The ability to elicit a fast intracellular signal leading to an adaptive response is crucial for the survival of microorganisms in response to changing environmental conditions. Therefore, in order to sense changes in nutrient availability, the yeast Saccharomyces cerevisiae has evolved three different classes of nutrient-sensing proteins acting at the plasma membrane: G protein-coupled receptors or classical receptor proteins, which detect the presence of certain nutrients and activate signal transduction in association with a G protein; nontransporting transceptors, i.e. nutrient carrier homologues with only a receptor function, previously called nutrient sensors; and transporting transceptors, i.e. active nutrient carriers that combine the functions of a nutrient transporter and receptor. Here, we provide an updated overview of the proteins involved in sensing nutrients for rapid activation of the protein kinase A pathway, which belong to the first and the third category, and we also provide a comparison with the best-known examples of the second category, the nontransporting transceptors, which control the expression of the regular transporters for the nutrient sensed by these proteins.
Besides their role as a source of energy and building blocks for the cell, nutrients also act as regulators of metabolism, growth, and development. The ability to adapt to changing nutritional conditions is a key process for survival, especially in microorganisms. This adaptation is mediated by a variety of signaling pathways, including pathways that initiate the sensing of nutrients directly at the plasma membrane (Holsbeeks, 2004). The yeast Saccharomyces cerevisiae has evolved three classes of nutrient-sensing plasma membrane proteins (Fig. 1).
The first class of sensors comprises the classical receptor proteins or G protein-coupled receptors (GPCRs), which, in yeast, detect the presence of glucose and sucrose or pheromones. A well-studied example is the GPCR system that is responsible for glucose and sucrose control of the protein kinase A (PKA) pathway (Thevelein & de Winde, 1999). PKA plays a central role in the nutritional control of metabolism, stress resistance, cell cycle, growth, and transcription. All these properties are tightly controlled by the availability of nutrients in the medium, especially by the presence of a rapidly fermentable sugar and other essential nutrients sustaining growth, such as amino acids and phosphate. In glucose-derepressed yeast cells, i.e. cells growing by respiration on a weakly or a nonfermentable carbon source, the PKA pathway is downregulated. Addition of rapidly fermentable sugars to derepressed yeast cells triggers an immediate increase in the cAMP level, which in turn causes rapid activation of PKA, resulting in drastic changes in its multiple targets.
The second class of sensors comprises the nontransporting nutrient carrier homologues with a sensor function. Here, we will focus on the glucose sensors, Snf3 and Rgt2, which are responsible for induction of genes encoding regular glucose/hexose transporters (Özcan, 1998), and a similar protein, Ssy1, which is a sensor required for amino acid-induced derepression of genes encoding specific amino acid permeases (AAPs) (Didion, 1998). Both types of sensors, Snf3/Rgt2 and Ssy1, have sequence similarity to either genuine glucose or amino acid transporters. However, these proteins have lost the capacity to transport any substrate.
The third class of sensors are active nutrient carriers that combine the functions of a nutrient transporter and receptor. Three examples of such proteins are provided in this review (Fig. 2b): the general AAP, Gap1 (Donaton, 2003); the ammonium permease, Mep2 (Van Nuland, 2006); and the phosphate carrier, Pho84 (Giots, 2003). Addition of essential nutrients, such as nitrogen or phosphate, to glucose-repressed, nitrogen-, or phosphate-starved cells, respectively, triggers rapid activation of the PKA pathway through these transceptors. In this case, cAMP is not involved as a second messenger (Hirimburegama, 1992; Durnez, 1994).
The second and third class of sensor proteins are called transceptors, for transporter-related receptors. Regulation of PKA signaling by nontransporting nutrient transceptors has so far not been reported. However, they may easily affect PKA signaling in an indirect way, by regulating the expression of nutrient carriers, whose substrates and/or metabolites can affect PKA signaling. Even though their signaling mechanisms may differ, these two categories are closely related in evolution. In fact, it is quite likely that in eukaryotes, no sharp distinction will exist between these two categories and intermediate forms will be found, i.e. transceptors with low residual transport activity. In prokaryotes, such an example has already been documented (Schwoppe, 2003).
Categories of nutrient-sensing plasma membrane proteins
GPCRs: the Gpr1-Gpa2 system
Yeast cells growing on a fermentable carbon source, such as glucose, display low levels of the storage carbohydrates trehalose and glycogen, as well as low expression of stress resistance genes. The latter effect, together with the low level of trehalose, which also acts as a stress-protective sugar, results in a general low tolerance to various stress conditions. On the other hand, in the stationary phase or when growing on a nonfermentable carbon source, such as glycerol or ethanol, the cells display the opposite phenotype. This high intrinsic stress tolerance is also a general characteristic of strains with impaired PKA activity, even when they are growing on a fermentable carbon source. The cAMP–PKA pathway has been implicated in the regulation of these phenotypes as a function of the nutrient conditions. The addition of glucose to glucose-deprived cells activates adenylate cyclase (Cyr1) and causes a rapid increase in cAMP, followed by a concomitant increase in PKA activity (Fig. 2a). For this, a dual glucose-responsive system is required: detection of the extracellular glucose occurs through a GPCR system, whereas intracellularly, the system depends on sugar phosphorylation. How sugar phosphorylation impinges on cAMP signaling is not yet fully understood, but it may occur through regulation of the Ras proteins (Colombo, 2004).
The sugar-sensing GPCR system consists of the receptor Gpr1, the Gα protein Gpa2 (Colombo, 1998) and its regulator of heterotrimeric G protein-signaling (RGS) protein, Rgs2 (Versele, 1999). The structural features that are commonly found in GPCRs are also present in Gpr1: it contains seven transmembrane domains, has an extended N- and C-terminus, and a large third intracellular loop. Gpr1 was originally isolated as an interaction partner of Gpa2, a Gα protein previously implicated in glucose-induced activation of the cAMP–PKA pathway (Xue, 1998; Kraakman, 1999). Gpr1 was also picked up in a separate screen for mutants with an increased stress resistance after initiation of fermentation. In this case, the mutant gene was designated as fil2 (for ‘fermentation-induced loss of stress resistance’; Van Dijck, 2000). The identification of fil2 as a nonsense mutation in the GPR1 gene revealed the importance of this receptor protein for rapid adaptation to the presence of glucose (Kraakman, 1999).
Among the array of sugars tested for their ability to trigger cAMP–PKA pathway activation via Gpr1, only glucose and sucrose were effective. The structurally very similar sugars fructose and galactose had no effect, whereas mannose had an antagonistic effect (Lemaire, 2004). This illustrates the very high specificity of Gpr1, a typical hallmark of GPCRs. The absence of Gpr1 activation by fructose, a rapidly fermentable sugar, implies that fructose only activates cAMP signaling via the glucose phosphorylation-dependent mechanism (Colombo, 2004; Paiardi, 2007).
To better understand how the aforementioned sugars act on Gpr1, substituted cysteine accessibility method (SCAM) analysis was applied to transmembrane domain VI, a domain thought to be important for ligand binding and for the subsequent conformational change occurring in GPCRs during receptor activation. This type of analysis identified several residues whose side chains are exposed into the sugar-binding pocket of Gpr1. Moreover, mutagenesis of certain residues exerted a differential effect on glucose and sucrose, indicating a different binding mechanism for these sugars (Lemaire, 2004). This finding also provided evidence for a direct binding of the sugars to Gpr1. Intriguingly, Gpr1 seems to have a higher affinity for sucrose than for glucose, although the latter has always appeared to be a much more preferred carbon source for yeast because of the repression of many sets of genes by high glucose levels. Nevertheless, high concentrations of glucose are rather scarce in nature: fruits and other glucose-rich environments are only available during a limited period of the year. Because sucrose is the transport sugar of plants, being present in very high concentrations in phloem tubes, sucrose is much more widespread in nature than glucose. Low concentrations of sucrose may thus be important for the survival of yeast in nature in seasons of the year in which no fruits are present.
Gpr1-Gpa2 are also required for pseudohyphal filamentous growth (Lorenz, 2000), a morphological switch from the unicellular budding form, to cells with an elongated cell morphology and a polarized budding pattern. This process is triggered when diploid cells are growing in the presence of a fermentable carbon source under nitrogen-limiting conditions. It has recently been shown that a reduction of glucose concentration in the medium causes induction of pseudohyphal filamentation in mep2Δgpr1Δ, but not in mep2Δgpa2Δ strains (Iyer, 2008). The authors have suggested that this fact may indicate that Gpr1 inhibits pseudohyphal growth by inhibiting Gpa2, whenever glucose levels decrease. On the other hand, sucrose has been shown to be the most potent inducer of pseudohyphal growth, acting through Gpr1, and even being capable of overriding the nitrogen deprivation requirement (Van de Velde & Thevelein, 2008). These facts again highlight the putative importance of the high sucrose affinity displayed by Gpr1 for yeast survival in nature. The receptor appears to play a crucial role in the detection of low concentrations of sucrose in the extracellular environment.
No classical Gβγ subunits for Gpa2 have been identified. The S. cerevisiae genome has only one gene encoding a classical Gβ protein, Ste4, and this protein functions in the pheromone pathway (Dohlman & Thorner, 2001). Extensive experiments in our laboratory have shown that this protein does not function in glucose-induced cAMP signaling (unpublished data). Initially, the kelch repeat proteins Krh1/Gpb2 and Krh2/Gpb1 were proposed to act as alternative Gβ subunits for Gpa2 (Harashima & Heitman, 2002). Arguments in favor of this theory were their physical binding to Gpa2, and the capacity of kelch repeat proteins to fold into a seven-bladed β propeller-like structure. This conformation resembles the 3D structure adopted by the WD-40 repeats in classical Gβ proteins. However, elucidation of the amino acid residues involved in the binding of Gpa2 to Krh1 has shown that this interaction does not resemble the binding observed between Gα and Gβ proteins (Niranjan, 2007). Based on interaction studies, Gpg1 has been proposed to act as the Gγ subunit for Gpa2 (Harashima & Heitman, 2002). More recently, the RACK1 orthologue Asc1, a 7 WD-40 repeat-containing protein without homology to classical Gβ proteins, has been suggested to function as the true substitute Gβ subunit for Gpa2 (Zeller, 2007). Asc1 binds directly to Gpa2 in a guanine nucleotide-dependent manner, inhibiting GDP–GTP exchange on Gpa2 and lowering the glucose-induced cAMP signal. So far, there have been no further reports of a substitute Gγ protein that would interact with Asc1.
Deletion of KRH1 and KRH2 causes a high PKA phenotype, suggesting a function of these proteins as PKA inhibitors (Peeters, 2006). The absence of both Krh1 and Krh2 lowers (but does not abolish) the extracellular cAMP requirement for growth in adenylate cyclase mutants. Moreover, both the PKA regulatory and the catalytic subunits are necessary for the Krh proteins to cause downregulation of known PKA targets, and deletion of KRH1 and KRH2 appears to reduce the interaction between the regulatory and the catalytic subunits of PKA in vivo. It is, therefore, likely that the Krh proteins function as PKA inhibitors by increasing the cAMP dependence of the holoenzyme. Based on these observations, Krh1 and Krh2 are now thought to function in an adenylate cyclase bypass pathway for regulation of PKA activity. According to the new model, addition of glucose to glucose-deprived cells causes Gpa2 to activate PKA via two distinct mechanisms: first, stimulation of the Gpr1-Gpa2 system leads to direct activation of adenylate cyclase with the increase in cAMP activating PKA, and second, stimulation of the Gpr1-Gpa2 system causes inhibition of the Krh proteins, resulting in a lower cAMP requirement for PKA and, thus, causing synergistic stimulation of PKA with the increase in the cAMP level.
Rapid signaling via Gpr1-Gpa2 after the addition of glucose or sucrose also requires intracellular glucose phosphorylation by Glk1, Hxk1, and/or Hxk2. Rolland (2000) created an efficient system to separate these two requirements. They used a yeast mutant incapable of transporting glucose while sustaining the glucose phosphorylation requirement by addition of a small amount of maltose, a sugar that does not interact with Gpr1 and is taken up by its own transporter (Rolland, 2000). In this way, they demonstrated that the role of the glucose transporters is confined to the uptake of the sugar and that the actual sensing of glucose occurs in two ways: extracellular glucose is sensed through Gpr1 while intracellular glucose is sensed by the glucose phosphorylation-dependent system.
The interdependency of the Gpr1-Gpa2 system and the glucose phosphorylation-dependent system might be explained by the fact that production of cAMP by adenylate cyclase is regulated by two G protein systems. Whereas Gpr1-Gpa2 is responsible for stimulating Cyr1 after the addition of glucose, the small G proteins Ras1 and Ras2 are necessary to maintain a sufficient level of basal Cyr1 activity. It cannot be excluded that the Ras proteins also function as signal transducers. Recent work showed a two- to threefold increase of GTP-bound Ras that is dependent on glucose phosphorylation (Colombo, 2004). In this way, glucose phosphorylation could result in a partial activation of adenylate cyclase by Ras, making it more susceptible to subsequent stimulation by the GPCR system, Gpr1-Gpa2.
Nontransporting nutrient carrier homologues
Because of the high sequence similarity of these nutrient sensors to their transporter counterparts, it is assumed that nontransporting nutrient carrier homologues have evolved from transporters through the acquisition of mutations that either conferred or maintained signaling capacity, as well as of mutations that caused loss of their transport capacity (Özcan, 1996; Özcan & Johnston, 1999).
Glucose transceptors Snf3 and Rgt2
Glucose is the primary carbon and energy source for yeast cells. Hence, yeast possesses a vast array of proteins responsible for transport and also for detection of this sugar. The plasma membrane proteins, Snf3 and Rgt2, are highly similar to hexose transporters in their primary amino acid sequence and deduced three-dimensional structure, and yet they are unable to transport glucose (Özcan, 1996, 1998). Instead, they act as nutrient sensors that generate an intracellular signal required for induction of hexose transporter (HXT) genes in response to glucose (extensively reviewed by Gancedo, 2008).
In the absence of glucose, a repressor complex containing Rgt1 binds to the promoters of the HXT genes, blocking their transcription (Fig. 3a). Addition of glucose results in hyperphosphorylation of Rgt1, causing its dissociation from the HXT promoters and subsequent HXT gene expression. Snf3 is a sensor for low glucose concentrations, responsible for induction of high-affinity transporters such as HXT2, HXT6, and HXT7. Rgt2 is instead a sensor for high glucose concentrations and is responsible for strong induction of HXT1 in abundance of this sugar. Both Snf3 and Rgt2 have an unusually long C-terminal cytosolic tail, which, although not absolutely required, plays an important role in signaling (Özcan, 1998; Moriya & Johnston, 2004). Both of them interact through this domain with the regulatory proteins, Mth1 and Std1. In the absence of glucose, a significant fraction of these two proteins is found in the nucleus associated with Rgt1 and the corepressor complex formed by Cyc8/Ssn6 and Tup1 (Polish, 2005). When glucose is sensed by Snf3 and/or Rgt2, they stimulate the phosphorylation of Mth1 and Std1 by the type I casein kinases Yck1/2, which results in SCF E3 ligase Grr1-mediated ubiquitination and subsequent degradation of Mth1 and Std1 by the proteasome. This releases Rgt1 from its repressive upstream binding sites, apparently also switching it into a transcriptional activator, which leads to derepression of downstream HXT genes (most recently reviewed by Santangelo, 2006).
Cross-talk between the two types of glucose sensors, Gpr1-Gpa2 and Snf3-Rgt2, has recently been uncovered at the level of Rgt1 regulation. Kim & Johnston (2006) have demonstrated that Gpr1-Gpa2-activated, Ras-dependent cAMP–PKA catalyzes direct phosphorylation of Rgt1. PKA and the consensus Ser residues of Rgt1 are required for glucose-induced removal of Rgt1 from the HXT promoters and for induction of HXT expression. Moreover, overexpression of the TPK genes leads to constitutive expression of the HXT genes. Conversely, simultaneous deletion of all glucose transporters overrides cAMP accumulation in response to glucose, which underscores the need for glucose uptake in the activation of PKA signaling (Rolland, 2001).
Although no direct binding of glucose to these sensors has been demonstrated, the current view is that binding of glucose to Snf3/Rgt2 causes a conformational change, leading to activation of the downstream signaling cascade. As for Ssy1 (see the next section), mutations in both Snf3 and Rgt2 have been identified that convert the proteins into constitutively activating sensors, causing constitutive activation of the pathway, even in the absence of glucose (Özcan, 1996).
Amino acid transceptor Ssy1
Another well-characterized model protein among the nontransporting nutrient sensors in S. cerevisiae is the member of the AAP family, Ssy1 (Andre, 1995). Together with Ptr3 and Ssy5, Ssy1 forms the plasma membrane SPS nutrient-sensing system (Forsberg & Ljungdahl, 2001), which, in response to extracellular amino acids, induces the transcription of a set of amino acid transporter genes and other genes involved in amino acid metabolism (latest reviewed by Ljungdahl, 2009).
The discovery of hyper-responsive and hyporesponsive mutant alleles strongly suggests that, in order to activate this sensing system, the extracellular amino acid must directly bind to the transporter homologue (Gaber, 2003; Poulsen, 2008). Amino acid binding to Ssy1 induces endoproteolytic cleavage of the cytosolic precursors of the transcription factors Stp1 and Stp2 (Fig. 3b). The processed forms of Stp1 and Stp2 are then translocated into the nucleus, where they act redundantly in the activation of SPS-sensor-regulated genes (Andreasson & Ljungdahl, 2002; Abdel-Sater, 2004a). Further steps involved in the activation of this signaling cascade have recently been elucidated. The SPS-sensor component Ssy5 is a chymotrypsin-like protease with an inhibitory prodomain and a catalytic domain. The prodomain is autocatalytically cleaved from the catalytic domain, but remains associated, forming an inactive protease complex that binds Stp1. Upon amino acid binding, Ssy1, together with Ptr3, promotes the release of the inhibitory prodomain, allowing processing of Stp1 and Stp2 by Ssy5 (Andreasson, 2006).
As for the Snf3-Rgt2 sensor system, the activation of downstream components, in this case Stp1 and Stp2, is also known to involve the activity of the Skip1/Cullin/F-box (SCF) Grr1 ubiquitin E3 ligase complex (Abdel-Sater, 2004b; Andreasson & Ljungdahl, 2004), and casein kinase-dependent phosphorylation (Abdel-Sater, 2004a). It is intriguing that signaling via the two classes of nontransporting transceptors discussed above (Snf3-Rgt2 and SPS sensor) shares several downstream components, such as those involved in phosphorylation and ubiquitination of downstream regulators. This indicates that, at least in yeast, this category of nutrient sensors has either functionally converged or diverged from a common ancestor in evolution in terms of the way in which they transduce the nutrient signal to their downstream components.
Although Ssy1 is a member of the AAP family, it does not transport amino acids and also differs from AAPs in its particularly long N-terminal cytosolic tail. This N-terminal region is highly conserved in putative SSY1 homologues in other yeast species (Souciet, 2000) and appears to be essential for the sensor-signal transducer functions of Ssy1 (Klasson, 1999). Based on the conformational changes occurring in transporters of the major facilitator superfamily, a model has been proposed for the signaling mechanism of Ssy1 (Wu, 2006). According to this model, Ssy1 is proposed to undergo similar transitions as regular transporters, i.e. from an outward-facing to an inward-facing conformation, and the outward-facing conformation has been suggested to act as the signaling conformation. Amino acid binding would inhibit the shift between the two conformations locking the sensor in the signaling conformation. Experiments in which the intracellular amino acid concentration was artificially increased and that resulted in a decline in the affinity of Ssy1 are in agreement with this model.
Nutrient transceptors and their role in the ‘fermentable growth medium’ (FGM)-induced pathway
During growth on a fermentable carbon source such as glucose, S. cerevisiae cells display a range of phenotypes indicating high activity of the PKA pathway. Starvation of the cells for an essential nutrient, such as nitrogen or phosphate, in the presence of a fermentable sugar, causes arrest in the G1 phase of the cell cycle. The arrested cells subsequently acquire a variety of characteristics that collectively define the G0 or the stationary phase, such as accumulation of the reserve carbohydrates trehalose and glycogen, induction of stress-responsive element (STRE)- and postdiauxic shift (PDS)-controlled genes, and higher stress resistance (reviewed by Gray, 2004). These characteristics are all typical for cells with low activity of the PKA pathway.
The readdition of a lacking essential nutrient to cells starved for this particular nutrient in the presence of a fermentable carbon source causes growth induction and rapid disappearance of the PKA-controlled stationary-phase characteristics. Hence, the cells rapidly switch to the ‘high-PKA phenotype.’ A convenient read-out for PKA activity, the neutral trehalase encoded by NTH1, is readily activated upon addition of the nutrient for which the cells were starved, leading to mobilization of trehalose (Hirimburegama, 1992). Activation of PKA targets further stimulates cells to proceed with fermentative growth. Efficient rapid activation by nitrogen sources or phosphate requires the presence of a rapidly fermented sugar, but not the other essential nutrient(s). However, maintenance of the high PKA phenotype requires the combination of a rapidly fermented sugar and a complete growth medium. For this reason, the signaling pathway involved in this process has been designated as the FGM-induced pathway (Thevelein, 1994).
In contrast to glucose activation of the Ras/PKA pathway, both nitrogen- and phosphate-induced activation of the FGM pathway do not appear to be mediated by an increase in the cAMP level. Although cAMP does not act as a second messenger, the free catalytic subunits of PKA (Tpk1, Tpk2, and Tpk3) are required (Hirimburegama, 1992; Durnez, 1994). The presence of a rapidly fermentable sugar (e.g. glucose), is also required for activation by amino acids, ammonium, or phosphate. However, the glucose-sensing process does not depend on sugar phosphorylation or metabolization (Durnez, 1994; Pernambuco, 1996; Giots, 2003). The same dual glucose-sensing system as that used in glucose-derepressed, respiring cells for activation of cAMP synthesis (Rolland, 2000) is also used for the sensing of glucose in the glucose-repressed cells for activation of the FGM pathway, but the two sensing systems can apparently function independently from each other.
Besides PKA, the Sch9 protein kinase activity is also required for rapid nitrogen-induced activation of the FGM pathway: sch9Δ cells are defective for both amino acid- and ammonium-induced activation of the FGM pathway (Crauwels, 1997). Although inorganic phosphate is also an activator of the FGM pathway, deletion of SCH9 does not prevent phosphate-induced activation of trehalase, repression of the STRE-controlled genes SSA3 and HSP12, or induction of the ribosomal protein gene RPL25 (Giots, 2003). Hence, nitrogen and phosphate activation seem to use, at least to some extent, different components of the signaling pathway downstream of the nitrogen and phosphate sensors.
How Sch9 impinges on PKA signaling exactly is still unclear, although genome-wide expression analyses indicate that Sch9 and PKA most likely act in parallel, partially redundant pathways (Roosen, 2005). Because the catalytic domains of Sch9 and the PKA catalytic subunits show high sequence similarity (Toda, 1988), it is indeed not surprising to find that they have several common target genes, probably regulated by proteins that can be phosphorylated by either Sch9 or PKA. For example, overexpression of Sch9 can recapitulate, to a large extent, the transcriptional changes caused by glucose-activated PKA. Deletion of SCH9, on the other hand, does not measurably affect this response (Zaman, 2009). The identification of genes that are specifically (and sometimes even antagonistically) regulated by PKA and Sch9 further demonstrates that these proteins are not completely functionally redundant (Roosen, 2005).
It has been shown recently that Sch9 is controlled by the nutrient-responsive, rapamycin-sensitive TORC1 complex (Urban, 2007). Inactivation of TORC1 by rapamycin mimics nitrogen deprivation in many aspects, such as repression of ribosomal protein genes and induction of STRE genes. Intriguingly, overactivation of the PKA pathway abrogates these rapamycin-induced effects (Schmelzle, 2004). However, several studies on the precise involvement of TORC1 and PKA in gene regulation indicate that these two protein kinases function in parallel to each other. One argument is that rapamycin still downregulates ribosomal gene expression in a strain without PKA activity (Zurita-Martinez & Cardenas, 2005). Moreover, expression profiling has shown that TOR and PKA kinases provide separate inputs in the control of the expression of amino acid biosynthetic genes (Chen & Powers, 2006). Additional differences between both pathways are further discussed in the recent review by Dechant & Peter (2008). Because a more direct connection between nutrient sensors and the TOR pathway has not yet been elucidated, the possibility that signals detected via PKA-connected sensors are upstream steps affecting TOR signaling remains open. More studies of the connection between both pathways will indeed prove useful to understand how external nutrients such as nitrogen are sensed by yeast cells.
Plasma membrane sensors for both nitrogen- and phosphate-induced activation of the FGM pathway have been found to be active nutrient transporters with an additional receptor function for nutrient sensing. Hence, they have subsequently been named transceptors (Holsbeeks, 2004). Next, we provide a detailed description of each of the best-characterized examples of S. cerevisiae nutrient transceptors that can currently be found in the literature: the amino acid transceptor, Gap1; the ammonium transceptors, Mep1 and Mep2; and the phosphate transceptor, Pho84.
Amino acid transceptor Gap1
Amino acids are transported into the yeast S. cerevisiae by general and specific transport systems. AAPs are all members of the conserved AAP family. This family contains both constitutive, specific permeases (e.g. the histidine permease, Hip1) and nitrogen-regulated permeases (Regenberg, 1999). The general AAP, Gap1, an example of the latter class, is a proton–amino acid symporter for all naturally occurring l-amino acids, 4-amino butyric acid, l-citrulline, some d-amino acids, and some toxic amino acid analogues (Jauniaux & Grenson, 1990).
Both the synthesis and the activity of Gap1 are tightly regulated according to the quality of nitrogen sources present in the medium. The presence of preferred nitrogen sources (e.g. glutamine, glutamate, or ammonia for certain strains) and/or high amino acid levels repress Gap1 both transcriptionally and post-translationally (reviewed in Magasanik & Kaiser, 2002). The transcription of Gap1 is positively regulated by the nitrogen catabolite repression (NCR) pathway (Hofman-Bang, 1999). Under nitrogen limitation, GAP1 expression is activated by two GATA-transcription factors: Gln3, which is repressed in a glutamine- and ammonia-containing medium, and Gat1/Nil1, which is repressed by elevated levels of glutamate or other amino acids (Stanbrough, 1995;,Stanbrough & Magasanik, 1996).
In a medium with a poor nitrogen source, such as proline, GAP1 is highly transcribed, and neosynthesized Gap1 accumulates at the plasma membrane in an active, stable form. Sorting of Gap1 to the plasma membrane is positively regulated in a poorly understood way by Npr1 kinase activity (Vandenbol, 1990; De Craene, 2001). Upon addition of a preferred nitrogen source, such as ammonia, or an excess of amino acids to the medium, plasma membrane Gap1 is internalized by endocytosis and targeted to the vacuole for degradation (Hein & André, 1997; Roberg, 1997). The intracellular fate of internal pools of neosynthesized Gap1 is also governed by the nitrogen source, such that part of the pool can either be rerouted to the vacuole for degradation without previous delivery to the plasma membrane or recycled back to the plasma membrane from endosomal compartments (Roberg, 1997; Gao & Kaiser, 2006; Rubio-Texeira & Kaiser, 2006).
Downregulation of Gap1 sorting at the plasma membrane and internal membranes requires ubiquitination of the permease on lysines 9 and 16 (Soetens, 2001). Gap1 undergoes monoubiquitination dependent on the ubiquitin E3 ligase Rsp5, and K63 polyubiquitination, mediated by the concerted action of Rsp5 and the E4-like redundant proteins, Bul1 and Bul2 (Helliwell, 2001; Soetens, 2001). A differential role of the two ubiquitin-acceptor lysines in endocytosis and internal vacuolar sorting has been described recently (Risinger & Kaiser, 2008). The amino acid transport activity of Gap1 can also be reversibly inactivated at the plasma membrane upon amino acid binding, presumably through some conformational change or reversible modification (Risinger, 2006).
In addition to its transport function, Gap1 was found to play a role as an amino acid sensor in the rapid activation of the PKA pathway upon addition of amino acids to nitrogen-starved cells (Donaton, 2003). Since then, and although its downstream signaling pathway still remains unclear, Gap1 has become the best-characterized example of a nutrient-transporting transceptor. Activation of PKA targets upon addition of amino acids to nitrogen-starved cells is mainly dependent on Gap1. For some amino acids, however, there is a residual activation in a gap1Δ strain, indicating that some other amino acid carriers may also have a weak transceptor function (Van Zeebroeck, 2009).
Activation of the PKA pathway can be triggered by low concentrations of l-citrulline, which, under such conditions, are exclusively transported by Gap1. High concentrations of l-citrulline can also be transported by other amino acid carriers, such as Can1, but in the latter case, activation of the PKA pathway does not take place. This result indicates, on the one hand, that l-citrulline carriers other than Gap1 cannot support signaling, and on the other, that intracellular l-citrulline is unable, by itself, to trigger the signaling through an intracellular amino acid sensor. Metabolism of the amino acid is also not required for signaling. This is shown by the observations that nonmetabolizable d-amino acids still trigger signaling, and that the deletion of the argininosuccinate synthetase-encoding ARG1 gene, catalyzing the first step of l-citrulline catabolism, does not prevent signaling (Donaton, 2003).
Truncation analysis of both N- and C-termini of Gap1, with the aim of identifying specific domains involved in signaling, unexpectedly revealed that short C-terminal truncations converted the transceptor into a constitutively active form that caused hyperactivation of the PKA pathway under all the conditions tested (Donaton, 2003). Further work showed that hyperactivation by the constitutively active alleles depends on a specific background mutation in the strain, which apparently causes mis-sorting of truncated Gap1 alleles to the plasma membrane. In a wild-type strain, such defective versions are instead eliminated by the endoplasmic reticulum-associated protein degradation quality control system (unpublished data).
SCAM analysis of Gap1 has recently identified the residues Ser388 and Val389 as being exposed into the amino acid-binding site of Gap1. Binding of MTSEA to the Ser388C and Val389C alleles blocked both transport and signaling, indicating that Gap1 uses the same amino acid-binding site for transport and signaling (Van Zeebroeck, 2009). Further insight into the function of Gap1 as a transceptor has emerged from the discovery of amino acid analogues that either bind to the amino acid-binding site without triggering signaling or that can trigger signaling without being transported. These compounds were found by screening a large collection of amino acid analogues, first for inhibition of Gap1 transport activity, and then for competitive or noncompetitive nature. Both categories were then tested for the capacity to trigger activation of the PKA pathway. All possible combinations were found: competitive inhibitors with and without agonist action, and noncompetitive inhibitors with and without agonist action. The competitive inhibitors without agonist action are particularly interesting, because they must bind to the same site as the transported amino acids, according to the competitive nature of their inhibition. Because they do not trigger signaling, mere binding to the amino acid-binding site of Gap1 is apparently not enough for signaling. Another interesting conclusion can be drawn from the competitive inhibitors with agonist action, because these were not transported by Gap1. They indicate that completion of the transport cycle is not required for signaling. Activation by these nontransported agonists, thus, creates a similar situation for Gap1 as that observed for the nontransporting sensors Snf3, Rgt2, and Ssy1. That is, under these conditions, Gap1 also acts like a pure receptor protein. This observation suggests that the transporting and nontransporting transceptors may in fact function in a quite similar way. Taken together, the results obtained with the amino acid analogues indicate that a compound must be able to trigger a specific conformational change in Gap1 in order to function as a signaling agonist. This conformational change may be part of, but does not require, the complete transport cycle (Van Zeebroeck, 2009).
The functioning of Gap1 as a receptor for amino acid activation of the PKA pathway suggests that the well-known amino acid-induced endocytic internalization and subsequent vacuolar degradation of Gap1 may actually represent a process similar to the well-known ligand-induced internalization of GPCRs (Sorkin & Von Zastrow, 2002). Evidence for reciprocal involvement of the PKA signaling pathway in the regulation of Gap1 transceptor expression and functioning is also starting to emerge. First, it has been shown that enhanced activity of the cAMP/PKA pathway due to the presence of the constitutively active Ras2Val19 allele blocks the increase in leucine uptake that takes place when yeast cells are transferred to a medium with the poor nitrogen source proline, whereas reduction of cAMP/PKA pathway activity by deletion of RAS2 enhances this response (Saenz, 1997). Subsequent work has shown that this effect is in fact due to altered regulation of Gap1 at the post-transcriptional rather than at the transcriptional level. Strains with an overactive cAMP/PKA pathway display reduced Gap1 activity, whereas a strain with a lower cAMP/PKA pathway activity is insensitive to Gap1 downregulation by ammonium. The cAMP/PKA pathway was suggested to act at the level of the ubiquitin-dependent downregulation of Gap1 (Garrett, 2008).
Yeast ammonium transceptors Mep1,2
Ammonium is, in most cases, a good source of nitrogen for yeast, because it can be used for the biosynthesis of glutamine and glutamate, which in turn are the main precursors for the biosynthesis of many other amino acids (Magasanik & Kaiser, 2002). Passive diffusion of NH3 across the plasma membrane may support growth, but the external conditions that yeast cells face usually do not favor NH3 diffusion. The extracellular environment is usually acidic, shifting the acid–base equilibrium toward the protonated NH4+, which cannot enter cells through diffusion (Kikeri, 1989; Lande, 1995). Moreover, the concentrations of ammonia are often too low to be able to support growth by simple diffusion. Under such conditions, ammonium internalization in yeast cells mostly relies on ammonia transporters located at the plasma membrane.
The S. cerevisiae genome encodes three ammonium transporters: Mep1, Mep2, and Mep3 (Marini, 1997). Mep proteins play a major role in the scavenging of extracellular ammonia and ammonia leakage compensation (Boeckstaens, 2007). From the three transporters, Mep2 shows the highest affinity for ammonia (Km 1–2 μM), followed by Mep1 (Km 5–10 μM) and Mep3 (Km 1.4–2.1 mM) (Marini, 1997). Like the GAP1 transceptor gene, MEP genes are controlled through NCR (Marini, 1997). In the presence of a good nitrogen source, all three genes are repressed. On a poor nitrogen source, MEP2 expression is much higher than MEP1 and MEP3 expression. Transcriptional activation of MEP2 is controlled by the Gln3 and Gat1 proteins, whereas transcriptional activation of MEP1 and MEP3 is only dependent on Gln3 and downregulated in a Gat1-dependent manner (Marini, 1997). Post-translational regulation of at least the Mep2 protein shares some components with that of Gap1. For instance, although Mep2-GFP has been observed to localize correctly at the plasma membrane in mutants lacking Npr1 and Ure2 (Rutherford, 2008), optimal ammonium transport by the Mep proteins still requires the Npr1 kinase (Boeckstaens, 2007). The latter group demonstrated that cells lacking the three Mep proteins or the Npr1 kinase displayed overlapping growth defects with a number of nitrogen sources, and that growth under such conditions could be restored by expression of a hyperactive mutant form of Mep2. The authors attributed this effect to regulated retrieval of excreted ammonia carried out by the Mep2 protein, and pointed to the dependence on Npr1 kinase for the maintenance of proper levels of Mep2 transporting activity.
Convergence of genetic and structural studies indicates that the Mep proteins are grouped into multimeric complexes at the plasma membrane. A triple mep1–3 null mutant is unable to grow on a solid medium at pH 6.1 containing ammonium below 5 mM as the only nitrogen source, but the expression of each of the MEP genes separately restores growth. Expression of wild-type Mep3, for instance, rescues this growth defect, but coexpression of Mep3 and a mutant, inactive form of Mep1 (G412D or G413D) does not, suggesting trans-inhibition (Marini, 2000). Structural studies have also shown that residues lining the interior of these channel-type permeases are predominantly nonpolar, suggesting that NH4+ must be deprotonated before its passage through the pore. The only two polar residues facing the pore are two highly conserved histidines, which some groups have suggested to be involved in this putative deprotonation event (Khademi, 2004). Replacement of the first of these two histidines by glutamate leads to conversion of Mep2 into a Mep1-like transporter (Boeckstaens, 2007).
More recently, it has been shown that Mep1 and Mep2 also function as sensors that modulate the activity of important nutrient-sensing pathways. Mep2 is required for induction of pseudohyphal growth (Lorenz & Heitman, 1998) and Mep2 and, to a lesser extent Mep1, are involved in signaling ammonium availability to the cAMP–PKA pathway (Van Nuland, 2006). Pseudohyphal growth is supposed to facilitate the search for nutrients by yeast colonies and has been studied mostly under conditions of nitrogen limitation. The activity of Mep2, but not that of Mep1 or Mep3, is required for induction of pseudohyphal growth (Lorenz & Heitman, 1998). Mep2 is strongly expressed at the cell surface under conditions supporting pseudohyphal growth and its further overexpression stimulates this differentiation process even in the presence of a nitrogen-rich medium (Rutherford, 2008). Two signaling pathways are involved in induction of pseudohyphal growth: a mitogen activated protein kinase (MAPK) pathway and the cAMP–PKA pathway (most recently reviewed by Bahn, 2007; Thevelein, 2008). The connection between Mep2 and the two pathways, however, remains unclear. Recent work has, nevertheless, shown that overexpression of Mep2 triggers a transcriptional profile consistent with activation of the MAPK pathway (Rutherford, 2008). Results from epistasis analysis confirmed that Mep2 functions upstream of the MAPK pathway rather than the cAMP–PKA pathway. These results fit with previous findings that involvement of Mep2 as a transceptor for activation of the PKA pathway is different from its role in the control of pseudohyphal growth (Van Nuland, 2006). Strong support for a transceptor role of Mep2 in triggering pseudohyphal growth was obtained with specific mutant alleles of Mep2 that maintained transport, but were deficient in signaling (Rutherford, 2008). The pair of histidines at the entrance of the translocation channel seem to play an important role in the sensor function because replacement of the first of these two histidines by glutamate leads to conversion of Mep2 into a Mep1-like transporter and eliminates Mep2's ability to activate pseudohyphal growth (Boeckstaens, 2007). How Mep2 precisely connects to the MAPK pathway, however, remains to be elucidated.
The MAPK cascade induced during pseudohyphal growth shares upstream components with the cAMP–PKA pathway, such as Ras2 (Mosch, 1999). Reducing cAMP levels by overexpression of the cAMP phosphodiesterase Pde2, or by deletion of GPR1 or GPA2, impairs transition to pseudohyphal growth (Lorenz & Heitman, 1997). Conversely, overstimulation of the PKA pathway by deletion of BCY1 or growth of a pde2Δ/pde2Δ diploid strain on a cAMP-containing medium induces pseudohyphal growth on a nitrogen-rich medium (Lorenz & Heitman, 1997; Pan & Heitman, 1999). Moreover, the different catalytic subunits of PKA have different effects on pseudohyphal growth: Tpk2 stimulates, but Tpk1 and Tpk3 have an inhibitory effect (Pan & Heitman, 1999). A putative PKA-phosphorylation site that, when substituted to alanine, abolishes pseudohyphal growth was found in the fourth intracellular loop of Mep proteins (Smith, 2003).
Besides their role in pseudohyphal growth, Mep2 and Mep1 also act as nutrient transceptors in signaling for PKA activation in response to the addition of ammonia to nitrogen-starved fermenting cells (FGM response; Van Nuland, 2006). A nonmetabolizable ammonia analogue, methylammonia, also triggers this Mep1,2-dependent response. Inhibition of ammonia metabolism by addition of a glutamine synthetase inhibitor to a gdh1Δ strain (GDH1 encodes glutamate dehydrogenase) did not prevent trehalase activation by methylammonia. Expression of Arabidopsis ammonium carriers in the triple mep1Δmep2Δmep3Δ strain restored the PKA response to some extent. Addition of 10 mM methylammonia to mep1ΔMEP2 mep3Δ cells or 300 mM to mep1Δmep2Δmep3Δ cells at acidic pH shows the same level of internalization, but trehalase activation only for the strain expressing Mep2. However, if the experiment is repeated at a basic pH, the triple mutant also shows a similar level of trehalase activation. This suggests the existence of a Mep2-independent, overlapping effect of pH in ammonia sensing through the PKA pathway (Vandormael et al., unpublished data).
The phosphate transceptor Pho84
Under good nutritional conditions, S. cerevisiae stores inorganic phosphate (Pi) in the form of polyphosphate (polyP) within the vacuole and other compartments. When the levels of external Pi are too low, the cells mobilize their internal stores of polyP, which, for a short period of time, will be able to sustain the intracellular requirement of Pi. However, continued exposure to phosphate starvation will trigger an emergency response consisting of full induction of the genes of the PHO regulon. The PHO pathway is a genetic regulatory circuit that controls responses and intracellular adaptations to variations in the external level of phosphate. Structural genes of this PHO regulon encode two main subgroups of proteins involved in the external scavenging of Pi: the phosphate transporters, Pho84 and Pho89, and the secreted acid phosphatases, Pho3, Pho5, Pho11, and Pho12 (latest reviewed by Mouillon & Persson, 2006).
Saccharomyces cerevisiae has five different permeases involved in the transport of inorganic phosphate. Apart from the high-affinity transporters Pho84 and Pho89, it also has the three low-affinity constitutive transporters Pho87, Pho90, and Pho91. All of these phosphate transporters are plasma membrane localized with the exception of the latter one, which resides at the vacuolar membrane (Bun-Ya, 1991; Persson, 1998, 1999; Hurlimann, 2007). No single phosphate transporter is essential for cell survival, but the quintuple deletion of all phosphate carriers is lethal (Wykoff & O'Shea, 2001). Overexpression of Git1, a glycerophosphoinositol transporter, rescues this lethality, suggesting an additional role for this transporter and phospholipid catabolic products in the retrieval of phosphate (Wykoff & O'Shea, 2001).
Deletion of PHO84 or triple deletion of PHO87, PHO90, and PHO91 causes constitutive derepression of the PHO genes. In the first case, overexpression of each of the other four transporters rescues the pho84 null mutant phenotype (Wykoff & O'Shea, 2001). These and further studies establishing a direct correlation between the levels of internal Pi and polyP with the expression of the secreted phosphatase gene PHO5 have led to the hypothesis that the signal controlling the activity of the PHO pathway primarily relies on the intracellular concentrations of Pi and polyP. They are thought to modulate the interactions between the corepressor Pho85–Pho80 and its substrate, the transcriptional activator Pho4 (Auesukaree, 2004; Thomas & O'Shea, 2005). The proposed models have excluded a more specific sensing/signaling role for either of the transporters for control of the PHO pathway, because overexpression of either of them suffices to restore internal Pi levels as well as normal regulation of the PHO genes (Wykoff & O'Shea, 2001; Wykoff, 2007).
On the other hand, it has recently been shown that rapid activation of the PKA pathway by addition of phosphate to phosphate-starved cells specifically requires Pho84 and/or Pho87 (Giots, 2003). This activation did not occur in deletion mutants of these two genes expressing the other three wild-type transporters, which implies a specific role of these two phosphate transporters in phosphate signaling. As in the case of nitrogen, activation of the PKA pathway through the activity of Pho84 and Pho87 does not result in an increased level of cAMP (Giots, 2003), confirming previous conclusions that cAMP is not involved as a secondary messenger in phosphate-induced activation of the PKA pathway (Hirimburegama, 1992). As for nitrogen signaling, the presence of a rapidly fermentable carbon source, such as glucose, is required for rapid phosphate signaling and the sugar is also sensed by the glucose-sensing GPCR Gpr1 and the sugar-phosphorylation-dependent system. However, as opposed to nitrogen signaling, phosphate signaling does not require the Sch9 protein kinase (Giots, 2003). Interestingly, activity of the PHO corepressor complex Pho85–Pho80 has also been linked to the presence of other nutrients such as carbon and nitrogen sources in the medium (Lee, 2000).
Recent work has shown that the nonmetabolizable phosphate analogue methylphosphonate can trigger transcriptional repression of the PHO genes, rapid activation of the PKA pathway through Pho84 as well as downregulation of Pho84 by endocytic internalization and vacuolar degradation. Addition of the PKA inhibitor H89 inhibited the latter process, suggesting that Pho84-mediated activation of the PKA pathway triggers Pho84 downregulation. Repression of the PHO genes, on the other hand, was not affected by H89, suggesting the existence of different phosphate-sensing mechanisms initiated by Pho84. The other four phosphate transporters were unable to trigger repression of PHO5 upon addition of methylphosphonate (Pratt, 2004; Mouillon & Persson, 2005).
Finally, sorting of the Pho84 transceptor is, in analogy to that of Gap1, regulated by phosphorylation and ubiquitination. Ubiquitination occurs in the middle intracellular loop and triggers Pho84 endocytosis. Interestingly, Pho84 endocytosis is delayed in strains with reduced PKA activity (Lundh, 2009).
The importance of the plasma membrane for the detection of nutrients by S. cerevisiae cells is demonstrated by the variety of plasma membrane-localized nutrient sensors. They can be divided into three categories: GPCRs, nontransporting nutrient carrier homologues, and proteins combining transporting and sensing capacities. Both the nontransporting and the transporting forms of the transporter-related sensors are termed ‘transceptors.’ It appears likely that intermediate forms, sensors with low, residual transport activity, will also be found. Equally likely is the possibility that transporting and nontransporting transceptors involved in the detection of the same type of nutrient may not only have parallel contributions but also synergistic interactions that contribute to the output signal elicited for this particular nutrient.
In two out of the three categories of plasma membrane nutrient sensors, there is a close connection between the signal detected and immediate activation of PKA targets. The PKA pathway seems, therefore, to be at the core of the process of translation of extracellular nutrient signals into an intracellular response. It remains to be elucidated whether a closer connection between the PKA pathway and the nontransporting transceptors exists. So far, no evidence has been found that directly links this transceptor class to PKA activation in response to nutrients. However, cross-talk and indirect effects because of the influence of these transceptors on nutrient uptake and thus, also on their metabolism, certainly exist. Hence, from this point of view, they have indirect implications in the activation of the PKA pathway in response to nutrients.
The presence of these three types of nutrient sensors is not restricted to S. cerevisiae. Many other fungi have genes encoding GPCRs with potential nutrient-sensing capacities (reviewed in Xue, 2008). Moreover, a variety of GPCRs, expressed in taste receptor cells in higher organisms, is involved in detecting bitter- and sweet-tasting nutrients (Chandrashekar, 2006). To date, only nontransporting transceptors for glucose and for amino acids have been identified in yeast. It remains to be elucidated whether additional nontransporting transceptors also exist for other nutrients. Homologues of both the glucose-sensing Snf3-Rgt2 transceptors and of components of the SPS amino acid sensor complex are also found in other fungi. The precise function of many of these proteins, with the exception of the Candida albicans Csy1, remains to be clarified. The latter has recently been identified to be involved in amino acid sensing in a similar way as Ssy1 in S. cerevisiae (Brega, 2004). There are still no clear-cut orthologues of the S. cerevisiae nontransporting transceptors found in mammalian cells. Recent work indicates that hSGLT3, a protein, based on its amino acid sequence, considered as belonging to the Na+/glucose cotransporter family, functions as a glucose sensor in smooth and skeletal muscle. SGLT3 does not transport sugars, but addition of glucose results in a Na+-dependent depolarization of the plasma membrane (Diez-Sampedro, 2003).
As for the class of transporting transceptors, examples have also been described in other eukaryotic organisms, from fungi, plants, and flies, up to mammals. For example, the homologues of the Gap1 protein in C. albicans seem to have an important regulatory effect on hyphae formation (Biswas, 2003). This suggests that CaGap1 may, as its S. cerevisiae counterpart, possess a sensing function in addition to its transport function. The Ustilago maydis Ump2 ammonium transporter can efficiently complement the pseudohyphal growth defect of the S. cerevisiae mep2Δ strain, suggesting that it may also have an additional ammonium-sensing role (Smith, 2003). In Drosophila, two amino acid transporters (PATs), CG3424 and CG1139, related to mammalian transporters, are regulators of growth, independent of transport (Goberdhan, 2005). The SNAT2 mammalian amino acid transporter has also been found to act as a sensor regulating its own expression (Hyde, 2003, 2007). Finally, evidence has been reported that interaction of the mammalian EAAT1 glutamate transporter with its substrate glutamate regulates glial signal transduction and morphology (Duan, 1999).
The plethora of recently, and some of them newly, identified categories of nutrient-responsive plasma membrane proteins already underscores the importance of nutrients as signaling molecules, not only for yeast but very likely also for all other organisms. The findings in yeast are likely only the tip of the iceberg of plasma membrane nutrient-sensing systems. Future major discoveries in this line, now increasingly likely for many organisms, will significantly help us to understand the intricate complexity of nutrient sensing and signaling, and its relationship with the control of growth and development both at the cellular and at the organismal levels.
Original research in our laboratory has been supported by grants from the Fund for Scientific Research – Flanders, Interuniversity Attraction Poles Network P6/14, and the Research Fund of the Katholieke Universiteit Leuven (Concerted Research Actions).