Environmental robustness of the global yeast genetic interaction network.

Phenotypes associated with genetic variants can be altered by interactions with other genetic variants (GxG), with the environment (GxE), or both (GxGxE). Yeast genetic interactions have been mapped on a global scale, but the environmental influence on the plasticity of genetic networks has not been examined systematically. To assess environmental rewiring of genetic networks, we examined 14 diverse conditions and scored 30,000 functionally representative yeast gene pairs for dynamic, differential interactions. Different conditions revealed novel differential interactions, which often uncovered functional connections between distantly related gene pairs. However, the majority of observed genetic interactions remained unchanged in different conditions, suggesting that the global yeast genetic interaction network is robust to environmental perturbation and captures the fundamental functional architecture of a eukaryotic cell.

Functional Analysis of Kinases and Transcription Factors in Saccharomyces cerevisiae Using an Integrated Overexpression Library.

Kinases and transcription factors (TFs) are key modulators of important signaling pathways and their activities underlie the proper function of many basic cellular processes such as cell division, differentiation, and development. Changes in kinase and TF dosage are often associated with disease, yet a systematic assessment of the cellular phenotypes caused by the combined perturbation of kinases and TFs has not been undertaken. We used a reverse-genetics approach to study the phenotypic consequences of kinase and TF overexpression (OE) in the budding yeast, Saccharomyces cerevisiae We constructed a collection of strains expressing stably integrated inducible alleles of kinases and TFs and used a variety of assays to characterize the phenotypes caused by TF and kinase OE. We used the Synthetic Genetic Array (SGA) method to examine dosage-dependent genetic interactions (GIs) between 239 gain-of-function (OE) alleles of TFs and six loss-of-function (LOF) and seven OE kinase alleles, the former identifying Synthetic Dosage Lethal (SDL) interactions and the latter testing a GI we call Double Dosage Lethality (DDL). We identified and confirmed 94 GIs between 65 OE alleles of TFs and 9 kinase alleles. Follow-up experiments validated regulatory relationships between genetically interacting pairs (Cdc28-Stb1 and Pho85-Pdr1), suggesting that GI studies involving OE alleles of regulatory proteins will be a rich source of new functional information.

The Dihydrofolate Reductase Protein-Fragment Complementation Assay: A Survival-Selection Assay for Large-Scale Analysis of Protein-Protein Interactions.

Protein-fragment complementation assays (PCAs) can be used to study protein-protein interactions (PPIs) in any living cell, in vivo or in vitro, in any subcellular compartment or membranes. Here, we present a detailed protocol for performing and analyzing a high-throughput PCA screening to study PPIs in yeast, using dihydrofolate reductase (DHFR) as the reporter protein. The DHFR PCA is a simple survival-selection assay in which Saccharomyces cerevisiae DHFR (scDHFR) is inhibited by methotrexate, thus preventing nucleotide synthesis and causing arrest of cell division. Complementation of cells with a methotrexate-insensitive murine DHFR restores nucleotide synthesis, allowing cell proliferation. The methotrexate-resistant DHFR has two mutations (L22F and F31S) and is 10,000 times less sensitive to methotrexate than wild-type scDHFR, but retains full catalytic activity. The DHFR PCA is sensitive enough for PPIs to be detected for open reading frame (ORF)-PCA fragments expressed off of their endogenous promoters.

Protein-Fragment Complementation Assays for Large-Scale Analysis, Functional Dissection, and Spatiotemporal Dynamic Studies of Protein-Protein Interactions in Living Cells.

Protein-fragment complementation assays (PCAs) comprise a family of assays that can be used to study protein-protein interactions (PPIs), conformation changes, and protein complex dimensions. We developed PCAs to provide simple and direct methods for the study of PPIs in any living cell, subcellular compartments or membranes, multicellular organisms, or in vitro. Because they are complete assays, requiring no cell-specific components other than reporter fragments, they can be applied in any context. PCAs provide a general strategy for the detection of proteins expressed at endogenous levels within appropriate subcellular compartments and with normal posttranslational modifications, in virtually any cell type or organism under any conditions. Here we introduce a number of applications of PCAs in budding yeast, Saccharomyces cerevisiae These applications represent the full range of PPI characteristics that might be studied, from simple detection on a large scale to visualization of spatiotemporal dynamics.

A nutrient-responsive pathway that determines M phase timing through control of B-cyclin mRNA stability.

The rate of cell-cycle progression must be tuned in response to nutrient levels to ensure that sufficient materials are synthesized to generate viable daughters. We report that accumulation of the yeast M phase B-cyclin CLB2 mRNA depends on assembly and activation of the heterogeneous nuclear RNA-binding protein (hnRNP) arginine methyltransferase Hmt1, which is promoted by the kinase Dbf2 and countered by the PP2A phosphatase Pph22. Activated Hmt1 methylates hnRNPs, which in turn stabilize CLB2 transcripts. Dbf2 activation of Hmt1 is highly cooperative, producing a sharp increase in CLB2, whereas Pph22 dephosphorylation is graded such that small changes in PP2A activity can cause large shifts in Dbf2-mediated Hmt1 activity. Starvation and rapamycin inhibition of TOR activate Pph22, causing a depletion of CLB2 and delay of M phase. We propose a general model wherein changes to Pph22 activity modulate cyclin mRNA stability to tune cell-cycle progression to environmental conditions.

Protein-fragment complementation assays for large-scale analysis, functional dissection and dynamic studies of protein-protein interactions in living cells.

Protein-fragment Complementation Assays (PCAs) are a family of assays for detecting protein-protein interactions (PPIs) that have been developed to provide simple and direct ways to study PPIs in any living cell, multicellular organism, or in vitro. PCAs can be used to detect PPI between proteins of any molecular weight and expressed at their endogenous levels. Proteins are expressed in their appropriate cellular compartments and can undergo any posttranslational modification or degradation that, barring effects of the PCA fragment fusion, they would normally undergo. Assays can be performed in any cell type or model organism that can be transformed or transfected with gene expression DNA constructs. Here we focus on recent applications of PCA in the budding yeast, Saccharomyces cerevisiae, that cover the gamut of applications one could envision for studying any aspect of PPIs. We present detailed protocols for large-scale analysis of PPIs with the survival-selection dihydrofolate reductase (DHFR), reporter PCA, and a new PCA based on a yeast cytosine deaminase reporter that allows for both survival and death selection. This PCA should prove a powerful way to dissect PPIs. We then present methods to study spatial localization and dynamics of PPIs based on fluorescent protein reporter PCAs.

A toolkit of protein-fragment complementation assays for studying and dissecting large-scale and dynamic protein-protein interactions in living cells.

Protein-fragment complementation assays (PCAs) are a family of assays for detecting protein-protein interactions (PPIs) that have been developed to provide simple and direct ways to study PPIs in any living cell, multicellular organism or in vitro. PCAs can be used to detect PPI between proteins of any molecular weight and expressed at their endogenous levels. Proteins are expressed in their appropriate cellular compartments and can undergo any posttranslational modification or degradation that, barring effects of the PCA fragment fusion, they would normally undergo. Applications of PCAs in yeast have been limited until recently, simply because appropriate expression plasmids or cassettes had not been developed. However, we have now developed and reported on several PCAs in Saccharomyces cerevisiae that cover the gamut of applications one could envision for studying any aspect of PPIs. Here, we present detailed protocols for large-scale analysis of PPIs with the survival-selection dihydrofolate reductase (DHFR) reporter PCA and a new PCA based on a yeast cytosine deaminase reporter that allows for both survival and death selection. This PCA should prove a powerful way to dissect PPIs. We then present a method to study spatial localization and dynamics of PPIs based on fluorescent protein reporter PCAs and finally, two luciferase reporter PCAs that have proved useful for studies of dynamics of PPIs.

An in vivo map of the yeast protein interactome.

Protein interactions regulate the systems-level behavior of cells; thus, deciphering the structure and dynamics of protein interaction networks in their cellular context is a central goal in biology. We have performed a genome-wide in vivo screen for protein-protein interactions in Saccharomyces cerevisiae by means of a protein-fragment complementation assay (PCA). We identified 2770 interactions among 1124 endogenously expressed proteins. Comparison with previous studies confirmed known interactions, but most were not known, revealing a previously unexplored subspace of the yeast protein interactome. The PCA detected structural and topological relationships between proteins, providing an 8-nanometer-resolution map of dynamically interacting complexes in vivo and extended networks that provide insights into fundamental cellular processes, including cell polarization and autophagy, pathways that are evolutionarily conserved and central to both development and human health.