP (Fig. 5C), suggesting that the corresponding Bul forms were not phosphorylated but had undergone another modification retarding their migration through the gel. In a recent systematic mass spectrometry analysis applied to yeast, both Bul1 and Bul2 were detected among purified ubiquitylated proteins (72). This observation prompted us to test whether the low-mobility, ALP-insensitive form of Bul1-FLAG appearing after Am addition is a ubiquitylated form of the protein. Two experimental results are in full support of this interpretation. First, in the rsp5 (npi1) mutant strain, the Bul1-FLAG signal migrated faster after Am addition, indicating that Bul1-FLAG is dephosphorylated, but the upper band was not detected (Fig. 6A). Second, we immunoprecipitated Bul1-FLAG in denatured cell extracts and tested by immunoblotting whether it was associated with Ub. The results show that Bul1 is indeed ubiquitylated and that this modification is induced by addition of Am to the cells (Fig. 6B). The signal detected with anti-Ub antibodies after Bul1-FLAG immunoprecipitation migrated as a single band, suggesting that Bul1 undergoes monoubiquitylation. This band most likely corresponds to the Am-induced upper band detected with anti-FLAG antibodies. Furthermore, that this upper band of Bul1 was not detected in the rsp5 (npi1) mutant indicates that Bul1 ubiquitylation depends on Rsp5 (Fig. 6A). We also analyzed the mutant Bul1 altered in its PY motif (Fig. 6C). In this case, the upper band did not appear after Am addition and the detected Bul1 signal seemed to migrate faster after Am addition, indicating that this mutant Bul1 form undergoes normal dephosphorylation but is not ubiquitylated (Fig. 6C). We finally immunodetected the Bul1 forms harboring several amino acid substitutions in the arrestin motif and observed that they were not ubiquitylated after Am addition (Fig. 6D).
Taken together, these results show that Bul1 is not only dephosphorylated but also ubiquitylated (probably monoubiquitylated) in response to Am addition. Ubiquitylation of Bul1, observed only for a fraction of the protein, depends on Rsp5 and on the PY motif in Bul1, i.e., on proper interaction between Bul1 and the WW domains of the Ub ligase. The conserved residues in the arrestin motif are also essential to Bul1 ubiquitylation. Under conditions where Bul1 cannot be ubiquitylated, it still undergoes dephosphorylation. Finally, Bul2 behaves like Bul1 on immunoblots, suggesting that it is also dephosphorylated and ubiquitylated in response to Am.
The Npr1 kinase protects Gap1 against ubiquitylation and degradation when cells grow on a poor N source (12). It is inactivated via TORC1 when amino acids are provided to the cells (63). As the Bul proteins are phosphorylated on Pro medium and dephosphorylated after Am addition, we hypothesized that their phosphorylation might depend on Npr1. We therefore immunodetected Bul1-FLAG in npr1Δ mutant cells growing on Pro medium (Fig. 7A). Interestingly, the protein migrated as two separated signals, as when Am is provided to wild-type cells, although the upper band in the npr1Δ mutant was less intense than that in Am-treated cells. Furthermore, the upper band was not detected in the npr1Δ rsp5 (npi1) mutant (Fig. 7A). These result suggest that Bul1 is not phosphorylated and is constitutively ubiquitylated in Npr1-lacking cells. In support of the view that the Bul proteins are constitutively active under these conditions, Gap1-GFP was constitutively delivered to the vacuole in npr1Δ cells growing on Pro medium but was stabilized at the cell surface in the npr1Δ bul1Δ bul2Δ strain (Fig. 7B). To further investigate the role of Npr1 in the posttranslational modifications of Bul1, we expressed Bul1-FLAG in an npr1(ts) mutant strain (22). The cells were first grown at 25°C on Pro medium and then shifted to the restrictive temperature of 35°C for 30 min to trigger Npr1 inactivation. In a previous study, we reported that this temperature shift induces Gap1 ubiquitylation (12). The results in Fig. 7C show that this temperature shift also caused separation of the immunodetected Bul1 signals into two distinct bands, i.e., the migration pattern observed in the wild type after Am addition. Furthermore, this temperature shift also led to endocytosis of Gap1-GFP and its delivery to the vacuole (Fig. 7D). These results suggest that phosphorylation of Bul1 in proline-grown cells depends on the Npr1 kinase and that Npr1 inactivation is sufficient to induce Bul1 dephosphorylation and ubiquitylation and Bul1-dependent Gap1 downregulation.
Inactivation of the Npr1 kinase triggered by good N sources correlates with its hyperphosphorylation and results from TORC1-dependent inhibition of the Sit4 phosphatase via the inhibitory subunit Tap42 (19, 32, 63). We constructed a sit4Δ mutant and observed that Npr1 is indeed hyperphosphorylated in this strain growing on Pro medium (Fig. 8A), as previously reported (19, 63). As Npr1 is hyperphosphorylated in the sit4Δ mutant, we expected that Gap1 would be constitutively delivered to the vacuole of this strain, as in the npr1Δ mutant. However, Gap1-GFP was present at the plasma membrane of sit4Δ mutant cells (Fig. 8B). This unexpected observation raised the possibility that Sit4 plays an additional role in Gap1 downregulation, e.g., in dephosphorylation of the Bul adaptors after Am addition. We thus immunodetected Bul1-FLAG in the wild type and in a sit4Δ mutant (Fig. 8C), using cell extracts treated or not with ALP. In sit4Δ cells, interestingly, Bul1 accumulated as a hyperphosphorylated form on Pro medium and was not dephosphorylated after Am addition. This lack of response is not due to some Am transport defect, as the Mep permeases were found to be highly active in the sit4Δ mutant (data not shown). Furthermore, treatment of cell extracts with ALP revealed that Am still induced Bul1 ubiquitylation in the sit4Δ mutant (Fig. 8C). We finally tested the role of Sit4 in Am-induced Gap1 endocytosis and observed that Gap1-GFP was not downregulated in response to Am (Fig. 8D). These results indicate that after Am addition, Sit4 dephosphorylates Bul1 or indirectly promotes its dephosphorylation and that this dephosphorylation is essential to promoting Gap1 downregulation. The fact that Bul1 is still ubiquitylated in the sit4Δ strain indicates that this modification does not require prior dephosphorylation of Bul1 and is not sufficient for Gap1 endocytosis.
The Bul adaptors are thus phosphorylated in an Npr1-dependent manner when cells grow on a poor N source such as Pro, and addition of Am leads to their Sit4-dependent dephosphorylation, a step that seems crucial for Gap1 downregulation. We next sought to determine why the Bul proteins do not target the Gap1 permease when phosphorylated. A proteomic study has revealed that Bul1, Bul2, and most of the arrestin-like Art proteins interact with 14-3-3 proteins (encoded by the redundant BMH1 and BMH2 genes) in a phosphorylation-dependent manner (35). Furthermore, a recent study has shown that the Bmh proteins bind to and inhibit the phosphorylated Art4/Rod1 adaptor required for ubiquitylation of the Jen1 permease (3). The 14-3-3 proteins are a family of conserved eukaryotic proteins involved in many and diverse cellular functions, including signaling, and interact with many other proteins when these are phosphorylated (9). We thus investigated whether 14-3-3 proteins might interact with Bul1 and whether this interaction might be under N control. For this, we expressed a Bmh2-GST plasmid in cells expressing Bul1-FLAG. As shown in Fig. 9A, Bul1 was found to interact with Bmh2 in cells growing on Pro medium, i.e., when Bul1 is phosphorylated, and to dissociate from it after addition of Am, i.e., when Bul1 is dephosphorylated. This strongly suggests that the phosphorylation state of Bul1 modulates its interaction with the 14-3-3 proteins.
We next examined the localization of Gap1 in the bmh1Δ bmh2Δ mutant. We used strain RRY1216, which, like all the mutants used in this study, derives from the Σ1278b genetic background (61). This strain and the corresponding wild type contain several amino acid auxotrophies, which were complemented by a plasmid (see Materials and Methods), so the cells could be grown with Pro as sole N source. The results show that in bmh1Δ bmh2Δ cells, a large fraction of the Gap1 was present in the vacuolar lumen (Fig. 9B). This phenotype is similar to that of the npr1Δ mutant (Fig. 7B), where the Bul proteins are not phosphorylated. In the bmh1Δ and bmh2Δ single-mutant strains, however, Gap1-GFP was normally present at the cell surface (data not shown). Hence, like the Npr1 kinase, the 14-3-3 proteins are required to protect Gap1 against downregulation when cells grow on a poor N source, most likely through binding and inhibition of the phosphorylated Bul adaptors.
Previous reports have shown that in cells growing on a poor N source like proline or urea, the Npr1 kinase (21, 69) prevents ubiquitylation of Gap1, thus stabilizing the permease at the plasma membrane (12, 22). We show in this study that Npr1 controls Gap1 ubiquitylation by promoting phosphorylation of the functionally redundant arrestin-like Bul1 and Bul2 proteins, which in this state are inhibited. Npr1 likely phosphorylates the Bul proteins directly, as it has been found to interact with them (8). We further show that Bul1, when phosphorylated, binds to the 14-3-3 proteins. This interaction appears to be the mechanism of Npr1-dependent Bul inhibition, as the 14-3-3 proteins—like the Npr1 kinase—are necessary to prevent Gap1 downregulation under poor N supply conditions. Furthermore, addition of Am, inducing Bul-dependent Gap1 ubiquitylation, coincides with dephosphorylation of the Bul proteins and their dissociation from the 14-3-3 factors. We also show that this dephosphorylation depends on the Sit4 PP2A-like phosphatase and seems essential to Bul activation, since Gap1 is not downregulated by Am in a sit4Δ mutant. Once dephosphorylated and released from the 14-3-3 proteins, the Bul proteins thus likely gain the ability to interact with Gap1 to promote Rsp5-dependent ubiquitylation. Remarkably, this mechanism strongly resembles that recently described for glucose-induced ubiquitylation and downregulation of the Jen1 lactate transporter (3). In the case of this permease, ubiquitylation depends on the Rod1/Art4 arrestin-like protein. On lactate medium, Rod1/Art4 is phosphorylated by the Snf1 kinase and bound to 14-3-3 proteins. When glucose is provided to the cells, Rod1/Art4 is dephosphorylated by the Reg1/Glc7 P1 phosphatase, dissociates from the 14-3-3 proteins, and promotes Jen1 ubiquitylation. Furthermore, in a reg1 mutant, Jen1 is not downregulated in response to glucose (3). Hence, there is a remarkable convergence between the mechanisms controlling permease ubiquitylation in response to carbon and nitrogen signals, as both systems involve control of the phosphorylation state of arrestin-like Rsp5 adaptors, modulating their inhibition by 14-3-3 proteins. Although further work is needed to determine to what extent this mechanism can be extrapolated to other situations where permease ubiquitylation is regulated, it is worth noting that yeast 14-3-3 proteins have been found to interact with most arrestin-like adaptors (35).
Our data showing that Npr1 targets the Bul adaptors brings further support to the emerging picture that this kinase is a key regulator of multiple arrestin-like adaptors. Among these is Art1, which, upon cycloheximide treatment (causing TORC1 activation and Npr1 inhibition), is dephosphorylated and promotes endocytosis of the Can1 arginine permease (46). Given the similarity to the mechanisms controlling the Bul proteins, it will be interesting to determine whether Npr1-dependent phosphoinhibition of Art1 also involves the 14-3-3 proteins and whether Art1 dephosphorylation depends on the Sit4 phosphatase. Npr1 is also reported to interact with and phosphorylate Aly2/Art3 (58). This Art adaptor promotes ubiquitylation of the Dip5 glutamate/aspartate permease in the presence of glutamate (25). Aly2/Art3 is also reported to control Gap1, but rather than promoting its downregulation, the adaptor, under poor N supply conditions, favors the endosome-to-Golgi complex or -cell-surface recycling of the permease. Furthermore, Npr1 favors this Aly2/Art3 function, i.e., Npr1 is proposed to promote Gap1 accumulation at the cell surface by stimulating (rather than inhibiting) an Art adaptor (58). Although it has not been determined whether this Npr1-dependent Art3 effect involves a change in Gap1 ubiquitylation, it seems unlikely that ubiquitylation of Gap1 favors its recycling to the plasma membrane. This raises the possibility that Npr1 uses different Art-dependent mechanisms to stabilize Gap1 at the plasma membrane. Another study even suggests that Npr1 might promote downregulation of other AAPs, e.g., the Tat2 tryptophan permease (2, 63). This permease is indeed downregulated when Npr1 is active, i.e., Gap1 and Tat2 are inversely regulated (2, 63). It would thus be interesting to determine whether some Art proteins are activated rather than inhibited by Npr1-dependent phosphorylation. Such a mechanism would allow cells to actively switch their battery of amino acid permeases in response to changed N supply conditions.
Although Npr1 clearly controls multiple arrestin-like adaptors, it might also phosphorylate the permeases themselves. The phosphorylation status of different amino acid permeases is indeed reported to be reduced in npr1Δ cells (46). In the yeast Hansenula polymorpha, furthermore, the Npr1 kinase ortholog is reported to phosphorylate the Ynt1 nitrate and nitrite permease under poor N supply conditions (50). This phosphorylation occurs at serine residues of a central intracellular loop and protects the permease against Ub-dependent downregulation (50, 54). Yeast Npr1 is also essential to the transport activity of the Mep transporters rather than to their stability at the cell surface, suggesting that it might also control permeases via other mechanisms (5).
Our work shows that the Bul adaptors are not only dephosphorylated but also ubiquitylated (likely monoubiquitylated) when Am is provided to cells. A lack of Npr1 function is in fact sufficient to induce this ubiquitylation (i.e., it is not due to the change in N supply conditions) as observed when the npr1(ts) mutant is shifted to 37°C. Furthermore, this ubiquitylation depends on Rsp5 and on the PY motif of the Bul proteins, i.e., on proper Bul-Rsp5 interaction. Rsp5-dependent ubiquitylation is a common property of Art proteins. It was first reported for Csr2/Art8 and Ecm21/Art2 (37, 57) and further illustrated for Art1 (44), Art3/Aly2 (25), Art9/Rim8 (28), and Art4/Rod1 (3). Studies on Art1 and Art4/Rod1 have revealed that this ubiquitylation is required for proper permease downregulation (3, 44), but the mechanisms controlling the ubiquitylation of Art proteins and its exact role in permease downregulation remain poorly known. In the case of Art4/Rod1, glucose-induced ubiquitylation requires prior dephosphorylation of the adaptor (3). In contrast, we observe that Bul1 is still ubiquitylated in a sit4Δ mutant, defective in Bul1 dephosphorylation. Yet another situation has been encountered for Art1, since a large fraction of the protein is ubiquitylated even under conditions where Art1 does not promote Can1 permease downregulation (44). Hence, the relationships between dephosphorylation and ubiquitylation of Art proteins seem to vary according to the proteins and conditions. Also intriguing is our observation that only a fraction of Bul1 and Bul2 is ubiquitylated after Am addition, and a similar situation has been observed for other arrestins as well (3, 44). This raises the possibility that different Bul forms coexist in the cell. For Bul1, we usually detect two phosphorylated forms under poor N supply conditions, both being dephosphorylated after Am addition. Hence, perhaps only one of the two phosphorylated forms undergoes ubiquitylation. Further studies are needed to determine whether these modified forms of Bul1 perform different functions and display different subcellular locations. Not only are the Bul proteins essential to ubiquitylation of Gap1 at the plasma membrane (64), but they are also required to induce this modification under conditions where the permease is deviated from the Golgi complex to the vacuole (27, 64). It will thus be interesting to study the role of Bul1 ubiquitylation and phosphorylation in each mechanism of Gap1 ubiquitylation.
Previous works have shown that the Npr1 kinase and the Sit4 phosphatase are under TORC1 control (45). On media where the N source is poor, when Npr1 is active, it is moderately phosphorylated (19, 63). In our experiments, at least two phosphorylated forms of Npr1 could be detected in proline-grown cells, in keeping with another study (19). These forms might result from the autophosphorylation activity of Npr1 (19). When amino acids are provided to cells, the TORC1 complex is activated and Npr1 is hyperphosphorylated (19, 63). The kinase catalyzing Npr1 hyperphosphorylation could be Tor1/2 of the TORC1 complex itself, which has been shown to interact with Npr1 (8). In our experiments, we typically induced Gap1 downregulation by providing Am to the cells, and we have confirmed that Am must enter via the Mep permeases and be converted to glutamate (the main N donor in reactions of amino acid biogenesis) to trigger Npr1 hyperphosphorylation and Gap1 downregulation. Note, however, that conversion of glutamate to glutamine is not essential to Am-induced downregulation of Gap1 (21) (our unpublished data) but that it is needed to activate TORC1-dependent transcriptional repression of the GAP1 gene (34) and of many others subject to nitrogen catabolite repression (10). This suggests that TORC1 may integrate different amino acid signals to orchestrate different responses (13, 45). Activation of TORC1 by internal amino acids requires the EGO complex, which includes the Gtr1 and Gtr2 GTPases (4, 14). Recently, it was shown that activation of EGO-TORC1 by leucine is mediated by an interaction between leucyl-tRNA synthetase and Gtr1 (6), and the mechanism seems conserved in mammalian cells (24). This remarkable finding opens the opportunity to study the influence of distinct amino acids on EGO-TORC1 function.
On a poor N source, the Sit4 phosphatase is needed to maintain Npr1 in a hypophosphorylated state (32, 63). We anticipated that in a sit4Δ mutant (where Npr1 is hyperphosphorylated), Gap1 would be constitutively targeted to the vacuole. Yet, the permease was not only present at the plasma membrane but also resistant to Am-induced downregulation. These observations are due to an additional role of Sit4 in controlling the Bul adaptors. We show that in a sit4Δ mutant, Bul1 fails to be dephosphorylated after Am addition. Hence, under poor N supply conditions, Sit4 acts on the Npr1 kinase (which in turn phosphoinhibits the Bul proteins), but after Am addition, Npr1 is inactivated (by TORC1) and Sit4 activates the Bul proteins by dephosphorylating them. Our observations suggest that if Sit4 is intrinsically less active after Am addition, the phosphorylation status of its target proteins is determined mainly by the activation level of the kinases acting on the same proteins, i.e., TORC1 (in the case of Npr1) and Npr1 (in the case of Bul1 and Bul2). Yet, we might also consider that Sit4 is similarly active when Pro or Am is the N source but that it targets different proteins, e.g., according to its association with regulatory subunits such as Tap42 (Fig. 10).
In conclusion, our study sheds light on key aspects of the molecular machinery inducing Gap1 permease ubiquitylation in response to TORC1 activation and brings further support to an emerging model: the view that control of permease ubiquitylation proceeds mainly via control by phosphorylation and dephosphorylation (and ubiquitylation) of arrestin-like adaptors promoting the interaction of these permeases with the Rsp5 Ub ligase.
We thank S. Léon and R. Haguenauer-Tsapis for regular and fruitful discussions, M. Hall for the pAS103 plasmid, Pascual Sanz for the pGST and pGST-Bmh2 plasmids, and Gerry Fink for the bmh strain. We also thank Catherine Jauniaux for her invaluable contribution to isolating strains and plasmids and Lydia Spedale for her technical assistance.
This work was supported by an FRSM grant (3.4.592.08.F) and an ARC grant (AUWB 2010-15-2) of the Fédération Wallonie-Bruxelles and by the CIBLES grant (grant 716760) of the Région Wallonne de Belgique.
Published ahead of print 10 September 2012