Role of the yeast Snf1 protein kinase in invasive growth.

The sucrose non-fermenting 1 (Snf1) protein kinase of Saccharomyces cerevisiae is important for transcriptional, metabolic and developmental responses to glucose limitation. Here we discuss the role of the Snf1 kinase in regulating filamentous invasive growth. Haploid invasive growth occurs in response to glucose limitation and requires FLO11, a gene encoding a cell-surface adhesin. Snf1 regulates transcription of FLO11 by antagonizing the function of two repressors, Nrg1 and Nrg2. Snf1 and the Nrg repressors also affect diploid pseudohyphal differentiation, which is a response to nitrogen limitation, suggesting an unexpected signalling role for the Snf1 kinase.

Interaction of the Srb10 kinase with Sip4, a transcriptional activator of gluconeogenic genes in Saccharomyces cerevisiae.

Sip4 is a Zn(2)Cys(6) 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.

Interaction of the repressors Nrg1 and Nrg2 with the Snf1 protein kinase in Saccharomyces cerevisiae.

The Snf1 protein kinase is essential for the transcription of glucose-repressed genes in Saccharomyces cerevisiae. We identified Nrg2 as a protein that interacts with Snf1 in the two-hybrid system. Nrg2 is a C(2)H(2) zinc-finger protein that is homologous to Nrg1, a repressor of the glucose- and Snf1-regulated STA1 (glucoamylase) gene. Snf1 also interacts with Nrg1 in the two-hybrid system and co-immunoprecipitates with both Nrg1 and Nrg2 from cell extracts. A LexA fusion to Nrg2 represses transcription from a promoter containing LexA binding sites, indicating that Nrg2 also functions as a repressor. An Nrg1 fusion to green fluorescent protein is localized to the nucleus, and this localization is not regulated by carbon source. Finally, we show that VP16 fusions to Nrg1 and Nrg2 allow low-level expression of SUC2 in glucose-grown cells, and we present evidence that Nrg1 and Nrg2 contribute to glucose repression of the DOG2 gene. These results suggest that Nrg1 and Nrg2 are direct or indirect targets of the Snf1 kinase and function in glucose repression of a subset of Snf1-regulated genes.

Subcellular localization of the Snf1 kinase is regulated by specific beta subunits and a novel glucose signaling mechanism.

The Snf1/AMP-activated protein kinase family has broad roles in transcriptional, metabolic, and developmental regulation in response to stress. In Saccharomyces cerevisiae, Snf1 is required for the response to glucose limitation. Snf1 kinase complexes contain the alpha (catalytic) subunit Snf1, one of the three related beta subunits Gal83, Sip1, or Sip2, and the gamma subunit Snf4. We present evidence that the beta subunits regulate the subcellular localization of the Snf1 kinase. Green fluorescent protein fusions to Gal83, Sip1, and Sip2 show different patterns of localization to the nucleus, vacuole, and/or cytoplasm. We show that Gal83 directs Snf1 to the nucleus in a glucose-regulated manner. We further identify a novel signaling pathway that controls this nuclear localization in response to glucose phosphorylation. This pathway is distinct from the glucose signaling pathway that inhibits Snf1 kinase activity and responds not only to glucose but also to galactose and sucrose. Such independent regulation of the localization and the activity of the Snf1 kinase, combined with the distinct localization of kinases containing different beta subunits, affords versatility in regulating physiological responses.

A regulatory shortcut between the Snf1 protein kinase and RNA polymerase II holoenzyme.

RNA polymerase II holoenzymes respond to activators and repressors that are regulated by signaling pathways. Here we present evidence for a "shortcut" mechanism in which the Snf1 protein kinase of the glucose signaling pathway directly regulates transcription by the yeast holoenzyme. In response to glucose limitation, the Snf1 kinase stimulates transcription by holoenzyme that has been artificially recruited to a reporter by a LexA fusion to a holoenzyme component. We show that Snf1 interacts physically with the Srb/mediator proteins of the holoenzyme in both two-hybrid and coimmunoprecipitation assays. We also show that a catalytically hyperactive Snf1, when bound to a promoter as a LexA fusion protein, activates transcription in a glucose-regulated manner; moreover, this activation depends on the integrity of the Srb/mediator complex. These results suggest that direct regulatory interactions between signal transduction pathways and RNA polymerase II holoenzyme provide a mechanism for transcriptional control in response to important signals.

Snf1 protein kinase regulates phosphorylation of the Mig1 repressor in Saccharomyces cerevisiae.

In glucose-grown cells, the Mig1 DNA-binding protein recruits the Ssn6-Tup1 corepressor to glucose-repressed promoters in the yeast Saccharomyces 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 and reg1 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.

Functional relationships of Srb10-Srb11 kinase, carboxy-terminal domain kinase CTDK-I, and transcriptional corepressor Ssn6-Tup1.

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 srb10delta and srb11delta 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 srb10delta and ctk1A 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.

[Prevention of respiratory muscles deconditioning and deterioration of aerobic working capacity in prolonged weightlessness and hypokinesia]. / Profilaktika detrenirovannosti dykhatel'nykh myshts i snizhenie aérobnoi rabotosposobnosti v usloviiakh dlitel'noi nevesomosti i gipokinezii.

According to the previous study [2], simulation of the physiological effects of weightlessness leads to deconditioning of the respiratory muscles which, in its turn, may be a factor impacting the aerobic working capacity. In the present work the experimental findings laid the basis for a physiological concept for medical/engineering requirements to countermeasures against deconditioning of the respiratory muscles, designing and laboratory and physiological testing of a prototype of training loading vest Elastik-R. The vest was shown to enhance speed and force qualities of the respiratory muscles in training athletes, to improve ventilation and gas-exchange functions of the lung, and to increase physical performance and aerobic capacity. Recommendations on utilization of the Elastik-R vest during space flight have been issued.

Genetic aspects of carbon catabolite repression of the STA2 glucoamylase gene in Saccharomyces cerevisiae.

Three regulatory genes, known to be required for glucose repression/derepression of some genes in Saccharomyces cerevisiae, were disrupted to study their effects on the carbon-source regulation of the STA2 glucoamylase gene expression. Using a STA2-lacZ fusion it was found that: (1) the MIG1 gene is dispensable for the repression of the STA2 gene; (2) there are two components in the carbon-source repression of STA2: HXK2-dependent and HXK2-independent; and (3) the HAP2 gene seems to be involved in repression rather than activation of the STA2 expression.

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.

Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast.

The SSN3 and SSN8 genes of Saccharomyces cerevisiae were identified by mutations that suppress a defect in SNF1, a protein kinase required for release from glucose repression. Mutations in SSN3 and SSN8 also act synergistically with a mutation of the MIG1 repressor protein to relieve glucose repression. We have cloned the SSN3 and SSN8 genes. SSN3 encodes a cyclin-dependent protein kinase (cdk) homolog and is identical to UME5. SSN8 encodes a cyclin homolog 35% identical to human cyclin C. SSN3 and SSN8 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. Using an immune complex assay, we detected protein kinase activity that depends on both SSN3 and SSN8. Thus, the two SSN proteins are likely to function as a cdk-cyclin pair. Genetic analysis indicates that the SSN3-SSN8 complex contributes to transcriptional repression of diversely regulated genes and also affects induction of the GAL1 promoter.

Genes required for derepression of an extracellular glucoamylase gene, STA2, in the yeast Saccharomyces.

A diastatic strain of Saccharomyces cerevisiae producing the STA2-encoded extracellular glucoamylase (GA) in a pronounced glucose-repressible fashion was used as a parent for generating mutants with reduced GA activity under normal conditions of derepression. In addition to mutations in STA2, five other recessive mutations were identified which fell into four complementation groups designated haf1 through haf4. RNA blot analysis suggested that the haf mutations confer defects in STA2 transcription. The haf mutants were pleiotropically defective in utilization of alternative carbon sources and resembled the snf (sucrose non-fermenting) mutants identified previously as unable to derepress the expression of the SUC2 gene encoding invertase. We present evidence strongly suggesting that haf1 = snf2, haf3 = snf1 and haf4 = snf5. By phenotypic criteria, the postulated HAF2 gene (which is none of the SNF genes tested) appears to be similar to SNF2, SNF5 and SNF6, and is possibly a non-redundant extension of this group of functionally related SNF genes.

Production of the STA2-encoded glucoamylase in Saccharomyces cerevisiae is subject to feed-back control.

Three modes of production of the extracellular glucoamylase (GA) in Saccharomyces cerevisiae have been identified; repressed, basal and induced. The repressed mode is found with cells grown in rich media containing non-limiting concentrations of monosaccharides or disaccharides, including GA-hydrolysable maltose, as a sole carbon source. Both the basal and the induced modes (spanned by some seven-fold differences in the rate of GA production) can be displayed by either glucose-limited or glycerol-plus ethanol-consuming cultures; the induced mode is switched over to the basal one due to a feed-back inhibition by extracellularly accumulated GA. It is proposed that the feed-back control involved in GA production can be attenuated by starch which can thus 'induce' higher rates of GA production compared to the basal mode.

[Mutational analysis of the starch utilization system in the yeast Saccharomyces cerevisiae]. / Mutatsionnyi analiz sistemy utilizatsii krakhmala u drozhzhei Saccharomyces cerevisiae.

Seven mutants of Saccharomyces cerevisiae deficient in production of extracellular glucoamylase have been analyzed. For each of the seven a monogenic pattern of inheriting the mutant phenotype has been observed. The mutations have been shown to map within five different genetic loci, three independent mutations affecting the STA2 locus and the other four residing in four formerly unidentified genes. As expected, the sta2 mutants recover the wild phenotype when transformed with a STA2-bearing multicopy plasmid. Such reversion has also been observed for the transformed stall mutant. Unlike the others, the sta16 mutant is unable to secrete heterologous alpha-amylase encoded by a plasmid-borne DNA fragment. All the mutants have a moderately reduced ability to secrete the invertase and acid phosphatase.