The central research subject in the Laboratory of Molecular Cell Biology and the VIB Department of Molecular Microbiology is nutrient-induced signal transduction in the yeast Saccharomyces cerevisiae and its role in the control of metabolism, stress resistance and growth. Most emphasis has been on the protein kinase A (PKA) pathway: the mechanisms of its activation by fermentable sugars such as glucose and sucrose and by other essential nutrients such as ammonium, amino acids and phosphate.

The studies on glucose-induced cAMP signaling have led to the identification of a G-protein coupled receptor (GPCR) system, consisting of Gpr1 (a 7-transmembrane receptor), Gpa2 (a Galpha protein) and Rgs2 (an RGS protein), that mediates sucrose and glucose activation of cAMP synthesis. This was the first nutrient-sensing G-protein coupled receptor system identified in eukaryotes. In addition, we found that two Gpa2- associated kelch-repeat proteins, Krh1 and Krh2, directly regulate PKA, thereby bypassing adenylate cyclase stimulation.

Research on the sensing of other nutrients has revealed a pathway closely related and probably also involving protein kinase A for regulation of the PKA targets as a function of the nutrient status. The so-called 'fermentable-growth-medium' induced pathway requires both a fermentable carbon source, like glucose, and a complete growth medium, i.e. all essential nutrients, for sustained activation. The nutrient-sensing and signal-transmission components of this pathway are being investigated in detail. We have shown that active nutrient carriers, like the Pho84 phosphate transporter, the Gap1 amino acid permease and the Mep2 ammonium permease act as sensors for phosphate, amino acid and ammonium activation, respectively, of the protein kinase A targets. Currently, the mechanisms by which these nutrient transporter-receptors activate the PKA pathway is under investigation.

One of the targets of the PKA pathway studied in our lab since many years is trehalose metabolism. In addition to a role as storage carbohydrate for survival during long-term starvation, trehalose also plays an important role in stress resistance of yeast cells. In glucose-fermenting cells the trehalose level and stress resistance are low while during growth on non-fermentable carbon sources and in stationary phase they are both high. This observation has been a starting point for the first applied research lines in the lab, namely the role of trehalose metabolism in the control of yeast fermentation and the development of stress resistant baker's and brewer's yeast strains. Nowadays the scope of the applied research has broadened to sugar sensing and signaling and trehalose metabolism in plants, ester biosynthesis in brewer's yeast, new antifungal targets in Candida species, the identification of new components involved in Parkinson's and Alzheimer's disease by screening in yeast, glucose sensing in pancreatic beta cells and connection with diabetes, and intestinal glucose sensing. In all these projects the yeast S. cerevisiae serves as the main research subject, model or tool.

Research on the glucose-sensing mechanism for activation of the PKA pathway has led to the identification of the trehalose-6-phosphate synthase enzyme (Tps1) as an essential controller of glucose influx into yeast glycolysis and therefore also of the fermentation pathway. Although trehalose-6-phosphate has been identified as an inhibitor of yeast hexokinase, other results indicate that additional mechanisms have to be involved in the Tps1 control of glycolysis.

Candida albicans is an opportunistic pathogen of humans. We are exploring whether its trehalose metabolism, in particular trehalose-6-phosphate phosphatase, can be used as a target for new antifungals. Trehalose metabolism is absent in humans and accumulation of trehalose-6-phosphate instead of trehalose under stressful conditions, like infection of a host organism, is toxic to the fungus. Other sugar phosphatases are also under investigation. We are also studying the role of the C. albicans nutrient sensors as potential antifungal targets. Currently we mainly focus on the C. albicans Gpr1 receptor. Contrary to the situation in S. cerevisiae, glucose or sucrose do not seem to be the ligands, but amino acids may be. Other nutrient receptors currently under investigation are the glucose sensors Snf3 and Snf31 and the methionine transporter Mup1, which may also function as a sensor. We are coordinating an EC-funded Marie Curie Research Training Network, called CanTrain, in which we are studying nutrient receptors as potential targets for antifungals and this both in C. albicans as well as in C. dubliniensis.

Alpha-synuclein belongs to a family of structurally related proteins that are prominently expressed in the central nervous system. In Parkinson's disease, aggregated alpha-synuclein proteins form brain lesions that are hallmarks of neurodegenerative synucleinopathies. We have developed a yeast mutant based screening system using a cDNA library from mouse brain to search for direct or indirect alpha synuclein inhibitor proteins. Identified proteins were shown to suppress the toxicity of alpha-SYN in yeast mutant cells and represented novel proteins or proteins that have been already described previously to interact with alpha-SYN or to play a role in Parkinson's disease. Further investigations are focused on the relationship between t-he different proteins, their function in mammalian cells and their possible genetic connection with Parkinson's disease.

The abundant deposition of an abnormal form of the Amyloid beta peptide (Abeta) in the brain is the hallmark of Alzheimer's disease. Two enzymatic activities, known as beta- and gamma-secretases, cleave the Abeta precursor protein to yield Abeta peptide. We have developed, in yeast cells, a reporter assay system for gamma-secretase. This system was used for screening a mouse brain cDNA library to search for direct or indirect gamma-secretase modulators. Identified proteins will be directly validated as modulator of gamma-secretase in mammalian cells by determining their effect on Abeta production. Further investigations will involve the study of the function of the proteins, their effect on the processing of other gamma-secretase substrates, and their valorization as new drug targets for the Alzheimer's disease.

We have already shown that inactivation of specific combinations of signaling components can lead to dramatic effects on the growth of yeast cells. These effects can be used as a screening tool to identify downstream components in screens for multi-copy suppressors but also for the identification of gene products from other eukaryotes possibly involved in nutrient-signaling pathways. For the latter purpose we have developed a screening system based on the severe growth deficiency caused by simultaneous inactivation of the Gpr1 receptor and the Sch9 protein kinase. Several mammalian genes have been isolated as suppressors of this growth defect using a cDNA library of mouse pancreatic cells. One of the genes isolated in this way probably acts as a transcription factor and was called MTAC, for 'mammalian transcription factor activating the cAMP pathway in yeast', because its overexpression causes strong stimulation of cAMP synthesis in yeast. Further studies are focusing on the precise action mechanism of MTAC in yeast, the characterization of MTAC in mammalian cells and in particular on a possible connection with the glucose-sensing system of pancreatic beta cells.

As they are responsible for the fruity character of fermented beverages, volatile esters constitute an important group of aromatic compounds in beer. In modern high-gravity fermentations, which are performed in tall cylindroconical vessels, the beer ester balance is often suboptimal, resulting in a clear decrease in beer quality. In order to obtain more control over ester production and counteract the negative consequences of high-gravity brewing and tall fermentors, intensive research has been carried out to elucidate the molecular genetics and biochemical pathways of yeast ester formation.

The most important aroma-active esters can be divided in two groups: the acetate esters and the medium-chain fatty acid (MCFA) ethyl esters. The most important acetate esters are isoamyl acetate (banana flavor) and ethyl acetate (solvent-like flavor). The group of MCFA ethyl esters consists of ethyl butanoate, ethyl hexanoate, ethyl octanoate and ethyl decanoate. These esters have a papaya, apple-like flavor and are important for the overall flavor of fermented beverages like beer and wine.

Up till now, most research has focused on the formation of acetate esters, which has led to the identification of three alcohol acetyl transferases, Atf1, its closely related homologue Lg-Atf1 and Atf2. In this lab, we are further investigating Atf1 and Atf2 and we have identified the enzymes responsible for the formation of the MCFA ethyl esters, Eht1 and Eeb1. Currently, research is conducted to elucidate the regulation of Eht1 and Eeb1 and to identify the true physiological function of ester biosynthesis in yeast.