Nutrient transceptors physically interact with the yeast S6/protein kinase B homolog, Sch9, a TOR kinase target.

Multiple starvation-induced, high-affinity nutrient transporters in yeast function as receptors for activation of the protein kinase A (PKA) pathway upon re-addition of their substrate. We now show that these transceptors may play more extended roles in nutrient regulation. The Gap1 amino acid, Mep2 ammonium, Pho84 phosphate and Sul1 sulfate transceptors physically interact in vitro and in vivo with the PKA-related Sch9 protein kinase, the yeast homolog of mammalian S6 protein kinase and protein kinase B. Sch9 is a phosphorylation target of TOR and well known to affect nutrient-controlled cellular processes, such as growth rate. Mapping with peptide microarrays suggests specific interaction domains in Gap1 for Sch9 binding. Mutagenesis of the major domain affects the upstart of growth upon the addition of L-citrulline to nitrogen-starved cells to different extents but apparently does not affect in vitro binding. It also does not correlate with the drop in L-citrulline uptake capacity or transceptor activation of the PKA target trehalase by the Gap1 mutant forms. Our results reveal a nutrient transceptor-Sch9-TOR axis in which Sch9 accessibility for phosphorylation by TOR may be affected by nutrient transceptor-Sch9 interaction under conditions of nutrient starvation or other environmental challenges.

The involvement of the Candida glabrata trehalase enzymes in stress resistance and gut colonization.

Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo to determine the role of these enzymes in virulence. Ath1 was found to be located in the periplasm and was essential for growth on trehalose as sole carbon source, while Nth1 on the other hand was important for oxidative stress resistance, an observation which was consistent by the lower survival rate of the NTH1 deletion strain in human macrophages. No significant phenotype was observed for Nth2. The triple deletion strain was unable to establish a stable colonization of the gastrointestinal (GI) tract in mice indicating the importance of having trehalase activity for colonization in the gut.

Aberrant Intracellular pH Regulation Limiting Glyceraldehyde-3-Phosphate Dehydrogenase Activity in the Glucose-Sensitive Yeast tps1Δ Mutant.

Whereas the yeast Saccharomyces cerevisiae shows great preference for glucose as a carbon source, a deletion mutant in trehalose-6-phosphate synthase, tps1Δ, is highly sensitive to even a few millimolar glucose, which triggers apoptosis and cell death. Glucose addition to tps1Δ cells causes deregulation of glycolysis with hyperaccumulation of metabolites upstream and depletion downstream of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The apparent metabolic barrier at the level of GAPDH has been difficult to explain. We show that GAPDH isozyme deletion, especially Tdh3, further aggravates glucose sensitivity and metabolic deregulation of tps1Δ cells, but overexpression does not rescue glucose sensitivity. GAPDH has an unusually high pH optimum of 8.0 to 8.5, which is not altered by tps1Δ. Whereas glucose causes short, transient intracellular acidification in wild-type cells, in tps1Δ cells, it causes permanent intracellular acidification. The hxk2Δ and snf1Δ suppressors of tps1Δ restore the transient acidification. These results suggest that GAPDH activity in the tps1Δ mutant may be compromised by the persistently low intracellular pH. Addition of NH4Cl together with glucose at high extracellular pH to tps1Δ cells abolishes the pH drop and reduces glucose-6-phosphate (Glu6P) and fructose-1,6-bisphosphate (Fru1,6bisP) hyperaccumulation. It also reduces the glucose uptake rate, but a similar reduction in glucose uptake rate in a tps1Δ hxt2,4,5,6,7Δ strain does not prevent glucose sensitivity and Fru1,6bisP hyperaccumulation. Hence, our results suggest that the glucose-induced intracellular acidification in tps1Δ cells may explain, at least in part, the apparent glycolytic bottleneck at GAPDH but does not appear to fully explain the extreme glucose sensitivity of the tps1Δ mutant.IMPORTANCE Glucose catabolism is the backbone of metabolism in most organisms. In spite of numerous studies and extensive knowledge, major controls on glycolysis and its connections to the other metabolic pathways remain to be discovered. A striking example is provided by the extreme glucose sensitivity of the yeast tps1Δ mutant, which undergoes apoptosis in the presence of just a few millimolar glucose. Previous work has shown that the conspicuous glucose-induced hyperaccumulation of the glycolytic metabolite fructose-1,6-bisphosphate (Fru1,6bisP) in tps1Δ cells triggers apoptosis through activation of the Ras-cAMP-protein kinase A (PKA) signaling pathway. However, the molecular cause of this Fru1,6bisP hyperaccumulation has remained unclear. We now provide evidence that the persistent drop in intracellular pH upon glucose addition to tps1Δ cells likely compromises the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a major glycolytic enzyme downstream of Fru1,6bisP, due to its unusually high pH optimum. Our work highlights the potential importance of intracellular pH fluctuations for control of major metabolic pathways.

Nutrient sensing and cAMP signaling in yeast: G-protein coupled receptor versus transceptor activation of PKA.

A major signal transduction pathway regulating cell growth and many associated physiological properties as a function of nutrient availability in the yeast Saccharomyces cerevisiae is the protein kinase A (PKA) pathway. Glucose activation of PKA is mediated by G-protein coupled receptor (GPCR) Gpr1, and secondary messenger cAMP. Other nutrients, including nitrogen, phosphate and sulfate, activate PKA in accordingly-starved cells through nutrient transceptors, but apparently without cAMP signaling. We have now used an optimized EPAC-based fluorescence resonance energy transfer (FRET) sensor to precisely monitor in vivo cAMP levels after nutrient addition. We show that GPCR-mediated glucose activation of PKA is correlated with a rapid transient increase in the cAMP level in vivo, whereas nutrient transceptor-mediated activation by nitrogen, phosphate or sulfate, is not associated with any significant increase in cAMP in vivo. We also demonstrate direct physical interaction between the Gap1 amino acid transceptor and the catalytic subunits of PKA, Tpk1, 2 and 3. In addition, we reveal a conserved consensus motif in the nutrient transceptors that is also present in Bcy1, the regulatory subunit of PKA. This suggests that nutrient transceptor activation of PKA may be mediated by direct release of bound PKA catalytic subunits, triggered by the conformational changes occurring during transport of the substrate by the transceptor. Our results support a model in which nutrient transceptors are evolutionary ancestors of GPCRs, employing a more primitive direct signaling mechanism compared to the indirect cAMP second-messenger signaling mechanism used by GPCRs for activation of PKA.

Multiple Transceptors for Macro- and Micro-Nutrients Control Diverse Cellular Properties Through the PKA Pathway in Yeast: A Paradigm for the Rapidly Expanding World of Eukaryotic Nutrient Transceptors Up to Those in Human Cells.

The nutrient composition of the medium has dramatic effects on many cellular properties in the yeast Saccharomyces cerevisiae. In addition to the well-known specific responses to starvation for an essential nutrient, like nitrogen or phosphate, the presence of fermentable sugar or a respirative carbon source leads to predominance of fermentation or respiration, respectively. Fermenting and respiring cells also show strong differences in other properties, like storage carbohydrate levels, general stress tolerance and cellular growth rate. However, the main glucose repression pathway, which controls the switch between respiration and fermentation, is not involved in control of these properties. They are controlled by the protein kinase A (PKA) pathway. Addition of glucose to respiring yeast cells triggers cAMP synthesis, activation of PKA and rapid modification of its targets, like storage carbohydrate levels, general stress tolerance and growth rate. However, starvation of fermenting cells in a glucose medium for any essential macro- or micro-nutrient counteracts this effect, leading to downregulation of PKA and its targets concomitant with growth arrest and entrance into G0. Re-addition of the lacking nutrient triggers rapid activation of the PKA pathway, without involvement of cAMP as second messenger. Investigation of the sensing mechanism has revealed that the specific high-affinity nutrient transporter(s) induced during starvation function as transporter-receptors or transceptors for rapid activation of PKA upon re-addition of the missing substrate. In this way, transceptors have been identified for amino acids, ammonium, phosphate, sulfate, iron, and zinc. We propose a hypothesis for regulation of PKA activity by nutrient transceptors to serve as a conceptual framework for future experimentation. Many properties of transceptors appear to be similar to those of classical receptors and nutrient transceptors may constitute intermediate forms in the development of receptors from nutrient transporters during evolution. The nutrient-sensing transceptor system in yeast for activation of the PKA pathway has served as a paradigm for similar studies on candidate nutrient transceptors in other eukaryotes and we succinctly discuss the many examples of transceptors that have already been documented in other yeast species, filamentous fungi, plants, and animals, including the examples in human cells.

Methionine is required for cAMP-PKA-mediated morphogenesis and virulence of Candida albicans.

Candida albicans is a major human fungal pathogen, causing superficial, as well as life-threatening invasive infections. Therefore, it has to adequately sense and respond to the host defense by expressing appropriate virulence attributes. The most important virulence factor of C. albicans is the yeast-to-hyphae morphogenetic switch, which can be induced by numerous environmental cues, including the amino acid methionine. Here, we show an essential role for methionine permease Mup1 in methionine-induced morphogenesis, biofilm formation, survival inside macrophages and virulence. Furthermore, we demonstrate that this process requires conversion of methionine into S-adenosyl methionine (SAM) and its decarboxylation by Spe2. The resulting amino-propyl group is then used for biosynthesis of polyamines, which have been shown to activate adenylate cyclase. Inhibition of the SPE2 SAM decarboxylase gene strongly impairs methionine-induced morphogenesis on specific media and significantly delays virulence in the mouse systemic infection model system. Further proof of the connection between methionine uptake and initial metabolism and the cAMP-PKA pathway was obtained by showing that both Mup1 and Spe2 are required for cAMP production in response to methionine. Our results suggest that amino acid transport and further metabolism are interesting therapeutic targets as inhibitors of this may prevent the morphogenetic switch, thereby preventing virulence.

The nutrient transceptor/PKA pathway functions independently of TOR and responds to leucine and Gcn2 in a TOR-independent manner.

Two nutrient-controlled signalling pathways, the PKA and TOR pathway, play a major role in nutrient regulation of growth as well as growth-correlated properties in yeast. The relationship between the two pathways is not well understood. We have used Gap1 and Pho84 transceptor-mediated activation of trehalase and phosphorylation of fragmented Sch9 as a read-out for rapid nutrient activation of PKA or TORC1, respectively. We have identified conditions in which L-citrulline-induced activation of Sch9 phosphorylation is compromised, but not activation of trehalase: addition of the TORC1 inhibitor, rapamycin and low levels of L-citrulline. The same disconnection was observed for phosphate activation in phosphate-starved cells. The leu2 auxotrophic mutation reduces amino acid activation of trehalase, which is counteracted by deletion of GCN2. Both effects were also independent of TORC1. Our results show that rapid activation of the TOR pathway by amino acids is not involved in rapid activation of the PKA pathway and that effects of Gcn2 inactivation as well as leu2 auxotrophy all act independently of the TOR pathway. Hence, rapid nutrient signalling to PKA and TOR in cells arrested by nutrient starvation acts through parallel pathways.

Nutrient Sensing at the Plasma Membrane of Fungal Cells.

To respond to the changing environment, cells must be able to sense external conditions. This is important for many processes including growth, mating, the expression of virulence factors, and several other regulatory effects. Nutrient sensing at the plasma membrane is mediated by different classes of membrane proteins that activate downstream signaling pathways: nontransporting receptors, transceptors, classical and nonclassical G-protein-coupled receptors, and the newly defined extracellular mucin receptors. Nontransporting receptors have the same structure as transport proteins, but have lost the capacity to transport while gaining a receptor function. Transceptors are transporters that also function as a receptor, because they can rapidly activate downstream signaling pathways. In this review, we focus on these four types of fungal membrane proteins. We mainly discuss the sensing mechanisms relating to sugars, ammonium, and amino acids. Mechanisms for other nutrients, such as phosphate and sulfate, are discussed briefly. Because the model yeast Saccharomyces cerevisiae has been the most studied, especially regarding these nutrient-sensing systems, each subsection will commence with what is known in this species.

Major sulfonate transporter Soa1 in Saccharomyces cerevisiae and considerable substrate diversity in its fungal family.

Sulfate is a well-established sulfur source for fungi; however, in soils sulfonates and sulfate esters, especially choline sulfate, are often much more prominent. Here we show that Saccharomyces cerevisiae YIL166C(SOA1) encodes an inorganic sulfur (sulfate, sulfite and thiosulfate) transporter that also catalyses sulfonate and choline sulfate uptake. Phylogenetic analysis of fungal SOA1 orthologues and expression of 20 members in the sul1Δ sul2Δ soa1Δ strain, which is deficient in inorganic and organic sulfur compound uptake, reveals that these transporters have diverse substrate preferences for sulfur compounds. We further show that SOA2, a S. cerevisiae SOA1 paralogue found in S. uvarum, S. eubayanus and S. arboricola is likely to be an evolutionary remnant of the uncharacterized open reading frames YOL163W and YOL162W. Our work highlights the importance of sulfonates and choline sulfate as sulfur sources in the natural environment of S. cerevisiae and other fungi by identifying fungal transporters for these compounds.

Characterization of the Candida albicans Amino Acid Permease Family: Gap2 Is the Only General Amino Acid Permease and Gap4 Is an S-Adenosylmethionine (SAM) Transporter Required for SAM-Induced Morphogenesis.

Amino acids are key sources of nitrogen for growth of Candida albicans. In order to detect and take up these amino acids from a broad range of different and changing nitrogen sources inside the host, this fungus must be able to adapt via its expression of genes for amino acid uptake and further metabolism. We analyzed six C. albicans putative general amino acid permeases based on their homology to the Saccharomyces cerevisiae Gap1 general amino acid permease. We generated single- and multiple-deletion strains and found that, based on growth assays and transcriptional or posttranscriptional regulation, Gap2 is the functional orthologue to ScGap1, with broad substrate specificity. Expression analysis showed that expression of all GAP genes is under control of the Csy1 amino acid sensor, which is different from the situation in S. cerevisiae, where the expression of ScGAP1 is not regulated by Ssy1. We show that Gap4 is the functional orthologue of ScSam3, the only S-adenosylmethionine (SAM) transporter in S. cerevisiae, and we report that Gap4 is required for SAM-induced morphogenesis. IMPORTANCECandida albicans is a commensal organism that can thrive in many niches in its human host. The environmental conditions at these different niches differ quite a bit, and this fungus must be able to sense these changes and adapt its metabolism to them. Apart from glucose and other sugars, the uptake of amino acids is very important. This is underscored by the fact that the C. albicans genome encodes 6 orthologues of the Saccharomyces. cerevisiae general amino acid permease Gap1 and many other amino acid transporters. In this work, we characterize these six permeases and we show that C. albicans Gap2 is the functional orthologue of ScGap1 and that C. albicans Gap4 is an orthologue of ScSam3, an S-adenosylmethionine (SAM) transporter. Furthermore, we show that Gap4 is required for SAM-induced morphogenesis, an important virulence factor of C. albicans.

Key Residues and Phosphate Release Routes in the Saccharomyces cerevisiae Pho84 Transceptor: THE ROLE OF TYR179 IN FUNCTIONAL REGULATION.

Pho84, a major facilitator superfamily (MFS) protein, is the main high-affinity Pi transceptor in Saccharomyces cerevisiae Although transport mechanisms have been suggested for other MFS members, the key residues and molecular events driving transport by Pi:H+ symporters are unclear. The current Pho84 transport model is based on the inward-facing occluded crystal structure of the Pho84 homologue PiPT in the fungus Piriformospora indica However, this model is limited by the lack of experimental data on the regulatory residues for each stage of the transport cycle. In this study, an open, inward-facing conformation of Pho84 was used to study the release of Pi A comparison of this conformation with the model for Pi release in PiPT revealed that Tyr179 in Pho84 (Tyr150 in PiPT) is not part of the Pi binding site. This difference may be due to a lack of detailed information on the Pi release step in PiPT. Molecular dynamics simulations of Pho84 in which a residue adjacent to Tyr179, Asp178, is protonated revealed a conformational change in Pho84 from an open, inward-facing state to an occluded state. Tyr179 then became part of the binding site as was observed in the PiPT crystal structure. The importance of Tyr179 in regulating Pi release was supported by site-directed mutagenesis and transport assays. Using trehalase activity measurements, we demonstrated that the release of Pi is a critical step for transceptor signaling. Our results add to previous studies on PiPT, creating a more complete picture of the proton-coupled Pi transport cycle of a transceptor.

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress.

Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress.

Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor.

The Saccharomyces cerevisiae amino acid transceptor Gap1 functions as receptor for signalling to the PKA pathway and concomitantly undergoes substrate-induced oligo-ubiquitination and endocytosis. We have identified specific amino acids and analogues that uncouple to certain extent signalling, transport, oligo-ubiquitination and endocytosis. L-lysine, L-histidine and L-tryptophan are transported by Gap1 but do not trigger signalling. Unlike L-histidine, L-lysine triggers Gap1 oligo-ubiquitination without substantial induction of endocytosis. Two transported, non-metabolizable signalling agonists, ß-alanine and D-histidine, are strong and weak inducers of Gap1 endocytosis, respectively, but both causing Gap1 oligo-ubiquitination. The non-signalling agonist, non-transported competitive inhibitor of Gap1 transport, L-Asp-γ-L-Phe, induces oligo-ubiquitination but no discernible endocytosis. The Km of L-citrulline transport is much lower than the threshold concentration for signalling and endocytosis. These results show that molecules can be transported without triggering signalling or substantial endocytosis, and that oligo-ubiquitination and endocytosis do not require signalling nor metabolism. Oligo-ubiquitination is required, but apparently not sufficient to trigger endocytosis. In addition, we demonstrate intracellular cross-induction of endocytosis of transport-defective Gap1(Y395C) by ubiquitination- and endocytosis-deficient Gap1(K9R,K16R). Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes.

Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has been a favorite organism for pioneering studies on nutrient-sensing and signaling mechanisms. Many specific nutrient responses have been elucidated in great detail. This has led to important new concepts and insight into nutrient-controlled cellular regulation. Major highlights include the central role of the Snf1 protein kinase in the glucose repression pathway, galactose induction, the discovery of a G-protein-coupled receptor system, and role of Ras in glucose-induced cAMP signaling, the role of the protein synthesis initiation machinery in general control of nitrogen metabolism, the cyclin-controlled protein kinase Pho85 in phosphate regulation, nitrogen catabolite repression and the nitrogen-sensing target of rapamycin pathway, and the discovery of transporter-like proteins acting as nutrient sensors. In addition, a number of cellular targets, like carbohydrate stores, stress tolerance, and ribosomal gene expression, are controlled by the presence of multiple nutrients. The protein kinase A signaling pathway plays a major role in this general nutrient response. It has led to the discovery of nutrient transceptors (transporter receptors) as nutrient sensors. Major shortcomings in our knowledge are the relationship between rapid and steady-state nutrient signaling, the role of metabolic intermediates in intracellular nutrient sensing, and the identity of the nutrient sensors controlling cellular growth.

Yeast nutrient transceptors provide novel insight in the functionality of membrane transporters.

In the yeast Saccharomyces cerevisiae several nutrient transporters have been identified that possess an additional function as nutrient receptor. These transporters are induced when yeast cells are starved for their substrate, which triggers entry into stationary phase and acquirement of a low protein kinase A (PKA) phenotype. Re-addition of the lacking nutrient triggers exit from stationary phase and sudden activation of the PKA pathway, the latter being mediated by the nutrient transceptors. At the same time, the transceptors are ubiquitinated, endocytosed and sorted to the vacuole for breakdown. Investigation of the signaling function of the transceptors has provided a new read-out and new tools for gaining insight into the functionality of transporters. Identification of amino acid residues that bind co-transported ions in symporters has been challenging because the inactivation of transport by site-directed mutagenesis is not conclusive with respect to the cause of the inactivation. The discovery of nontransported agonists of the signaling function in transceptors has shown that transport is not required for signaling. Inactivation of transport with maintenance of signaling in transceptors supports that a true proton-binding residue was mutagenised. Determining the relationship between transport and induction of endocytosis has also been challenging, since inactivation of transport by mutagenesis easily causes loss of all affinity for the substrate. The use of analogues with different combinations of transport and signaling capacities has revealed that transport, ubiquitination and endocytosis can be uncoupled in several unexpected ways. The results obtained are consistent with transporters undergoing multiple substrate-induced conformational changes, which allow interaction with different accessory proteins to trigger specific downstream events.

In vivo phosphorylation of Ser21 and Ser83 during nutrient-induced activation of the yeast protein kinase A (PKA) target trehalase.

The readdition of an essential nutrient to starved, fermenting cells of the yeast Saccharomyces cerevisiae triggers rapid activation of the protein kinase A (PKA) pathway. Trehalase is activated 5-10-fold within minutes and has been used as a convenient reporter for rapid activation of PKA in vivo. Although trehalase can be phosphorylated and activated by PKA in vitro, demonstration of phosphorylation during nutrient activation in vivo has been lacking. We now show, using phosphospecific antibodies, that glucose and nitrogen activation of trehalase in vivo is associated with phosphorylation of Ser(21) and Ser(83). Unexpectedly, mutants with reduced PKA activity show constitutive phosphorylation despite reduced trehalase activation. The same phenotype was observed upon deletion of the catalytic subunits of yeast protein phosphatase 2A, suggesting that lower PKA activity causes reduced trehalase dephosphorylation. Hence, phosphorylation of trehalase in vivo is not sufficient for activation. Deletion of the inhibitor Dcs1 causes constitutive trehalase activation and phosphorylation. It also enhances binding of trehalase to the 14-3-3 proteins Bmh1 and Bmh2, suggesting that Dcs1 inhibits by preventing 14-3-3 binding. Deletion of Bmh1 and Bmh2 eliminates both trehalase activation and phosphorylation. Our results reveal that trehalase activation in vivo is associated with phosphorylation of typical PKA sites and thus establish the enzyme as a reliable read-out for nutrient activation of PKA in vivo.

Peptides induce persistent signaling from endosomes by a nutrient transceptor.

The yeast Gap1 transceptor mediates amino acid activation of the protein kinase A pathway and undergoes endocytic internalization following amino acid transport. We identified three specific γ-glutamyl dipeptides that cause persistent cyclic AMP-independent activation of protein kinase A, prevent Gap1 vacuolar sorting and cause Gap1 accumulation in endosomes. To our knowledge, these are the first examples of persistent agonists of a transceptor. In yeast mutants blocked in multivesicular body sorting, L-citrulline mimicked persistent signaling, further supporting that the internalized Gap1 transceptor keeps signaling. Unexpectedly, these dipeptides were transported by Gap1 and not by the regular dipeptide transporters. Their uptake was unusually sensitive to external pH and caused transient intracellular acidification. High external pH, NHA1 deletion or V-ATPase inhibition overcame the vacuolar sorting defect. Hence, this work has identified specific dipeptides that cause enhanced proton influx through the Gap1 symporter, resulting in its defective vacuolar sorting, and independently transform it into a persistently signaling transceptor.

A split-ubiquitin two-hybrid screen for proteins physically interacting with the yeast amino acid transceptor Gap1 and ammonium transceptor Mep2.

Several nutrient permeases have been identified in yeast, which combine a transport and receptor function, and are called transceptors. The Gap1 general amino acid permease and the Mep2 ammonium permease mediate rapid activation by amino acids and by ammonium, respectively, of the protein kinase A (PKA) pathway in nitrogen-starved cells. Their mode of action is not well understood. Both proteins are subject to complex controls governing their intracellular trafficking. Using a split-ubiquitin yeast two-hybrid screen with Gap1 or Mep2 as bait, we identified proteins putatively interacting with Gap1 and/or Mep2. They are involved in glycosylation, the secretory pathway, sphingolipid biosynthesis, cell wall biosynthesis and other processes. For several candidate interactors, determination of transport and signaling capacity, as well as localization of Gap1 or Mep2 in the corresponding deletion strains, confirmed a functional interaction with Gap1 and/or Mep2. Also common interacting proteins were identified. Transport and signaling were differentially affected in specific deletion strains, clearly separating the two functions of the transceptors and confirming that signaling does not require transport. We identified two new proteins, Bsc6 and Yir014w, that affect trafficking or downregulation of Gap1. Deletion of EGD2, YNL024c or SPC2 inactivates Gap1 transport and signaling, while its plasma membrane level appears normal.. Vma4 is required for Mep2 expression, while Gup1 appears to be required for proper distribution of Mep2 over the plasma membrane. Some of the interactions were confirmed by GST pull-down assay, using the C-terminal tail of Gap1 or Mep2 expressed in E.coli. Our results reveal the effectiveness of split-ubiquitin two-hybrid screening for identification of proteins functionally interacting with membrane proteins. They provide several candidate proteins involved in the transport and signaling function or in the complex trafficking control of the Gap1 and Mep2 transceptors.

From transporter to transceptor: signaling from transporters provokes re-evaluation of complex trafficking and regulatory controls: endocytic internalization and intracellular trafficking of nutrient transceptors may, at least in part, be governed by their signaling function.

When cells are starved of their substrate, many nutrient transporters are induced. These undergo rapid endocytosis and redirection of their intracellular trafficking when their substrate becomes available again. The discovery that some of these transporters also act as receptors, or transceptors, suggests that at least part of the sophisticated controls governing the trafficking of these proteins has to do with their signaling function rather than with control of transport. In yeast, the general amino acid permease Gap1 mediates signaling to the protein kinase A pathway. Its endocytic internalization and intracellular trafficking are subject to amino acid control. Other nutrient transceptors controlling this signal transduction pathway appear to be subject to similar trafficking regulation. Transporters with complex regulatory control have also been suggested to function as transceptors in other organisms. Hence, precise regulation of intracellular trafficking in nutrient transporters may be related to the need for tight control of nutrient-induced signaling.