Sergei Kuchin

Snf1 Protein Kinase Regulates Phosphorylation of the Mig1 Repressor in Saccharomyces cerevisiae

ABSTRACT In glucose-grown cells, the Mig1 DNA-binding protein recruits the Ssn6-Tup1 corepressor to glucose-repressed promoters in the yeastSaccharomyces cerevisiae. Previous work showed that Mig1 is differentially phosphorylated in response to glucose. Here we examine the role of Mig1 in regulating repression and the role of the Snf1 protein kinase in regulating Mig1 function. Immunoblot analysis of Mig1 protein from a snf1 mutant showed that Snf1 is required for the phosphorylation of Mig1; moreover, hxk2 andreg1 mutations, which relieve glucose inhibition of Snf1, correspondingly affect phosphorylation of Mig1. We show that Snf1 and Mig1 interact in the two-hybrid system and also coimmunoprecipitate from cell extracts, indicating that the two proteins interact in vivo. In immune complex assays of Snf1, coprecipitating Mig1 is phosphorylated in a Snf1-dependent reaction. Mutation of four putative Snf1 recognition sites in Mig1 eliminated most of the differential phosphorylation of Mig1 in response to glucose in vivo and improved the two-hybrid interaction with Snf1. These studies, together with previous genetic findings, indicate that the Snf1 protein kinase regulates phosphorylation of Mig1 in response to glucose.

Snf1 Protein Kinase and the Repressors Nrg1 and Nrg2 Regulate FLO11, Haploid Invasive Growth, and Diploid Pseudohyphal Differentiation

ABSTRACT The Snf1 protein kinase of Saccharomyces cerevisiae is important for many cellular responses to glucose limitation, including haploid invasive growth. We show here that Snf1 regulates transcription of FLO11, which encodes a cell surface glycoprotein required for invasive growth. We further show that Nrg1 and Nrg2, two repressor proteins that interact with Snf1, function as negative regulators of invasive growth and as repressors of FLO11. We also examined the role of Snf1, Nrg1, and Nrg2 in two other Flo11-dependent processes. Mutations affected the initiation of biofilm formation, which is glucose sensitive, but also affected diploid pseudohyphal differentiation, thereby unexpectedly implicating Snf1 in a response to nitrogen limitation. Deletion of the NRG1 and NRG2 genes suppressed the defects of a snf1 mutant in all of these processes. These findings suggest a model in which the Snf1 kinase positively regulates Flo11-dependent developmental events by antagonizing Nrg-mediated repression of the FLO11 gene.

Functional Relationships of Srb10-Srb11 Kinase, Carboxy-Terminal Domain Kinase CTDK-I, and Transcriptional Corepressor Ssn6-Tup1

ABSTRACT The Srb10-Srb11 protein kinase of Saccharomyces cerevisiae is a cyclin-dependent kinase (cdk)-cyclin pair which has been found associated with the carboxy-terminal domain (CTD) of RNA polymerase II holoenzyme forms. Previous genetic findings implicated the Srb10-Srb11 kinase in transcriptional repression. Here we use synthetic promoters and LexA fusion proteins to test the requirement for Srb10-Srb11 in repression by Ssn6-Tup1, a global corepressor. We show that srb10Δ and srb11Δ mutations reduce repression by DNA-bound LexA-Ssn6 and LexA-Tup1. A point mutation in a conserved subdomain of the kinase similarly reduced repression, indicating that the catalytic activity is required. These findings establish a functional link between Ssn6-Tup1 and the Srb10-Srb11 kinase in vivo. We also explored the relationship between Srb10-Srb11 and CTD kinase I (CTDK-I), another member of the cdk-cyclin family that has been implicated in CTD phosphorylation. We show that mutation of CTK1, encoding the cdk subunit, causes defects in transcriptional repression by LexA-Tup1 and in transcriptional activation. Analysis of the mutant phenotypes and the genetic interactions of srb10Δ and ctk1Δ suggests that the two kinases have related but distinct roles in transcriptional control. These genetic findings, together with previous biochemical evidence, suggest that one mechanism of repression by Ssn6-Tup1 involves functional interaction with RNA polymerase II holoenzyme.

SSN genes that affect transcriptional repression in Saccharomyces cerevisiae encode SIN4, ROX3, and SRB proteins associated with RNA polymerase II.

The RNA polymerase II of Saccharomyces cerevisiae exists in holoenzyme forms containing a complex, known as the mediator, associated with the carboxyl-terminal domain. The mediator includes several SRB proteins and is required for transcriptional activation. Previous work showed that a cyclin-dependent kinase-cyclin pair encoded by SSN3 and SSN8, two members of the SSN suppressor family, are identical to two SRB proteins in the mediator. Here we have identified the remaining SSN genes by cloning and genetic analysis. SSN2 and SSN5 are identical to SRB9 and SRB8, respectively, which encode additional components of the mediator. Genetic evidence implicates the SSN genes in transcriptional repression. Thus, these identities provide genetic insight into mediator and carboxyl-terminal domain function, strongly suggesting a role in mediating transcriptional repression as well as activation. We also show that SSN4 and SSN7 are the same as SIN4 and ROX3, respectively, raising the possibility that these genes also encode mediator proteins.

Interaction of the Srb10 Kinase with Sip4, a Transcriptional Activator of Gluconeogenic Genes in Saccharomyces cerevisiae

ABSTRACT Sip4 is a Zn2Cys6 transcriptional activator that binds to the carbon source-responsive elements of gluconeogenic genes in Saccharomyces cerevisiae. The Snf1 protein kinase interacts with Sip4 and regulates its phosphorylation and activator function in response to glucose limitation; however, evidence suggested that another kinase also regulates Sip4. Here we examine the role of the Srb10 kinase, a component of the RNA polymerase II holoenzyme that has been primarily implicated in transcriptional repression but also positively regulates Gal4. We show that Srb10 is required for phosphorylation of Sip4 during growth in nonfermentable carbon sources and that the catalytic activity of Srb10 stimulates the ability of LexA-Sip4 to activate transcription of a reporter. Srb10 and Sip4 coimmunoprecipitate from cell extracts and interact in two-hybrid assays, suggesting that Srb10 regulates Sip4 directly. We also present evidence that the Srb10 and Snf1 kinases interact with different regions of Sip4. These findings support the view that the Srb10 kinase not only plays negative roles in transcriptional control but also has broad positive roles during growth in carbon sources other than glucose.

Analysis of Transcriptional Repression by Mig1 in Saccharomyces cerevisiae Using a Reporter Assay

Publisher Summary This chapter provides useful approach for analysis of the function and regulation of a repressor. The advantages include the assessment of function in vivo, the variation of physiological conditions and genetic background of the host, and no requirement of knowledge of the DNA sequence recognized by the native repressor. The major limitations are inherent in the fact that the fusion protein and the reporter may not display all of the regulatory properties exhibited by the native protein at its natural target promoter. Findings require confirmation by other methods that assess the function of the native repressor at its natural target sites. The application of this approach to such repressors as Mig1 has provided valuable insights to the repressor function, regulation, and relationships to corepressors and to RNA polymerase II.