Sul1 and Sul2 sulfate transceptors signal to protein kinase A upon exit of sulfur starvation.

Sulfate is an essential nutrient with pronounced regulatory effects on cellular metabolism and proliferation. Little is known, however, about how sulfate is sensed by cells. Sul1 and Sul2 are sulfate transporters in the yeast Saccharomyces cerevisiae, strongly induced upon sulfur starvation and endocytosed upon the addition of sulfate. We reveal Sul1,2-dependent activation of PKA targets upon sulfate-induced exit from growth arrest after sulfur starvation. We provide two major arguments in favor of Sul1 and Sul2 acting as transceptors for signaling to PKA. First, the sulfate analogue, d-glucosamine 2-sulfate, acted as a non-transported agonist of signaling by Sul1 and Sul2. Second, mutagenesis to Gln of putative H(+)-binding residues, Glu-427 in Sul1 or Glu-443 in Sul2, abolished transport without affecting signaling. Hence, Sul1,2 can function as pure sulfate sensors. Sul1(E427Q) and Sul2(E443Q) are also deficient in sulfate-induced endocytosis, which can therefore be uncoupled from signaling. Overall, our data suggest that transceptors can undergo independent conformational changes, each responsible for triggering different downstream processes. The Sul1 and Sul2 transceptors are the first identified plasma membrane sensors for extracellular sulfate. High affinity transporters induced upon starvation for their substrate may generally act as transceptors during exit from starvation.

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.

Agp2, a member of the yeast amino acid permease family, positively regulates polyamine transport at the transcriptional level.

Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3Δ nor sam3Δ single mutant is defective in polyamine transport, while the dur3Δ sam3Δ double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2Δ mutant. Studies of Agp2 localization indicate that in the double mutant dur3Δ sam3Δ, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport.

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.

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.

Saccharomyces cerevisiae plasma membrane nutrient sensors and their role in PKA signaling.

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.

Novel mechanisms in nutrient activation of the yeast protein kinase A pathway.

In yeast the Protein Kinase A (PKA) pathway can be activated by a variety of nutrients. Fermentable sugars, like glucose and sucrose, trigger a spike in the cAMP level, followed by activation of PKA and phosphorylation of target proteins causing a.o. mobilization of reserve carbohydrates, repression of stress-related genes and induction of growth-related genes. Glucose and sucrose are sensed by a G-protein coupled receptor system that activates adenylate cyclase and also activates a bypass pathway causing direct activation of PKA. Addition of other essential nutrients, like nitrogen sources or phosphate, to glucose-repressed nitrogen- or phosphate-starved cells, also triggers rapid activation of the PKA pathway. In these cases cAMP is not involved as a second messenger. Amino acids are sensed by the Gap1 transceptor, previously considered only as an amino acid transporter. Recent results indicate that the amino acid ligand has to induce a specific conformational change for signaling. The same amino acid binding site is involved in transport and signaling. Similar results have been obtained for Pho84 which acts as a transceptor for phosphate activation of the PKA pathway. Ammonium activation of the PKA pathway in nitrogen-starved cells is mediated mainly by the Mep2 transceptor, which belongs to a different class of transporter proteins. Hence, different types of sensing systems are involved in control of the yeast PKA pathway by nutrients.

Urmylation controls Nil1p and Gln3p-dependent expression of nitrogen-catabolite repressed genes in Saccharomyces cerevisiae.

Urm1 is a modifier protein that is conjugated to substrate proteins through thioester formation with the E1-like enzyme, Uba4. Here is shown that the lack of urmylation causes derepression of the GAP1 gene (encoding a nitrogen-regulated broad-spectrum amino acid-scavenging permease) in the presence of rich nitrogen sources, and simultaneous inhibition of the expression of CIT2, a TCA-cycle gene involved in the production of glutamate and glutamine. This effect is dependent on the TORC1- and nutrient-regulated transcriptional factors, Nil1p and Gln3p. Evidence is provided that, in the absence of urmylation, nuclear/cytosolic shuffling of both transcriptional factors is altered, ultimately leading to inability to repress GAP1 gene in the presence of a rich nitrogen source. Altogether, the data presented here indicate an important role of the urmylation pathway in regulating the expression of genes involved in sensing and controlling amino acids levels.

Amino acids regulate retrieval of the yeast general amino acid permease from the vacuolar targeting pathway.

Intracellular sorting of the general amino acid permease (Gap1p) in Saccharomyces cerevisiae depends on availability of amino acids such that at low amino acid concentrations Gap1p is sorted to the plasma membrane, whereas at high concentrations Gap1p is sorted to the vacuole. In a genome-wide screen for mutations that affect Gap1p sorting we identified deletions in a subset of components of the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular endosome (MVE). Gap1p-GFP is delivered to the vacuolar interior by the MVE pathway in wild-type cells, but when formation of the MVE is blocked by mutation, Gap1p-GFP efficiently cycles from this compartment to the plasma membrane, resulting in unusually high permease activity at the cell surface. Importantly, cycling of Gap1p-GFP to the plasma membrane is blocked by high amino acid concentrations, defining recycling from the endosome as a major step in Gap1p trafficking under physiological control. Mutations in LST4 and LST7 genes, previously identified for their role in Gap1p sorting, similarly block MVE to plasma membrane trafficking of Gap1p. However, mutations in other recycling complexes such as the retromer had no significant effect on the intracellular sorting of Gap1p, suggesting that Gap1p follows a genetically distinct pathway for recycling. We previously found that Gap1p sorting from the Golgi to the endosome requires ubiquitination of Gap1p by an Rsp5p ubiquitin ligase complex, but amino acid abundance does not appear to significantly alter the accumulation of polyubiquitinated Gap1p. Thus the role of ubiquitination appears to be a signal for delivery of Gap1p to the MVE, whereas amino acid abundance appears to control the cycling of Gap1p from the MVE to the plasma membrane.

Endless versatility in the biotechnological applications of Kluyveromyces LAC genes.

Most microorganisms adapted to life in milk owe their ability to thrive in this habitat to the evolution of mechanisms for the use of the most abundant sugar present on it, lactose, as a carbon source. Because of their lactose-assimilating ability, Kluyveromyces yeasts have long been used in industrial processes involved in the elimination of this sugar. The identification of the genes conferring Kluyveromyces with a system for permeabilization and intracellular hydrolysis of lactose (LAC genes), along with the current possibilities for their transfer into alternative organisms through genetic engineering, has significantly broadened the industrial profitability of lactic yeasts. This review provides an updated overview of the general properties of Kluyveromyces LAC genes, and the multiple techniques involving their biotechnological utilization. Emphasis is also made on the potential that some of the latest technologies, such as the generation of transgenics, will have for a further benefit in the use of these and related genes.

A comparative analysis of the GAL genetic switch between not-so-distant cousins: Saccharomyces cerevisiae versus Kluyveromyces lactis.

Despite their close phylogenetic relationship, Kluyveromyces lactis and Saccharomyces cerevisiae have adapted their carbon utilization systems to different environments. Although they share identities in the arrangement, sequence and functionality of their GAL gene set, both yeasts have evolved important differences in the GAL genetic switch in accordance to their relative preference for the utilization of galactose as a carbon source. This review provides a comparative overview of the GAL-specific regulatory network in S. cerevisiae and K. lactis, discusses the latest models proposed to explain the transduction of the galactose signal, and describes some of the particularities that both microorganisms display in their regulatory response to different carbon sources. Emphasis is placed on the potential for improved strategies in biotechnological applications using yeasts.

Lactose: the milk sugar from a biotechnological perspective.

Lactose is a very important sugar because of its abundance in the milk of humans and domestic animals. Lactose is a valuable asset as a basic nutrient and the main substrate in fermentative processes that led to the production of fermented milk products, such as yogurt and kefir. In some instances, lactose also can be a problem as the causative agent of some diseases, such as lactose intolerance and galactosemia, or for being a by-product generated in huge amounts by the cheese industry. The study of the biochemical reactions leading to the synthesis and assimilation of lactose has provided valuable models for the understanding of biosynthetic and catabolic processes. Lactose-hydrolyzing enzymes are structurally and phylogenetically related to different types of beta-galactosidases and bacterial cellobiases involved in the enzymatic degradation of cellulose. Biotransformation of lactose, by either enzymatic or fermentative procedures, is important for different types of industrial applications in dairy and pharmaceutical industries.

Control of dinucleoside polyphosphates by the FHIT-homologous HNT2 gene, adenine biosynthesis and heat shock in Saccharomyces cerevisiae.

BACKGROUND: The FHIT gene is lost early in the development of many tumors. Fhit possesses intrinsic ApppA hydrolase activity though ApppA cleavage is not required for tumor suppression. Because a mutant form of Fhit that is functional in tumor suppression and defective in catalysis binds ApppA well, it was hypothesized that Fhit-substrate complexes are the active, signaling form of Fhit. Which substrates are most important for Fhit signaling remain unknown. RESULTS: Here we demonstrate that dinucleoside polyphosphate levels increase 500-fold to hundreds of micromolar in strains devoid of the Saccharomyces cerevisiae homolog of Fhit, Hnt2. Accumulation of dinucleoside polyphosphates is reversed by re-expression of Hnt2 and is active site-dependent. Dinucleoside polyphosphate levels depend on an intact adenine biosynthetic pathway and time in liquid culture, and are induced by heat shock to greater than 0.1 millimolar even in Hnt2+ cells. CONCLUSIONS: The data indicate that Hnt2 hydrolyzes both ApppN and AppppN in vivo and that, in heat-shocked, adenine prototrophic yeast strains, dinucleoside polyphosphates accumulate to levels in which they may saturate Hnt2.