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1.
Int. microbiol ; 25(3): 639-647, Ago. 2022. ilus
Artigo em Inglês | IBECS | ID: ibc-216221

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.(AU)


Assuntos
Humanos , Retículo Endoplasmático , Tunicamicina , Glicosilação , Transcriptoma , Microbiologia
2.
Int Microbiol ; 25(3): 639-647, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35597864

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/farmacologia
3.
Crit Rev Microbiol ; 48(6): 657-695, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34893006

RESUMO

While fungi are widely occupying nature, many species are responsible for devastating mycosis in humans. Such niche diversity explains how quick fungal adaptation is necessary to endow the capacity of withstanding fluctuating environments and to cope with host-imposed conditions. Among all the molecular mechanisms evolved by fungi, the most studied one is the activation of the phosphorelay signalling pathways, of which the high osmolarity glycerol (HOG) pathway constitutes one of the key molecular apparatus underpinning fungal adaptation and virulence. In this review, we summarize the seminal knowledge of the HOG pathway with its more recent developments. We specifically described the HOG-mediated stress adaptation, with a particular focus on osmotic and oxidative stress, and point out some lags in our understanding of its involvement in the virulence of pathogenic species including, the medically important fungi Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, compared to the model yeast Saccharomyces cerevisiae. Finally, we also highlighted some possible applications of the HOG pathway modifications to improve the fungal-based production of natural products in the industry.


Assuntos
Produtos Biológicos , Glicerol , Humanos , Glicerol/metabolismo , Proteínas Fúngicas/metabolismo , Pressão Osmótica , Aspergillus fumigatus/metabolismo , Concentração Osmolar , Saccharomyces cerevisiae/metabolismo
4.
Curr Genet ; 66(5): 867-880, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32564133

RESUMO

The pheromone response and the high osmolarity glycerol (HOG) pathways are considered the prototypical MAPK signaling systems. They are the best-understood pathways in eukaryotic cells, yet they continue to provide insights in how cells relate with the environment. These systems are subjected to tight regulatory circuits to prevent hyperactivation in length and intensity. Failure to do this may be a matter of life or death specially for unicellular organisms such as Saccharomyces cerevisiae. The signaling pathways are fine-tuned by positive and negative feedback loops exerted by pivotal control elements that allow precise responses to specific stimuli, despite the fact that some elements of the systems are common to different signaling pathways. Here we describe the experimentally proven negative feedback loops that modulate the pheromone response and the HOG pathways. As described in this review, MAP kinases are central mechanistic components of these feedback loops. They have the capacity to modulate basal signaling activity, a fast extranuclear response, and a longer-lasting transcriptional process.


Assuntos
Retroalimentação Fisiológica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Feromônios/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Concentração Osmolar , Saccharomyces cerevisiae/crescimento & desenvolvimento
5.
Cells ; 8(7)2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31336877

RESUMO

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 Dobradas
6.
Cells ; 7(8)2018 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-30110882

RESUMO

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.

7.
Mol Cell Biol ; 38(13)2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29632077

RESUMO

The unfolded protein response (UPR) is an adaptive pathway that restores cellular homeostasis after endoplasmic reticulum (ER) stress. The ER-resident kinase/RNase Ire1 is the only UPR sensor conserved during evolution. Autophagy, a lysosomal degradative pathway, also contributes to the recovery of cell homeostasis after ER stress, but the interplay between these two pathways is still poorly understood. We describe the Dictyostelium discoideum ER stress response and characterize its single bona fide Ire1 orthologue, IreA. We found that tunicamycin (TN) triggers a gene-expression reprogramming that increases the protein folding capacity of the ER and alleviates ER protein load. Further, IreA is required for cell survival after TN-induced ER stress and is responsible for nearly 40% of the transcriptional changes induced by TN. The response of Dictyostelium cells to ER stress involves the combined activation of an IreA-dependent gene expression program and the autophagy pathway. These two pathways are independently activated in response to ER stress but, interestingly, autophagy requires IreA at a later stage for proper autophagosome formation. We propose that unresolved ER stress in cells lacking IreA causes structural alterations of the ER, leading to a late-stage blockade of autophagy clearance. This unexpected functional link may critically affect eukaryotic cell survival under ER stress.


Assuntos
Dictyostelium/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Autofagia/genética , Autofagia/fisiologia , Dictyostelium/citologia , Dictyostelium/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica , Genes de Protozoários , Homeostase , Modelos Biológicos , Mutagênese Sítio-Dirigida , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Ribonucleases/deficiência , Ribonucleases/genética , Ribonucleases/metabolismo , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas
8.
FEBS J ; 285(6): 1079-1096, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29341399

RESUMO

Yeast cells respond to hyperosmotic stress by activating the high-osmolarity glycerol (HOG) pathway, which consists of two branches, Hkr1/Msb2-Sho1 and Sln1, which trigger phosphorylation and nuclear internalization of the Hog1 mitogen-activated protein kinase. In the nucleus, Hog1 regulates gene transcription and cell cycle progression, which allows the cell to respond and adapt to hyperosmotic conditions. This study demonstrates that the uncoupling of the known sensors of both branches of the pathway at the level of Ssk1 and Ste11 impairs cell growth in hyperosmotic medium. However, under these conditions, Hog1 was still phosphorylated and internalized into the nucleus, suggesting the existence of an alternative Hog1 activation mechanism. In the ssk1ste11 mutant, phosphorylated Hog1 failed to associate with chromatin and to activate transcription of canonical hyperosmolarity-responsive genes. Accordingly, Hog1 also failed to induce glycerol production at the levels of a wild-type strain. Inactivation of the Ptp2 phosphatase moderately rescued growth impairment of the ssk1ste11 mutant under hyperosmotic conditions, indicating that downregulation of the HOG pathway only partially explains the phenotypes displayed by the ssk1ste11 mutant. Cell cycle defects were also observed in response to stress when Hog1 was phosphorylated in the ssk1ste11 mutant. Taken together, these observations indicate that Hog1 phosphorylation by noncanonical upstream mechanisms is not sufficient to trigger a protective response to hyperosmotic stress.


Assuntos
MAP Quinase Quinase Quinases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adaptação Fisiológica/genética , Regulação Fúngica da Expressão Gênica , Glicerol/metabolismo , MAP Quinase Quinase Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Concentração Osmolar , Pressão Osmótica , Fosforilação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico
9.
World J Microbiol Biotechnol ; 33(6): 111, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28470426

RESUMO

Cells contain signal transduction pathways that mediate communication between the extracellular environment and the cell interior. These pathways control transcriptional programs and posttranscriptional processes that modify cell metabolism in order to maintain homeostasis. One type of these signal transduction systems are the so-called Two Component Systems (TCS), which conduct the transfer of phosphate groups between specific and conserved histidine and aspartate residues present in at least two proteins; the first protein is a sensor kinase which autophosphorylates a histidine residue in response to a stimulus, this phosphate is then transferred to an aspartic residue located in a response regulator protein. There are classical and hybrid TCS, whose difference consists in the number of proteins and functional domains involved in the phosphorelay. The TCS are widespread in bacteria where the sensor and its response regulator are mostly specific for a given stimulus. In eukaryotic organisms such as fungi, slime molds, and plants, TCS are present as hybrid multistep phosphorelays, with a variety of arrangements (Stock et al. in Annu Rev Biochem 69:183-215, 2000; Wuichet et al. in Curr Opin Microbiol 292:1039-1050, 2010). In these multistep phosphorelay systems, several phosphotransfer events take place between different histidine and aspartate residues localized in specific domains present in more than two proteins (Thomason and Kay, in J Cell Sci 113:3141-3150, 2000; Robinson et al. in Nat Struct Biol 7:626-633, 2000). This review presents a brief and succinct description of the Two-component systems of model yeasts, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Cryptococcus neoformans and Kluyveromyces lactis. We have focused on the comparison of domain organization and functions of each component present in these phosphorelay systems.


Assuntos
Fosfatos/metabolismo , Transdução de Sinais/fisiologia , Leveduras/metabolismo , Ácido Aspártico/metabolismo , Candida albicans/metabolismo , Cryptococcus neoformans/metabolismo , Proteínas Fúngicas , Histidina/metabolismo , Histidina Quinase , Kluyveromyces/metabolismo , Fosforilação/fisiologia , Fosfotransferases/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo
10.
Mol Microbiol ; 104(5): 822-836, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28295748

RESUMO

The Kluyveromyces lactis SLN1 phosphorelay system includes the osmosensor histidine kinase Sln1, the phosphotransfer protein Ypd1 and the response regulator Ssk1. Here we show that K. lactis has a functional phosphorelay system. In vitro assays, using a heterologous histidine kinase, show that the phosphate group is accepted by KlYpd1 and transferred to KlSsk1. Upon hyperosmotic stress the phosphorelay is inactivated, KlYpd1 is dephosphorylated in a KlSln1 dependent manner, and only the version of KlSsk1 that lacks the phosphate group interacts with the MAPKKK KlSsk2. Interestingly, inactivation of the KlPtp2 phosphatase in a ΔKlsln1 mutant did not lead to KlHog1 constitutive phosphorylation. KlHog1 can replace ScHog1p and activate the hyperosmotic response in Saccharomyces cerevisiae, and when ScSln1 is inactivated, KlHog1 becomes phosphorylated and induces cell lethality. All these observations indicate that the phosphorelay negatively regulates KlHog1. Nevertheless, in the absence of KlSln1 or KlYpd1, no constitutive phosphorylation is detected and cells are viable, suggesting that a strong negative feedback that is independent of KlPtp2 operates in K. lactis. Compared with S. cerevisiae, K. lactis has only a moderate accumulation of glycerol and fails to produce trehalose under hyperosmotic stress, indicating that regulation of osmolyte production is different in K. lactis.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Kluyveromyces/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Concentração Osmolar , Fosforilação , Proteínas Quinases/metabolismo , Transdução de Sinais , Estresse Fisiológico
11.
Eukaryot Cell ; 14(9): 922-30, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26150414

RESUMO

When treated with a hyperosmotic stimulus, Kluyveromyces lactis cells respond by activating the mitogen-activated protein kinase (MAPK) K. lactis Hog1 (KlHog1) protein via two conserved branches, SLN1 and SHO1. Mutants affected in only one branch can cope with external hyperosmolarity by activating KlHog1p by phosphorylation, except for single ΔKlste11 and ΔKlste50 mutants, which showed high sensitivity to osmotic stress, even though the other branch (SLN1) was intact. Inactivation of both branches by deletion of KlSHO1 and KlSSK2 also produced sensitivity to high salt. Interestingly, we have observed that in ΔKlste11 and ΔKlsho1 ΔKlssk2 mutants, which exhibit sensitivity to hyperosmotic stress, and contrary to what would be expected, KlHog1p becomes phosphorylated. Additionally, in mutants lacking both MAPK kinase kinases (MAPKKKs) present in K. lactis (KlSte11p and KlSsk2p), the hyperosmotic stress induced the phosphorylation and nuclear internalization of KlHog1p, but it failed to induce the transcriptional expression of KlSTL1 and the cell was unable to grow in high-osmolarity medium. KlHog1p phosphorylation via the canonical HOG pathway or in mutants where the SHO1 and SLN1 branches have been inactivated requires not only the presence of KlPbs2p but also its kinase activity. This indicates that when the SHO1 and SLN1 branches are inactivated, high-osmotic-stress conditions activate an independent input that yields active KlPbs2p, which, in turn, renders KlHog1p phosphorylation ineffective. Finally, we found that KlSte11p can alleviate the sensitivity to hyperosmotic stress displayed by a ΔKlsho1 ΔKlssk2 mutant when it is anchored to the plasma membrane by adding the KlSho1p transmembrane segments, indicating that this chimeric protein can substitute for KlSho1p and KlSsk2p.


Assuntos
Kluyveromyces/genética , Sistema de Sinalização das MAP Quinases , Pressão Osmótica , Estresse Fisiológico , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Kluyveromyces/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo
12.
PLoS One ; 7(9): e45702, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23049841

RESUMO

BACKGROUND: Gene duplication and the subsequent divergence of paralogous pairs play a central role in the evolution of novel gene functions. S. cerevisiae possesses two paralogous genes (ALT1/ALT2) which presumably encode alanine aminotransferases. It has been previously shown that Alt1 encodes an alanine aminotransferase, involved in alanine metabolism; however the physiological role of Alt2 is not known. Here we investigate whether ALT2 encodes an active alanine aminotransferase. PRINCIPAL FINDINGS: Our results show that although ALT1 and ALT2 encode 65% identical proteins, only Alt1 displays alanine aminotransferase activity; in contrast ALT2 encodes a catalytically inert protein. ALT1 and ALT2 expression is modulated by Nrg1 and by the intracellular alanine pool. ALT1 is alanine-induced showing a regulatory profile of a gene encoding an enzyme involved in amino acid catabolism, in agreement with the fact that Alt1 is the sole pathway for alanine catabolism present in S. cerevisiae. Conversely, ALT2 expression is alanine-repressed, indicating a role in alanine biosynthesis, although the encoded-protein has no alanine aminotransferase enzymatic activity. In the ancestral-like yeast L. kluyveri, the alanine aminotransferase activity was higher in the presence of alanine than in the presence of ammonium, suggesting that as for ALT1, LkALT1 expression could be alanine-induced. ALT2 retention poses the questions of whether the encoded protein plays a particular function, and if this function was present in the ancestral gene. It could be hypotesized that ALT2 diverged after duplication, through neo-functionalization or that ALT2 function was present in the ancestral gene, with a yet undiscovered function. CONCLUSIONS: ALT1 and ALT2 divergence has resulted in delegation of alanine aminotransferase activity to Alt1. These genes display opposed regulatory profiles: ALT1 is alanine-induced, while ALT2 is alanine repressed. Both genes are negatively regulated by the Nrg1 repressor. Presented results indicate that alanine could act as ALT2 Nrg1-co-repressor.


Assuntos
Alanina Transaminase/química , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/enzimologia , Alanina/química , Catálise , Sistema Livre de Células , Proteínas Correpressoras , Ativação Enzimática , Evolução Molecular , Glucose/química , Modelos Biológicos , Modelos Químicos , Oligonucleotídeos/genética , Filogenia
13.
Eukaryot Cell ; 10(4): 540-6, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21335532

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-Híbrido
14.
Mol Biol Cell ; 21(3): 489-98, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20016006

RESUMO

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-Híbrido
15.
Eukaryot Cell ; 7(1): 78-85, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18024598

RESUMO

Systematic disruption of genes encoding kinases and mitogen-activated protein kinases (MAPKs) was performed in Kluyveromyces lactis haploid cells. The mutated strains were assayed by their capacity to mate and to respond to hyperosmotic stress. The K. lactis Ste11p (KlSte11p) MAPK kinase kinase (MAPKKK) was found to act in both mating and osmoresponse pathways while the scaffold KlSte5p and the MAPK KlFus3p appeared to be specific for mating. The p21-activated kinase KlSte20p and the kinase KlSte50p participated in both pathways. Protein association experiments showed interaction of KlSte50p and KlSte20p with Galpha and Gbeta, respectively, the G protein subunits involved in the mating pathway. Both KlSte50p and KlSte20p also showed interaction with KlSte11p. Disruption mutants of the K. lactis PBS2 (KlPBS2) and KlHOG1 genes of the canonical osmotic response pathway resulted in mutations sensitive to high salt and high sorbitol but dispensable for mating. Mutations that eliminate the MAPKK KlSte7p activity had a strong effect on mating and also showed sensitivity to osmotic stress. Finally, we found evidence of physical interaction between KlSte7p and KlHog1p, in addition to diminished Hog1p phosphorylation after a hyperosmotic shock in cells lacking KlSte7p. This study reveals novel roles for components of transduction systems in yeast.


Assuntos
Proteínas Fúngicas/metabolismo , Genes Fúngicos Tipo Acasalamento , Kluyveromyces/enzimologia , Pressão Osmótica , Proteínas Quinases/metabolismo , Proteínas Fúngicas/genética , Kluyveromyces/genética , 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 , Mutação , Concentração Osmolar , Osmose , Fosforilação , Proteínas Quinases/genética , Transdução de Sinais
16.
FEMS Yeast Res ; 7(5): 740-7, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17506833

RESUMO

The Kluyveromyces lactis genes for sexual pheromones have been analyzed. The alpha-factor gene encodes a predicted polypeptide of 187 amino acid residues containing four tridecapeptide repeats (WSWITLRPGQPIF). A nucleotide blast search of the entire K. lactis genome sequence allowed the identification of the nonannotated putative a-pheromone gene that encodes a predicted protein of 33 residues containing one copy of the dodecapeptide a-factor (WIIPGFVWVPQC). The role of the K. lactis structural genes KlMFalpha1 and KlMFA1 in mating has been investigated by the construction of disruption mutations that totally eliminate gene functions. Mutants of both alleles showed sex-dependent sterility, indicating that these are single-copy genes and essential for mating. MATalpha, Klsst2 mutants, which, by analogy to Saccharomyces cerevisiae, are defective in Galpha-GTPase activity, showed increased sensitivity to synthetic alpha-factor and increased capacity to mate. Additionally, Klbar1 mutants (putatively defective in alpha-pheromone proteolysis) showed delay in mating but sensitivity to alpha-pheromone. From these results, it can be deduced that the K. lactis MATa cell produces the homolog of the S. cerevisiaealpha-pheromone, whereas the MATalpha cell produces the a-pheromone.


Assuntos
Regulação Fúngica da Expressão Gênica , Kluyveromyces/efeitos dos fármacos , Kluyveromyces/genética , Peptídeos/farmacologia , Feromônios , Transdução de Sinais , Sequência de Aminoácidos , Sequência de Bases , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacologia , Deleção de Genes , Genes Fúngicos , Kluyveromyces/metabolismo , Kluyveromyces/fisiologia , Fator de Acasalamento , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/genética , Peptídeos/metabolismo , Feromônios/síntese química , Feromônios/genética , Feromônios/metabolismo , Feromônios/farmacologia
17.
Can J Microbiol ; 52(7): 627-35, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16917518

RESUMO

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 Aminoácidos
18.
FEMS Yeast Res ; 6(3): 336-44, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16630274

RESUMO

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/genética
19.
Arch Med Res ; 37(1): 102-8, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16314194

RESUMO

BACKGROUND: Familial hypercholesterolemia (FH) and familial defective apolipoprotein B-100 (FDB) are relatively common lipid disorders caused by mutations of the low-density lipoprotein receptor (LDLR) and apolipoprotein B (apoB) genes, respectively. A third locus on chromosome 1p34.1-p32 was recently linked to FH and the responsible gene has been identified [protein convertase subtilisin/kexin type 9 (PCSK9)]. METHODS: We assessed the contribution of the LDLR, apoB, and PCSK9 genes as cause of FH in Mexico. Forty six unrelated probands, as well as 68 affected and 60 healthy relatives, were included. RESULTS: All index cases were diagnosed as having heterozygous autosomal dominant FH. Seventeen of the 46 index cases had LDLR gene mutations, four of which were novel (Fs92ter108, C268R, Q718X, and Fs736ter743); and only one patient had an apoB mutation (R3500Q). We sequenced the PCSK9 gene in the remainder of the 28 probands with no identified LDLR or APOB gene defects; however, no PCSK9 mutations were found, including one large kindred with positive linkage to the 1p34.1-32 locus (multipoint LOD score of 3.3) and two small pedigrees. Linkage was excluded from these three loci in at least four kindreds suggesting that other yet uncharacterized genes are involved. CONCLUSIONS: Our results underline substantial genetic heterogeneity for FH in the Mexican population.


Assuntos
Apolipoproteínas B/genética , Cromossomos Humanos Par 1/genética , Heterogeneidade Genética , Hiperlipoproteinemia Tipo II/genética , Receptores de LDL/genética , Serina Endopeptidases/genética , Adulto , Apolipoproteína B-100 , Feminino , Humanos , Escore Lod , Masculino , México , Pessoa de Meia-Idade , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Locos de Características Quantitativas
20.
Yeast ; 22(12): 947-56, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16134098

RESUMO

In the yeast Saccharomyces cerevisiae the Gbetagamma dimer of the heterotrimeric G protein transduces a pheromone signal from serpentine receptor to a MAP kinase cascade that activates the mating response pathway. Haploid cells lacking the Gbeta subunit do not respond to sexual pheromone, leading to sterility. In this work we demonstrate that the beta-subunit of Kluyveromyces lactis, encoded by the KlSTE4 gene, is a component of the G protein, and that its disruption gives rise to sterile cells. However, unlike Ste4p in S. cerevisiae, its overexpression does not induce growth arrest or promote mating. It has been shown that in K. lactis, the Galpha subunit has a positive role in the mating process, hence the resulting double GalphaDelta GbetaDelta mutant was viable and sterile. Here we show that the overproduction of Gbeta subunit fails to rescue GalphaDelta mutant from sterility and that expression of a constitutive active allele of Galpha enhances transcription of the KlSTE4 gene. The mating pathway triggered by the Gbeta-subunit requires a functional KlSte12p transcription factor. Gbeta has a 10-fold higher association rate with the Galpha1 subunit involved in pheromone response than with Galpha2, the protein involved in cAMP regulation in K. lactis. Additionally, the Gbeta-subunit from K. lactis is able to interact with the Galpha-subunit from S. cerevisiae but fails to restore the mating deficiency of Scste4Delta mutant. The data presented indicate that the mating pathway of K. lactis is positively and cooperatively regulated by both the Galpha and the Gbeta subunits.


Assuntos
Proteínas Fúngicas/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Kluyveromyces/metabolismo , Sequência de Aminoácidos , Proteínas Fúngicas/genética , 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/genética , Genes Fúngicos , Kluyveromyces/genética , Kluyveromyces/crescimento & desenvolvimento , Dados de Sequência Molecular , Mutação , Feromônios/genética , Feromônios/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Especificidade da Espécie
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