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Department of Physiology, Carlsberg Laboratory, DK-2500 Copenhagen Valby, Denmark,1 Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-35002
Received 4 July 2003/ Accepted 8 July 2003
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ABSTRACT |
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INTRODUCTION |
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Key components of the amino acid sensing signal transduction pathway, including Ssy1, Ptr3, Ssy5, and Stp1, were identified in a genetic screen for mutants that failed to take up branched-chain amino acids (16). Because several genes in S. cerevisiae encode permeases capable of transporting branched-chain amino acids, the screen revealed mutations that reduced the expression of all of them simultaneously, thereby focusing on components of the signaling pathway itself. Thus, SSY1 was found to be required for normal amino acid-dependent transcriptional induction of several target permease genes, including BAP2, BAP3, PTR2, and TAT1 (6).
Consistent with their regulation of multiple transporter genes, components of the amino acid signaling pathway were independently discovered through other investigations, including screens for mutations that confer resistance to toxic dipeptides (14), amino acid analogues (4), or high concentrations of histidine (17).
Ssy1 is a plasma membrane protein (17) evolutionarily related to the amino acid permeases in S. cerevisiae (22) and thus is positioned at the presumed head of the amino acid signal transduction pathway. Ssy1 interacts at the plasma membrane with Ptr3 and Ssy5, forming a complex designated SPS for Ssy1-Ptr3-Ssy5 (8). Presumably, amino acid binding confers a conformational alteration in Ssy1 producing a signal that is transmitted to various target promoters. Proteolytic activation of the transcription factor Stp1 (or Stp2) is a key event in the signal transduction process (2). The discovery that overexpression of the amino terminus of Ssy1 interferes with amino acid signaling (4, 8) revealed the importance of this part of Ssy1.
Although the structure of Ssy1 is related to that of amino acid permeases, available evidence supports the notion that Ssy1 functions as a sensor rather than as a transporter. First, transport of amino acids by bona fide permeases fails to elicit signaling in ssy1 mutant cells (6, 13, 16, 17). In addition, Ssy1 itself seems to be unable to mediate amino acid transport. Although citrulline elicits the signaling response in SSY1 gap1 mutant cells (13), such cells are unable to take up enough citrulline as a sole nitrogen source to confer growth (11). Similarly, although SSY1 is required for maximal uptake of branched-chain amino acids, SSY1 overexpression fails to restore their uptake in cells in which the four genes known to encode branched-chain amino acid permeases have been deleted (6). Nor does the expression of SSY1 restore uptake of tryptophan in cells in which the genes for the authentic tryptophan permeases have been deleted (13). However, such tests cannot rule out the possibility that Ssy1 transports small quantities of the signaling molecule, and if any transport occurs, the sensor that actually recognizes the signaling amino acid would not have to be Ssy1 but could be a closely associated protein. Thus, a key question is: does signaling require the uptake and intracellular appearance of an inducer?
To answer this question, we sought to determine if Ssy1 could be converted genetically into a sensor capable of activating the signaling pathway during growth on medium lacking amino acids. By using a novel genetic screen, we identified gain-of-function mutations in Ssy1 that conferred constitutive signaling. Some alleles also increased the apparent affinity for inducers, supporting the hypothesis that Ssy1 interacts directly with extracellular amino acids to initiate signaling.
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MATERIALS AND METHODS |
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The strains used are presented in Table 1. From an initial cross between HY438 (H. Liang and R. F. Gaber, unpublished data) and M4871, a recipient strain suitable for the expression of an SPS-dependent AGP1::KAT1 reporter construct was generated by backcrossing appropriate ssy1
trk1 trk2 meiotic progeny with the HY438 parent. The resulting strain, RGY4990, harbors a wild-type GAP1 gene. All strains containing the trk1 trk2 mutations were derived from this strain. Strain M5003, containing the PAGP1-KAT1 reporter construct integrated at the TRP1 locus, was constructed by transformation of strain RGY4990 to Trp+ with XbaI-digested plasmid pOK6 (described below). M5025, containing the PAGP1-KAT1 reporter integrated at the AGP1 locus, was constructed by transformation of strain RGY4990 to Trp+ with BssHII-digested plasmid pOK6. All strains harboring the wild-type TRK1 and TRK2 genes are congenic with S288c.
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Strains harboring null alleles of SSY1, PTR3, or SSY5 were constructed by transformation of strain M4054 with the loxP-KanMX4-loxP gene disruption cassette system (12). Successful integration of the disruption cassettes was verified by diagnostic PCR, and the inserted KanMX4 element was evicted upon introduction of CRE-recombinase expressed from plasmid pSH47 (12).
Plasmid pSSY1 (16; also referred to as pMB5, M. B. Bruun, M.S. thesis, University of Copenhagen, Copenhagen, Denmark, 1997) contains the 3.6-kb XhoI-NcoI fragment encompassing the wild-type SSY1 gene cloned into pRS316[CEN/URA3] (25). pAGP1-KAT1 was constructed as follows. The 1-kb region upstream of the AGP1 ORF was amplified from S. cerevisiae chromosomal DNA by PCR with primers AGP1F-SacI and AGP1R-BamHI. The amplified DNA was cloned into SacI- and BamHI-digested plasmid pHXT6-KAT1 (R. Kurtz and R. F. Gaber, unpublished data), producing plasmid pRG806-12, in which the KAT1 cDNA is placed under control of the amino acid-inducible AGP1 promoter (PAGP1-KAT1). The entire PAGP1-KAT1 gene fusion was subcloned into pRS314[CEN/TRP1] (25) by in vivo homologous recombination. To accomplish this, PAGP1-KAT1 from pRG806-12 was amplified by PCR with primers 316F and 316R and the resulting DNA fragment was cotransformed into RGY5424 with ClaI-digested pRS314. Plasmid DNA rescued from one of the resulting Trp+ transformants was analyzed by restriction digestion and found to represent a successful subclone of PAGP1-KAT1 into pRS314. The resulting plasmid, pRG807a, was designated pAGP1-KAT1.
PAGP1-KAT1 from pAGP1-KAT1 was subcloned into integrative vector pRS304[TRP1] (25) to produce plasmid pOK6. The 3.6-kb KpnI/SacII fragment of SSY1 from pSSY1 was cloned into pRS306[URA3] (25) to produce pOK13, and the corresponding fragment from pSSY1-101 was inserted into pRS306 to produce pOK14.
SSY1 mutagenesis strategies. To generate random mutations in SSY1 for the mutant screen, a 2.76-kb fragment of DNA encompassing the SSY1 ORF was subjected to mutagenic PCR with the Mutazyme polymerase and the GeneMorph kit from Stratagene under the conditions recommended by the manufacturer. Primers used to generate the amplified fragment were SSY1Check.for and SSY1ORF.rev. Plasmid pRG819-7, consisting of the XhoI-BamHI SSY1 fragment inserted into pRS314, was used as template.
A separate library of plasmids with mutations at codon 382 of SSY1 was generated by a two-step PCR with primers that contained degenerate nucleotides at the appropriate sites (316R, Oligo21 through Oligo23; details are available upon request). The plasmids that produced mutant phenotypes were sequenced and show identity to SSY1 except for the differences at codon 382.
Specific mutations in SSY1 were generated by site-directed mutagenesis with appropriate oligonucleotide primers to generate the desired mutation and to eliminate a unique restriction site (primer URA3-StuI-Elim, USE mutagenesis) in the target plasmid. Mutagenic primers SSY1-A237T, SSY1-T382K, and SSY1-T382D included restriction sites for diagnostic purposes. The double mutation in pSSY1-104 was constructed in a single round of mutagenesis. The sequences of the mutagenic primers used are listed in Table S1 in the supplemental material.
Genetic screen for constitutive SSY1 alleles. To obtain yeast transformants that harbor potential mutant alleles of SSY1, 0.1 µg of EcoRI-HindIII-digested pSSY1 and 0.1 µg of the Mutazyme-amplified SSY1 ORF were used to transform recipient strain RGY5000 to Ura+ on medium containing permissive concentrations of potassium (SDHP-URA). Ura+ colonies were replica plated to non-potassium-supplemented medium (SD), either with or without various amino acids as inducers. Defective alleles of SSY1 were detected by the inability (or decreased ability) to grow on SD plus an inducer. The single gain-of-function allele, SSY1-101, conferred the ability to grow on SD in the absence of amino acid.
Screening of the SSY1382 degenerate library. Strain M5025, carrying the PAGP1-KAT1 reporter gene integrated at the AGP1 locus, was transformed with plasmid DNA from the SSY1382 degenerate library with selection on SD containing 100 mM KCl. Randomly picked colonies were suspended in water and diluted to an optical density at 600 nm (OD600) of 0.1, and 4-µl droplets of each were applied to SD medium and tested for growth. Colonies that exhibited stronger growth than the wild-type control were selected for rescue of the resident library plasmid into E. coli. All rescued plasmids were reintroduced into strains M5025 and M4941 to confirm the phenotypes.
Sequence analysis was performed with a MegaBACE1000 (Amersham Life Science). Alleles SSY1-108 through SSY1-120 from the SSY1382 degenerate library were identified by determination of the sequence of the region immediately surrounding codon 382 of the SSY1 ORF.
ß-Galactosidase assays. For ß-galactosidase assays, a modified protocol based on those of Didion et al. (7) and Ausubel et al. (3) was used. Typically, 1.2 ml of a saturated overnight culture was used to inoculate 60 ml of fresh medium. Fifty milliliters of culture at an OD600 between 0.5 and 1.0 was chilled, and cells were harvested for the assay. For the affinity experiment (see Fig. 5), however, cultures were inoculated with exponential-phase precultures and into minimal medium containing L-citrulline at appropriate concentrations; the cells were allowed to grow for an additional 5 h before being harvested at an OD600 between 0.5 and 0.7; the data for this experiment were fitted by nonlinear regression with the Sigma2000 software (SPSS Inc., Chicago, Ill.).
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RESULTS |
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trk2 background, growth is nearly abolished unless the growth medium is supplemented with high concentrations of potassium (18). Expression of the Kat1 channel restores potassium uptake, permitting growth on normal yeast medium (Fig. 1A). The AGP1 promoter was chosen to drive KAT1 expression because the basal level of AGP1 transcription is low but becomes high in the presence of certain amino acids (13).
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trk2 recipient cells conferred upon them the ability to grow on restrictive (non-potassium-supplemented) media such as SD and MP, but in a manner strictly dependent on the presence of inducers, such as leucine (Fig. 1A). To confirm that expression of AGP1::KAT1 relied on Ssy1-dependent signaling, pAGP1-KAT1 was introduced into ssy1 trk1 trk2 cells. Although the ssy1 trk1 trk2 pAGP1-KAT1 cells grew normally on medium supplemented with 100 mM KCl (SDHP), they were unable to grow on the restrictive medium either in the presence or in the absence of amino acids (Fig. 1A). The coupling of amino acid signaling to the uptake of potassium resulted in a growth assay that could be used to assess the relative efficacies of various amino acids with regard to their ability to induce the AGP1::KAT1 reporter. In these tests, strain RG5004, which expresses SSY1 and AGP1::KAT1 from plasmids, was tested for the ability to grow on SD supplemented with different concentrations of various amino acids as inducers. The results of this experiment generated a profile of the relative potencies of the amino acids tested (Fig. 1B). Leucine was most potent, conferring growth at concentrations of less than 5 µM. In contrast, even at concentrations more than 1,000-fold higher, glutamate and aspartate were only marginally effective. Still others, including glutamine, lysine, glycine, proline, arginine, and cysteine, were completely ineffective. Although the potency profile shown in Fig. 1B differs somewhat from a profile reported previously (13) in which the abilities of different amino acids (at 5 mM) to induce an AGP1::lacZ reporter were compared, AGP1::KAT1-dependent growth proved to be an exquisitely sensitive measure of the relative potencies of different inducers and thus served as a convenient assay for the specificity of Ssy1-dependent signaling.
Identification of a constitutive signaling mutation in SSY1.
To identify sites within Ssy1 that may be directly involved with the amino acid sensing or signaling mechanism, the SSY1 ORF was amplified by PCR under mutagenic conditions and introduced into an ssy1 trk1 trk2 pAGP1-KAT1 recipient by cotransformation with a linearized plasmid containing regions of sequence overlap with the amplified SSY1 ORF. Transformants were selected on SC medium supplemented with a high potassium concentration but lacking uracil (SCHP-URA). Colonies that developed were tested for their Ssy phenotype by replica plating to non-potassium-supplemented medium either with (SD plus Leu) or without (SD) leucine as an inducer. Ssy- transformants were detected by their inability to grow on SD plus Leu. Under the most mutagenic conditions, approximately 25% of the Ura+ transformants were also Ssy-. In contrast, less than 1% of transformants exhibited the Ssy- phenotype in a control experiment in which a high-fidelity polymerase was used. A broad range of Ssy- phenotype severity was observed among the transformants.
Among the approximately 10,000 Ura+ transformants screened, a single colony was identified that grew on both SD plus Leu and SD, suggesting that AGP1::KAT1 expression was no longer dependent on the presence of an inducer. Plasmid pSSY1-101, isolated from this transformant, was reintroduced into the ssy1 trk1 trk2 pAGP1-KAT1 recipient and found to confer the ability to grow on SD in the absence of amino acids (Fig. 2A).
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trk2-encoded phenotype can occur by mutations in transporter genes that produce increased membrane permeability to potassium (21, 26). Thus, to determine whether growth on SD was a direct effect of the mutant Ssy1 protein expressed from pSSY1-101, e.g., because of potassium leakage through the mutant Ssy1 protein, or if it resulted in increased expression of the AGP1::KAT1 reporter, pSSY1-101 was introduced into a trk1 trk2 mutant host lacking the pAGP1-KAT1 reporter. All of the transformants grew on SDHP but failed to grow on SD, even in the presence of an inducer (data not shown). Thus, pSSY1-101 conferred the ability of ssy1 trk1 trk2 mutant cells to grow on SD in a manner independent of an inducer but dependent on the pAGP1-KAT1 reporter.
Sequence analysis of pSSY1-101 revealed two mutations compared to the published SSY1 ORF sequence. A transition from G to A at position 709 resulted in a codon change from alanine to threonine (A237T), and a C-to-A transversion at position 1145 resulted in a change from threonine to lysine (T382K). To determine which mutation(s) was responsible for the mutant phenotype, the two single mutations and the double mutant were constructed by site-directed mutagenesis of an otherwise wild-type SSY1 gene expressed from plasmid pSSY1. Plasmids with the SSY1-102 (Ssy1T382K), SSY1-103 (Ssy1A237T), SSY1-104 (Ssy1A237T, T382K), or wild-type alleles were introduced into an ssy1 trk1 trk2 mutant recipient in which the pAGP1-KAT1 reporter was integrated at the AGP1 locus, and the transformants were tested for the ability to grow on SD (Fig. 2A). Both pSSY1-102 and pSSY1-104 conferred growth on SD lacking an inducer, indicating that the T382K mutation is sufficient to confer the mutant phenotype. The A237T mutant had no discernible phenotype by itself and did not strengthen the phenotype conferred by the T382K mutation.
Suppression of the trk1 trk2-encoded phenotype in a manner dependent on the AGP1::KAT1 reporter suggested that SSY1-102 conferred increased expression of the amino acid-responsive AGP1 promoter. To further test this and to quantify their effect(s) on signaling, plasmids encoding the SSY1-102, SSY1-103, SSY1-104, and wild-type SSY1 alleles were introduced into ssy1 mutant cells harboring an integrated AGP1::lacZ reporter construct and ß-galactosidase activity was measured in the absence or presence of leucine (Fig. 2B). Inducer-independent expression of the integrated reporter in wild-type SSY1 cells was low, but expression increased approximately fivefold upon induction with leucine. As expected from the growth tests, SSY1-103 conferred no discernible effect in the absence of amino acids but responded normally to an inducer. In contrast, cells harboring the SSY1-102 allele produced maximal levels of ß-galactosidase activity even in the absence of an inducer, revealing that the T382K mutation confers constitutive activity of the signaling pathway.
Constitutive induction by SSY1-102 was not limited to the AGP1 promoter. Quantification of ß-galactosidase produced in cells expressing the amino acid-responsive BAP2::lacZ reporter revealed that SSY1-102 conferred inducer-independent expression from the BAP2 promoter as well (data not shown).
Ssy1382K confers a dominant gain of function. As a test for dominance, wild-type SSY1 cells expressing a second wild-type or mutant copy of SSY1 integrated at the URA3 locus were generated with plasmids pOK13 and pOK14, respectively, and both inducer-dependent and inducer-independent signaling was quantified with the AGP1::lacZ reporter. Compared to the cells harboring the single SSY1 gene, cells expressing an additional wild-type gene produced a modest increase in both inducer-independent (from 0.006 to 0.012 Miller units) and inducer-dependent (from 0.025 to 0.065 Miller units) ß-galactosidase activities. In contrast, when the second copy of SSY1 contained the T382K mutation, full activity of the AGP1::lacZ reporter (0.12 Miller units) occurred even in the absence of an inducer (in the presence of an inducer, 0.11 Miller units). Thus, T382K confers a dominant gain of function with regard to signal transduction.
Constitutive signaling by Ssy1382K requires Ptr3 and Ssy5. Amino acid signaling requires the participation of other proteins, including Ptr3 and Ssy5, both of which exist in a complex with Ssy1 (8). Two tests were performed to determine if the requirement for Ptr3 or Ssy5 was bypassed by the constitutive SSY1-102 mutation. In one, SSY1-102 cells were tested for the ability to grow on YPD+MM (15). Branched-chain amino acid biosynthesis is inhibited by MM, so growth depends on SPS induction of BAP2 and other permeases. Wild-type SSY1 and mutant SSY1-102 cells grew normally on YPD+MM, but growth was impaired upon deletion of either PTR3 or SSY5 (Fig. 3A).
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or ssy5 mutation were fully resistant. Thus, the SSY1-102 allele is unable to induce PTR2 in cells with either Ptr3 or Ssy5 deleted. We conclude from these experiments that constitutive signaling via Ssy1382K does not bypass the requirement for the other components of the SPS complex.
Ssy1382K, Ssy1382R, Ssy1382H, and Ssy1382L confer amino acid-independent signaling.
On the basis of the topology of the related permease Gap1p (9) and the online prediction facility www.cbs.dtu.dk/services/TMHMM, the amino acid altered by SSY1-102 is predicted to lie on the extracellular face of the plasma membrane. Since SSY1-102 resulted in constitutive signaling, we reasoned that other mutations at this site might produce interesting phenotypes. Additional codons at position 382 were therefore generated and assessed for their effects on signaling. Most of these mutations were obtained by screening of two independently generated libraries of mutations at codon 382 made by degenerate primer-mediated PCR. Each SSY1 allele was introduced into an ssy1 trk1 trk2 mutant recipient in which the AGP1::KAT1 reporter construct had been integrated at the AGP1 locus, and the transformants were tested for the ability to grow on SD in the absence of an inducer.
A screen of 90 yeast transformants from the library pools identified 17 that were capable of growth on SD without an inducer. Plasmids rescued from these transformants were retested for the ability to confer the constitutive phenotype, and the SSY1 alleles were sequenced. The gain-of-function alleles that were found encode Lys, Arg, His, or Leu at codon 382. Sequence analysis of 96 randomly selected library plasmids that were recovered in E. coli identified 11 additional SSY1 alleles, encoding Ala, Cys, Gly, Ile, Met, Phe, Pro, Ser, Trp, Tyr, or Val at codon 382. A mutant allele containing an Asp codon at this site was independently generated by site-directed mutagenesis. Plasmids encoding each of these 12 new SSY1 alleles were introduced into an ssy1 trk1 trk2 AGP1::KAT1 mutant recipient and tested for the ability to confer growth on non-potassium-supplemented medium in the presence or absence of an inducer. On SD, the growth of these transformants was not greater than the growth of wild-type SSY1 cells. On the other hand, all of the transformants grew well on SD plus leucine, indicating that these mutations did not impair the ability of Ssy1 to initiate signaling in response to amino acid (not shown).
Ssy1382H and Ssy1382L respond to subthreshold concentrations of inducers.
Growth tests in which trk1 trk2 pAGP1-KAT1 cells expressing the gain-of-function SSY1 alleles were compared revealed two phenotypic classes. Cells expressing either the T382K or the T382R allele grew strongly even in the absence of an inducer. In fact, growth of either mutant on SD in the absence of an inducer was nearly indistinguishable from its growth in the presence of amino acids (Fig. 4), suggesting that Ssy1382R, like Ssy1382K, is fully constitutive. In contrast, cells expressing the T382H or T382L mutant form exhibited growth that was only marginally stronger than that conferred by wild-type SSY1 cells in the absence of amino acids. Both of these mutant forms retained the ability to respond to the presence of amino acids (Fig. 4). Interestingly, however, compared to cells expressing the wild-type sensor, Ssy1382H- and Ssy1382L-expressing cells were capable of responding to concentrations of amino acids that are normally too low to produce a strong signaling response by this assay (compare relevant inducer concentrations in Fig. 4 with those in Fig. 1B). This suggested the possibility that the T382H and T382L mutations increased the sensitivity of Ssy1 to inducers.
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Ssy1382H exhibits greater apparent affinity for an inducer.
To better understand the basis of this phenotype, the ability of cells expressing either the wild-type or Ssy1382H sensor to respond to an inducer was quantified by measuring ß-galactosidase produced from AGP1::lacZ during growth in the presence of different concentrations of citrulline. Citrulline was chosen because it is not taken up by cells with a deletion of the general amino acid permease, Gap1 (11). The results of these experiments were striking (Fig. 5). First, at the lower inducer concentrations (basal level), signaling was increased in the T382H mutant compared with that in the wild type. Second, citrulline was capable of increasing signaling to levels equivalent to that of the wild type at the higher concentrations, confirming that the mutant Ssy1382H sensor did indeed retain the ability to fully respond to an inducer. Third, Ssy1382H-expressing cells responded to significantly lower levels of an inducer than did wild-type cells. Whereas cells expressing wild-type Ssy1 exhibited an apparent Kd of approximately 800 µM citrulline, cells expressing Ssy1382H exhibited an apparent Kd of only 
80 µM citrulline. Thus, the inducer concentration needed to induce a strong signaling response is reduced 10-fold by the T382H mutation. Because Ssy1382H exhibited a general increase in sensitivity to different amino acids, we favor a model in which the T382H mutation results in a shift in equilibrium between the nonsignaling and signaling-competent conformations of Ssy1. Taken together with our results showing that mutations at T382 confer constitutive signaling, the data strongly support the notion that Ssy1 functions as a receptor rather than a transporter and that Ssy1 interacts directly with the extracellular signaling molecules to initiate signaling.
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DISCUSSION |
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Ssy1 has been hypothesized to function at the head of the amino acid signaling pathway largely because of the fact that it is an integral plasma membrane protein that resembles amino acid permeases and is required for signaling, yet it is apparently unable to transport amino acids. A similar situation has been described for the glucose sensors Snf3 and Rgt2. These are proteins that resemble glucose transporters and are required for glucose sensing yet apparently do not transport glucose (20, 24). Indeed, constitutive inducer-independent signaling mutations have been obtained for both Snf3 and Rgt2 (23). In addition, a mutation in SSY1 has been reported (4) that abolishes the response to a high concentration of phenylalanine while the response to a high concentration of leucine is maintained; this mutation could be affected in the relative potency of amino acids, as suggested by the authors, or it could be generally hyporesponsive. Amino acid and glucose sensors may detect the extracellular presence of their respective nutrients directly and initiate signal transduction through interactions with other components of the signaling complex. However, the inability to detect amino acid or glucose uptake does not rule out the possibility that Ssy1 or Snf3/Rgt2 can indeed transport small quantities of the nutrients. If this is so, signaling might actually rely on the intracellular detection of these signaling molecules by other proteins. In the case of amino acid signaling, Ptr3 and Ssy5 would be excellent candidates as they have been shown to be intimately associated with Ssy1 and are responsible for mediating downstream effects (2, 8). Thus, a key aspect that we wished to resolve was whether it is the extracellular or intracellular presence of amino acids that is required for signaling by Ssy1.
The discovery that mutation of SSY1 codon 382 from threonine to lysine (or arginine) results in constitutive signaling in the absence of extracellular amino acids reveals that Ssy1 can initiate signaling in the absence of an intracellular, i.e., transported, inducer. These results effectively preclude a model in which any other downstream factor is required to recognize an internalized inducer and are consistent with the notion that Ssy1 functions as the protein that directly senses an inducer. Although the argument could still be made that some as yet unidentified factor is the actual amino acid sensor and that constitutive forms of Ssy1 simply bypass the need for this putative factor, SSY1 mutations such as T382H, which confer a 10-fold increase in the apparent affinity for an inducer, argue strongly against this. The simplest interpretation of the occurrence of hyperresponsive mutations such as T382H and T382L is that Ssy1 interacts directly with extracellular amino acids as part of the signaling mechanism. Thus, some alleles of SSY1 can confer constitutive signaling while others increase its ligand sensitivity. Taken together, our data support a model in which Ssy1 functions as a receptor, initiating signaling through direct interaction with extracellular amino acids. Constitutive signaling requires Ptr3 and Ssy5, indicating that Ssy1382K is unable to induce AGP1 in the absence of the full SPS complex. Thus, as far as we can tell, Ssy1382K confers normal, albeit constitutive activation of the SPS complex and the appropriate downstream transcription factors.
The sequence conservation between Ssy1 and other members of the large family of amino acid permeases in S. cerevisiae indicates that Ssy1 may have evolved from an ancestral transporter. If this is so, signal transduction by this sensor may rely on structural and functional aspects intrinsic to bona fide transporters. This is likely to include the abilities to bind amino acids and undergo subsequent conformational changes that are then translated into productive interactions with downstream factors such as Ptr3 and Ssy5. As mentioned, this might also be accompanied by undetected amino acid transport. The evolutionary recruitment of sensors from transporters appears to have occurred at least twice in fungi, but such sensors have not been described in other eukaryotes. Nutrient availability for free-living fungi can change drastically, over many orders of magnitude, in extracellular concentration. Thus, microorganisms such as S. cerevisiae appear to have adapted to such wide disparity in nutrient availability through the evolutionary accrual of at least 15 different amino acid permease genes and at least 17 sugar transporters. Within each family, individual transporters exhibit substrate specificities and affinities that overlap those of other members, suggesting that different transporters are best able to cope with specific conditions of nutrient availability. In the case of the HXT family, this has been largely borne out with regard to the differential expression of high-, moderate-, and low-affinity transporters under suitable conditions of glucose availability (23). The fact that constitutive mutations have now been identified in both glucose and amino acid sensors has helped to place constraints on both the nature of the intracellular signals and models of how these sensors might function. Gain-of-function mutations in downstream factors can also be very informative, as illustrated by a constitutively active truncated version of Stp1 (2).
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ACKNOWLEDGMENTS |
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The experiments described in this report were initiated in the laboratory of M.C.K.-B. by R.F.G. during a sabbatical leave granted by Northwestern University. This work was supported by a grant from the National Science Foundation (R.F.G.).
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FOOTNOTES |
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The supplemental material for this article may be found at http://ec.asm.org/.
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