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© The Rockefeller University Press,
0021-9525/1999//1227 $5.00
The Journal of Cell Biology, Volume 146, Number 6,
, 1999 1227-1238
Original Article |
Starvation Induces Vacuolar Targeting and Degradation of the Tryptophan Permease in Yeast
hall{at}ubaclu.unibas.ch
In Saccharomyces cerevisiae, amino acid permeases are divided into two classes. One class, represented by the general amino acid permease GAP1, contains permeases regulated in response to the nitrogen source. The other class, including the high affinity tryptophan permease, TAT2, consists of the so-called constitutive permeases. We show that TAT2 is regulated at the level of protein stability. In exponentially growing cells, TAT2 is in the plasma membrane and also accumulates in internal compartments of the secretory pathway. Upon nutrient deprivation or rapamycin treatment, TAT2 is transported to and degraded in the vacuole. The ubiquitination machinery and lysine residues within the NH2-terminal 31 amino acids of TAT2 mediate ubiquitination and degradation of the permease. Starvation-induced degradation of internal TAT2 is blocked in sec18, sec23, pep12, and vps27 mutants, but not in sec4, end4, and apg1 mutants, suggesting that, upon nutrient limitation, internal TAT2 is diverted from the late secretory pathway to the vacuolar pathway. Furthermore, our results suggest that TAT2 stability and sorting are controlled by the TOR signaling pathway, and regulated inversely to that of GAP1.
Key Words: GAP1 • TOR • rapamycin • ubiquitin • endocytosis
© 1999 The Rockefeller University Press
AMINO acid permeases and homologues in yeast form a family of 23 members with demonstrated or suspected transport activity, and an additional member with a presumed regulatory function (Nelissen et al. 1997; Didion et al. 1998). Based on their function and regulation, the amino acid permeases are divided into two classes (Sophianopoulou and Diallinas 1995). Permeases of one class, including the general amino acid permease, GAP1,1 and the proline permease, PUT4, are regulated in response to the available nitrogen source (Jauniaux et al. 1987; Jauniaux and Grenson 1990; Vandenbol et al. 1990). In the presence of a good nitrogen source, such as ammonium or glutamine, the uptake activity by these permeases is low, whereas in media containing a poor nitrogen source, such as proline or urea, transport activity is strongly induced. Both permeases transport amino acids that can be used as a nitrogen source. The nitrogen-dependent regulation of GAP1 is complex, occurring at the levels of GAP1 transcription and GAP1 sorting and degradation (Stanbrough and Magasanik 1995; Roberg et al. 1997b; Springael and Andre 1998). The second class of amino acid permeases consists of mainly high affinity transporters that are specific for single amino acids or a small set of structurally related amino acids. These permeases, including the histidine permease, HIP1 (Tanaka and Fink 1985), and the tryptophan permease, TAT2 (Schmidt et al. 1994), import amino acids primarily for use in protein synthesis. They are thought not to be regulated and are thus often referred to as the constitutive permeases.
Newly made amino acid permeases, despite lacking an NH2-terminal signal sequence, are inserted into the membrane of the endoplasmic reticulum (Ljungdahl et al. 1992). Assisted by the ER-resident protein SHR3, the permeases are then packaged into coatomer protein (COP)-II vesicles (Kuehn et al. 1996), and routed through the secretory pathway to the plasma membrane, where they actively import their amino acid substrates. For GAP1, it has recently been shown that transport from the Golgi to the plasma membrane is not a default process. Depending on the nitrogen source, GAP1 is delivered from the Golgi to either the vacuole or the plasma membrane. In the presence of a good nitrogen source, GAP1 is routed to the vacuole and degraded, whereas, in the presence of a poor nitrogen source, GAP1 is targeted to the plasma membrane (Roberg et al. 1997a,Roberg et al. 1997b).
The GAP1 permease, like many other integral plasma membrane proteins, is removed from the plasma membrane via ubiquitin-triggered internalization (Hicke and Riezman 1996; Hicke 1997; Springael and Andre 1998). The NPI1/RSP5 ubiquitin-protein ligase is required for GAP1 ubiquitination. NPI1/RSP5 was originally identified as the nitrogen permease inactivator for GAP1 (Hein et al. 1995; Jauniaux et al. 1987). After internalization, GAP1 is transported to the vacuole for degradation.
The immunosuppressant rapamycin induces a starvation state in yeast cells, stationary phase or G0, by binding and inhibiting target of rapamycin (TOR) 1 and TOR2 (Heitman et al. 1991; Stan et al. 1994; Zheng et al. 1995; Barbet et al. 1996). The TOR proteins, two highly homologous phosphatidylinositol kinase-related kinases (Kunz et al. 1993; Helliwell et al. 1994), normally control cell growth by signaling activation of translation initiation and early G1 progression in response to nutrients (Barbet et al. 1996; Di Como and Arndt 1996; Berset et al. 1998). TOR2 also activates a Rho-type GTPase switch and thereby controls the cell cycle–dependent organization of the actin cytoskeleton (Schmidt et al. 1996, Schmidt et al. 1997; Bickle et al. 1998). However, this TOR2 unique function is not rapamycin sensitive (Zheng et al. 1995; Schmidt et al. 1996).
Here we show that the constitutive permease TAT2 is indeed regulated. Inversely to GAP1, TAT2 is targeted to the vacuole and degraded upon nutrient deprivation. Surprisingly, an internal pool of TAT2 appears to be routed directly to the vacuole independently of the plasma membrane. Furthermore, the regulated sorting of TAT2 is controlled by the TOR proteins.
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Materials and Methods |
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was used for propagation and isolation of plasmids as described (Ausubel et al. 1998).
DNA Manipulations
Restriction enzyme digests and ligations were done by standard methods. Enzymes and buffers were obtained commercially (Boehringer Mannheim). DNA was sequenced by the dideoxy-chain termination method with the T7 sequencing system (Pharmacia).
Construction of HA-tagged and Mutant TAT2
HA-TAT2 (in pAS55, pAS64, or pTB287) encodes an NH2-terminally HA-tagged, fully functional TAT2 protein under control of its own promoter. HA-TAT2 was constructed by ligating a 0.65-kb PstI-XbaI PCR product, containing the TAT2 promoter, 5' untranslated region, initiation codon, and double HA tag, to a 2.3-kb XbaI-EcoRI PCR product containing the TAT2 open reading frame and 3' noncoding region. The PstI and EcoRI sites were natural sites flanking the TAT2 gene. The PCR primers used to generate the 0.65-kb fragment were 5'-CGTCTAGATGCATAGTCCGGGACGTCATAGGGATAGCCCGCATAGTCAGGAACA-TCGTATGGGTACATATGAGAGTGTGTTGCGTAATTTG-3' (XbaI site in italics, antisense 2xHA open reading frame underlined and antisense initiation codon in bold) and the 1233 primer (New England Biolabs). The primers used to generate the 2.3-kb fragment were 5'-CGTCTAGAACCGAAGACTTTATTTCTTCTGTC-3' (XbaI site in italics) and the –20 primer (New England Biolabs). To create deletion variants, the following primers were used in combination with the –20 primer (New England Biolabs) to generate alternative 2.3-kb fragments: 5'-GGTCTAGAATGCGTTCAAATGAGGAGCTG-3' (TAT2
10), 5'-GGTCTAGAATGGAGCGAAAATCTAACTTTGG-3' (TAT217), 5'-CGTCTAGAATGTCTAACTTTGGATTTGTAG-3' (TAT220), 5'-CGTCTAGATCCAAGCAATTAACATCATC-3' (TAT229), 5'-CGTCTAGATCCAGGCAATTAACATCATC-3' (TAT229K31R) and 5'-GGTCTAGACAATTAATATCATCCTCATC-3' (TAT231). An internal deletion (TAT217-31) was constructed in a two-step PCR with the complementary primers 5'-TCAAATGAGGAGCTGCAATTAACATCATCC-3' and 5'-GGATGATGTTAATTGCAGCTCCTCATTTGA-3', each in combination with a flanking primer and pTB287 as the template. TAT25K>R was reconstituted from two PCR fragments generated with primers 5'-GCGAGCTCCTCATTTGAACGCCTGACAGAAGA-3' (antisense) and 5'-GAGGAGCTCAGGGAGCGAAGATCTAACTTTGGATTTGTAGAATACAGATCCAGGCAATTAACATCAT-CCTCATC-3' (sense), each in combination with a flanking primer and pTB287 as the template. A silent nucleotide substitution that created a SacI site (italics) is indicated in bold, and point mutations that changed lysine codons to arginine codons are underlined. Vent DNA Polymerase (New England Biolabs) or Taq polymerase (Boehringer Mannheim) was used in PCR. Mutations, deletions, and introduction of the HA tag-encoding sequence in TAT2 were verified by sequencing.
Amino Acid Import Studies
The import rates of radiolabeled amino acids (L-[5-3H]-tryptophan (33 Ci/mmol), L-[4,5-3H]-leucine (58 Ci/mmol), and L-[2,5-3H]-histidine (42 Ci/mmol; Amersham) were measured as described (Heitman et al. 1993), with the following modifications. Wild-type (JK9-3d) cells were grown to early logarithmic phase in YPD medium at 30°C, and the culture was split into six equal aliquots. Individual aliquots were incubated at 30°C in the presence of rapamycin for either 0, 15, 30, 45, 60, or 90 min. Uptake of labeled amino acids (% import/OD over time) was determined for cells of each aliquot. For the graph in Fig. 1 A the 10-min timepoint of each curve was plotted over the rapamycin preincubation time of the given aliquot.
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Indirect Immunofluorescence
Untreated logarithmically growing cells or cells treated with rapamycin for 60 min were fixed for 2 h in the growth medium supplemented with formaldehyde (3.7% final) and potassium phosphate buffer (100 mM final, pH 6.5). Cells were washed and resuspended in sorbitol buffer (1.2 M sorbitol and 100 mM potassium phosphate, pH 6.5). Cell walls were digested for 30 min at 37°C in sorbitol buffer supplemented with β-mercaptoethanol (20 mM final) and recombinant lyticase (5 mg/ml) or zymolyase 20T (12.5 mg/ml; Seigagaku Corporation), yielding identical results. Spheroblasts were fixed on poly-L-lysine–coated glass slides and permeabilized with PBT (53 mM Na2HPO4, 13 mM NaH2PO4, 75 mM NaCl, 1% BSA, and 0.1% Triton X-100). Immunofluorescence directed against the HA-epitope was performed by application of a high affinity monoclonal anti-HA antibody (clone 16B12; Babco) at a dilution of 1:1,000 in PBT for 2 h, and subsequently of a Cy3-conjugated rabbit anti–mouse IgG (Molecular Probes), diluted 1:1,000 in PBT, for 60 min. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI; Sigma) at a concentration of 1 µg/ml. Cells were visualized with a Zeiss Axiophot microscope (100x objective) and a video imaging system (MWG Biotech).
Cell Labeling and Immunoprecipitation
Logarithmically growing wild-type cells (JK9-3d) expressing HA-TAT2 were diluted in 400 ml SC medium (starting OD600 = 0.1) containing 2 mCi of L-[35S]methionine (Easytag protein labeling system; NEN). After 4 h (OD600 = 0.3), a 1,000-fold excess of cold methionine was added. The culture was split, and rapamycin or empty drug vehicle was added. Temperature-sensitive sec4 cells (RH1552) grown at 24°C to early logarithmic phase and expressing HA-TAT2 or HA-TAT25K>R were chilled on ice, harvested by centrifugation, and immediately resuspended at an OD600 = 5 in 12 ml of SC medium containing 2 mCi of L-[35S]methionine instead of cold methionine. Cells were incubated for 3 min at 24°C, then shifted to 37°C for an additional 5 min. The cultures were chilled on ice, harvested, and resuspended at an OD600 = 0.5 in prewarmed (37°C) SC medium containing cold methionine and rapamycin (200 ng/ml). Culture aliquots equal to 15 OD600 of cells were removed after 0, 30, 60, and 90 min, and kept on ice for 10 min in the presence of 10 mM NaN3 and 10 mM NaF. Cells were harvested and cell extracts prepared as described above. Extracts were precleared for 30 min with Sepharose CL-4B, and incubated for 2 h at 4°C with protein G immobilized on Sepharose CL-4B (Sigma) and monoclonal anti-HA antibodies (12CA5 or 16B12; Babco), in a volume of 1 ml. The beads were collected and washed extensively with extraction buffer. Bound protein was solubilized by incubating in SDS sample buffer (Ausubel et al. 1998) for 10 min at 37°C. Gels were scanned and signals quantified using a PhosphorImager (Molecular Dynamics) and ImageQuant software. Ubiquitinated TAT2 was visualized in Western blots by use of monoclonal anti-myc antibody (clone 9E10). For this purpose, myc-tagged ubiquitin was expressed as described (Ellison and Hochstrasser 1991), in wild-type cells (JK9-3d) also expressing HA-TAT2, HA-TAT25K>R, or untagged TAT2. The permease was immunoprecipitated from extracts of these cells (from a total of 1 mg of protein) as described above.
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As rapamycin causes starvation in yeast cells, the rapamycin-induced downregulation of TAT2 appeared to be a novel starvation response. To confirm that the downregulation of TAT2 is indeed a starvation response, we examined if nutrient deprivation causes a similar downregulation. Wild-type cells grown to early logarithmic phase in medium containing glucose, ammonium, and required amino acids (SC medium) were harvested by filtration and resuspended in normal SC medium and in nutrient-modified SC medium. TAT2 protein levels were then examined after 60 min of incubation in the new media. First, the medium was modified by increasing, decreasing, or removing tryptophan. Whereas increasing (20x; 800 µg/ml) and decreasing (0.1x; 4 µg/ml) the tryptophan concentration had little or no effect, tryptophan starvation resulted in an almost complete loss of TAT2 protein (Fig. 1 B, upper panel). Second, cells were shifted to medium modified in the nitrogen source, i.e., medium containing a reduced amount of ammonium (0.1x; 1 mM), no ammonium, or proline instead of ammonium. As shown in Fig. 1 B (middle panel), nitrogen limitation correlated with a decrease in the level of TAT2 protein, up to a complete loss of the permease in ammonium-free and proline media. In contrast to TAT2, GAP1 is activated upon shift from ammonium to the poor nitrogen source proline, and inactivated (degraded) upon shift from proline to ammonium (Magasanik 1992; Springael and Andre 1998), suggesting that TAT2 and GAP1 are inversely regulated in response to the nitrogen source. Third, cells were shifted to media with reduced (0.1x; 0.2%) or no glucose. The levels of TAT2 protein again correlated with nutrient availability, dropping to undetectable levels in the absence of glucose (Fig. 1 B, lower panel). Thus, the downregulation of TAT2 protein is a novel starvation response, caused by either rapamycin treatment or amino acid, nitrogen or carbon deprivation.
To determine whether the nutrient regulation observed for TAT2 also occurs for other high affinity constitutive permeases, we measured histidine import in rapamycin-treated cells. The effect of rapamycin on histidine uptake was similar to that observed for tryptophan uptake (not shown). We also examined the abundance of myc-tagged histidine permease HIP1 (Tanaka and Fink 1985; Kuehn et al. 1996) in rapamycin-treated cells. Like TAT2, HIP1 almost completely disappeared within 60 min of rapamycin treatment (Fig. 1 C). Thus, starvation-induced downregulation is not unique to TAT2, but also applies to another, and possibly to all, so-called constitutive amino acid permeases. The starvation program in yeast may involve a global downregulation of high specificity permeases.
In contrast to TAT2 and HIP1, rapamycin treatment caused a significant increase in GAP1 protein, even in the presence of nutrients (Fig. 1 C). The abundance of SHR3, an ER-resident amino acid permease chaperone (Ljungdahl et al. 1992), was not significantly affected by rapamycin treatment (Fig. 1 C). Thus, the rapamycin-sensitive TOR proteins appear to regulate inversely the high specificity permeases, such as TAT2 and HIP1, and the broad specificity permease GAP1, in response to nutrient availability.
Starvation Induces Turnover of TAT2
Rapamycin also causes a downregulation in translation initiation (Barbet et al. 1996). Because the downregulation of translation is earlier than the observed downregulation of TAT2, the decrease in TAT2 levels could reflect a loss of de novo protein synthesis in combination with a normally short half-life of the permease. We performed a pulse–chase experiment to determine whether the loss of TAT2 resulted from increased turnover or solely from reduced synthesis of the protein. Cells expressing HA-tagged TAT2 were grown to early logarithmic phase in the presence of radiolabeled methionine, and chased with an excess of cold methionine in the presence or absence of rapamycin. Culture aliquots were taken at 15-min intervals and processed for immunoprecipitation of the permease. As shown in Fig. 2, the half-life of TAT2 in the rapamycin-treated cells was 
30 min, whereas in the rapamycin-untreated cells the half-life was >90 min. Thus, the TAT2 protein is significantly more stable in exponentially growing cells than in starved cells, indicating that starvation induces degradation of TAT2.
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10%) resistant to rapamycin-induced degradation. In a pep4 mutant lacking vacuolar proteases, TAT2 was completely resistant to rapamycin-induced degradation.
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To investigate further the involvement of ubiquitination in TAT2 degradation, we examined if TAT2 itself is ubiquitinated. The permease was immunoprecipitated from rapamycin-treated cells expressing HA-TAT2 alone or coexpressing HA-TAT2 and myc-tagged ubiquitin (myc-Ub) (Ellison and Hochstrasser 1991). Probing with an anti-HA antibody revealed the signal for TAT2 (50 kD) in both immunoprecipitates, whereas probing with an anti-myc antibody revealed only >50 kD proteins solely in the immunoprecipitate derived from cells coexpressing myc-Ub (Fig. 3 B). Neither signal was detected in immunoprecipitates from cells expressing an untagged version of TAT2. This suggests that TAT2 is ubiquitinated. Taken together, the above results indicate that TAT2 degradation involves ubiquitination, vacuolar proteases, and, in part, endocytosis.
The NH2 Terminus of TAT2 Is Required for Ubiquitination and Degradation
Ligand-induced ubiquitination and internalization of the pheromone receptor STE2 involves the sequence SINDAKSS within the cytosolic tail of the receptor (Hicke et al. 1998). Replacement of the aspartic acid to alanine or the lysine residue to an arginine in the DAKS core of this motif impairs the internalization of a truncated form of the receptor, whereas mutation of the aspartic acid to glutamic acid, or substitution of the first serine by an alanine, did not interfere with endocytosis. According to a structure prediction by the TOP PRED algorithm, both the NH2-terminal and COOH-terminal sequences of TAT2 face the cytoplasm. In the extreme NH2 terminus of TAT2, two ExKS motifs, similar to the DxKS core of the STE2 SINDAKSS motif, are present (Fig. 5 A, amino acids 18–21 and 27–30).
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29) had no effect, deletion of the NH2-terminal 31 amino acids (TAT231) completely stabilized the protein (Fig. 5 A). To further characterize the sequence required for degradation, a smaller deletion that completely removed the two ExKS motifs within the NH2-terminal 31 amino acids was constructed (TAT217-31, Fig. 5 A). However, this deletion did not result in a degradation-resistant variant of TAT2, suggesting that the ExKS motifs are not essential for TAT2 degradation.
Ubiquitin is attached to lysine residues in target proteins. There are five lysine residues in the NH2-terminal 31 amino acids of TAT2, and the above analysis suggests that all five lysines must be deleted to prevent TAT2 degradation. To determine if one or more of the lysines is required for degradation, possibly as an acceptor for ubiquitin, all five lysines were changed to arginines. Single lysine mutants and several combinations, including a combination of all five substitutions, were examined for stability upon rapamycin treatment (Fig. 5 A for TAT25K>R, and data not shown). Stabilization of TAT2 was observed only when all five lysine residues were exchanged for arginines. Furthermore, the TAT25K>R protein was approximately threefold more abundant (in rapamycin-treated and untreated cells) than wild-type TAT2 in rapamycin-untreated cells (data not shown). This increase in the level of TAT25K>R was similar to the increase of wild-type TAT2 caused by a defect in the ubiquitination machinery, as described above, and may again reflect a loss of basal turnover of the permease. Immunofluorescence on rapamycin-treated and -untreated cells revealed a pronounced accumulation of TAT25K>R in the plasma membrane, with no detectable signal elsewhere in the cell (Fig. 5 B, and data not shown). As observed for wild-type TAT2 in ubiquitination mutants, the similar pattern for rapamycin-treated and -untreated cells may reflect an aberrantly high accumulation of the stabilized permease in the plasma membrane even in growing cells. The TAT229K31R and TAT231 proteins behaved identically to TAT25K>R with regard to both abundance and localization (data not shown). Finally, probing immunoprecipitated TAT25K>R for ubiquitin failed to reveal any ubiquitinated forms of TAT25K>R (Fig. 3 B). Taken together, these results suggest that at least one lysine residue within the NH2-terminal 31 amino acids of TAT2 is necessary for ubiquitination and degradation of at least the plasma membrane pool of the permease.
Upon Starvation, TAT2 Is Sorted from Internal Compartments to the Vacuole Independently of the Plasma Membrane
If internal TAT2 is transported to the vacuole via the plasma membrane, TAT2 should be completely stabilized in an end4 mutant, due to a block in plasma membrane internalization. However, results presented above indicate that the large intracellular pool of TAT2 is degraded in an end4 mutant, suggesting that internal TAT2 is sorted to the vacuole independently of the plasma membrane. To investigate if TAT2 is targeted from the ER and the Golgi to the vacuole without passing through the plasma membrane, we examined the stability of TAT2 in sec4, sec18, and sec23 mutants. SEC4 (Goud et al. 1988) is a small GTPase required for fusion of Golgi-derived vesicles with the plasma membrane. SEC23 (Hicke and Schekman 1989) is a subunit of the COPII complex required for ER to Golgi transport. SEC18 (Eakle et al. 1988) is the yeast NSF homologue and is generally required for fusion of vesicles with acceptor membranes (reviewed in Kaiser et al. 1997). The amounts of TAT2 in extracts of rapamycin-treated wild-type cells and the sec mutants were compared by Western analysis (Fig. 6). In the sec4 mutant, TAT2 was degraded like in wild-type cells. In contrast, TAT2 was completely stable in the sec18 mutant and mostly stable in the sec23 mutant. Taken together, the above results suggest that TAT2 is targeted from the ER and the Golgi to the vacuole independently of the plasma membrane. Thus, upon starvation, TAT2 appears to be diverted from the secretory pathway to the vacuolar pathway.
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Because starvation-induced degradation of intracellular TAT2 does not rely on passage through the cell surface or on autophagy, a likely transport route to the vacuole is via the PEP12-, VPS45-, and VPS27-dependent pathway (Cowles et al. 1994; Piper et al. 1994, Piper et al. 1995; Becherer et al. 1996). In this VPS pathway, proteins are delivered from the Golgi to the vacuole via a prevacuolar/endosomal compartment. If TAT2 is transported to the vacuole via the VPS pathway, the permease should be stable in a rapamycin-treated mutant defective in this pathway. Indeed, TAT2 was still present in extracts of rapamycin-treated pep12, vps27, and vps45 cells (Fig. 6, data not shown). Taken together, the above results suggest that upon nutrient depletion, TAT2 is transported from the ER and Golgi to the vacuole via the VPS pathway, without passing through the cell surface.
Mutation of the five NH2-terminal lysine residues in TAT2 results in an apparently complete stabilization of the permease (see Fig. 5 A), suggesting that the intracellular pool of TAT2, in addition to the plasma membrane pool (see above), requires ubiquitination for rapamycin-induced targeting to the vacuole and degradation. To investigate whether the NH2-terminal lysine residues are required for degradation of the internal pool of TAT2, temperature-sensitive sec4 cells expressing either HA-TAT2 or HA-TAT25K>R were pulse labeled for 3 min at permissive temperature, shifted to nonpermissive temperature, and chased in the presence of rapamycin (see Materials and Methods). This experiment was designed to investigate the fate of newly made intracellular TAT2 and TAT25K>R proteins that are unable to reach the plasma membrane due to the sec4 mutation. Whereas newly made HA-TAT2 was rapidly degraded in rapamycin-treated cells, HA-TAT25K>R was stable over a 90-min chase period (Fig. 7), suggesting that the NH2-terminal lysine residues of TAT2 and ubiquitination are also required for rapamycin-induced degradation of the internal pool of TAT2.
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Discussion |
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TAT2, and at least the histidine permease HIP1, are turned over upon starvation. This is a novel aspect of the starvation response in yeast cells. Why are specific permeases turned over upon starvation? The specific permease TAT2 and the broad-range permease GAP1 are regulated inversely, at least in response to the quality and quantity of the nitrogen source. There are several specific permeases but only two known broad-range permeases, GAP1 and AGP1, in yeast (Grenson et al. 1970; Jauniaux and Grenson 1990; Nelissen et al. 1997; Schreve et al. 1998). We suggest that specific amino acid permeases are expressed and functionally maintained under nutrient-rich conditions, and are probably fine-regulated by the availability of their substrates. Once nutrients become limiting, cells may express and maintain only a couple broad-range permeases, instead of the several specific permeases, as a means to reduce energy consumption. The inverse regulation of specific and broad-range permeases provides a mechanism to optimize import with regard to the quality and quantity of nutrients.
Upon rapamycin treatment or upon shift from a good nitrogen source (ammonium) to a poor nitrogen source (proline), GAP1 is stabilized and TAT2 is degraded. How is this inverse regulation of TAT2 and GAP1 achieved? To date, two posttranslational regulators of GAP1 are known, NPI1 and NPR1. NPI1, a ubiquitin ligase, presumably ubiquitinates GAP1 and thereby triggers the internalization and degradation of the permease (Springael and Andre 1998). NPR1, a Ser/Thr protein kinase, is a positive regulator of GAP1 (Vandenbol et al. 1987). The mechanism by which NPR1 activates GAP1 is unknown, but the finding that GAP1 is phosphorylated when active suggests that NPR1 directly phosphorylates GAP1 and thereby protects it from NPI1-dependent degradation (Stanbrough and Magasanik 1995). NPI1 is also responsible for inactivation of TAT2. Thus, we consider an inverse regulation via this ubiquitin ligase unlikely. However, the inverse regulation may be achieved through NPR1. Indeed, we have found that NPR1 mediates the destruction of TAT2 under conditions when the kinase protects GAP1 (Schmidt et al. 1998). Furthermore, we have shown that NPR1 is controlled by TOR and the TOR downstream effector TAP42 (Schmidt et al. 1998). Because TAT2 is regulated in response to nitrogen, carbon and amino acid availability, it is of interest to determine whether GAP1 is also regulated in response to nutrients other than nitrogen. Furthermore, it remains to be determined how the TOR nutrient–signaling pathway senses the availability of nutrients.
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Acknowledgments |
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T. Beck was the recipient of fellowships from the Roche Research Foundation and the Krebsliga beider Basel. This work was supported by grants of the Swiss National Science Foundation and the Canton of Basel to M.N. Hall.
Submitted: 21 December 1998
Revised: 19 July 1999
Accepted: 12 August 1999
1.used in this paper: COP, coatomer protein; GAP, general amino acid permease; HIP, histidine permease; PUT, proline permease; TAT, tryptophan permease; TOR, target of rapamycin
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References |
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|---|
Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A. & Struhl K., Current Protocols in Molecular Biology, 1998, John Wiley & Sons, Inc, New York.
Barbet N.C., Schneider U., Helliwell S.B., Stansfield I., Tuite M.F. & Hall M.N.. TOR controls translation initiation and early G1 progression in yeast, Mol. Biol. Cell., 7, 1996, 25–42.[Abstract]
Becherer K.A., Rieder S.E., Emr S.D. & Jones E.W.. Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast, Mol. Biol. Cell., 7, 1996, 579–594.[Abstract]
Berset C., Trachsel H. & Altmann M.. The TOR (target of rapamycin) signal transduction pathway regulates the stability of translation initiation factor eIF4G in the yeast Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. USA., 95, 1998, 4264–4269.
Bickle M., Delley P.A., Schmidt A. & Hall M.N.. Cell wall integrity modulates RHO1 activity via the exchange factor ROM2, EMBO J., 17, 1998, 2235–2245.[Medline]
Cowles C.R., Emr S.D. & Horazdovsky B.F.. Mutations in the VPS45 gene, a SEC1 homologue, result in vacuolar protein sorting defects and accumulation of membrane vesicles, J. Cell Sci., 107, 1994, 3449–3459.[Abstract]
Di Como C.J. & Arndt K.T.. Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases, Genes Dev, 10, 1996, 1904–1916.
Didion T., Regenberg B., Jorgensen M.U., Kielland-Brandt M.C. & Andersen H.A.. The permease homologue Ssy1p controls the expression of amino acid and peptide transporter genes in Saccharomyces cerevisiae, Mol. Microbiol., 27, 1998, 643–650.[Medline]
Eakle K.A., Bernstein M. & Emr S.D.. Characterization of a component of the yeast secretion machineryidentification of the SEC18 gene product, Mol. Cell. Biol., 8, 1988, 4098–4109.
Ellison M.J. & Hochstrasser M.. Epitope-tagged ubiquitin. A new probe for analyzing ubiquitin function, J. Biol. Chem., 266, 1991, 21150–21157.
Galan J. & Haguenauer-Tsapis R.. Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein, EMBO (Eur. Mol. Biol. Organ.) J., 16, 1997, 5847–5854.[Medline]
Galan J.M., Moreau V., Andre B., Volland C. & Haguenauer-Tsapis R.. Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease, J. Biol. Chem., 271, 1996, 10946–10952.
Goud B., Salminen A., Walworth N.C. & Novick P.J.. A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast, Cell, 53, 1988, 753–768.[Medline]
Grenson M., Hou C. & Crabeel M.. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease, J. Bacteriol., 103, 1970, 770–777.
Hein C., Springael J.Y., Volland C., Haguenauer-Tsapis R. & Andre B.. NPl1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase, Mol. Microbiol., 18, 1995, 77–87.[Medline]
Heitman J., Movva N.R. & Hall M.N.. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast, Science, 253, 1991, 905–909.
Heitman J., Koller A., Kunz J., Henriquez R., Schmidt A., Movva N.R. & Hall M.N.. The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae, Mol. Cell. Biol., 13, 1993, 5010–5019.
Helliwell S.B., Wagner P., Kunz J., Deuter-Reinhard M., Henriquez R. & Hall M.N.. TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast, Mol. Biol. Cell., 5, 1994, 105–118.[Abstract]
Hicke L.. Ubiquitin-dependent internalization and down-regulation of plasma membrane proteins, FASEB J., 11, 1997, 1215–1226.[Abstract]
Hicke L. & Schekman R.. Yeast Sec23p acts in the cytoplasm to promote protein transport from the endoplasmic reticulum to the Golgi complex in vivo and in vitro, EMBO (Eur. Mol. Biol. Organ.) J., 8, 1989, 1677–1684.[Medline]
Hicke L. & Riezman H.. Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis, Cell, 84, 1996, 277–287.[Medline]
Hicke L., Zanolari B. & Riezman H.. Cytoplasmic tail phosphorylation of the alpha-factor receptor is required for its ubiquitination and internalization, J. Cell Biol., 141, 1998, 349–358.
Horak J. & Wolf D.H.. Catabolite inactivation of the galactose transporter in the yeast Saccharomyces cerevisiaeubiquitination, endocytosis, and degradation in the vacuole, J. Bacteriol., 179, 1997, 1541–1549.
Ito H., Fukuda Y., Murata K. & Kimura A.. Transformation of intact yeast cells treated with alkali cations, J. Bacteriol., 153, 1983, 163–168.
Jauniaux J.C. & Grenson M.. GAP1, the general amino acid permease gene of Saccharomyces cerevisiae. Nucleotide sequence, protein similarity with the other bakers yeast amino acid permeases, and nitrogen catabolite repression, Eur. J. Biochem., 190, 1990, 39–44.[Medline]
Jauniaux J.C., Vandenbol M., Vissers S., Broman K. & Grenson M.. Nitrogen catabolite regulation of proline permease in Saccharomyces cerevisiae. Cloning of the PUT4 gene and study of PUT4 RNA levels in wild-type and mutant strains, Eur. J. Biochem., 164, 1987, 601–606.[Medline]
Jenness D.D., Li Y., Tipper C. & Spatrick P.. Elimination of defective -factor pheromone receptor, Mol. Cell. Biol., 17, 1997, 6236–6245.[Abstract]
Kaiser C.A., Gimeno R.E. & Shaywitz D.A.. Protein Secretion, membrane biogenesis, and endocytosis, Pringle J.R., Broach J.R. & Jones E.W., The Molecular and Cellular Biology of the Yeast Saccharomyces cerevisiae. Vol. 3, 1997, 91–227, Cold Spring Harbor Laboratory Press, NY.
Kuehn M.J., Schekman R. & Ljungdahl P.O.. Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro, J. Cell Biol., 135, 1996, 585–595.
Kunz J., Henriquez R., Schneider U., Deuter-Reinhard M., Movva N.R. & Hall M.N.. Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression, Cell, 73, 1993, 585–596.[Medline]
Latterich M. & Watson M.D.. Isolation and characterization of osmosensitive vacuolar mutants of Saccharomyces cerevisiae, Mol. Microbiol., 5, 1991, 2417–2426.[Medline]
Ljungdahl P.O., Gimeno C.J., Styles C.A. & Fink G.R.. SHR3a novel component of the secretory pathway specifically required for localization of amino acid permeases in yeast, Cell, 71, 1992, 463–478.[Medline]
Lucero P. & Lagunas R.. Catabolite inactivation of the yeast maltose transporter requires ubiquitin-ligase npi1/rsp5 and ubiquitin-hydrolase npi2/doa4, FEMS Microbiol. Lett., 147, 1997, 273–277.[Medline]
Magasanik B.. Regulation of nitrogen utilization, Jones E.W., Pringle J.R. & Broach J.R., The Molecular and Cellular Biology of the Yeast Saccharomyces cerevisiae. Vol. 2. Gene Expression, 1992, 283–317, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Matsuura A., Tsukada M., Wada Y. & Ohsumi Y.. Apg1p, a novel protein kinase required for the autophagic process in Saccharomyces cerevisiae, Gene, 192, 1997, 245–250.[Medline]
Nelissen B., De Wachter R. & Goffeau A.. Classification of all putative permeases and other membrane plurispanners of the major facilitator superfamily encoded by the complete genome of Saccharomyces cerevisiae, FEMS Microbiol. Rev., 21, 1997, 113–134.[Medline]
Noda T. & Ohsumi Y.. Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast, J. Biol. Chem., 273, 1998, 3963–3966.
Papa F.R. & Hochstrasser M.. The yeast DOA4 gene encodes a deubiquitinating enzyme related to a product of the human tre-2 oncogene, Nature, 366, 1993, 313–319.[Medline]
Piper R.C., Whitters E.A. & Stevens T.H.. Yeast Vps45p is a Sec1p-like protein required for the consumption of vacuole-targeted, post-Golgi transport vesicles, Eur. J. Cell Biol., 65, 1994, 305–318.[Medline]
Piper R.C., Cooper A.A., Yang H. & Stevens T.H.. VPS27 controls vacuolar and endocytic traffic through a prevacuolar compartment in Saccharomyces cerevisiae, J. Cell Biol., 131, 1995, 603–617.
Raths S., Rohrer J., Crausaz F. & Riezman H.. end3 and end4two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae, J. Cell Biol., 120, 1993, 55–65.
Roberg K.J., Bickel S., Rowley N. & Kaiser C.A.. Control of amino acid permease sorting in the late secretory pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7, and LST8, Genetics, 147, 1997, 1569–1584a.[Abstract]
Roberg K.J., Rowley N. & Kaiser C.A.. Physiological regulation of membrane protein sorting late in the secretory pathway of Saccharomyces cerevisiae, J. Cell Biol., 137, 1997, 1469–1482b.
Schmidt A., Hall M.N. & Koller A.. Two FK506 resistance-conferring genes in Saccharomyces cerevisiae, TAT1 and TAT2, encode amino acid permeases mediating tyrosine and tryptophan uptake, Mol. Cell. Biol., 14, 1994, 6597–6606.
Schmidt A., Kunz J. & Hall M.N.. TOR2 is required for organization of the actin cytoskeleton in yeast, Proc. Natl. Acad. Sci. USA., 93, 1996, 13780–13785.
Schmidt A., Bickle M., Beck T. & Hall M.N.. The yeast phosphatidylinositol kinase homolog TOR2 activates RHO1 and RHO2 via the exchange factor ROM2, Cell, 88, 1997, 531–542.[Medline]
Schmidt A., Beck T., Koller A., Kunz J. & Hall M.N.. The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease, EMBO (Eur. Mol. Biol. Organ.) J., 17, 1998, 6924–6931.[Medline]
Schreve J.L., Sin J.K. & Garrett J.M.. The Saccharomyces cerevisiae YCC5 (YCL025c) gene encodes an amino acid permease, Agp1, which transports asparagine and glutamine, J. Bacteriol., 180, 1998, 2556–2559.
Sherman F.. Getting started with yeast, Guthrie C. & Fink G.R., Guide to Yeast Genetics and Molecular Biology. Vol. 194, 1991, 3–21, Academic Press, San Diego.
Singer J.D., Manning B.M. & Formosa T.. Coordinating DNA replication to produce one copy of the genome requires genes that act in ubiquitin metabolism, Mol. Cell. Biol., 16, 1996, 1356–1366.[Abstract]
Sophianopoulou V. & Diallinas G.. Amino acid transporters of lower eukaryotesregulation, structure and topogenesis, FEMS Microbiol. Rev., 16, 1995, 53–75.[Medline]
Springael J.Y. & Andre B.. Nitrogen-regulated ubiquitination of the Gap1 permease of Saccharomyces cerevisiae, Mol. Biol. Cell., 9, 1998, 1253–1263.
Stan R., McLaughlin M.M., Cafferkey R., Johnson R.K., Rosenberg M. & Livi G.P.. Interaction between FKBP12–rapamycin and TOR involves a conserved serine residue, J. Biol. Chem., 269, 1994, 32027–32030.
Stanbrough M. & Magasanik B.. Transcriptional and posttranslational regulation of the general amino acid permease of Saccharomyces cerevisiae, J. Bacteriol., 177, 1995, 94–102.
Tanaka J. & Fink G.R.. The histidine permease gene (HIP1) of Saccharomyces cerevisiae, Gene, 38, 1985, 205–214.[Medline]
Vandenbol M., Jauniaux J.C., Vissers S. & Grenson M.. Isolation of the NPR1 gene responsible for the reactivation of ammonia-sensitive amino-acid permeases in Saccharomyces cerevisiae. RNA analysis and gene dosage effects, Eur. J. Biochem., 164, 1987, 607–612.[Medline]
Vandenbol M., Jauniaux J.C. & Grenson M.. The Saccharomyces cerevisiae NPR1 gene required for the activity of ammonia-sensitive amino acid permeases encodes a protein kinase homologue, Mol. Gen. Genet., 222, 1990, 393–399.[Medline]
Zheng X.F., Florentino D., Chen J., Crabtree G.R. & Schreiber S.L.. TOR kinase domains are required for two distinct functions, only one of which is inhibited by rapamycin, Cell, 82, 1995, 121–130.[Medline]
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