A Ste6p/P-glycoprotein homologue from the asexual yeast Candida albicans transports the a-factor mating pheromone in Saccharomyces cerevisiae

Abstract In the yeast Saccharomyces cerevisiae, mating pheromone response is initiated by activation of a G protein- and mitogen-activated protein (MAP) kinase-dependent signaling pathway and attenuated by several mechanisms that promote adaptation or desensitization. To identify genes whose products negatively regulate pheromone signaling, we screened for mutations that suppress the hyperadaptive phenotype of wild-type cells overexpressing signaling-defective G protein β subunits. This identified recessive mutations in MOT3, which encodes a nuclear protein with two Cys2-His2 Zn fingers. MOT3 was found to be a dosage-dependent inhibitor of pheromone response and pheromone-induced gene expression and to require an intact signaling pathway to exert its effects. Several results suggested that Mot3 attenuates expression of pheromone-responsive genes by mechanisms distinct from those used by the negative transcriptional regulators Cdc36, Cdc39, and Mot2. First, a Mot3-lexA fusion functions as a transcriptional activator. Second, Mot3 is a dose-dependent activator of several genes unrelated to pheromone response, including CYC1, SUC2, and LEU2. Third, insertion of consensus Mot3 binding sites (C/A/T)AGG(T/C)A activates a promoter in a MOT3-dependent manner. These findings, and the fact that consensus binding sites are found in the 5′ flanking regions of many yeast genes, suggest that Mot3 is a globally acting transcriptional regulator. We hypothesize that Mot3 regulates expression of factors that attenuate signaling by the pheromone response pathway.

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Divergent Subunit Interactions among Fungal mRNA 5′-Capping Machineries

Eukaryotic Cell ◽

10.1128/ec.1.3.448-457.2002 ◽

2002 ◽

Vol 1 (3) ◽

pp. 448-457 ◽

Cited By ~ 17

Author(s):

Toshimitsu Takagi ◽

Eun-Jung Cho ◽

Rozmin T. K. Janoo ◽

Vladimir Polodny ◽

Yasutaka Takase ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Candida Albicans ◽

Rna Polymerase Ii ◽

Subunit Interactions ◽

Pol Ii ◽

Mrna Capping ◽

Capping Enzyme ◽

Enzyme Subunits ◽

Carboxyl Terminal

ABSTRACT The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5′-triphosphatase (RTPase) and GTP::mRNA guanylyltransferase (GTase). The GTase subunit (Ceg1) binds to the phosphorylated carboxyl-terminal domain of the largest subunit (CTD-P) of RNA polymerase II (pol II), coupling capping with transcription. Ceg1 bound to the CTD-P is inactive unless allosterically activated by interaction with the RTPase subunit (Cet1). For purposes of comparison, we characterize here the related GTases and RTPases from the yeasts Schizosaccharomyces pombe and Candida albicans. Surprisingly, the S. pombe capping enzyme subunits do not interact with each other. Both can independently interact with CTD-P of pol II, and the GTase is not repressed by CTD-P binding. The S. pombe RTPase gene (pct1 +) is essential for viability. Pct1 can replace the S. cerevisiae RTPase when GTase activity is supplied by the S. pombe or mouse enzymes but not by the S. cerevisiae GTase. The C. albicans capping enzyme subunits do interact with each other. However, this interaction is not essential in vivo. Our results reveal an unexpected diversity among the fungal capping machineries.

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Overexpression of Candida albicans secretory aspartyl proteinase 2 and its expression in Saccharomyces cerevisiae do not augment virulence in mice

Microbiology ◽

10.1099/00221287-144-8-2299 ◽

1998 ◽

Vol 144 (8) ◽

pp. 2299-2310 ◽

Cited By ~ 13

Author(s):

N. Dubois ◽

A. R. Colina ◽

F. Aumont ◽

P. Belhumeur ◽

L. de Repentigny

Keyword(s):

Saccharomyces Cerevisiae ◽

Candida Albicans

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Essential role for induced Ca2+ influx followed by [Ca2+]i rise in maintaining viability of yeast cells late in the mating pheromone response pathway. A study of [Ca2+]i in single Saccharomyces cerevisiae cells with imaging of fura-2.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)38311-5 ◽

1990 ◽

Vol 265 (22) ◽

pp. 13391-13399

Author(s):

H Iida ◽

Y Yagawa ◽

Y Anraku

Keyword(s):

Saccharomyces Cerevisiae ◽

Essential Role ◽

Yeast Cells ◽

Pheromone Response ◽

Mating Pheromone ◽

Fura 2 ◽

Pheromone Response Pathway

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CaNdt80 Is Involved in Drug Resistance in Candida albicans by Regulating CDR1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.12.4505-4512.2004 ◽

2004 ◽

Vol 48 (12) ◽

pp. 4505-4512 ◽

Cited By ~ 66

Author(s):

Chia-Geun Chen ◽

Yun-Liang Yang ◽

Hsin-I Shih ◽

Chia-Li Su ◽

Hsiu-Jung Lo

Keyword(s):

Saccharomyces Cerevisiae ◽

Drug Resistance ◽

Candida Albicans ◽

Type Strain ◽

Antifungal Agents ◽

Wild Type Strain ◽

Efflux Pump ◽

Antifungal Drugs ◽

Galactosidase Activity ◽

Wild Type

ABSTRACT Overexpression of CDR1, an efflux pump, is one of the major mechanisms contributing to drug resistance in Candida albicans. CDR1 p-lacZ was constructed and transformed into a Saccharomyces cerevisiae strain so that the lacZ gene could be used as the reporter to monitor the activity of the CDR1 promoter. Overexpression of CaNDT80, the C. albicans homolog of S. cerevisiae NDT80, increases the β-galactosidase activity of the CDR1 p-lacZ construct in S. cerevisiae. Furthermore, mutations in CaNDT80 abolish the induction of CDR1 expression by antifungal agents in C. albicans. Consistently, the Candt80/Candt80 mutant is also more susceptible to antifungal drugs than the wild-type strain. Thus, the gene for CaNdt80 may be the first gene among the regulatory factors involved in drug resistance in C. albicans whose function has been identified.

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Cell Wall-Related Bionumbers and Bioestimates of Saccharomyces cerevisiae and Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00250-13 ◽

2013 ◽

Vol 13 (1) ◽

pp. 2-9 ◽

Cited By ~ 58

Author(s):

Frans M. Klis ◽

Chris G. de Koster ◽

Stanley Brul

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Candida Albicans ◽

Cell Size ◽

Pathogenic Fungus ◽

Turgor Pressure ◽

Hyphal Growth ◽

Biological Research ◽

Content Type ◽

Starting Point

ABSTRACTBionumbers and bioestimates are valuable tools in biological research. Here we focus on cell wall-related bionumbers and bioestimates of the budding yeastSaccharomyces cerevisiaeand the polymorphic, pathogenic fungusCandida albicans. We discuss the linear relationship between cell size and cell ploidy, the correlation between cell size and specific growth rate, the effect of turgor pressure on cell size, and the reason why using fixed cells for measuring cellular dimensions can result in serious underestimation ofin vivovalues. We further consider the evidence that individual buds and hyphae grow linearly and that exponential growth of the population results from regular formation of new daughter cells and regular hyphal branching. Our calculations show that hyphal growth allowsC. albicansto cover much larger distances per unit of time than the yeast mode of growth and that this is accompanied by strongly increased surface expansion rates. We therefore predict that the transcript levels of genes involved in wall formation increase during hyphal growth. Interestingly, wall proteins and polysaccharides seem barely, if at all, subject to turnover and replacement. A general lesson is how strongly most bionumbers and bioestimates depend on environmental conditions and genetic background, thus reemphasizing the importance of well-defined and carefully chosen culture conditions and experimental approaches. Finally, we propose that the numbers and estimates described here offer a solid starting point for similar studies of other cell compartments and other yeast species.

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Efecto proapoptótico y antimetastásico en líneas tumorales humanas colorrectales de una proteína secretada por la bacteria Rizosférica Antártica Bacillus sp. K2I17

10.32457/20.500.12728/87432019dcbm4 ◽

2019 ◽

Author(s):

Alequis Tomás Pavón Oro

Keyword(s):

Saccharomyces Cerevisiae ◽

Staphylococcus Aureus ◽

Candida Albicans ◽

Deschampsia Antarctica ◽

Bacillus Sp ◽

E Coli ◽

Cáncer Colorrectal

El cáncer es la segunda causa de muerte en el mundo, y específicamente en Chile el cáncer colorrectal es el único que presenta un aumento sostenido de la mortalidad en la última década. La búsqueda de nuevos agentes quimioterapeúticos anticancerígenos ha propuesto a los microorganismos extremófilos como una fuente potencial para obtener moléculas citotóxicas, que induzcan apoptosis en las células tumorales. Las condiciones extremas del continente antártico y las presiones selectivas por el espacio y los nutrientes que se producen entre los microorganismos del rizobioma de la planta Deschampsia antarctica Desv sugirieron como hipótesis que las bacterias rizosféricas aisladas en la Antártica secretan al sobrenadante de cultivo moléculas bioactivas que inhiben la invasión y proliferación de líneas tumorales humanas de origen colorrectal mediante un mecanismo apoptótico. En este sentido, el objetivo general del trabajo fue identificar y caracterizar a moléculas bioactivas con acción antinvasiva y antiproliferativa, además, determinar el mecanismo inhibitorio de la proliferación en líneas tumorales humanas de origen colorrectal. Los resultados del primer objetivo específico demostraron que los sobrenadantes de cultivo de los aislados rizosféricos antárticos K2 y MI disminuyeron la viabilidad de la línea celular de adenocarcinoma colorrectal LoVo en el ensayo de reducción metabólica del MTT. Además, como los sobrenadantes no tuvieron efecto en la viabilidad de las bacterias E. coli y Staphylococcus aureus, y tampoco en los hongos unicelulares Candida albicans y Saccharomyces cerevisiae, el resultado indicó que la actividad antiproliferativa fue selectiva hacia la línea celular LoVo.

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Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3603-3612.1991 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3603-3612

Author(s):

S Marcus ◽

G A Caldwell ◽

D Miller ◽

C B Xue ◽

F Naider ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Biological Activity ◽

Methyl Ester ◽

Total Synthesis ◽

Biologically Active ◽

Structural Genes ◽

Wild Type ◽

Mating Pheromone ◽

Carboxy Terminal

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

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The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.120.5.1203 ◽

1993 ◽

Vol 120 (5) ◽

pp. 1203-1215 ◽

Cited By ~ 59

Author(s):

K Kuchler ◽

H G Dohlman ◽

J Thorner

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Gene Product ◽

Cell Polarity ◽

Polyclonal Antibodies ◽

Apparent Molecular Weight ◽

Subcellular Fractionation ◽

Mating Pheromone ◽

Yeast Saccharomyces Cerevisiae ◽

Factor Secretion

STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.

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Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3603 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3603-3612 ◽

Cited By ~ 74

Author(s):

S Marcus ◽

G A Caldwell ◽

D Miller ◽

C B Xue ◽

F Naider ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Biological Activity ◽

Methyl Ester ◽

Total Synthesis ◽

Biologically Active ◽

Structural Genes ◽

Wild Type ◽

Mating Pheromone ◽

Carboxy Terminal

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

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