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The protein–protein interaction (PPI) network of the Mediator complex is very tightly regulated and depends on different developmental and environmental cues. Here, we present an interactive platform for comparative analysis of the Mediator subunits from humans, baker's yeast Saccharomyces cerevisiae, and model plant Arabidopsis thaliana in a user‐friendly web‐interface database called MediatorWeb. MediatorWeb provides an interface to visualize and analyze the PPI network of Mediator subunits. The database facilitates downloading the untargeted and unweighted network of Mediator complex, its submodules, and individual Mediator subunits to better visualize the importance of individual Mediator subunits or their submodules. Further, MediatorWeb offers network visualization of the Mediator complex and interacting proteins that are functionally annotated. This feature provides clues to understand functions of Mediator subunits in different processes. In an additional tab, MediatorWeb provides quick access to secondary and tertiary structures, as well as residue–level contact information for Mediator subunits in each of the three model organisms. Another useful feature of MediatorWeb is detection of interologs based on orthologous analyses, which can provide clues to understand the functions of Mediator complex in less explored kingdoms. Thus, MediatorWeb and its features can help the user to understand the role of Mediator complex and its subunits in the transcription regulation of gene expression. [ABSTRACT FROM AUTHOR]

The propensity for Saccharomyces cerevisiae yeast to ferment sugars into ethanol and CO2 has long been useful in the production of a wide range of food and drink. In the production of alcoholic beverages, the yeast strain selected for fermentation is crucial because not all strains are equally proficient in tolerating fermentation stresses. One potential mechanism by which domesticated yeast may have adapted to fermentation stresses is through changes in the expression of stress response genes. MED15 is a general transcriptional regulator and RNA Pol II Mediator complex subunit which modulates the expression of many metabolic and stress response genes. In this study, we explore the role of MED15 in alcoholic fermentation. In addition, we ask whether MED15 alleles from wine, sake or palm wine yeast improve fermentation activity and grape juice fermentation stress responses. And last, we investigate to what extent any differences in activity are due to allelic differences in the lengths of three polyglutamine tracts in MED15. We find that strains lacking MED15 are deficient in fermentation and fermentation stress responses and that MED15 alleles from alcoholic beverage yeast strains can improve both the fermentation capacity and the response to ethanol stresses when transplanted into a standard laboratory strain. Finally, we find that polyglutamine tract length in the Med15 protein is one determinant in the efficiency of the alcoholic fermentation process. These data lead to a working model in which polyglutamine tract length and other types of variability within transcriptional hubs like the Mediator subunit, Med15, may contribute to a reservoir of transcriptional profiles that may provide a fitness benefit in the face of environmental fluctuations. [ABSTRACT FROM AUTHOR]

The Mediator complex is required for basal activity of the RNA polymerase (Pol) II transcriptional apparatus and for responsiveness to some activator proteins. Med15, situated in the Mediator tail, plays a role in transmitting regulatory information from distant DNA-bound transcription factors to the transcriptional apparatus poised at promoters. Yeast Med15 and its orthologs share an unusual, glutamine-rich amino acid composition. Here, we discuss this sequence feature and the tendency of polyglutamine tracts to vary in length among strains of Saccharomyces cerevisiae , and we propose that different polyglutamine tract lengths may be adaptive within certain domestication habitats. With the benefit of insight from over 1000 completed genome sequences from different yeast strains that are now archived in sequence databases, it has become clear that there is substantial variation within glutamine-rich regions of the yeast proteome. The ecological diversity of sequenced yeast genomes including yeast from wine, beer, biofuel, food, and clinical and laboratory settings, among others, provides an opportunity to evaluate correlations in variant polyglutamine alleles and specific domestication traits. In general, the subunits of the tail module of the RNA Pol II mediator complex are fast evolving. This property may be important for contacting species-specific transcription factors. Like many large complexes upon which eukaryotic cells rely, the subunit composition of the RNA Pol II mediator complex varies with the organism and, in animals there is variation in different cell types. Specialized complexes may have specific functions in gene expression that are not yet fully understood. Here, we raise the possibility that additional levels of specialization might be afforded by variation in Q tract length and other low-complexity regions found in mediator subunits. [ABSTRACT FROM AUTHOR]

The evolution of animals involved acquisition of an emergent gene repertoire for gastrulation. Whether loss of genes also co-evolved with this developmental reprogramming has not yet been addressed. Here, we identify twenty-four genetic functions that are retained in fungi and choanoflagellates but undetectable in animals. These lost genes encode: (i) sixteen distinct biosynthetic functions; (ii) the two ancestral eukaryotic ClpB disaggregases, Hsp78 and Hsp104, which function in the mitochondria and cytosol, respectively; and (iii) six other assorted functions. We present computational and experimental data that are consistent with a joint function for the differentially localized ClpB disaggregases, and with the possibility of a shared client/chaperone relationship between the mitochondrial Fe/S homoaconitase encoded by the lost LYS4 gene and the two ClpBs. Our analyses lead to the hypothesis that the evolution of gastrulation-based multicellularity in animals led to efficient extraction of nutrients from dietary sources, loss of natural selection for maintenance of energetically expensive biosynthetic pathways, and subsequent loss of their attendant ClpB chaperones. [ABSTRACT FROM AUTHOR]

Opportunistic yeast pathogens arose multiple times in the Saccharomycetes class, including the recently emerged, multidrug-resistant (MDR) Candida auris. We show that homologs of a known yeast adhesin family in Candida albicans, the Hyr/Iff-like (Hil) family, are enriched in distinct clades of Candida species as a result of multiple, independent expansions. Following gene duplication, the tandem repeat–rich region in these proteins diverged extremely rapidly and generated large variations in length and β-aggregation potential, both of which are known to directly affect adhesion. The conserved N-terminal effector domain was predicted to adopt a β-helical fold followed by an α-crystallin domain, making it structurally similar to a group of unrelated bacterial adhesins. Evolutionary analyses of the effector domain in C. auris revealed relaxed selective constraint combined with signatures of positive selection, suggesting functional diversification after gene duplication. Lastly, we found the Hil family genes to be enriched at chromosomal ends, which likely contributed to their expansion via ectopic recombination and break-induced replication. Combined, these results suggest that the expansion and diversification of adhesin families generate variation in adhesion and virulence within and between species and are a key step toward the emergence of fungal pathogens. [ABSTRACT FROM AUTHOR]

[Display omitted] • Regulated aggregation affects protein storage, activity and spatial distribution. • Many "bodies" historically connected to cell and development are protein condensates. • Physiological aggregates are regulated, reversible, and often multifunctional. • Chaperones, protein modification, local milieu and co-aggregates alter aggregation. • Exploring multiple experimental systems helps identify regulators of aggregation. Protein aggregation is a feature of numerous neurodegenerative diseases. However, regulated, often reversible, formation of protein aggregates, also known as condensates, helps control a wide range of cellular activities including stress response, gene expression, memory, cell development and differentiation. This review presents examples of aggregates found in biological systems, how they are used, and cellular strategies that control aggregation and disaggregation. We include features of the aggregating proteins themselves, environmental factors, co-aggregates, post-translational modifications and well-known aggregation-directed activities that influence their formation, material state, stability and dissolution. We highlight the emerging roles of biomolecular condensates in early animal development, and disaggregation processing proteins that have recently been shown to play key roles in gametogenesis and embryogenesis. [ABSTRACT FROM AUTHOR]

The Saccharomyces cerevisiae Sln1 protein is a ‘twocomponent’ regulator involved in osmotolerance. Two component regulators are a family of signal-transduction molecules with histidine kinase activity common in prokaryotes and recently identified in eukaryotes. Phosphorylation of Sln1p inhibits the HOG1 MAP kinase osmosensing pathway via a phosphorelay mechanism including Ypd1p and the response regulator, Ssk1p. SLN1 also activates an MCM1-dependent reporter gene, P-lacZ, but this function is independent of Ssk1p. We present genetic and biochemical evidence that Skn7p is the response regulator for this alternative Sln1p signaling pathway. Thus, the yeast Sln1 phosphorelay is actually more complex than appreciated previously; the Sln1 kinase and Ypd1 phosphorelay intermediate regulate the activity of two distinct response regulators, Ssk1p and Skn7p. The established role of Skn7p in oxidative stress is independent of the conserved receiver domain aspartate, D427. In contrast, we show that Sln1p activation of Skn7p requires phosphorylation of D427. The expression of TRX2, previously shown to exhibit Skn7p-dependent oxidative-stress activation, is also regulated by the SLN1 phosphorelay functions of Skn7p. The identification of genes responsive to both classes of Skn7p function suggests a central role for Skn7p and the SLN1—SKN7 pathway in integrating and coordinating cellular response to various types of environmental stress. [ABSTRACT FROM AUTHOR]

The yeast Sln1p sensor kinase is best known as an osmosensor involved in the regulation of the hyperosmolarity glycerol mitogen-activated protein kinase cascade. Down-regulation of Sln1 kinase activity occurs under hypertonic conditions and leads to phosphorylation of the Hog1p mitogen-activated protein kinase and increased osmotic stress-response gene expression. Conditions leading to kinase up-regulation include osmotic imbalance caused by glycerol retention in the glycerol channel mutant, fps1 (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367). The hypothesis that Sln1p kinase activity is responsive to turgor was first suggested by the increased Sln1p kinase activity in mutants lacking Fps1p in which glycerol accumulation leads to water uptake. Also consistent with the turgor hypothesis is the observation that reduced turgor caused by treatment of cells with nystatin, a drug that increases membrane permeability and causes cell shrinkage, reduced Sln1p kinase activity (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367; Reiser, V., Raitt, D. C., and Saito, H. (2003) J. Cell Biol. 161, 1035-1040). The turgor hypothesis is revisited here in the context of the identification and characterization of the cell wall gene, CCW12, as a determinant of Sln1p activity. Results of this analysis suggest that the activity of the plasma membrane localized Sln1p is affected by the presence or absence of specific outer cell wall proteins and that this effect is independent of turgor. [ABSTRACT FROM AUTHOR]

The Wnt/β-catenin signaling pathway is central to metazoan development and routinely dysregulated in cancer. Wnt/β-catenin signaling initiates transcriptional reprogramming upon stabilization of the transcription factor β-catenin, which is otherwise posttranslationally processed by a destruction complex and degraded by the proteasome. Since various Wnt signaling components are enriched at centrosomes, we examined the functional contribution of centrosomes to Wnt signaling, β-catenin regulation, and posttranslational modifications. In HEK293 cells depleted of centrosomes we find that β-catenin synthesis and degradation rates are unaffected but that the normal accumulation of β-catenin in response to Wnt signaling is attenuated. This is due to accumulation of a novel high-molecular-weight form of phosphorylated β-catenin that is constitutively degraded in the absence of Wnt. Wnt signaling operates by inhibiting the destruction complex and thereby reducing destruction complex-phosphorylated β-catenin, but high-molecular-weight β-catenin is unexpectedly increased by Wnt signaling. Therefore these studies have identified a pool of β-catenin effectively shielded from regulation by Wnt. We present a model whereby centrosomes prevent inappropriate β-catenin modifications that antagonize normal stabilization by Wnt signals.

Protein aggregation, once believed to be a harbinger and/or consequence of stress, age, and pathological conditions, is emerging as a novel concept in cellular regulation. Normal versus pathological aggregation may be distinguished by the capacity of cells to regulate the formation, modification, and dissolution of aggregates. We find that Caenorhabditis elegans aggregates are observed in large cells/blastomeres (oocytes, embryos) and in smaller, further differentiated cells (primordial germ cells), and their analysis using cell biological and genetic tools is straightforward. These observations are consistent with the hypothesis that aggregates are involved in normal development. Using cross-platform analysis in Saccharomyces cerevisiae, C. elegans , and Xenopus laevis , we present studies identifying a novel disaggregase family encoded by animal genomes and expressed embryonically. Our initial analysis of yeast Arb1/Abcf2 in disaggregation and animal ABCF proteins in embryogenesis is consistent with the possibility that members of the ABCF gene family may encode disaggregases needed for aggregate processing during the earliest stages of animal development.

The histidine phosphotransfer (HPt) protein Ypd1 is an important participant in the Saccharomyces cerevisiae multistep two-component signal transduction pathway and, unlike the expanded histidine kinase gene family, is encoded by a single gene in nearly all model and pathogenic fungi. Ypd1 is essential for viability in both S. cerevisiae and in Cryptococcus neoformans. These and other aspects of Ypd1 biology, combined with the availability of structural and mutational data in S. cerevisiae, suggest that the essential interactions between Ypd1 and response regulator domains would be a good target for antifungal drug development. The goal of this minireview is to summarize the wealth of data on S. cerevisiae Ypd1 and to consider the potential benefits of conducting related studies in pathogenic fungi.

The histidine kinase-based phosphorelay has emerged as a common strategy among bacteria, fungi, protozoa, and plants for triggering important stress responses and interpreting developmental cues in response to environmental as well as chemical, nutritional, and hormone signals. The absence of this type of signaling mechanism in animals makes the so-called "two-component" pathway an attractive target for development of antimicrobial agents. The best-studied eukaryotic example of a two-component pathway is the SLN1 pathway in Saccharomyces cerevisiae, which responds to turgor and other physical properties associated with the fungal cell wall. One of the two phosphoreceiver proteins known as response regulators in this pathway is Skn7, a highly conserved stress-responsive transcription factor with a subset of activities that are dependent on SLN1 pathway phosphorylation and another subset that are independent. Interest in Skn7as a determinant in fungal virulence stems primarily from its well-established role in the oxidative stress response; however, the involvement of Skn7 in maintenance of cell wall integrity may also be relevant. Since the cell wall is crucial for fungal survival, structural and biosynthetic proteins affecting wall composition and signaling pathways that respond to wall stress are likely to play key roles in virulence. Here we review the molecular and phenotypic characteristics of different fungal Skn7 proteins and consider how each of these properties may contribute to fungal virulence.

The histidine kinase-based signal transduction pathway was first uncovered in bacteria and is a prominent form of regulation in prokaryotes. However, this type of signal transduction is not unique to prokaryotes; over the last decade two-component signal transduction pathways have been identified and characterized in diverse eukaryotes, from unicellular yeasts to multicellular land plants. A number of small but important differences have been noted in the architecture and function of eukaryotic pathways. Because of the powerful genetic approaches and facile molecular analysis associated with the yeast system, the SLN1 osmotic response pathway in Saccharomyces cerevisiae is particularly useful as a eukaryotic pathway model. This chapter provides an overview of genetic and biochemical methods that have been important in elucidating the stimulus-response events that underlie this pathway and in understanding the details of a eukaryotic His-Asp phosphorelay., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The bifunctional Saccharomyces cerevisiae Skn7 transcription factor regulates osmotic stress response genes as well as oxidative stress response genes; however, the mechanisms involved in these two types of regulation differ. Skn7 osmotic stress activity depends on the phosphorylation of the receiver domain aspartate, D427, by the Sln1 histidine kinase. In contrast, D427 and the SLN1-SKN7 phosphorelay are dispensable for the oxidative stress response, although the receiver domain is required. The majority of oxidative stress response genes regulated by Skn7 also are regulated by the redox-responsive transcription factor Yap1. It is therefore possible that the nuclearly localized Skn7 does not itself respond to the oxidant but simply cooperates with Yap1 when it translocates to the nucleus. We report here that oxidative stress leads to a phosphatase-sensitive, slow-mobility Skn7 variant. This suggests that Skn7 undergoes a posttranslational modification by phosphorylation following exposure to oxidant. Oxidant-dependent Skn7 phosphorylation was eliminated in strains lacking the Yap1 transcription factor. This suggests that the phosphorylation of Skn7 is regulated by Yap1. Mutations in the receiver domain of Skn7 were identified that affect its oxidative stress function. These mutations were found to compromise the association of Yap1 and Skn7 at oxidative stress response gene promoters. A working model is proposed in which the association of Yap1 with Skn7 in the nucleus is a prerequisite for Skn7 phosphorylation and the activation of oxidative stress response genes.

The yeast Sln1p sensor kinase is best known as an osmosensor involved in the regulation of the hyperosmolarity glycerol mitogen-activated protein kinase cascade. Down-regulation of Sln1 kinase activity occurs under hypertonic conditions and leads to phosphorylation of the Hog1p mitogen-activated protein kinase and increased osmotic stress-response gene expression. Conditions leading to kinase up-regulation include osmotic imbalance caused by glycerol retention in the glycerol channel mutant, fps1 (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367). The hypothesis that Sln1p kinase activity is responsive to turgor was first suggested by the increased Sln1p kinase activity in mutants lacking Fps1p in which glycerol accumulation leads to water uptake. Also consistent with the turgor hypothesis is the observation that reduced turgor caused by treatment of cells with nystatin, a drug that increases membrane permeability and causes cell shrinkage, reduced Sln1p kinase activity (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367; Reiser, V., Raitt, D. C., and Saito, H. (2003) J. Cell Biol. 161, 1035-1040). The turgor hypothesis is revisited here in the context of the identification and characterization of the cell wall gene, CCW12, as a determinant of Sln1p activity. Results of this analysis suggest that the activity of the plasma membrane localized Sln1p is affected by the presence or absence of specific outer cell wall proteins and that this effect is independent of turgor.

Yeast Sln1p is an osmotic stress sensor with histidine kinase activity. Modulation of Sln1 kinase activity in response to changes in the osmotic environment regulates the activity of the osmotic response mitogen-activated protein kinase pathway and the activity of the Skn7p transcription factor, both important for adaptation to changing osmotic stress conditions. Many aspects of Sln1 function, such as how kinase activity is regulated to allow a rapid response to the continually changing osmotic environment, are not understood. To gain insight into Sln1p function, we conducted a two-hybrid screen to identify interactors. Mog1p, a protein that interacts with the yeast Ran1 homolog, Gsp1p, was identified in this screen. The interaction with Mog1p was characterized in vitro, and its importance was assessed in vivo. mog1 mutants exhibit defects in SLN1-SKN7 signal transduction and mislocalization of the Skn7p transcription factor. The requirement for Mog1p in normal localization of Skn7p to the nucleus does not fully account for the mog1-related defects in SLN1-SKN7 signal transduction, raising the possibility that Mog1p may play a role in Skn7 binding and activation of osmotic response genes.

Sln1p is a plasma membrane-localized two-component histidine kinase that functions as an osmotic stress sensor in Saccharomyces cerevisiae. Changes in osmotic pressure modulate Sln1p kinase activity, which, together with Ypd1p, a phosphorelay intermediate, changes the phosphorylation status of two response regulators, Ssk1p and Skn7p. Ssk1p controls the activity of the HOG1 mitogen-activated protein kinase pathway. Skn7p is a nuclearly localized transcription factor that regulates genes involved in cell wall integrity and other processes. Subcellular compartmentalization may therefore play an important role in eukaryotic two-component pathway regulation. We have studied the subcellular localization of SLN1 pathway components and find that Ypd1p is a dynamic protein with a role in shuttling the osmotic stress signal from Sln1p to Ssk1p in the cytosol and to Skn7p in the nucleus. The need to translocate the signal into different intracellular compartments contributes a spatial dimension to eukaryotic two-component pathways compared to the prototypical two-component pathways of prokaryotes.

The yeast "two-component" osmotic stress phosphorelay consists of the histidine kinase, Sln1p, the phosphorelay intermediate, Ypd1p and two response regulators, Ssk1p and Skn7p, whose activities are regulated by phosphorylation of a conserved aspartyl residue in the receiver domain. Dephospho-Ssk1p leads to activation of the hyper-osmotic response (HOG) pathway, whereas phospho-Skn7p presumably leads to activation of hypo-osmotic response genes. The multifunctional Skn7 protein is important in oxidative as well as osmotic stress; however, the Skn7p receiver domain aspartate that is the phosphoacceptor in the SLN1 pathway is dispensable for oxidative stress. Like many well-characterized bacterial response regulators, Skn7p is a transcription factor. In this report we investigate the role of Skn7p in osmotic response gene activation. Our studies reveal that the Skn7p HSF-like DNA binding domain interacts with a cis-acting element identified upstream of OCH1 that is distinct from the previously defined HSE-like Skn7p binding site. Our data support a model in which Skn7p receiver domain phosphorylation affects transcriptional activation rather than DNA binding to this class of DNA binding site.