The PhosphoGRID Saccharomyces cerevisiae protein phosphorylation site database: version 2.0 update.

PhosphoGRID is an online database that curates and houses experimentally verified in vivo phosphorylation sites in the Saccharomyces cerevisiae proteome (www.phosphogrid.org). Phosphosites are annotated with specific protein kinases and/or phosphatases, along with the condition(s) under which the phosphorylation occurs and/or the effects on protein function. We report here an updated data set, including nine additional high-throughput (HTP) mass spectrometry studies. The version 2.0 data set contains information on 20 177 unique phosphorylated residues, representing a 4-fold increase from version 1.0, and includes 1614 unique phosphosites derived from focused low-throughput (LTP) studies. The overlap between HTP and LTP studies represents only ∼3% of the total unique sites, but importantly 45% of sites from LTP studies with defined function were discovered in at least two independent HTP studies. The majority of new phosphosites in this update occur on previously documented proteins, suggesting that coverage of phosphoproteins in the yeast proteome is approaching saturation. We will continue to update the PhosphoGRID data set, with the expectation that the integration of information from LTP and HTP studies will enable the development of predictive models of phosphorylation-based signaling networks. Database URL: http://www.phosphogrid.org/

Cdk8 regulates stability of the transcription factor Phd1 to control pseudohyphal differentiation of Saccharomyces cerevisiae.

The yeast Saccharomyces differentiates into filamentous pseudohyphae when exposed to a poor source of nitrogen in a process involving a collection of transcription factors regulated by nutrient signaling pathways. Phd1 is important for this process in that it regulates expression of most other transcription factors involved in differentiation and can induce filamentation on its own when overproduced. In this article, we show that Phd1 is an unstable protein whose degradation is initiated through phosphorylation by Cdk8 of the RNA polymerase II mediator subcomplex. Phd1 is stabilized by cdk8 disruption, and the naturally filamenting Σ1278b strain was found to have a sequence polymorphism that eliminates a Cdk8 phosphorylation site, which both stabilizes the protein and contributes to enhanced differentiation. In nitrogen-starved cells, PHD1 expression is upregulated and the Phd1 protein becomes stabilized, which causes its accumulation during differentiation. PHD1 expression is partially dependent upon Ste12, which was also previously shown to be destabilized by Cdk8-dependent phosphorylations, but to a significantly smaller extent than Phd1. These observations demonstrate the central role that Cdk8 plays in initiation of differentiation. Cdk8 activity is inhibited in cells shifted to limiting nutrient conditions, and we argue that this effect drives the initiation of differentiation through stabilization of multiple transcription factors, including Phd1, that cause activation of genes necessary for filamentous response.

Organizational constraints on Ste12 cis-elements for a pheromone response in Saccharomyces cerevisiae.

Ste12 of Saccharomyces cerevisiae binds to pheromone-response cis-elements (PREs) to regulate several classes of genes. Genes induced by pheromones require multimerization of Ste12 for binding of at least two PREs on responsive promoters. We have systematically examined nucleotides of the consensus PRE for binding of wild-type Ste12 to DNA in vitro, as well as the organizational requirements of PREs to produce a pheromone response in vivo. Ste12 binds as a monomer to a single PRE in vitro, and two PREs upstream of a minimal core promoter cause induction that is proportional to their relative affinity for Ste12 in vitro. Although consensus PREs are arranged in a variety of configurations in the promoters of responsive genes, we find that there are severe constraints with respect to how they can be positioned in an artificial promoter to cause induction. Two closely-spaced PREs can induce transcription in a directly-repeated or tail-to-tail orientation, although PREs separated by at least 40 nucleotides are capable of inducing transcription when oriented in a head-to-head or tail-to-tail configuration. We characterize several examples of promoters that bear multiple consensus PREs or a single PRE in combination with a PRE-like sequence that match these requirements. A significant number of responsive genes appear to possess only a single PRE, or PREs in configurations that would not be expected to enable induction, and we suggest that, for many pheromone-responsive genes, Ste12 must activate transcription by binding to cryptic or sub-optimal sites on DNA, or may require interaction with additional uncharacterized DNA bound factors.

PhosphoGRID: a database of experimentally verified in vivo protein phosphorylation sites from the budding yeast Saccharomyces cerevisiae.

Protein phosphorylation plays a central role in cellular regulation. Recent proteomics strategies for identifying phosphopeptides have been developed using the model organism Saccharomyces cerevisiae, and consequently, when combined with studies of individual gene products, the number of reported specific phosphorylation sites for this organism has expanded enormously. In order to systematically document and integrate these various data types, we have developed a database of experimentally verified in vivo phosphorylation sites curated from the S. cerevisiae primary literature. PhosphoGRID (www.phosphogrid.org) records the positions of over 5000 specific phosphorylated residues on 1495 gene products. Nearly 900 phosphorylated residues are reported from detailed studies of individual proteins; these in vivo phosphorylation sites are documented by a hierarchy of experimental evidence codes. Where available for specific sites, we have also noted the relevant protein kinases and/or phosphatases, the specific condition(s) under which phosphorylation occurs, and the effect(s) that phosphorylation has on protein function. The unique features of PhosphoGRID that assign both function and specific physiological conditions to each phosphorylated residue will provide a valuable benchmark for proteome-level studies and will facilitate bioinformatic analysis of cellular signal transduction networks. Database URL: http://phosphogrid.org/

Disintegrator vectors for single-copy yeast chromosomal integration.

Vectors were developed for two-step chromosomal integration of reporter genes or expression constructs. With these vectors, integration produces a disruption of the ADE8, LYS2, MET15, LEU2, HIS3 or FCY1 genes, and integrants can be easily identified by replica-plating on selective media. Integration using these 'disintegrator' vectors produces a single-copy integration of the construct of interest at the junction of the marker deletion, and removes the additional plasmid sequences. Importantly, the integrated constructs do not contain flanking sequence duplications, and therefore should be highly stable. Each of the vectors was shown to reliably integrate a TEF1-KAN expression cassette and/or GAL1-HIS3 and STE12-LacZ reporter genes.