RESUMO
In addition to the UPR pathway, yeast cells require components of the HOG pathway to respond to ER stress. In this work, we found that unphosphorylated Sln1 and Ssk1 are required to mount an appropriate response to Tn. We also found that the MAPKKKs Ssk2 participates in the Tn response, but its osmo-redundant protein Ssk22 does not. We also found that the Pbs2 docking sites for Ssk2 (RDS-I and KD) are partially dispensable when mutated separately; however, the prevention of Ssk2 binding to Pbs2, by the simultaneous mutation of RDS-I and KD, caused strong sensitivity to Tn. In agreement with the lack of Hog1 phosphorylation during Tn treatment, a moderate resistance to Tn is obtained when a Pbs2 version lacking its kinase activity is expressed; however, the presence of mutual Pbs2-Hog1 docking sites is essential for the Tn response. Finally, we detected that Tn induced a transcriptional activation of some components of the SLN1 branch. These results indicate that the Tn response requires a complex formed by the MAPK module and components of the SLN1 branch but not their canonical osmoregulatory activities.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Estresse do Retículo Endoplasmático , MAP Quinase Quinase Quinases/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Tunicamicina/metabolismo , Tunicamicina/farmacologiaRESUMO
In the yeast Saccharomyces cerevisiae, components of the High Osmolarity Glycerol (HOG) pathway are important for the response to diverse stresses including response to endoplasmic reticulum stress (ER stress), which is produced by the accumulation of unfolded proteins in the lumen of this organelle. Accumulation of unfolded proteins may be due to the inhibition of protein N-glycosylation, which can be achieved by treatment with the antibiotic tunicamycin (Tn). In this work we were interested in finding proteins involved in the ER stress response regulated by Hog1, the mitogen activated protein kinase (MAPK) of the HOG pathway. A high gene dosage suppression screening allowed us to identify genes that suppressed the sensitivity to Tn shown by a hog1Δ mutant. The suppressors participate in a limited number of cellular processes, including lipid/carbohydrate biosynthesis and protein glycosylation, vesicle-mediated transport and exocytosis, cell wall organization and biogenesis, and cell detoxification processes. The finding of suppressors Rer2 and Srt1, which participate in the dolichol biosynthesis pathway revealed that the hog1Δ strain has a defective polyprenol metabolism. This work uncovers new genetic and functional interactors of Hog1 and contributes to a better understanding of the participation of this MAPK in the ER stress response.
Assuntos
Farmacorresistência Fúngica/genética , Estresse do Retículo Endoplasmático/genética , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Supressão Genética , Tunicamicina/farmacologia , Alquil e Aril Transferases/metabolismo , Dimetilaliltranstransferase/metabolismo , Dosagem de Genes , Regulação Fúngica da Expressão Gênica/genética , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Resposta a Proteínas não DobradasRESUMO
Eukaryotic cells have evolved signalling pathways that allow adaptation to harmful conditions that disrupt endoplasmic reticulum (ER) homeostasis. When the function of the ER is compromised in a condition known as ER stress, the cell triggers the unfolded protein response (UPR) in order to restore ER homeostasis. Accumulation of misfolded proteins due to stress conditions activates the UPR pathway. In mammalian cells, the UPR is composed of three branches, each containing an ER sensor (PERK, ATF6 and IRE1). However, in yeast species, the only sensor present is the inositol-requiring enzyme Ire1. To cope with unfolded protein accumulation, Ire1 triggers either a transcriptional response mediated by a transcriptional factor that belongs to the bZIP transcription factor family or an mRNA degradation process. In this review, we address the current knowledge of the UPR pathway in several yeast species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida glabrata, Cryptococcus neoformans, and Candida albicans. We also include unpublished data on the UPR pathway of the budding yeast Kluyveromyces lactis. We describe the basic components of the UPR pathway along with similarities and differences in the UPR mechanism that are present in these yeast species.
RESUMO
Yeast mating signal transduction pathways require a heterotrimeric G protein composed of Gα, Gß, and Gγ subunits connected to a mitogen-activated protein kinase (MAPK) module. While in Saccharomyces cerevisiae elimination of Gα induces constitutive activation of the mating pathway, in Kluyveromyces lactis it produces partial sterility, which indicates that K. lactis Gα (KlGα) is required to positively activate mating. We use physical interaction experiments to determine that KlGα interacts with the adaptor protein KlSte50p. The Ras association (RA) domain of KlSte50p favored interaction with the GDP-bound KlGα subunit, and when the KlGα protein is constitutively activated, the interaction drops significantly. Additionally, KlSte50p strongly associates with the MAPK kinase kinase (MAPKKK) KlSte11p through its sterile alpha motif (SAM) domain. Genetic experiments placed KlSte50p downstream of the G protein α subunit, indicating that KlGα may stimulate the mating pathway via KlSte50p. Fusion of KlSte50p to the KlGß subunit partially eliminated the requirement of KlGα for mating, indicating that one contribution of KlGα to the mating pathway is to facilitate plasma membrane anchoring of KlSte50p.
Assuntos
Proteínas Fúngicas/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Kluyveromyces/fisiologia , Feromônios/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Proteínas Fúngicas/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Kluyveromyces/genética , Dados de Sequência Molecular , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The Kluyveromyces lactis heterotrimeric G protein is a canonical Galphabetagamma complex; however, in contrast to Saccharomyces cerevisiae, where the Ggamma subunit is essential for mating, disruption of the KlGgamma gene yielded cells with almost intact mating capacity. Expression of a nonfarnesylated Ggamma, which behaves as a dominant-negative in S. cerevisiae, did not affect mating in wild-type and DeltaGgamma cells of K. lactis. In contrast to the moderate sterility shown by the single DeltaKlGalpha, the double DeltaKlGalpha DeltaKlGgamma mutant displayed full sterility. A partial sterile phenotype of the DeltaKlGgamma mutant was obtained in conditions where the KlGbeta subunit interacted defectively with the Galpha subunit. The addition of a CCAAX motif to the C-end of KlGbeta, partially suppressed the lack of both KlGalpha and KlGgamma subunits. In cells lacking KlGgamma, the KlGbeta subunit cofractionated with KlGalpha in the plasma membrane, but in the DeltaKlGalpha DeltaKlGgamma strain was located in the cytosol. When the KlGbeta-KlGalpha interaction was affected in the DeltaKlGgamma mutant, most KlGbeta fractionated to the cytosol. In contrast to the generic model of G-protein function, the Gbeta subunit of K. lactis has the capacity to attach to the membrane and to activate mating effectors in absence of the Ggamma subunit.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Kluyveromyces/fisiologia , Feromônios/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
Genes encoding the Galpha subunit were cloned from Mucor circinelloides, a zygomycete dimorphic fungus. There are at least four genes that encode for Galpha subunits, gpa1, gpa2, gpa3, and gpa4. The genes gpa1 and gpa3 were isolated and characterized, and their predicted products showed 36%-67% identity with Galpha subunits from diverse fungi. Northern blot analysis of gpa3 showed that it is present in spores and constitutively expressed during mycelium development and during yeast-mycelium and mycelium-yeast transitions. However, during yeast cell growth, decreased levels of mRNA were observed. Sequence analysis of gpa3 cDNA revealed that Gpa3 encodes a polypeptide of 356 amino acids with a calculated molecular mass of 40.8 kDa. The deduced sequence of Gpa3 protein contains all the consensus regions of Galpha subunits of the Galpha(i/o/t) subfamily except the cysteine near the C terminus for potential ADP-ribosylation by pertussis toxin. This cDNA was expressed in Escherichia coli and purified by affinity chromatography. Based on its electrophoretic mobility in SDS-PAGE, the molecular mass of the His6-tagged Gpa3 was 45 kDa. The recombinant protein was recognized by a polyclonal antibody against a fragment of a human Galpha(i/o/t). Furthermore, the recombinant Gpa3 was ADP-ribosylated by activated cholera toxin and [32P]NAD but not by pertussis toxin. These results indicate that in M. circinelloides the Galpha subunit Gpa3 is expressed constitutively during differentiation.
Assuntos
Proteínas Fúngicas/biossíntese , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Mucor/genética , Proteínas Recombinantes/biossíntese , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/isolamento & purificação , Dados de Sequência Molecular , Mucor/metabolismo , Filogenia , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
The mating pheromone response pathway in Saccharomyces cerevisiae is one of the best understood signalling pathways in eukaryotes. Comparison of this system with pathways in other fungal species has generated surprises and insights. Cloning and targetted disruption of genes encoding components of the pheromone response pathway has allowed the attribution of specific functions to these signal transduction components. In this review we describe current knowledge of the Kluyveromyces lactis mating system, and compare it with the well-understood S. cerevisiae pathway, emphasizing the similarities and differences in the heterotrimeric G protein activity. This mating pathway is controlled positively by both the Galpha and the Gbeta subunits of the heterotrimeric G protein.
Assuntos
Kluyveromyces/fisiologia , Feromônios/fisiologia , Transdução de Sinais , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Kluyveromyces/genética , Feromônios/genética , Receptores de Feromônios/fisiologia , Elementos de Resposta , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais/genéticaRESUMO
The Kluyveromyces lactis FUS1 gene was cloned, physically characterized and its role in the mating response pathway was determined. The gene encodes a putative membrane protein, whose structure shows a single membrane-spanning segment, a short extracellular amino-terminus and a long carboxy-terminus, located in the cytoplasmic side. The predicted primary structure of the protein shows a number of serine and threonine residues in the amino-terminus, which in analogy to Fus1p of Saccharomyces cerevisiae might be O-glycosylated. A fus1-GFP hybrid protein was tentatively located in the plasma membrane of dividing cells and upon activation of the pheromone response pathway, the protein seems to be relocated at the tip of elongated cells. KlFus1p is required for optimal conjugation of sexual partners and its expression is significantly enhanced by overexpression of both a constitutively active form of KlGpa1p, the G protein alpha subunit that triggers the mating response in this strain, and the KlSte12p transcription factor. Inactivation of the KlSte12 protein strongly reduces mating and affects KlFUS1 gene expression. The KlFUS1 gene has been deposited in the GenBank under accession number AF519444.