RESUMEN
In the yeast Saccharomyces cerevisiae and other ascomycetes, the maintenance of cell wall integrity is governed by a family of plasma-membrane spanning sensors that include the Wsc-type proteins. These cell wall proteins apparently sense stress-induced mechanical forces at the cell surface and target the cell wall integrity (CWI) signaling pathway, but the structural base for their sensor function is yet unknown. Here, we solved a high-resolution crystal structure of the extracellular cysteine-rich domain (CRD) of yeast Wsc1, which shows the characteristic PAN/Apple domain fold with two of the four Wsc1 disulfide bridges being conserved in other PAN domain cores. Given the general function of PAN domains in mediating protein-protein and protein-carbohydrate interactions, this finding underpins the importance of Wsc domains in conferring sensing and localization functions. Our Wsc1 CRD structure reveals an unusually high number of surface-exposed aromatic residues that are conserved in other fungal CRDs, and can be arranged into three solvent-exposed clusters. Mutational analysis demonstrates that two of the aromatic clusters are required for conferring S. cerevisiae Wsc1-dependent resistance to the glucan synthase inhibitor caspofungin, and the chitin-binding agents Congo red and Calcofluor white. These findings suggest an essential role of surface-exposed aromatic clusters in fungal Wsc-type sensors that might include an involvement in stress-induced sensor-clustering required to elicit appropriate cellular responses via the downstream CWI pathway.
RESUMEN
Selective adhesion of fungal cells to one another and to foreign surfaces is fundamental for the development of multicellular growth forms and the successful colonization of substrates and host organisms. Accordingly, fungi possess diverse cell wall-associated adhesins, mostly large glycoproteins, which present N-terminal adhesion domains at the cell surface for ligand recognition and binding. In order to function as robust adhesins, these glycoproteins must be covalently linkedto the cell wall via C-terminal glycosylphosphatidylinositol (GPI) anchors by transglycosylation. In this review, we summarize the current knowledge on the structural and functional diversity of so far characterized protein families of adhesion domains and set it into a broad context by an in-depth bioinformatics analysis using sequence similarity networks. In addition, we discuss possible mechanisms for the membrane-to-cell wall transfer of fungal adhesins by membrane-anchored Dfg5 transglycosidases.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adhesión Celular , Saccharomyces cerevisiae/citologíaRESUMEN
The correct distribution and trafficking of proteins are essential for all organisms. Eukaryotes evolved a sophisticated trafficking system which allows proteins to reach their destination within highly compartmentalized cells. One eukaryotic hallmark is the attachment of a glycosylphosphatidylinositol (GPI) anchor to C-terminal ω-peptides, which are used as a zip code to guide a subset of membrane-anchored proteins through the secretory pathway to the plasma membrane. In fungi, the final destination of many GPI-anchored proteins is their outermost compartment, the cell wall. Enzymes of the Dfg5 subfamily catalyze the essential transfer of GPI-anchored substrates from the plasma membrane to the cell wall and discriminate between plasma membrane-resident GPI-anchored proteins and those transferred to the cell wall (GPI-CWP). We solved the structure of Dfg5 from a filamentous fungus and used in crystallo glycan fragment screening to reassemble the GPI-core glycan in a U-shaped conformation within its binding pocket. The resulting model of the membrane-bound Dfg5â¢GPI-CWP complex is validated by molecular dynamics (MD) simulations and in vivo mutants in yeast. The latter show that impaired transfer of GPI-CWPs causes distorted cell-wall integrity as indicated by increased chitin levels. The structure of a Dfg5â¢ß1,3-glycoside complex predicts transfer of GPI-CWP toward the nonreducing ends of acceptor glycans in the cell wall. In addition to our molecular model for Dfg5-mediated transglycosylation, we provide a rationale for how GPI-CWPs are specifically sorted toward the cell wall by using GPI-core glycan modifications.
Asunto(s)
Pared Celular/metabolismo , Proteínas Fúngicas/metabolismo , Hongos/metabolismo , Glicoproteínas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Pared Celular/química , Pared Celular/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Hongos/química , Hongos/clasificación , Hongos/genética , Glicoproteínas/química , Glicoproteínas/genética , Glicosilfosfatidilinositoles/química , Transporte de ProteínasRESUMEN
For host-cell interaction, the human fungal pathogen Candida glabrata harbors a large family of more than 20 cell wall-attached epithelial adhesins (Epas). Epa family members are lectins with binding pockets containing several conserved and variable structural hot spots, which were implicated in mediating functional diversity. In this study, we have performed an elaborate structure-based mutational analysis of numerous Epa paralogs to generally determine the role of diverse structural hot spots in conferring host cell binding and ligand binding specificity. Our study reveals that several conserved structural motifs contribute to efficient host cell binding. Moreover, our directed motif exchange experiments reveal that the variable loop CBL2 is key for programming ligand binding specificity, albeit with limited predictability. In contrast, we find that the variable loop L1 affects host cell binding without significantly influencing the specificity of ligand binding. Our data strongly suggest that variation of numerous structural hot spots in the ligand binding pocket of Epa proteins is a main driver of their functional diversification and evolution.
Asunto(s)
Candida glabrata , Proteínas Fúngicas , Lectinas , Secuencias de Aminoácidos , Células CACO-2 , Candida glabrata/química , Candida glabrata/genética , Candida glabrata/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Lectinas/química , Lectinas/genética , Lectinas/metabolismo , Dominios ProteicosRESUMEN
Microorganisms have evolved specific cell surface molecules that enable discrimination between cells from the same and from a different kind. Here, we investigate the role of Flo11-type cell surface adhesins from social yeasts in kin discrimination. We measure the adhesion forces mediated by Flo11A-type domains using single-cell force spectroscopy, quantify Flo11A-based cell aggregation in populations and determine the Flo11A-dependent segregation of competing yeast strains in biofilms. We find that Flo11A domains from diverse yeast species confer remarkably strong adhesion forces by establishing homotypic interactions between single cells, leading to efficient cell aggregation and biofilm formation in homogenous populations. Heterotypic interactions between Flo11A domains from different yeast species or Saccharomyces cerevisiae strains confer weak adhesive forces and lead to efficient strain segregation in heterogenous populations, indicating that in social yeasts Flo11A-mediated cell adhesion is a major mechanism for kin discrimination at species and sub-species levels. These findings, together with our structure and mutation analysis of selected Flo11A domains, provide a rationale of how cell surface receptors have evolved in microorganisms to mediate kin discrimination.
Asunto(s)
Adhesión Celular/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Biopelículas , Comunicación Celular/fisiologíaRESUMEN
Cell-cell and cell-substrate based adhesion of yeasts are major determinants of their adoption of different life styles. Genome-mining of ascomycetous GPI-anchored cell wall proteins with lectin-like PA14 domains identified a unique class of putative adhesins in the clade of methylotrophic Komagataella yeasts, many of which are known to colonize plants and insects involving yet unknown adhesion mechanisms. Here, we report the functional and structural analysis of two of its members: KpFlo1 (=Cea1), that is highly specific for terminal N-acetylglucosamine moieties, and KpFlo2, which represents an orphan lectin with intact binding site but unknown specificity. Crystal structures of the Cea1 adhesion domain complexed to N-acetylglucosamine and N,N'-diacetylchitobiose reveal a Ca2+-dependent binding mode that differs from other members of the PA14/Flo5 adhesin family. Heterologous expression of Cea1A in Saccharomyces cerevisiae promotes cellular adhesion to non-reducing ends of non-crystalline chitin. Overall, our data suggest that high-affinity recognition of ß-GlcNAc-capped glycans by Cea1 enable Komagataella species to interact with surface cues present in fungi and insects.
RESUMEN
The ubiquitin-proteasome system (UPS) controls cellular functions by maintenance of a functional proteome and degradation of key regulatory proteins. Central to the UPS is the proteasome that adjusts the abundance of numerous proteins, thereby safeguarding their activity or initiating regulatory events. Here, we demonstrate that the essential Saccharomyces cerevisiae protein Yjr141w/Ipa1 (Important for cleavage and PolyAdenylation) belongs to the HECT_2 (homologous to E6-AP carboxyl terminus_2) family. We found that five cysteine residues within the HECT_2 family signature and the C-terminus are essential for Ipa1 activity. Furthermore, Ipa1 interacts with several ubiquitin-conjugating enzymes in vivo and localizes to the cytosol and nucleus. Importantly, Ipa1 has an impact on proteasome activity, which is indicated by the activation of the Rpn4 regulon as well as by decreased turnover of destabilized proteasome substrates in an IPA1 mutant. These changes in proteasome activity might be connected to reduced maturation or modification of proteasomal core particle proteins. Our results highlight the influence of Ipa1 on the UPS. The conservation within the HECT_2 family and the connection of the human HECT_2 family member to an age-related degeneration disease might suggest that HECT_2 family members share a conserved function linked to proteasome activity.
Asunto(s)
Proteínas Fúngicas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Saccharomycetales/metabolismo , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Secuencia Conservada , Citosol/metabolismo , Daño del ADN , Proteínas Fúngicas/química , Familia de Multigenes , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteolisis , Estrés Fisiológico , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
For host colonization, the human fungal pathogen Candida glabrata is known to utilize a large family of highly related surface-exposed cell wall proteins, the lectin-like epithelial adhesins (Epas). To reveal the structure-function relationships within the entire Epa family, we have performed a large scale functional analysis of the adhesion (A) domains of 17 Epa paralogs in combination with three-dimensional structural studies of selected members with cognate ligands. Our study shows that most EpaA domains exert lectin-like functions and together recognize a wide variety of glycans with terminal galactosides for conferring epithelial cell adhesion. We further identify several conserved and variable structural features within the diverse Epa ligand binding pockets, which affect affinity and specificity. These features rationalize why mere phylogenetic relationships within the Epa family are weak indicators for functional classification and explain how Epa-like adhesins have evolved in C. glabrata and related fungal species.
Asunto(s)
Candida glabrata/química , Moléculas de Adhesión Celular/química , Proteínas Fúngicas/química , Lectinas/química , Polisacáridos/química , Secuencia de Aminoácidos , Sitios de Unión , Evolución Biológica , Candida glabrata/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Variación Genética , Lectinas/genética , Lectinas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Polisacáridos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Saccharomyces cerevisiae harbors a family of GPI-anchored cell wall proteins for interaction with its environment. The flocculin Flo11, a major representative of these fungal adhesins, confers formation of different types of multicellular structures such as biofilms, flors, or filaments. To understand these environment-dependent growth phenotypes on a molecular level, we solved the crystal structure of the N-terminal Flo11A domain at 0.89-Å resolution. Besides a hydrophobic apical region, the Flo11A domain consists of a ß sandwich of the fibronectin type III domain (FN3). We further show that homophilic Flo11-Flo11 interactions and heterophilic Flo11-plastic interactions solely depend on the Flo11A domain and are strongly pH dependent. These functions of Flo11A involve an apical region with its surface-exposed aromatic band, which is accompanied by acidic stretches. Together with electron microscopic reconstructions of yeast cell-cell contact sites, our data suggest that Flo11 acts as a spacer-like, pH-sensitive adhesin that resembles a membrane-tethered hydrophobin.
Asunto(s)
Adhesión Celular/fisiología , Fibronectinas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Familia de Multigenes/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Aminoácidos Aromáticos/química , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In Saccharomyces cerevisiae and related yeast species, the TEA transcription factor Tec1, together with a second transcription factor, Ste12, controls development, including cell adhesion and filament formation. Tec1-Ste12 complexes control target genes through Tec1 binding sites (TEA consensus sequences [TCSs]) that can be further combined with Ste12 binding sites (pheromone response elements [PREs]) for cooperative DNA binding. The activity of Tec1-Ste12 complexes is known to be under negative control of the Dig1 and Dig2 (Dig1/2) transcriptional corepressors that confer regulation by upstream signaling pathways. Here, we found that Tec1 and Ste12 can associate with the transcriptional coregulators Msa1 and Msa2 (Msa1/2), which were previously found to associate with the cell cycle transcription factor complexes SBF (Swi4/Swi6 cell cycle box binding factor) and MBF (Mbp1/Swi6 cell cycle box binding factor) to control G1-specific transcription. We further show that Tec1-Ste12-Msa1/2 complexes (i) do not contain Swi4 or Mbp1, (ii) assemble at single TCSs or combined TCS-PREs in vitro, and (iii) coregulate genes involved in adhesive and filamentous growth by direct promoter binding in vivo. Finally, we found that, in contrast to Dig proteins, Msa1/2 seem to act as coactivators that enhance the transcriptional activity of Tec1-Ste12. Taken together, our findings add an additional layer of complexity to our understanding of the control mechanisms exerted by the evolutionarily conserved TEA domain and Ste12-like transcription factors.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Adhesión Celular , Proteínas de Ciclo Celular/metabolismo , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Modelos Biológicos , Regiones Promotoras Genéticas/genética , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
In Saccharomyces cerevisiae, the RNA-binding protein Whi3 controls cell cycle progression, biofilm formation, and stress response by post-transcriptional regulation of the Cdc28-Cln3 cyclin-dependent protein kinase and the dual-specificity protein kinase Yak1. Previous work has indicated that Whi3 might govern these processes by additional, yet unknown mechanisms. In this study, we have identified additional effectors of Whi3 that include the G1 cyclins Cln1/Cln2 and two known regulators of biofilm formation, the catalytic PKA subunit Tpk1 and the transcriptional activator Tec1. We also provide evidence that Whi3 regulates production of these factors by post-transcriptional control and might exert this function by affecting translational elongation. Unexpectedly, we also discovered that Whi3 is a key regulator of cellular ploidy, because haploid whi3Δ mutant strains exhibit a significant increase-in-ploidy phenotype that depends on environmental conditions. Our data further suggest that Whi3 might control stability of ploidy by affecting the expression of many key genes involved in sister chromatid cohesion and of NIP100 that encodes a component of the yeast dynactin complex for chromosome distribution. Finally, we show that absence of Whi3 induces a transcriptional stress response in haploid cells that is relieved by whole-genome duplication. In summary, our study suggests that the RNA-binding protein Whi3 acts as a central regulator of cell division and development by post-transcriptional control of key genes involved in chromosome distribution and cell signaling.
Asunto(s)
Ploidias , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transducción de Señal , División Celular , Ciclinas/genética , Ciclinas/metabolismo , Complejo Dinactina , Regulación del Desarrollo de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The human pathogenic yeast Candida glabrata harbors more than 20 surface-exposed, epithelial adhesins (Epas) for host cell adhesion. The Epa family recognizes host glycans and discriminates between target tissues by their adhesin (A) domains, but a detailed structural basis for ligand-binding specificity of Epa proteins has been lacking so far. In this study, we provide high-resolution crystal structures of the Epa1A domain in complex with different carbohydrate ligands that reveal how host cell mucin-type O-glycans are recognized and allow a structure-guided classification of the Epa family into specific subtypes. Further detailed structural and functional characterization of subtype-switched Epa1 variants shows that specificity is governed by two inner loops, CBL1 and CBL2, involved in calcium binding as well as by three outer loops, L1, L2, and L3. In summary, our study provides the structural basis for promiscuity and specificity of Epa adhesins, which might further contribute to developing anti-adhesive antimycotics and combating Candida colonization.
Asunto(s)
Candida glabrata/química , Proteínas Fúngicas/química , Lectinas/química , Modelos Moleculares , Familia de Multigenes/genética , Filogenia , Conformación Proteica , Calcio/metabolismo , Candida glabrata/fisiología , Análisis por Conglomerados , Biología Computacional , Cristalografía por Rayos X , Fluorescencia , Proteínas Fúngicas/genética , Lectinas/genética , Polisacáridos , Unión ProteicaRESUMEN
In Saccharomyces cerevisiae, the Ras/cyclic AMP (cAMP)/protein kinase A (PKA) pathway is a nutrient-sensitive signaling cascade that regulates vegetative growth, carbohydrate metabolism, and entry into meiosis. How this pathway controls later steps of meiotic development is largely unknown. Here, we have analyzed the role of the Ras/cAMP/PKA pathway in spore formation by the meiosis-specific manipulation of Ras and PKA or by the disturbance of cAMP production. We found that the regulation of spore formation by acetate takes place after commitment to meiosis and depends on PKA and appropriate A kinase activation by Ras/Cyr1 adenylyl cyclase but not by activation through the Gpa2/Gpr1 branch. We further discovered that spore formation is regulated by carbon dioxide/bicarbonate, and an analysis of mutants defective in acetate transport (ady2Δ) or carbonic anhydrase (nce103Δ) provided evidence that these metabolites are involved in connecting the nutritional state of the meiotic cell to spore number control. Finally, we observed that the potential PKA target Ady1 is required for the proper localization of the meiotic plaque proteins Mpc70 and Spo74 at spindle pole bodies and for the ability of these proteins to initiate spore formation. Overall, our investigation suggests that the Ras/cAMP/PKA pathway plays a crucial role in the regulation of spore formation by acetate and indicates that the control of meiotic development by this signaling cascade takes places at several steps and is more complex than previously anticipated.
Asunto(s)
Acetatos/metabolismo , Dióxido de Carbono/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas ras/metabolismo , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Eliminación de Gen , Meiosis , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismo , Proteínas ras/genéticaRESUMEN
The budding yeast Saccharomyces cerevisiae is a eukaryotic microorganism that is able to choose between different unicellular and multicellular lifestyles. The potential of individual yeast cells to switch between different growth modes is advantageous for optimal dissemination, protection and substrate colonization at the population level. A crucial step in lifestyle adaptation is the control of self- and foreign adhesion. For this purpose, S. cerevisiae contains a set of cell wall-associated proteins, which confer adhesion to diverse biotic and abiotic surfaces. Here, we provide an overview of different aspects of S. cerevisiae adhesion, including a detailed description of known lifestyles, recent insights into adhesin structure and function and an outline of the complex regulatory network for adhesin gene regulation. Our review shows that S. cerevisiae is a model system suitable for studying not only the mechanisms and regulation of cell adhesion, but also the role of this process in microbial development, ecology and evolution.
Asunto(s)
Adhesión Celular , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Regulación Fúngica de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In Saccharomyces cerevisiae, the TEA transcription factor Tec1 controls several developmental programs in response to nutrients and pheromones. Tec1 is targeted by the pheromone-responsive Fus3/Kss1 mitogen-activated protein kinase (MAPK) cascade, which destabilizes the transcription factor to ensure efficient mating of sexual partner cells. The regulation of Tec1 by signaling pathways that control cell division and development in response to nutrients, however, is not known. Here, we show that Tec1 protein stability is under control of the nutrient-sensitive target of rapamycin complex 1 (TORC1) signaling pathway via the Tip41-Tap42-Sit4 branch. We further show that degradation of Tec1 upon inhibition of TORC1 by rapamycin does not involve polyubiquitylation and appears to be proteasome independent. However, rapamycin-induced Tec1 degradation depends on the HECT ubiquitin ligase Rsp5, which physically interacts with Tec1 via conserved PxY motives. We further demonstrate that rapamycin and mating pheromone control Tec1 protein stability through distinct mechanisms by targeting different domains of the transcription factor. Finally, we show that Tec1 is a positive regulator of yeast chronological lifespan (CLS), a known TORC1-regulated process. Our findings indicate that in yeast, Tec1 links TORC1 and MAPK signaling pathways to coordinate control of cellular development in response to different stimuli.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antifúngicos/farmacología , Northern Blotting , Ciclinas/genética , Ciclinas/metabolismo , Proteínas de Unión al ADN/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microscopía Fluorescente , Unión Proteica , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genética , Sirolimus/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
In Saccharomyces cerevisiae, adhesive growth on solid surfaces is mediated by the flocculin Flo11 to confer biofilm and filament formation. Expression of FLO11 is governed by a complex regulatory network that includes, e.g., the protein kinase A (PKA) signaling pathway. In addition, numerous regulatory genes, which have not been integrated into regulatory networks, affect adhesive growth, including WHI3 encoding an RNA-binding protein and YAK1 coding for a dual-specificity tyrosine-regulated protein kinase. In this study, we present evidence that Whi3 and Yak1 form part of a signaling pathway that regulates FLO11-mediated surface adhesion and is involved in stress resistance. Our study further suggests that Whi3 controls YAK1 expression at the post-transcriptional level and that Yak1 targets the transcriptional regulators Sok2 and Phd1 to control FLO11. We also discovered that Yak1 regulates acidic stress resistance and adhesion via the transcription factor Haa1. Finally, we provide evidence that the catalytic PKA subunit Tpk1 inhibits Yak1 by targeting specific serine residues to suppress FLO11. In summary, our data suggest that Yak1 is at the center of a regulatory cascade for adhesive growth and stress resistance, which is under dual control of Whi3 and the PKA subunit Tpk1.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/crecimiento & desarrollo , Estrés Fisiológico/genética , Adhesión Celular/genética , Adhesión Celular/fisiología , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas de Unión al ADN/genética , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiología , Glucógeno Sintasa Quinasa 3/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Proteínas Represoras/genética , Saccharomyces cerevisiae/genética , Serina/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Transcripción/genéticaRESUMEN
In the budding yeast Saccharomyces cerevisiae, self-recognition and the thereby promoted aggregation of thousands of cells into protective flocs is mediated by a family of cell-surface adhesins, the flocculins (Flo). Based on this social behavior FLO genes fulfill the definition of "greenbeard" genes, which direct cooperation toward other carriers of the same gene. The process of flocculation plays an eminent role in the food industry for the production of beer and wine. However, the precise mode of flocculin-mediated surface recognition and the exact structure of cognate ligands have remained elusive. Here, we present structures of the adhesion domain of a flocculin complexed to its cognate ligands derived from yeast high-mannose oligosaccharides at resolutions up to 0.95 Å. Besides a PA14-like architecture, the Flo5A domain reveals a previously undescribed lectin fold that utilizes a unique DcisD calcium-binding motif for carbohydrate binding and that is widely spread among pro- and eukaryotes. Given the high abundance of high-mannose oligosaccharides in yeast cell walls, the Flo5A structure suggests a model for recognition, where social non-self- instead of unsocial self-interactions are favored.
Asunto(s)
Lectinas/química , Oligosacáridos/química , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Sitios de Unión/genética , Calcio/metabolismo , Secuencia de Carbohidratos , Cristalografía por Rayos X , Lectinas/genética , Lectinas/metabolismo , Manosa/química , Manosa/metabolismo , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/metabolismo , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oligosacáridos/metabolismo , Unión Proteica , Pliegue de Proteína , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
In Saccharomyces cerevisiae, the TEA transcription factor Tec1 is known to regulate target genes together with a second transcription factor, Ste12. Tec1-Ste12 complexes can activate transcription through Tec1 binding sites (TCSs), which can be further combined with Ste12 binding sites (PREs) for cooperative DNA binding. However, previous studies have hinted that Tec1 might regulate transcription also without Ste12. Here, we show that in vivo, physiological amounts of Tec1 are sufficient to stimulate TCS-mediated gene expression and transcription of the FLO11 gene in the absence of Ste12. In vitro, Tec1 is able to bind TCS elements with high affinity and specificity without Ste12. Furthermore, Tec1 contains a C-terminal transcriptional activation domain that confers Ste12-independent activation of TCS-regulated gene expression. On a genome-wide scale, we identified 302 Tec1 target genes that constitute two distinct classes. A first class of 254 genes is regulated by Tec1 in a Ste12-dependent manner and is enriched for genes that are bound by Tec1 and Ste12 in vivo. In contrast, a second class of 48 genes can be regulated by Tec1 independently of Ste12 and is enriched for genes that are bound by the stress transcription factors Yap6, Nrg1, Cin5, Skn7, Hsf1, and Msn4. Finally, we find that combinatorial control by Tec1-Ste12 complexes stabilizes Tec1 against degradation. Our study suggests that Tec1 is able to regulate TCS-mediated gene expression by Ste12-dependent and Ste12-independent mechanisms that enable promoter-specific transcriptional control.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Adhesión Celular , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Genes Reporteros , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Análisis por Micromatrices , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Factores de Transcripción/genéticaRESUMEN
TM9 proteins constitute a well defined family, characterized by the presence of a large variable extracellular domain and nine putative transmembrane domains. This family is highly conserved throughout evolution and comprises three members in Dictyostelium discoideum and Saccharomyces cerevisiae and four in humans and mice. In Dictyostelium, previous analysis demonstrated that TM9 proteins are implicated in cellular adhesion. In this study, we generated TM9 mutants in S. cerevisiae and analyzed their phenotype with particular attention to cellular adhesion. S. cerevisiae strains lacking any one of the three TM9 proteins were severely suppressed for adhesive growth and filamentous growth under conditions of nitrogen starvation. In these mutants, expression of the FLO11-lacZ reporter gene was strongly reduced, whereas expression of FRE(Ty1)-lacZ was not, suggesting that TM9 proteins are implicated at a late stage of nutrient-controlled signaling pathways. We also reexamined the phenotype of Dictyostelium TM9 mutant cells, focusing on nutrient-controlled cellular functions. Although the initiation of multicellular development and autophagy was unaltered in Dictyostelium TM9 mutants, nutrient-controlled secretion of lysosomal enzymes was dysregulated in these cells. These results suggest that in both yeast and amoebae, TM9 proteins participate in the control of specific cellular functions in response to changing nutrient conditions.
Asunto(s)
Dictyostelium/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiología , Regulación Fúngica de la Expresión Génica , Regulación de la Expresión Génica , Proteínas de la Membrana/fisiología , Animales , Autofagia , Adhesión Celular , Proteínas Fúngicas/genética , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Modelos Biológicos , Mutación , Plásmidos/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Especificidad de la EspecieRESUMEN
In Saccharomyces cerevisiae, diploid yeast cells follow a bipolar budding program, which depends on the two transmembrane glycoproteins Bud8p and Bud9p that potentially act as cortical tags to mark the cell poles. Here, we have performed systematic structure-function analyses of Bud8p and Bud9p to identify functional domains. We find that polar transport of Bud8p and Bud9p does not depend on N-terminal sequences but instead on sequences in the median part of the proteins and on the C-terminal parts that contain the transmembrane domains. We show that the guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange factor Bud5p, which is essential for bud site selection and physically interacts with Bud8p, also interacts with Bud9p. Regions of Bud8p and Bud9p predicted to reside in the extracellular space are likely to confer interaction with the N-terminal region of Bud5p, implicating indirect interactions between the cortical tags and the GDP/GTP exchange factor. Finally, we have identified regions of Bud8p and Bud9p that are required for interaction with the cortical tag protein Rax1p. In summary, our study suggests that Bud8p and Bud9p carry distinct domains for delivery of the proteins to the cell poles, for interaction with the general budding machinery and for association with other cortical tag proteins.