When yeast cells change their mind: cell cycle "Start" is reversible under starvation.

Eukaryotic cells decide in late G1 phase of the cell cycle whether to commit to another round of division. This point of cell cycle commitment is termed "Restriction Point" in mammals and "Start" in the budding yeast Saccharomyces cerevisiae. At Start, yeast cells integrate multiple signals such as pheromones and nutrients, and will not pass Start if nutrients are lacking.

Rewiring of the protein-protein-metabolite interactome during the diauxic shift in yeast.

In budding yeast Saccharomyces cerevisiae, the switch from aerobic fermentation to respiratory growth is separated by a period of growth arrest, known as the diauxic shift, accompanied by a significant metabolic rewiring, including the derepression of gluconeogenesis and the establishment of mitochondrial respiration. Previous studies reported hundreds of proteins and tens of metabolites accumulating differentially across the diauxic shift transition. To assess the differences in the protein-protein (PPIs) and protein-metabolite interactions (PMIs) yeast samples harvested in the glucose-utilizing, fermentative phase, ethanol-utilizing and early stationary respiratory phases were analysed using isothermal shift assay (iTSA) and a co-fractionation mass spectrometry approach, PROMIS.

Protein-Metabolite Interactions Shape Cellular Metabolism and Physiology.

Protein-metabolite interactions regulate many important cellular processes but still remain understudied. Recent technological advancements are gradually uncovering the complexity of the protein-metabolite interactome. Here, we highlight some classic and recent examples of how protein metabolite interactions regulate metabolism, both locally and globally, and how this contributes to cellular physiology.

Live cell microscopy: From image to insight.

Live-cell microscopy is a powerful tool that can reveal cellular behavior as well as the underlying molecular processes. A key advantage of microscopy is that by visualizing biological processes, it can provide direct insights. Nevertheless, live-cell imaging can be technically challenging and prone to artifacts.

Global mapping of protein-metabolite interactions in Saccharomyces cerevisiae reveals that Ser-Leu dipeptide regulates phosphoglycerate kinase activity.

Protein-metabolite interactions are of crucial importance for all cellular processes but remain understudied. Here, we applied a biochemical approach named PROMIS, to address the complexity of the protein-small molecule interactome in the model yeast Saccharomyces cerevisiae. By doing so, we provide a unique dataset, which can be queried for interactions between 74 small molecules and 3982 proteins using a user-friendly interface available at https://promis.

Regulation of trehalase activity by multi-site phosphorylation and 14-3-3 interaction.

Protein phosphorylation enables a rapid adjustment of cellular activities to diverse intracellular and environmental stimuli. Many phosphoproteins are targeted on more than one site, which allows the integration of multiple signals and the implementation of complex responses. However, the hierarchy and interplay between multiple phospho-sites are often unknown.

Multiple Layers of Phospho-Regulation Coordinate Metabolism and the Cell Cycle in Budding Yeast.

The coordination of metabolism and growth with cell division is crucial for proliferation. While it has long been known that cell metabolism regulates the cell division cycle, it is becoming increasingly clear that the cell division cycle also regulates metabolism. In budding yeast, we previously showed that over half of all measured metabolites change concentration through the cell cycle indicating that metabolic fluxes are extensively regulated during cell cycle progression.

Fueling the Cycle: CDKs in Carbon and Energy Metabolism.

Cyclin-dependent kinases (CDKs) are the central regulators of the eukaryotic cell cycle, and are conserved across eukaryotes. Their main and well-studied function lies in the regulation and the time-keeping of cell cycle entry and progression. Additionally, more and more non canonical functions of CDKs are being uncovered.

How yeast coordinates metabolism, growth and division.

All cells, especially single cell organisms, need to adapt their metabolism, growth and division coordinately to the available nutrients. This coordination is mediated by extensive cross-talk between nutrient signaling, metabolism, growth, and the cell division cycle, which is only gradually being uncovered: Nutrient signaling not only controls entry into the cell cycle at the G1/S transition, but all phases of the cell cycle. Metabolites are even sensed directly by cell cycle regulators to prevent cell cycle progression in absence of sufficient metabolic fluxes.

The Yeast Cyclin-Dependent Kinase Routes Carbon Fluxes to Fuel Cell Cycle Progression.

Cell division entails a sequence of processes whose specific demands for biosynthetic precursors and energy place dynamic requirements on metabolism. However, little is known about how metabolic fluxes are coordinated with the cell division cycle. Here, we examine budding yeast to show that more than half of all measured metabolites change significantly through the cell division cycle.

A genetically encoded Förster resonance energy transfer sensor for monitoring in vivo trehalose-6-phosphate dynamics.

Trehalose-6-phosphate is a pivotal regulator of sugar metabolism, growth, and osmotic equilibrium in bacteria, yeasts, and plants. To directly visualize the intracellular levels of intracellular trehalose-6-phosphate, we developed a series of specific Förster resonance energy transfer (FRET) sensors for in vivo microscopy. We demonstrated real-time monitoring of regulation in the trehalose pathway of Escherichia coli.

Temporal system-level organization of the switch from glycolytic to gluconeogenic operation in yeast.

The diauxic shift in Saccharomyces cerevisiae is an ideal model to study how eukaryotic cells readjust their metabolism from glycolytic to gluconeogenic operation. In this work, we generated time-resolved physiological data, quantitative metabolome (69 intracellular metabolites) and proteome (72 enzymes) profiles. We found that the diauxic shift is accomplished by three key events that are temporally organized: (i) a reduction in the glycolytic flux and the production of storage compounds before glucose depletion, mediated by downregulation of phosphofructokinase and pyruvate kinase reactions; (ii) upon glucose exhaustion, the reversion of carbon flow through glycolysis and onset of the glyoxylate cycle operation triggered by an increased expression of the enzymes that catalyze the malate synthase and cytosolic citrate synthase reactions; and (iii) in the later stages of the adaptation, the shutting down of the pentose phosphate pathway with a change in NADPH regeneration.

The integrated response of primary metabolites to gene deletions and the environment.

Intracellular metabolites arise from the molecular integration of genomic and environmental factors that jointly determine metabolic activity. However, it is not clear how the interplay of genotype, nutrients, growth, and fluxes affect metabolite concentrations globally. Here we used quantitative metabolomics to assess the combined effect of environment and genotype on the metabolite composition of a yeast cell.

Cell size control in yeast.

Cell size is an important adaptive trait that influences nearly all aspects of cellular physiology. Despite extensive characterization of the cell-cycle regulatory network, the molecular mechanisms coupling cell growth to division, and thereby controlling cell size, have remained elusive. Recent work in yeast has reinvigorated the size control field and suggested provocative mechanisms for the distinct functions of setting and sensing cell size.

Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations.

Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo.

In vivo biochemistry: quantifying ion and metabolite levels in individual cells or cultures of yeast.

Over the past decade, we have learned that cellular processes, including signalling and metabolism, are highly compartmentalized, and that relevant changes in metabolic state can occur at sub-second timescales. Moreover, we have learned that individual cells in populations, or as part of a tissue, exist in different states. If we want to understand metabolic processes and signalling better, it will be necessary to measure biochemical and biophysical responses of individual cells with high temporal and spatial resolution.

Integrated multilaboratory systems biology reveals differences in protein metabolism between two reference yeast strains.

The field of systems biology is often held back by difficulties in obtaining comprehensive, high-quality, quantitative data sets. In this paper, we undertook an interlaboratory effort to generate such a data set for a very large number of cellular components in the yeast Saccharomyces cerevisiae, a widely used model organism that is also used in the production of fuels, chemicals, food ingredients and pharmaceuticals. With the current focus on biofuels and sustainability, there is much interest in harnessing this species as a general cell factory.

Differential glucose repression in common yeast strains in response to HXK2 deletion.

Under aerobic, high glucose conditions, Saccharomyces cerevisiae exhibits glucose repression and thus a predominantly fermentative metabolism. Here, we show that two commonly used prototrophic representatives of the CEN.PK and S288C strain families respond differently to deletion of the hexokinase 2 (HXK2) - a key player in glucose repression: In CEN.

Prediction of kinetic parameters from DNA-binding site sequences for modeling global transcription dynamics in Escherichia coli.

The majority of dynamic gene regulatory network (GRN) models are comprised of only a few genes and do not take multiple transcription regulation into account. The models are conceived in this way in order to minimize the number of kinetic parameters. In this paper, we propose a new approach for predicting kinetic parameters from DNA-binding site sequences by correlating the protein-DNA-binding affinities with nucleotide sequence conservation.

High-throughput quantitative metabolomics: workflow for cultivation, quenching, and analysis of yeast in a multiwell format.

Metabolomics is a founding pillar of quantitative biology and a valuable tool for studying metabolism and its regulation. Here we present a workflow for metabolomics in microplate format which affords high-throughput and yet quantitative monitoring of primary metabolism in microorganisms and in particular yeast. First, the most critical step of rapid sampling was adapted to a multiplex format by using fritted 96-well plates for cultivation, which ensure fast sample transfer and permit us to use well-established quenching in cold solvents.

Cross-platform comparison of methods for quantitative metabolomics of primary metabolism.

Quantitative metabolomics is under intense development, and no commonly accepted standard analytical technique has emerged, yet. The employed analytical methods were mostly chosen based on educated guesses. So far, there has been no systematic cross-platform comparison of different separation and detection methods for quantitative metabolomics.

In vitro synthesis and characterization of guanosine 3',5'-bis(diphosphate).

The intracellular alarmone guanosine 3',5'-bis(diphosphate) (ppGpp) has been thoroughly investigated over the past 40 years and has become one of the best-known effectors in bacterial physiology. ppGpp is also of great importance for biotechnological applications. Systems biology research, involving experimental and mathematical approaches, has contributed a great deal to uncovering the alarmone's complex regulatory effects.

Quantification of rRNA in Escherichia coli using capillary gel electrophoresis with laser-induced fluorescence detection.

Over the past 10 years, sophisticated powerful techniques have been developed for the quantification of messenger RNA (mRNA) and ribosomal RNA (rRNA), enabling researchers in science, industry, and molecular medicine to explore gene expression. These techniques require the (reverse) transcription of analyte RNA, hybridization with synthetic oligonucleotides, and other additional steps that make them costly, time-consuming, and quantitatively difficult to perform. The current work demonstrates how 16S and 23S rRNA can be quantified precisely using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) directly after the extraction of total RNA without requiring further reactions or calibration.