Nutrient sensing through the plasma membrane of eukaryotic cells, Cirencester, UK, 25–29 September, 2004

Yeast has been at the forefront of research on nutrient signaling for many years. Also in many other eukaryotic cell types, nutrients exert a wide variety of regulatory effects. Up till recently, however, the literature on nutrient-induced regulatory phenomena was very diverse. The discovery of several types of plasma membrane-based nutrient sensors in yeast has dramatically changed this situation. Non-transporting nutrient carrier homologues, G-protein-coupled receptors and actively-transporting nutrient carriers have all been identified as nutrient sensors in the yeast plasma membrane and in several cases this has rapidly been extended to other yeast species. These striking discoveries have resulted in an intensive search in other eukaryotic systems, fungal, plant as well as mammalian types, for similar nutrient sensors and several examples have recently been identified (for a review see: Holsbeeks et al., 2004, Trends Biochem. Sci. 29, 556–564).

The conference in Cirencester, organized by Soraya Shirazi-Beechey, Johan Thevelein and Frank Stolz and managed by the UK. Biochemical Society as a focused meeting, concentrated on these novel types of nutrient-sensing systems present in or functionally closely linked to the eukaryotic plasma membrane.

The meeting was divided in three sections with basically one day each devoted to nutrient sensing in yeasts, plants and mammalian cells. The first day had lectures by Mark Johnston explaining the rationale of glucose sensing in yeast with respect to its fermentative lifestyle and further concentrating on the Snf3-Rgt2 signaling pathway which controls the induction of the glucose carrier genes by glucose. It was the first example discovered in eukaryotic cells of a nutrient sensing system employing non-transporting nutrient carrier homologues as nutrient sensors. The next talk was given by Johan Thevelein who concentrated on the remarkable variety of nutrient sensors involved in rapid nutrient activation of the protein kinase A (PKA) pathway. Glucose activation of cAMP synthesis involves the Gpr1 G-protein-coupled receptor, the first GPCR discovered with a function in nutrient sensing. Several actively-transporting nutrient carriers, e.g., Gap1, Mep2 and Pho84, apparently function as nutrient sensors for activation of the PKA pathway by amino acids, ammonium and phosphate, respectively. In Schizosaccharomyces pombe Git3, a homologue of Gpr1, also functions in glucose activation of cAMP synthesis. This topic was covered by Charlie Hoffman who presented an overview of the complete pathway, which differs in several respects from the pathway in Saccharomyces cerevisiae and even seems to involve several proteins without clear homologues in other organisms. He also presented the first evidence for domains involved in interaction between the Gpa2, Gα protein and adenylate cyclase, which is apparently different from what has previously been found for this type of interaction in mammalian cells. Oleh Stasyk highlighted the first example of a nutrient carrier homologue involved in a nutrient repression pathway. The Gcr1 protein in Hansenula polymorpha is required for glucose repression of alcohol oxidase, involved in methanol utilization and peroxisome development. It clearly belongs to the glucose carrier superfamily and is apparently most closely related to the Snf3 and Rgt2 glucose sensors of S. cerevisiae. It also seems to be a non-transporting glucose carrier homologue, functioning as glucose sensor. Morten Kielland-Brandt presented the story of Ssy1, the first discovered non-transporting amino-acid carrier homologue functioning as an amino- acid sensor discovered in eukaryotic cells. He presented an intriguing model proposing a connection between the outward and inward conformations and the signaling capacity of Ssy1 and other transporter/receptors. The prediction of the model that intracellular amino acids would inhibit the sensing of extracellular amino acids was supported by several lines of experimental evidence. In the next presentation, Fernando Moreno concentrated on the long-standing issue of the precise role of hexokinase-2 in the yeast glucose repression pathway. He elaborated on the discovery that hexokinase-2 is also present in the nucleus and serves to maintain the Mig1 repressor in this location. He also presented novel data on the connection between Snf1 and the control of the hexokinase – Mig1 nuclear localization. It appears that hexokinase-2 might act at several points in the glucose repression pathway and that its catalytic activity may be important only for part of these interactions. This would at least explain why it has been so difficult to get a clear answer on the requirement of hexokinase catalytic activity for glucose repression.

In the plant section, Jen Sheen explained the dramatic regulatory effects of sugars on gene expression in photosynthesis, carbon metabolism and plant development. Among the apparently great diversity of glucose sensing mechanisms, the dual role of Arabidopsis thaliana hexokinase-1 in glucose phosphorylation and as a sensor for glucose repression has been clearly established. This contrasts with the situation in yeast, where the precise role of hexokinase-2 in glucose repression remains unclear. A. thaliana hexokinase-1 is localized both in the cytosol and in the nucleus, and the identification of proteins in the nucleus interacting with hexokinase,which upon inactivation also render the plant glucose-insensitive, provides strong further support for a role of hexokinase-1 as a glucose sensor in plants. She also highlighted the connection between the glucose- sensing system and the plant hormone-signaling network. Sucrose is the major transport sugar of plants and Jef Smeekens concentrated on sucrose sensing mechanisms. Sucrose was shown to specifically repress the expression of certain genes, including a bZIP transcription factor gene, at the translational level through a small upstream ORF. Sucrose sensing seems to be exerted at the level of the plasma membrane, possibly by one or more sucrose transporters. Gabriel Iturriaga demonstrated a connection between trehalose metabolism in plants and glucose signaling. Most plants do not accumulate trehalose in appreciable quantities but in spite of this they contain a whole family of trehalose-6-phosphate synthase genes. Modification of trehalose-6-phosphate levels strongly affects plant morphology and development. The same is true for the glucose- sensing machinery and evidence has now been obtained that the two systems might be connected. Ian Graham continued with the trehalose metabolism story in plants. He showed that the embryo lethality of the tps1 mutant can be rescued partially by addition of trehalose-6-phosphate. In S. cerevisiae trehalose-6-phosphate inhibits hexokinase allosterically, but A. thaliana hexokinase-1 and -2 are not affected by trehalose-6-phosphate. Other arguments also seem to indicate that Tps1 does not work through hexokinase in plants. On the other hand, the hyperaccumulation of sugar reserves in the torpedo stage in a tps1 mutant is reminiscent of free sugar accumulation in yeast tps1 mutants and might hint to a flux problem in glycolysis. Brian Forde discussed nitrate and glutamate sensing by plant cells. For stimulation of root elongation nitrate as such is apparently sensed, since its metabolites ammonium and glutamate cannot exert the same effect. A downstream factor Anr1 has been identified that can stimulate root elongation. He also dealt with the surprising finding that plants contain glutamate-sensitive ion channels like those involved in neurotransmission in animals. Addition of glutamate but not of other amino acids also causes specific effects on root development, which might be due to sensing by the ion channels. Marcus Fehr presented an intriguing novel system to measure intracellular metabolite levels in real time, possibly overcoming an important obstacle in the whole field of nutrient signaling and regulation. It makes use of bacterial periplasmic proteins that bind specific substrates, which are then presented to the transporter. They are known to undergo a large conformational change upon binding of the substrate, that brings two lobes of the protein into each other's proximity. The proteins have been engineered with chromophores on the two lobes, triggering fluorescence resonance energy transfer (FRET) after the conformational change of the protein brings the two lobes close to each other. The system was developed with the maltose-binding protein and then extended to a glucose- and a ribose-binding protein. The different systems allowed to measure in real time the intracellular level of maltose, glucose and ribose, respectively, after their expression in different cell types.

The mammalian section was started by Robert Margolskee who discussed the receptors involved in taste perception, and in particular the G-protein-coupled receptors responsible for sweet, bitter and umami (glutamate) taste. He showed how the differences in taste perception between humans and mice could be exploited through the construction of chimaeric receptors to identify domains responsible for binding of specific ligands. He also revealed that several of the taste receptors as well as their cognate Gα protein, gustducin, are expressed in the gut. Soraya Shirazi-Beechey followed up on this, explaining the evidence for existence of a glucose-sensing system in the gut that is responsible for the induction of SGLT1. This is the sodium-linked glucose transporter responsible for glucose uptake from the gut. All evidence points to a G-protein-coupled receptor linked to the cAMP pathway. She also presented detailed evidence for the presence of the taste receptors and gustducin in the epithelial cells of the intestine, making the sweet taste receptor a good candidate to function in SGLT1 induction by glucose. Franz Matschinsky concentrated on the pivotal role of glucokinase in the control of glucose-induced insulin release in pancreatic β cells. The latter are responsible for monitoring the glucose level in the blood. They secrete insulin when glucose is in excess in the blood. Nearly 200 glucokinase mutations have been discovered in patients with hereditary forms of diabetes and they strongly support the critical role of glucokinase as the pacemaker of glucose catabolism and therefore of the ATP/ADP ratio which controls the ATP-sensitive K⁺ channels in the plasma membrane. He also discussed the small-molecule activators of glucokinase, which are being developed as potential diabetes drugs. Nada Abumrad dealt with the nature of the fatty-acid-sensing system. She discussed extensively the function of the CD36 protein, a putative transport and/or sensor protein in the plasma membrane of a variety of cell types. Evidence from CD36 knock-out and overexpression mice clearly supports a pivotal role of this protein in controlling lipid catabolism, clearance and storage. However, whether the protein is able to transport fatty acids itself or whether it only binds the fatty acids as a sensor is not clear yet. Finally, Daniela Riccardi presented the story of the Ca²⁺ receptor CaR, which functions to detect Ca²⁺ as a primary messenger in the extracellular fluids. This role of Ca²⁺ is much less known, compared to the role of Ca²⁺ as an intracellular second messenger. The low affinity for Ca²⁺ of the receptor fits with its role in detection of the mM-concentrations of extracellular Ca²⁺. CaR is a G-protein-coupled receptor which binds Ca²⁺ in the N-terminal extracellular domain. It has been suggested to function also as an amino-acid sensor because the sensitivity for Ca²⁺ is enhanced strongly by many amino acids.

In addition to the poster session the conference also had a workshop with oral presentations selected from the poster abstracts, in which several other examples of nutrient-sensing systems in eukaryotic cells were highlighted.

The meeting took place in the beautiful setting of the Royal Agricultural College in Cirencester, where about fifty delegates enjoyed a highly interactive and lively meeting with plenty of time for discussion. Clearly, research on nutrient sensing in eukaryotic cells has developed to a point where people working on fungal, plant and animal systems can learn a lot from each other. The first general principles and mechanisms for nutrient sensing in eukaryotic cells are now emerging. This conference with its unusual combination of diversity and focus has for the first time brought the different players together. It was a highly stimulating experience, worthwile of being repeated at a regular basis in view of the rapid progress in this field.