Academic literature on the topic 'Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Signal Transduction'

SUMMARY Eukaryotic cells possess an exquisitely interwoven and fine-tuned series of signal transduction mechanisms with which to sense and respond to the ubiquitous fermentable carbon source glucose. The budding yeast Saccharomyces cerevisiae has proven to be a fertile model system with which to identify glucose signaling factors, determine the relevant functional and physical interrelationships, and characterize the corresponding metabolic, transcriptomic, and proteomic readouts. The early events in glucose signaling appear to require both extracellular sensing by transmembrane proteins and intracellular sensing by G proteins. Intermediate steps involve cAMP-dependent stimulation of protein kinase A (PKA) as well as one or more redundant PKA-independent pathways. The final steps are mediated by a relatively small collection of transcriptional regulators that collaborate closely to maximize the cellular rates of energy generation and growth. Understanding the nuclear events in this process may necessitate the further elaboration of a new model for eukaryotic gene regulation, called “reverse recruitment.” An essential feature of this idea is that fine-structure mapping of nuclear architecture will be required to understand the reception of regulatory signals that emanate from the plasma membrane and cytoplasm. Completion of this task should result in a much improved understanding of eukaryotic growth, differentiation, and carcinogenesis.

SUMMARY The yeast cell wall is a highly dynamic structure that is responsible for protecting the cell from rapid changes in external osmotic potential. The wall is also critical for cell expansion during growth and morphogenesis. This review discusses recent advances in understanding the various signal transduction pathways that allow cells to monitor the state of the cell wall and respond to environmental challenges to this structure. The cell wall integrity signaling pathway controlled by the small G-protein Rho1 is principally responsible for orchestrating changes to the cell wall periodically through the cell cycle and in response to various forms of cell wall stress. This signaling pathway acts through direct control of wall biosynthetic enzymes, transcriptional regulation of cell wall-related genes, and polarization of the actin cytoskeleton. However, additional signaling pathways interface both with the cell wall integrity signaling pathway and with the actin cytoskeleton to coordinate polarized secretion with cell wall expansion. These include Ca2+ signaling, phosphatidylinositide signaling at the plasma membrane, sphingoid base signaling through the Pkh1 and -2 protein kinases, Tor kinase signaling, and pathways controlled by the Rho3, Rho4, and Cdc42 G-proteins.

Saccharomyces cerevisiae Ste5 is a scaffold protein that recruits many pheromone signaling molecules to sequester the pheromone pathway from other homologous mitogen-activated protein kinase pathways. G1 cell cycle arrest and mating are two different physiological consequences of pheromone signal transduction and Ste5 is required for both processes. However, the roles of Ste5 in G1 arrest and mating are not fully understood. To understand the roles of Ste5 better, we isolated 150 G1 cell cycle arrest defective STE5 mutants by chemical mutagenesis of the gene. Here, we found that two G1 cell cycle arrest defective STE5 mutants (ste5MD248V and ste5delta-776) retained mating capacity. When overproduced in a wild-type strain, several ste5 mutants also showed different dominant phenotypes for G1 arrest and mating. Isolation and characterization of the mutants suggested separable roles of Ste5 in G1 arrest and mating of S. cerevisiae. In addition, the roles of Asp-248 and Tyr-421, which are important for pheromone signal transduction were further characterized by site-directed mutagenesis studies.Key words: Ste5, Saccharomyces cerevisiae, signal transduction, mating, G1 cell cycle arrest.

Abstract In eukaryotes, control of transcription by extracellular signals involves the translocation to the nucleus of at least one component of the signal transduction pathway. Transport through the nuclear envelope requires the activity of an import or export receptor that interacts with the small GTPase Ran. We have cloned the MSN5 gene of the yeast Saccharomyces cerevisiae that is postulated to encode one of these receptors. Msn5p belongs to a family of proteins with a conserved N-terminal sequence that acts as a RanGTP-binding domain. The results presented here provide genetic data supporting Msn5p involvement in several different signal transduction pathways. All of these pathways include changes in gene expression, and regulated nucleocytoplasmic redistribution of a component in response to external conditions has already been described in some of them. We have cloned MSN5 following two different strategies. Msn5p was constitutively localized in the nucleus. Phenotypic analysis of the msn5 mutant demonstrated that this protein participates in processes such as catabolite repression, calcium signaling, mating, and cell proliferation, as well as being involved in previously characterized phosphate utilization. Therefore, Msn5p could be a receptor for several proteins involved in different signaling pathways.

ABSTRACT CDC42 encodes a highly conserved GTPase of the Rho family that is best known for its role in regulating cell polarity and actin organization. In addition, various studies of both yeast and mammalian cells have suggested that Cdc42p, through its interaction with p21-activated kinases (PAKs), plays a role in signaling pathways that regulate target gene transcription. However, recent studies of the yeast pheromone response pathway suggested that prior results with temperature-sensitive cdc42 mutants were misleading and that Cdc42p and the Cdc42p-PAK interaction are not involved in signaling. To clarify this issue, we have identified and characterized novel viable pheromone-resistant cdc42 alleles that retain the ability to perform polarity-related functions. Mutation of the Cdc42p residue Val36 or Tyr40 caused defects in pheromone signaling and in the localization of the Ste20p PAK in vivo and affected binding to the Ste20p Cdc42p-Rac interactive binding (CRIB) domain in vitro. Epistasis analysis suggested that they affect the signaling step at which Ste20p acts, and overproduction of Ste20p rescued the defect. These results suggest that Cdc42p is in fact required for pheromone response and that interaction with the PAK Ste20p is critical for that role. Furthermore, the ste20ΔCRIB allele, previously used to disrupt the Cdc42p-Ste20p interaction, behaved as an activated allele, largely bypassing the signaling defect of thecdc42 mutants. Additional observations lead us to suggest that Cdc42p collaborates with the SH3-domain protein Bem1p to facilitate signal transduction, possibly by providing a cell surface scaffold that aids in the local concentration of signaling kinases, thus promoting activation of a mitogen-activated protein kinase cascade by Ste20p.

ABSTRACT The yeast Saccharomyces cerevisiae senses glucose, its preferred carbon source, through multiple signal transduction pathways. In one pathway, glucose represses the expression of many genes through the Mig1 transcriptional repressor, which is regulated by the Snf1 protein kinase. In another pathway, glucose induces the expression of HXT genes encoding glucose transporters through two glucose sensors on the cell surface that generate an intracellular signal that affects function of the Rgt1 transcription factor. We profiled the yeast transcriptome to determine the range of genes targeted by this second pathway. Candidate target genes were verified by testing for Rgt1 binding to their promoters by chromatin immunoprecipitation and by measuring the regulation of the expression of promoter lacZ fusions. Relatively few genes could be validated as targets of this pathway, suggesting that this pathway is primarily dedicated to regulating the expression of HXT genes. Among the genes regulated by this glucose signaling pathway are several genes involved in the glucose induction and glucose repression pathways. The Snf3/Rgt2-Rgt1 glucose induction pathway contributes to glucose repression by inducing the transcription of MIG2, which encodes a repressor of glucose-repressed genes, and regulates itself by inducing the expression of STD1, which encodes a regulator of the Rgt1 transcription factor. The Snf1-Mig1 glucose repression pathway contributes to glucose induction by repressing the expression of SNF3 and MTH1, which encodes another regulator of Rgt1, and also regulates itself by repressing the transcription of MIG1. Thus, these two glucose signaling pathways are intertwined in a regulatory network that serves to integrate the different glucose signals operating in these two pathways.

CAP, a protein from Saccharomyces cerevisiae that copurifies with adenylyl cyclase, appears to be required for yeast cells to be fully responsive to RAS proteins. CAP also appears to be required for normal cell morphology and responsiveness to nutrient deprivation and excess. We describe here a molecular and phenotypic analysis of the CAP protein. The N-terminal domain is necessary and sufficient for cellular response to activated RAS protein, while the C-terminal domain is necessary and sufficient for normal cellular morphology and responses to nutrient extremes. Thus, CAP is a novel example of a bifunctional component involved in the regulation of diverse signal transduction pathways.

CAP, a protein from Saccharomyces cerevisiae that copurifies with adenylyl cyclase, appears to be required for yeast cells to be fully responsive to RAS proteins. CAP also appears to be required for normal cell morphology and responsiveness to nutrient deprivation and excess. We describe here a molecular and phenotypic analysis of the CAP protein. The N-terminal domain is necessary and sufficient for cellular response to activated RAS protein, while the C-terminal domain is necessary and sufficient for normal cellular morphology and responses to nutrient extremes. Thus, CAP is a novel example of a bifunctional component involved in the regulation of diverse signal transduction pathways.

The yeast STE18 gene product has sequence and functional similarity to the γ subunits of G proteins. The cloned STE18 gene was subjected to a saturation mutagenesis using doped oligonucleotides. The populations of mutant genes were screened for two classes of STE18 mutations, those that allowed for increased mating of a strain containing a defective STE4 gene (compensators) and those that inhibited mating even in the presence of a functional STE18 gene (dominant negatives). Three amino acid substitutions that enhanced mating in a specific STE4 (Gβ) point mutant background were identified. These compensatory mutations were allele specific and had no detectable phenotype of their own; they may define residues that mediate an association between the Gβ and Gγ subunits or in the association of the Gβγ subunit with other components of the signalling pathway. Several dominant negative mutations were also identified, including two C terminal truncations. These mutant proteins were unable to function in signal transduction by themselves, but they prevented signal transduction mediated by pheromone, as well as the constitutive signalling which is present in cells defective in the GPAI (Gα) gene. These mutant proteins may sequester Gβ or some other component of the signalling machinery in a nonfunctional complex. Key wordsi yeast, G protein, STE18, mutagenesis, pheromone response.

Diploid Saccharomyces cerevisiae strains starved for nitrogen undergo a developmental transition from growth as single yeast form (YF) cells to a multicellular form consisting of filaments of pseudohyphal (PH) cells. Filamentous growth is regulated by an evolutionarily conserved signaling pathway that includes the small GTP-binding proteins Ras2p and Cdc42p, the protein kinases Ste20p, Ste11p and Ste7p, and the transcription factor Ste12p. Here, we designed a genetic screen for mutant strains defective for filamentous growth (dfg) to identify novel targets of the filamentation signaling pathway, and we thereby identified 16 different genes, CDC39, STE12, TEC1, WH13, NAB1, DBR1, CDC55, SRV2, TPM1, SPA2, BNI1, DFG5, DFG9, DFG10, BUD8 and DFG16, mutations that block filamentous growth. Phenotypic analysis of dfg mutant strains genetically dissects filamentous growth into the cellular processes of signal transduction, bud site selection, cell morphogenesis and invasive growth. Epistasis tests between dfg mutant alleles and dominant activated alleles of the RAS2 and STE11 genes, RAS2Val19 and STE11-4, respectively, identify putative targets for the filamentation signaling pathway. Several of the genes described here have homologues in filamentous fungi, where they also regulate fungal development.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Apr. 25, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics." Discipline: Biochemistry and Biophysics; Department/School: Medicine. On title page, [beta] appears as Greek character.

In response to external stimuli, many intracellular signaling proteins undergo dynamic changes in localization to the plasma membrane. Using the Saccharomyces cerevisiaemating pathway as a model, I investigated the molecular interactions that govern plasma membrane localization of signaling proteins, and how the plasma membrane compartmentalization of a signaling complex influences the overall signaling behavior of the pathway. Signaling proteins often consist of multiple interaction domains that collectively dictate their localization and function. Ste20 is a p21-activated kinase (PAK) that functions downstream of the Rho-type GTPase Cdc42 to activate several mitogen-activated protein (MAP) kinase pathways in budding yeast, including the mating pathway. I identified a short domain in Ste20 that directly binds to membrane lipids via electrostatic interaction. A mutation in this domain abolishes both the localization and function of Ste20. Thus, the previously known Cdc42 binding is necessary but not sufficient; instead, direct membrane binding by Ste20 is also critical. By replacing this domain with heterologous membranebinding domains, I demonstrated that phospholipid specificity is not essential in vivo. Functionally important short membrane-binding domains were also found in the Cdc42 effectors Gic1 and Gic2, indicating that generic membrane binding can work in concert with the CRIB domain to regulate activation of Cdc42 targets. These results underscore the importance of cooperation between protein-protein and protein-membrane interaction in achieving proper localization of signaling proteins at the cell cortex. At the system level, MAP kinase cascades can be graded or switch-like. The budding yeast mating pathway exhibits a graded response to increasing levels of pheromone. Previously the scaffold protein Ste5 was hypothesized to contribute to this graded response. To test this idea, I activated the pathway in a variety of ways and measured the response at the single cell level. I found that the graded response is not perturbed by the deletion of negative regulators of the pathway whereas the response became switch-like when the pathway was activated by a crosstalk stimulus that bypasses the upstream components. Interestingly, activation of the pathway in the cytoplasm using the graded expression of MAPKKK resulted in an ultrasensitive response. In contrast, activation of the pathway at the plasma membrane using the graded expression of membranetargeted active pathway components remained graded. In these settings, the scaffold protein Ste5 increased ultrasensitivity when limited to the cytosol; however, if Ste5 was allowed to function at the plasma membrane, signaling was graded. The results suggest that, in the mating pathway, the inherently ultrasensitive MAPK cascade is converted to a graded system by the scaffoldmediated assembly of signaling complexes at the plasma membrane. Therefore, the plasma membrane localization of Ste5 helps shape the input-output properties of the mating MAPK pathway in a manner that is suitable for the biology of mating. Taken together, this thesis underscores the importance of plasma membrane localization during mating pathway signaling in yeast. The examples described here provide further appreciation of how multiple interaction domains can function together to achieve specific targeting of the signaling proteins, as well as advances in understanding the role of scaffold proteins in modulating signaling behavior to promote graded signaling at the plasma membrane.

Dissertation (PhD)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Cells of the yeast Saccharomyces cerevisiae are able to change their morphological appearance in response to a variety of extracellular and intracellular signals. The processes involved in morphogenesis are well characterised in this organism, but the exact mechanism by which information emanating from the environment is integrated into the regulation of the actin cytoskeleton and the yeast cell cycle, is still not clearly understood. Considerable progress has, however, been made. The processes are investigated on various levels including: (i) the nature of the signals required to elicit a morphological adaptation, (ii) the mechanism by which these signals are perceived and transmitted to the nucleus for gene transcription regulation (signal transduction pathways), (iii) the role of the cytoskeleton, particularly actin, in morphogenesis, and (iv) the relationship between cell cycle regulators and factors required for alterations in cellular shape. The focus of this study was on elements involved in the regulation of one of these morphological processes, pseudohyphal formation, in S. cerevisiae. During pseudohyphal differentiation normal oval yeast cells become elongated and mother and daughter cells stay attached after cytokinesis to give rise to filaments. These filaments are able to penetrate the growth substrate, a phenomenon referred to as invasive growth. Actin remodelling is a prerequisite for the formation of elongated cells during pseudohyphal development and invasive growth. Its main contribution to this event is the directing of vesicles, containing cell wall constituents and enzymes, to specific sites of cell wall growth at the cell periphery. In order to fulfil this cellular function, actin is regulated on several levels. Signal transduction pathways that are activated in response to external nutritional signals play important roles in the regulation of the actin cytoskeleton during pseudohyphal differentiation. For this reason a literature review was compiled to introduce various aspects of actin-structure, the regulation of this structure and the functions actin performs during morphogenesis. The connection between signal transduction elements involved in morphological processes and actin remodelling is also reviewed. This study entailed the genetic analysis of numerous factors involved in the regulation of pseudohyphal differentiation, invasive growth and starch metabolism. Several transcriptional regulators playing a role in these phenomena were investigated. Apart from the transcription factors, which include Mss11p, Msn1p, Ste12p, F108p,Phd1p and Tec1p, additional elements ranging from transporters to G-proteins, were also investigated. Mutant strains deleted for one or more of these factors were constructed and tested to assess their abilities to form filaments that penetrate the growth substrate, and to utilise starch as a carbon source. Complex genetic relationships were observed for various combinations of these factors. Specifically, F108p,Msn1p and Ste12p were shown to act independently in controlling invasive growth and starch metabolism, suggesting that these factors are regulated by different signal transduction pathways. Mss11p, on the other hand, was found to play an indispensable role and seems to act as a downstream factor of Msn1 p, Fl08p, Ste12p and Tec1 p. The exception to this is Phd1 p, since multiple copies of PHD1 partially suppress the effect of a MSS11 deletion. The data suggests that Mss11 p functions at the confluence of several signalling pathways controlling the transcriptional regulation of genes required for invasive growth and starch degradation. Different nutritional signals were also found to differentially regulate specific signalling elements during the invasive growth response. For example, Tec1 p requires Msn1 p activity in response to growth on media containing a limited nitrogen source. This dependency, however, was absent when invasive growth was tested on glucose and starch media. Evidence was also obtained that confirmed the transcriptional co-regulation of MUC1 and STA2. MUC1 encodes a mucin-like protein that is required for invasive growth and pseudohyphal differentiation, whereas STA2 encodes a glucoamylase required for starch degradation. Unpublished results indicated that several transcriptional regulators of invasive growth also exert an effect on starch metabolism. The data generated during this study complemented and confirmed published results. It also contributed to the compilation of a more detailed model, integrating the numerous factors involved in these signalling processes.
AFRIKAANSE OPSOMMING: Saccharomyces cerevisiae gisselle beskik oor die vermoë om hul morfologiese voorkoms in responstot 'n verskeidenheid van ekstrasellulêre en intrasellulêre seine te verander. Die prosesse betrokke by morfogenese is goed gekarakteriseerd in hierdie organisme, maar die presiese meganisme waardeur inligting vanuit die omgewing geïntegreer word in die reguleringvan die aktien-sitoskelet en die gisselsiklus, word nog nie ten volle verstaan nie. Aansienlike vordering in die verband is egter gemaak. Die prosesse word op verskeie vlakke ondersoek, insluitende: (i) die aard van die seine wat benodig word om 'n morfologiese aanpassing te inisïeer; (ii) die meganisme waardeur hierdie seine waargeneem en herlei word na die selkern vir die regulering van geen-transkripsie (seintransduksie paaie); (iii) die rol van die sitoskelet, spesifiek aktien, in morfogenese en (iv) die verhouding tussen selsiklusreguleerders en faktore wat benodig word vir verandering in selvorm. Hierdie navorsing fokus op elemente betrokke by die regulering van een van hierdie morfologiese prosesse in S. cerevisiae, naamlik pseudohife-vorming. Gedurende pseudohife-differensiëring neem tipiese ovaalvormige selle 'n verlengde voorkoms aan wat tot die vorming van filamente lei. Hierdie filamente is in staat om die groeisubstraat te penetreer, 'n verskynsel bekend as penetrasie-groei. Aktienherrangskikking is 'n voorvereiste vir die vorming van verlengde selle tydens pseudohife-ontwikkeling. Die hoofbydrae van aktien tot hierdie verskynsel is die oriëntering van uitskeidingsvesikels, wat selwandkomponente en ensieme bevat, na spesifieke areas van selwandgroei op die seloppervlak. Aktien word op verskeie vlakke gereguleer om hierdie sellulêre funksie te vervul. Seintransduksiepaaie wat geaktiveer word in respons tot ekstrasellulêre voedingsseine speel 'n belangrike rol in die regulering van die aktien-sitoskelet tydens pseudohife-differensiëring. Op grond hiervan is 'n literatuuroorsig saamgestel vir die bekendstelling van verskeie aspekte van aktienstruktuur, die regulering van hierdie strukture en die funksies wat deur aktien gedurende morfogenese vervul word. Die verband tussen seintransduksie-elemente betrokke by morfologiese prosesse en aktien herrangskikkingword ook behandel. Hierdie studie het die genetiese analisering van verskeie faktore betrokke by pseudohife-differensiëring, penetrasie-groei en styselmetabolisme, behels. Verskeie transkripsionele reguleerders wat In rol speel in hierdie prosesse was bestudeer. Buiten die transkripsiefaktore Mss11p, Msn1p, Ste12p, F108p,Phd1P en Tec1p, was addisionele faktore, wat gewissel het van transporters tot G-proteïene, ook ondersoek. Mutante-rasse met geendelesies vir een of meer van hierdie faktore is gekonstrueer en getoets om vas te stel hoe dit hul vermoë raak om penetrerende filamente te vorm, asook om te bepaal of stysel as koolstofbron gebruik kan word. Komplekse genetiese interaksies vir verskeie kombinasies van hierdie faktore is waargeneem. Dit was waargeneem dat F108p,Msn1p en Ste12p onafhanklik funksioneer tydens die regulering van penetrasie-groei en styselmetabolisme, wat impliseer dat hierdie faktore deur verskillende seintransduksiepaaie gereguleer word. Mss11 p word beskou as In onmisbare rolspeler in hierdie prosesse en dit kom voor asof hierdie protein as 'n stroom-af faktor is en vereis word vir die funksionering van Msn1p, F108p, Ste12p en Tec1p. Phd1p is egter 'n uitsondering, aangesien veelvuldige kopieë van PHD1 die effek van 'n MSS11-delesie gedeeltelik oorkom. Die data impliseer dat Mss11 p by die samevloei van verskeie seintransduksiepaaie, benodig vir die transkripsionele regulering van gene betrokke by penetrasie-groei en styselmetabolisme, funksioneer. Dit was ook waargeneem dat verskillende voedingsseine die faktore betrokke by die penetrasie-groeirespons differensieel reguleer. Tec1 p byvoorbeeld benodig Msn1paktiwitieit in respons tot groei op media met 'n beperkte stikstofbron. Hierdie afhanklike interaksie is egter afwesig wanneer penetrasie-groei bestudeer word op glukose- en styselmedia. Resultate wat die gesamentlike transkripsionele regulering van MUC1 en STA2 bevestig, is ook verkry. MUC1 kodeer vir 'n mukienagtige proteïen wat benodig word vir pseudohife-vorming en penetrasie-groei, terwyl STA2 kodeer vir 'n glukoamilase essensieël vir styselafbraak. Ongepubliseerde resultate dui daarop dat verskeie transkripsionele reguleerders van penetrasie-groei ook In effek uitoefen op styselmetabolisme. Die data wat gegenereer is tydens hierdie studie komplementeer en bevestig reeds gepubliseerde resultate. Dit het ook bygedra tot die samestelling van 'n gedetaileerde model wat die verskillende faktore, betrokke by hierdie seintransduksieprosesse, integreer.