A novel yeast hybrid modeling framework integrating Boolean and enzyme-constrained networks enables exploration of the interplay between signaling and metabolism.

The interplay between nutrient-induced signaling and metabolism plays an important role in maintaining homeostasis and its malfunction has been implicated in many different human diseases such as obesity, type 2 diabetes, cancer, and neurological disorders. Therefore, unraveling the role of nutrients as signaling molecules and metabolites together with their interconnectivity may provide a deeper understanding of how these conditions occur. Both signaling and metabolism have been extensively studied using various systems biology approaches. However, they are mainly studied individually and in addition, current models lack both the complexity of the dynamics and the effects of the crosstalk in the signaling system. To gain a better understanding of the interconnectivity between nutrient signaling and metabolism in yeast cells, we developed a hybrid model, combining a Boolean module, describing the main pathways of glucose and nitrogen signaling, and an enzyme-constrained model accounting for the central carbon metabolism of Saccharomyces cerevisiae, using a regulatory network as a link. The resulting hybrid model was able to capture a diverse utalization of isoenzymes and to our knowledge outperforms constraint-based models in the prediction of individual enzymes for both respiratory and mixed metabolism. The model showed that during fermentation, enzyme utilization has a major contribution in governing protein allocation, while in low glucose conditions robustness and control are prioritized. In addition, the model was capable of reproducing the regulatory effects that are associated with the Crabtree effect and glucose repression, as well as regulatory effects associated with lifespan increase during caloric restriction. Overall, we show that our hybrid model provides a comprehensive framework for the study of the non-trivial effects of the interplay between signaling and metabolism, suggesting connections between the Snf1 signaling pathways and processes that have been related to chronological lifespan of yeast cells.

Correlating single-molecule characteristics of the yeast aquaglyceroporin Fps1 with environmental perturbations directly in living cells.

Membrane proteins play key roles at the interface between the cell and its environment by mediating selective import and export of molecules via plasma membrane channels. Despite a multitude of studies on transmembrane channels, understanding of their dynamics directly within living systems is limited. To address this, we correlated molecular scale information from living cells with real time changes to their microenvironment. We employed super-resolved millisecond fluorescence microscopy with a single-molecule sensitivity, to track labelled molecules of interest in real time. We use as example the aquaglyceroporin Fps1 in the yeast Saccharomyces cerevisiae to dissect and correlate its stoichiometry and molecular turnover kinetics with various extracellular conditions. We show that Fps1 resides in multi tetrameric clusters while hyperosmotic and oxidative stress conditions cause Fps1 reorganization. Moreover, we demonstrate that rapid exposure to hydrogen peroxide causes Fps1 degradation. In this way we shed new light on aspects of architecture and dynamics of glycerol-permeable plasma membrane channels.

Mig1 localization exhibits biphasic behavior which is controlled by both metabolic and regulatory roles of the sugar kinases.

Glucose, fructose and mannose are the preferred carbon/energy sources for the yeast Saccharomyces cerevisiae. Absence of preferred energy sources activates glucose derepression, which is regulated by the kinase Snf1. Snf1 phosphorylates the transcriptional repressor Mig1, which results in its exit from the nucleus and subsequent derepression of genes. In contrast, Snf1 is inactive when preferred carbon sources are available, which leads to dephosphorylation of Mig1 and its translocation to the nucleus where Mig1 acts as a transcription repressor. Here we revisit the role of the three hexose kinases, Hxk1, Hxk2 and Glk1, in glucose de/repression. We demonstrate that all three sugar kinases initially affect Mig1 nuclear localization upon addition of glucose, fructose and mannose. This initial import of Mig1 into the nucleus was temporary; for continuous nucleocytoplasmic shuttling of Mig1, Hxk2 is required in the presence of glucose and mannose and in the presence of fructose Hxk2 or Hxk1 is required. Our data suggest that Mig1 import following exposure to preferred energy sources is controlled via two different pathways, where (1) the initial import is regulated by signals derived from metabolism and (2) continuous shuttling is regulated by the Hxk2 and Hxk1 proteins. Mig1 nucleocytoplasmic shuttling appears to be important for the maintenance of the repressed state in which Hxk1/2 seems to play an essential role.

Distributed biological computation with multicellular engineered networks.

Ongoing efforts within synthetic and systems biology have been directed towards the building of artificial computational devices using engineered biological units as basic building blocks. Such efforts, inspired in the standard design of electronic circuits, are limited by the difficulties arising from wiring the basic computational units (logic gates) through the appropriate connections, each one to be implemented by a different molecule. Here, we show that there is a logically different form of implementing complex Boolean logic computations that reduces wiring constraints thanks to a redundant distribution of the desired output among engineered cells. A practical implementation is presented using a library of engineered yeast cells, which can be combined in multiple ways. Each construct defines a logic function and combining cells and their connections allow building more complex synthetic devices. As a proof of principle, we have implemented many logic functions by using just a few engineered cells. Of note, small modifications and combination of those cells allowed for implementing more complex circuits such as a multiplexer or a 1-bit adder with carry, showing the great potential for re-utilization of small parts of the circuit. Our results support the approach of using cellular consortia as an efficient way of engineering complex tasks not easily solvable using single-cell implementations.

A fungicide-responsive kinase as a tool for synthetic cell fate regulation.

Engineered biological systems that precisely execute defined tasks have major potential for medicine and biotechnology. For instance, gene- or cell-based therapies targeting pathogenic cells may replace time- and resource-intensive drug development. Engineering signal transduction systems is a promising, yet presently underexplored approach. Here, we exploit a fungicide-responsive heterologous histidine kinase for pathway engineering and synthetic cell fate regulation in the budding yeast Saccharomyces cerevisiae. Rewiring the osmoregulatory Hog1 MAPK signalling system generates yeast cells programmed to execute three different tasks. First, a synthetic negative feedback loop implemented by employing the fungicide-responsive kinase and a fungicide-resistant derivative reshapes the Hog1 activation profile, demonstrating how signalling dynamics can be engineered. Second, combinatorial integration of different genetic parts including the histidine kinases, a pathway activator and chemically regulated promoters enables control of yeast growth and/or gene expression in a two-input Boolean logic manner. Finally, we implemented a genetic 'suicide attack' system, in which engineered cells eliminate target cells and themselves in a specific and controllable manner. Taken together, fungicide-responsive kinases can be applied in different constellations to engineer signalling behaviour. Sensitizing engineered cells to existing chemicals may be generally useful for future medical and biotechnological applications.

Yeast AMP-activated protein kinase monitors glucose concentration changes and absolute glucose levels.

Analysis of the time-dependent behavior of a signaling system can provide insight into its dynamic properties. We employed the nucleocytoplasmic shuttling of the transcriptional repressor Mig1 as readout to characterize Snf1-Mig1 dynamics in single yeast cells. Mig1 binds to promoters of target genes and mediates glucose repression. Mig1 is predominantly located in the nucleus when glucose is abundant. Upon glucose depletion, Mig1 is phosphorylated by the yeast AMP-activated kinase Snf1 and exported into the cytoplasm. We used a three-channel microfluidic device to establish a high degree of control over the glucose concentration exposed to cells. Following regimes of glucose up- and downshifts, we observed a very rapid response reaching a new steady state within less than 1 min, different glucose threshold concentrations depending on glucose up- or downshifts, a graded profile with increased cell-to-cell variation at threshold glucose concentrations, and biphasic behavior with a transient translocation of Mig1 upon the shift from high to intermediate glucose concentrations. Fluorescence loss in photobleaching and fluorescence recovery after photobleaching data demonstrate that Mig1 shuttles constantly between the nucleus and cytoplasm, although with different rates, depending on the presence of glucose. Taken together, our data suggest that the Snf1-Mig1 system has the ability to monitor glucose concentration changes as well as absolute glucose levels. The sensitivity over a wide range of glucose levels and different glucose concentration-dependent response profiles are likely determined by the close integration of signaling with the metabolism and may provide for a highly flexible and fast adaptation to an altered nutritional status.

Differential role of HAMP-like linkers in regulating the functionality of the group III histidine kinase DhNik1p.

Nik1 orthologs are sensor kinases that function upstream of the high osmolarity glycerol/p38 MAPK pathway in fungi. They contain a poly-HAMP module at their N terminus, which plays a pivotal role in osmosensing as well as fungal death upon exposure to fludioxonil. DhNik1p is a typical member of this class that contains five HAMP domains and four HAMP-like linkers. We investigated the contribution of each of the HAMP-like linker regions to the functionality of DhNik1p and found that the HAMP4b linker was essential as its deletion resulted in the complete loss of activity. Replacement of this linker with flexible peptide sequences did not restore DhNik1p activity. Thus, the HAMP-like sequence and possibly structural features of this linker region are indispensable for the kinase activity of DhNik1p. To gain insight into the global shape of the poly-HAMP module in DhNik1p (HAMP1­5), multi-angle laser light and small angle x-ray scattering studies were carried out. Those data demonstrate that the maltose-binding protein-tagged HAMP1­5 protein exist as a dimer in solution with an elongated shape of maximum linear dimension ∼365 Å. Placement of a sequence similarity based model of the HAMP1­5 protein inside experimental data-based models showed how two chains of HAMP1­5 are entwined on each other and the overall structure retained a periodicity. Normal mode analysis of the structural model is consistent with the H4b linker being a key to native-like collective motion in the protein. Overall, our shape-function studies reveal how different elements in the HAMP1­5 structure mediate its function.

Yeast reveals unexpected roles and regulatory features of aquaporins and aquaglyceroporins.

BACKGROUND: The yeast Saccharomyces cerevisiae provides unique opportunities to study roles and regulation of aqua/glyceroporins using frontline tools of genetics and genomics as well as molecular cell and systems biology. SCOPE OF REVIEW: S. cerevisiae has two similar orthodox aquaporins. Based on phenotypes mediated by gene deletion or overexpression as well as on their expression pattern, the yeast aquaporins play important roles in key aspects of yeast biology: establishment of freeze tolerance, during spore formation as well as determination of cell surface properties for substrate adhesion and colony formation. Exactly how the aquaporins perform those roles and the mechanisms that regulate their function under such conditions remain to be elucidated. S. cerevisiae also has two different aquaglyceroporins. While the role of one of them, Yfl054c, remains to be determined, Fps1 plays critical roles in osmoregulation by controlling the accumulation of the osmolyte glycerol. Fps1 communicates with two osmo-sensing MAPK signalling pathways to perform its functions but the details of Fps1 regulation remain to be determined. MAJOR CONCLUSIONS: Several phenotypes associated with aqua/glyceroporin function in yeasts have been established. However, how water and glycerol transport contribute to the observed effects is not understood in detail. Also many of the basic principles of regulation of yeast aqua/glyceroporins remain to be elucidated. GENERAL SIGNIFICANCE: Studying the yeast aquaporins and aquaglyceroporins offers rich insight into the life style, evolution and adaptive responses of yeast and rewards us with discoveries of unexpected roles and regulatory mechanisms of members of this ancient protein family. This article is part of a Special Issue entitled Aquaporins.

An integrated view on a eukaryotic osmoregulation system.

Osmoregulation encompasses active homeostatic processes that ensure proper cell volume, shape and turgor as well as an intercellular milieu optimal for the diverse biochemical processes. Recent studies demonstrate that yeast cells operate within a tight window of cellular water concentrations that still allows rapid diffusion of biomolecules while already moderate cell compression following hyper-osmotic stress leads to macromolecular crowding and a slow-down of cellular processes. Yeast cells accumulate glycerol as compatible osmolyte under hyper-osmotic stress to regain cell volume and turgor and release glycerol following a hypo-osmotic shock. The high osmolarity glycerol (HOG) response pathway controls glycerol accumulation at various levels, where each mechanism contributes to the temporal and quantitative pattern of volume recovery: inhibition of glycerol efflux, direct activation of the first enzyme in glycerol biosynthesis, stimulation of glycolytic flux as well as upregulation of expression of genes encoding enzymes in glycerol biosynthesis and an active glycerol uptake system. The HOG mitogen-activated protein kinase (MAPK) pathway communicates with the other yeast MAPK pathways to control cell morphogenesis. Cross-talk between the MAPK pathways has recently been used to re-wire osmostress-controlled expression of glycerol biosynthesis genes from Hog1 to Kss1-Fus3. The results of this study further illustrate the key importance of glycerol accumulation under osmostress and allow studying Hog1-dependent and independent processes as well as redundancy and robustness of the MAPK system.

Molecular communication: crosstalk between the Snf1 and other signaling pathways.

The yeast Saccharomyces cerevisiae employs different conserved signaling pathways to adapt to altered availability of nutrient and energy sources. Crosstalk between the pathways occurs to integrate different internal and external stimuli and adjust cellular metabolism, growth and proliferation to altered environmental conditions. The main glucose repression pathway, Snf1/Mig1, plays an essential role in adaptation to glucose limitation. However, the Snf1 protein kinase is also involved in regulation of many other cellular processes. We summarize evidence that Snf1 is part of a network of communicating pathways, and we suggest research directions that may help elucidating signal flow within this network.

A novel chloroplast localized Rab GTPase protein CPRabA5e is involved in stress, development, thylakoid biogenesis and vesicle transport in Arabidopsis.

A novel Rab GTPase protein in Arabidopsis thaliana, CPRabA5e (CP = chloroplast localized) is located in chloroplasts and has a role in transport. Transient expression of CPRabA5e:EGFP fusion protein in tobacco (Nicotiana tabacum) leaves, and immunoblotting using Arabidopsis showed localization of CPRabA5e in chloroplasts (stroma and thylakoids). Ypt31/32 in the yeast Saccharomyces cerevisiae are involved in regulating vesicle transport, and CPRabA5e a close homolog of Ypt31/32, restores the growth of the ypt31Δ ypt32(ts) mutant at 37 °C in yeast complementation. Knockout mutants of CPRabA5e displayed delayed seed germination and growth arrest during oxidative stress. Ultrastructural studies revealed that after preincubation at 4 °C mutant chloroplasts contained larger plastoglobules, lower grana, and more vesicles close to the envelopes compared to wild type, and vesicle formation being enhanced under oxidative stress. This indicated altered thylakoid development and organization of the mutants. A yeast-two-hybrid screen with CPRabA5e as bait revealed 13 interacting partner proteins, mainly located in thylakoids and plastoglobules. These proteins are known or predicted to be involved in development, stress responses, and photosynthesis related processes, consistent with the stress phenotypes observed. The results observed suggest a role of CPRabA5e in transport to and from thylakoids, similar to cytosolic Rab proteins involved in vesicle transport.

Synthetic biology: lessons from engineering yeast MAPK signalling pathways.

All living cells respond to external stimuli and execute specific physiological responses through signal transduction pathways. Understanding the mechanisms controlling signalling pathways is important for diagnosing and treating diseases and for reprogramming cells with desired functions. Although many of the signalling components in the budding yeast Saccharomyces cerevisiae have been identified by genetic studies, many features concerning the dynamic control of pathway activity, cross-talk, cell-to-cell variability or robustness against perturbation are still incompletely understood. Comparing the behaviour of engineered and natural signalling pathways offers insight complementary to that achievable with standard genetic and molecular studies. Here, we review studies that aim at a deeper understanding of signalling design principles and generation of novel signalling properties by engineering the yeast mitogen-activated protein kinase (MAPK) pathways. The underlying approaches can be applied to other organisms including mammalian cells and offer opportunities for building synthetic pathways and functionalities useful in medicine and biotechnology.

Bridging the gaps in systems biology.

Systems biology aims at creating mathematical models, i.e., computational reconstructions of biological systems and processes that will result in a new level of understanding-the elucidation of the basic and presumably conserved "design" and "engineering" principles of biomolecular systems. Thus, systems biology will move biology from a phenomenological to a predictive science. Mathematical modeling of biological networks and processes has already greatly improved our understanding of many cellular processes. However, given the massive amount of qualitative and quantitative data currently produced and number of burning questions in health care and biotechnology needed to be solved is still in its early phases. The field requires novel approaches for abstraction, for modeling bioprocesses that follow different biochemical and biophysical rules, and for combining different modules into larger models that still allow realistic simulation with the computational power available today. We have identified and discussed currently most prominent problems in systems biology: (1) how to bridge different scales of modeling abstraction, (2) how to bridge the gap between topological and mechanistic modeling, and (3) how to bridge the wet and dry laboratory gap. The future success of systems biology largely depends on bridging the recognized gaps.

Quantitative analysis of glycerol accumulation, glycolysis and growth under hyper osmotic stress.

We provide an integrated dynamic view on a eukaryotic osmolyte system, linking signaling with regulation of gene expression, metabolic control and growth. Adaptation to osmotic changes enables cells to adjust cellular activity and turgor pressure to an altered environment. The yeast Saccharomyces cerevisiae adapts to hyperosmotic stress by activating the HOG signaling cascade, which controls glycerol accumulation. The Hog1 kinase stimulates transcription of genes encoding enzymes required for glycerol production (Gpd1, Gpp2) and glycerol import (Stl1) and activates a regulatory enzyme in glycolysis (Pfk26/27). In addition, glycerol outflow is prevented by closure of the Fps1 glycerol facilitator. In order to better understand the contributions to glycerol accumulation of these different mechanisms and how redox and energy metabolism as well as biomass production are maintained under such conditions we collected an extensive dataset. Over a period of 180 min after hyperosmotic shock we monitored in wild type and different mutant cells the concentrations of key metabolites and proteins relevant for osmoadaptation. The dataset was used to parameterize an ODE model that reproduces the generated data very well. A detailed computational analysis using time-dependent response coefficients showed that Pfk26/27 contributes to rerouting glycolytic flux towards lower glycolysis. The transient growth arrest following hyperosmotic shock further adds to redirecting almost all glycolytic flux from biomass towards glycerol production. Osmoadaptation is robust to loss of individual adaptation pathways because of the existence and upregulation of alternative routes of glycerol accumulation. For instance, the Stl1 glycerol importer contributes to glycerol accumulation in a mutant with diminished glycerol production capacity. In addition, our observations suggest a role for trehalose accumulation in osmoadaptation and that Hog1 probably directly contributes to the regulation of the Fps1 glycerol facilitator. Taken together, we elucidated how different metabolic adaptation mechanisms cooperate and provide hypotheses for further experimental studies.

Yeast aquaglyceroporins use the transmembrane core to restrict glycerol transport.

Aquaglyceroporins are transmembrane proteins belonging to the family of aquaporins, which facilitate the passage of specific uncharged solutes across membranes of cells. The yeast aquaglyceroporin Fps1 is important for osmoadaptation by regulating intracellular glycerol levels during changes in external osmolarity. Upon high osmolarity conditions, yeast accumulates glycerol by increased production of the osmolyte and by restricting glycerol efflux through Fps1. The extended cytosolic termini of Fps1 contain short domains that are important for regulating glycerol flux through the channel. Here we show that the transmembrane core of the protein plays an equally important role. The evidence is based on results from an intragenic suppressor mutation screen and domain swapping between the regulated variant of Fps1 from Saccharomyces cerevisiae and the hyperactive Fps1 ortholog from Ashbya gossypii. This suggests a novel mechanism for regulation of glycerol flux in yeast, where the termini alone are not sufficient to restrict Fps1 transport. We propose that glycerol flux through the channel is regulated by interplay between the transmembrane helices and the termini. This mechanism enables yeast cells to fine-tune intracellular glycerol levels at a wide range of extracellular osmolarities.

A framework for mapping, visualisation and automatic model creation of signal-transduction networks.

Intracellular signalling systems are highly complex. This complexity makes handling, analysis and visualisation of available knowledge a major challenge in current signalling research. Here, we present a novel framework for mapping signal-transduction networks that avoids the combinatorial explosion by breaking down the network in reaction and contingency information. It provides two new visualisation methods and automatic export to mathematical models. We use this framework to compile the presently most comprehensive map of the yeast MAP kinase network. Our method improves previous strategies by combining (I) more concise mapping adapted to empirical data, (II) individual referencing for each piece of information, (III) visualisation without simplifications or added uncertainty, (IV) automatic visualisation in multiple formats, (V) automatic export to mathematical models and (VI) compatibility with established formats. The framework is supported by an open source software tool that facilitates integration of the three levels of network analysis: definition, visualisation and mathematical modelling. The framework is species independent and we expect that it will have wider impact in signalling research on any system.

Time course gene expression profiling of yeast spore germination reveals a network of transcription factors orchestrating the global response.

BACKGROUND: Spore germination of the yeast Saccharomyces cerevisiae is a multi-step developmental path on which dormant spores re-enter the mitotic cell cycle and resume vegetative growth. Upon addition of a fermentable carbon source and nutrients, the outer layers of the protective spore wall are locally degraded, the tightly packed spore gains volume and an elongated shape, and eventually the germinating spore re-enters the cell cycle. The regulatory pathways driving this process are still largely unknown. Here we characterize the global gene expression profiles of germinating spores and identify potential transcriptional regulators of this process with the aim to increase our understanding of the mechanisms that control the transition from cellular dormancy to proliferation. RESULTS: Employing detailed gene expression time course data we have analysed the reprogramming of dormant spores during the transition to proliferation stimulated by a rich growth medium or pure glucose. Exit from dormancy results in rapid and global changes consisting of different sequential gene expression subprograms. The regulated genes reflect the transition towards glucose metabolism, the resumption of growth and the release of stress, similar to cells exiting a stationary growth phase. High resolution time course analysis during the onset of germination allowed us to identify a transient up-regulation of genes involved in protein folding and transport. We also identified a network of transcription factors that may be regulating the global response. While the expression outputs following stimulation by rich glucose medium or by glucose alone are qualitatively similar, the response to rich medium is stronger. Moreover, spores sense and react to amino acid starvation within the first 30 min after germination initiation, and this response can be linked to specific transcription factors. CONCLUSIONS: Resumption of growth in germinating spores is characterized by a highly synchronized temporal organisation of up- and down-regulated genes which reflects the metabolic reshaping of the quickening spores.

The genome of the xerotolerant mold Wallemia sebi reveals adaptations to osmotic stress and suggests cryptic sexual reproduction.

Wallemia (Wallemiales, Wallemiomycetes) is a genus of xerophilic Fungi of uncertain phylogenetic position within Basidiomycota. Most commonly found as food contaminants, species of Wallemia have also been isolated from hypersaline environments. The ability to tolerate environments with reduced water activity is rare in Basidiomycota. We sequenced the genome of W. sebi in order to understand its adaptations for surviving in osmotically challenging environments, and we performed phylogenomic and ultrastructural analyses to address its systematic placement and reproductive biology. W. sebi has a compact genome (9.8 Mb), with few repeats and the largest fraction of genes with functional domains compared with other Basidiomycota. We applied several approaches to searching for osmotic stress-related proteins. In silico analyses identified 93 putative osmotic stress proteins; homology searches showed the HOG (High Osmolarity Glycerol) pathway to be mostly conserved. Despite the seemingly reduced genome, several gene family expansions and a high number of transporters (549) were found that also provide clues to the ability of W. sebi to colonize harsh environments. Phylogenetic analyses of a 71-protein dataset support the position of Wallemia as the earliest diverging lineage of Agaricomycotina, which is confirmed by septal pore ultrastructure that shows the septal pore apparatus as a variant of the Tremella-type. Mating type gene homologs were identified although we found no evidence of meiosis during conidiogenesis, suggesting there may be aspects of the life cycle of W. sebi that remain cryptic.

Crystal structure of a yeast aquaporin at 1.15 angstrom reveals a novel gating mechanism.

Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 A resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.