Crosstalk between lipd and central carbon metabolism

Lipids are essential components of all cells. Besides their major function as constituents of biological membranes, they contribute to post-translational modification of proteins and to the cellular signaling network. In addition, almost all eukaryotic cells use neutral lipids to store energy and carbon in intracellular lipid droplets. Importantly, the metabolic pathways for lipid synthesis and storage are highly conserved from unicellular yeast to higher organisms. In this project we propose experimental strategies that will contribute to a better understanding of the regulation of these processes. We will characterize hitherto unknown players in the regulatory network governing the metabolism and storage of fat in the model yeast Saccharomyces cerevisiae. In most cases, alterations in lipid metabolism, such as increased fat storage, are not caused by a single mutated gene but are rather polygenic traits. Therefore, we used a method to identify genome- wide quantitative trait loci (QTL) in a natural isolate that has elevated levels of storage lipids as compared to standard laboratory yeast strains. The genes that were identified in this study are not yet reported to play a role in lipid metabolism but are rather known for their involvement in other cellular processes, like glycogen metabolism. Two of the major hits in the study point at an important role for acetyl-CoA in regulation of lipid metabolism. Acetyl-CoA is a central metabolite at the crossroads between anabolic and catabolic pathways. In addition to its metabolic role, acetyl-CoA is also an important regulator of non-histone protein activities by acetylation of lysine residues. We confirmed this importance of acetyl-CoA in regulation of lipid synthesis by conducting carbon flux and metabolome analyses. These studies provided several possible candidate proteins with known enzymatic function in central carbon metabolism that could be regulated by acetyl-CoA, either by lysine acetylation or through allosteric regulation. This project deals with the characterization of the proteins that were identified in the QTL study. We will determine whether these proteins or their encoding genes interact with each other and we will elucidate the mechanisms by which they contribute to lipid metabolic pathways. Furthermore, we will conduct experiments that are intended to clarify the role of acetyl-CoA in the regulation of glucose catabolic enzymes. The aim of these studies is to obtain a better understanding how lipid metabolism and central carbon metabolism are connected and how they influence each other.

The yeast Saccharomyces cerevisiae is one of the best studied eukaryotes because it is used as a model organism in basic research and as a host in biotechnology for the synthesis of many products, from fine chemicals and pharmaceuticals to bulk products like bioethanol. In this project, we investigated two yeast strains with regard to their lipid metabolism. These two strains were selected because they have significantly different abilities to accumulate storage lipids. The aim of the project was to identify the differences on the genome level that result in the observed difference in the phenotype and to gain a better understanding of the regulation of storage lipid metabolism. First, we performed a quantitative trait loci (QTL) study. In this experiment, the two strains were crossed and, after meiosis, a large number of daughter cells, all with individual combinations of the parent genomes, were obtained. From this population, the cells with the highest fat content were selected and their genomes were sequenced to identify those genetic variations that were enriched from one of the parents, assuming that these enriched loci are causal for the phenotype. In this QTL study, we found ca. ten genes that contribute to the high lipid content in one of the parent strains. We selected three of them, PIG1, PHO23 and RML2, for further investigations. Lipid analysis of deletion mutants and of strains in which the alleles were exchanged to the ones from the other strain confirmed that these three genes are involved in the storage of lipids. However, we also found that the effects of these alleles on lipid storage are dependent on additional genes, suggesting extensive genetic interactions and a complex network of genes that determine the lipid content of a strain. Therefore, the storage of lipids is a so-called quantitative trait, which means that the phenotype is determined by many genes, in contrast to a Mendelian trait, which is determined by one single gene. The genes that we found in the QTL study play roles in many diverse processes, such as glycogen storage, histone deacetylation and mitochondrial protein synthesis. In a second approach, we investigated the role of acetyl-CoA in the regulation of lipid metabolism. Acetyl-CoA is the major substrate for the synthesis of the most important lipid classes in a cell. The synthesis of acetyl-CoA itself depends on pyruvate, the end product of glycolysis. We worked on the hypothesis that acetyl-CoA is involved in the regulation of glycolysis and lipid metabolic pathways. Work on this aspect resulted in the identification of several enzymes in these pathways that are acetylated, a process that requires acetyl-CoA. For one of these proteins we confirmed that this acetylation has a strong effect on enzymatic activity.