Regulation of Cell Polarity through Phosphorylation of Bni4 by Pho85 G1 Cyclin-dependent Kinases in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the G1-specific cyclin-dependent kinases (Cdks) Cln1,2-Cdc28 and Pcl1,2-Pho85 are essential for ensuring that DNA replication and cell division are properly linked to cell polarity and bud morphogenesis. However, the redundancy of Cdks and cyclins means that identification of relevant Cdk substrates remains a significant challenge. We used array-based genetic screens (synthetic genetic array or SGA analysis) to dissect redundant pathways associated with G1 cyclins and identified Bni4 as a substrate of the Pcl1- and Pcl2-Pho85 kinases. BNI4 encodes an adaptor protein that targets several proteins to the bud neck. Deletion of BNI4 results in severe growth defects in the absence of the Cdc28 cyclins Cln1 and Cln2, and overexpression of BNI4 is toxic in yeast cells lacking the Pho85 cyclins Pcl1 and Pcl2. Phosphorylation of Bni4 by Pcl-Pho85 is necessary for its localization to the bud neck, and the bud neck structure can be disrupted by overexpressing BNI4 in pcl1Δpcl2Δ mutant cells. Our data suggest that misregulated Bni4 may bind in an uncontrolled manner to an essential component that resides at the bud neck, causing catastrophic morphogenesis defects.

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Pulsed Electric Field (PEF) Enhances Iron Uptake by the Yeast Saccharomyces cerevisiae

Biomolecules ◽

10.3390/biom11060850 ◽

2021 ◽

Vol 11 (6) ◽

pp. 850

Author(s):

Karolina Nowosad ◽

Monika Sujka ◽

Urszula Pankiewicz ◽

Damijan Miklavčič ◽

Marta Arczewska

Keyword(s):

Saccharomyces Cerevisiae ◽

Electric Field ◽

Metal Ion ◽

Treatment Time ◽

Control Sample ◽

Pulsed Electric Field ◽

Yeast Cells ◽

Iron Ions ◽

Metal Ion Binding ◽

Yeast Saccharomyces Cerevisiae

The aim of the study was to investigate the influence of a pulsed electric field (PEF) on the level of iron ion accumulation in Saccharomyces cerevisiae cells and to select PEF conditions optimal for the highest uptake of this element. Iron ions were accumulated most efficiently when their source was iron (III) nitrate. When the following conditions of PEF treatment were used: voltage 1500 V, pulse width 10 μs, treatment time 20 min, and a number of pulses 1200, accumulation of iron ions in the cells from a 20 h-culture reached a maximum value of 48.01 mg/g dry mass. Application of the optimal PEF conditions thus increased iron accumulation in cells by 157% as compared to the sample enriched with iron without PEF. The second derivative of the FTIR spectra of iron-loaded and -unloaded yeast cells allowed us to determine the functional groups which may be involved in metal ion binding. The exposure of cells to PEF treatment only slightly influenced the biomass and cell viability. However, iron-enriched yeast (both with or without PEF) showed lower fermentative activity than a control sample. Thus obtained yeast biomass containing a high amount of incorporated iron may serve as an alternative to pharmacological supplementation in the state of iron deficiency.

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A New Saccharomyces cerevisiae Strain with a Mutant Smt3-Deconjugating Ulp1 Protein Is Affected in DNA Replication and Requires Srs2 and Homologous Recombination for Its Viability

Molecular and Cellular Biology ◽

10.1128/mcb.24.12.5130-5143.2004 ◽

2004 ◽

Vol 24 (12) ◽

pp. 5130-5143 ◽

Cited By ~ 32

Author(s):

Christine Soustelle ◽

Laurence Vernis ◽

Karine Fréon ◽

Anne Reynaud-Angelin ◽

Roland Chanet ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Nonhomologous End Joining ◽

End Joining ◽

Recombination Mechanism ◽

Growth Defects ◽

Protein Conjugates ◽

Synthetically Lethal ◽

Mutant Cells ◽

Insight Into

ABSTRACT The Saccharomyces cerevisiae Srs2 protein is involved in DNA repair and recombination. In order to gain better insight into the roles of Srs2, we performed a screen to identify mutations that are synthetically lethal with an srs2 deletion. One of them is a mutated allele of the ULP1 gene that encodes a protease specifically cleaving Smt3-protein conjugates. This allele, ulp1-I615N, is responsible for an accumulation of Smt3-conjugated proteins. The mutant is unable to grow at 37°C. At permissive temperatures, it still shows severe growth defects together with a strong hyperrecombination phenotype and is impaired in meiosis. Genetic interactions between ulp1 and mutations that affect different repair pathways indicated that the RAD51-dependent homologous recombination mechanism, but not excision resynthesis, translesion synthesis, or nonhomologous end-joining processes, is required for the viability of the mutant. Thus, both Srs2, believed to negatively control homologous recombination, and the process of recombination per se are essential for the viability of the ulp1 mutant. Upon replication, mutant cells accumulate single-stranded DNA interruptions. These structures are believed to generate different recombination intermediates. Some of them are fixed by recombination, and others require Srs2 to be reversed and fixed by an alternate pathway.

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Rsp5p, a New Link between the Actin Cytoskeleton and Endocytosis in the Yeast Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.22.20.6946-6958.2002 ◽

2002 ◽

Vol 22 (20) ◽

pp. 6946-6948 ◽

Cited By ~ 42

Author(s):

Joanna Kamińska ◽

Beata Gajewska ◽

Anita K. Hopper ◽

Teresa ˙Zołądek

Keyword(s):

Saccharomyces Cerevisiae ◽

Actin Cytoskeleton ◽

Cytoskeletal Proteins ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Ww Domains ◽

Ubiquitin Protein Ligase ◽

Growth Defects ◽

Genes Encoding ◽

Cytoskeleton Dynamics

ABSTRACT Rsp5p is an ubiquitin-protein ligase of Saccharomyces cerevisiae that has been implicated in numerous processes including transcription, mitochondrial inheritance, and endocytosis. Rsp5p functions at multiple steps of endocytosis, including ubiquitination of substrates and other undefined steps. We propose that one of the roles of Rsp5p in endocytosis involves maintenance and remodeling of the actin cytoskeleton. We report the following. (i) There are genetic interactions between rsp5 and several mutant genes encoding actin cytoskeletal proteins. rsp5 arp2, rsp5 end3, and rsp5 sla2 double mutants all show synthetic growth defects. Overexpressed wild-type RSP5 or mutant rsp5 genes with lesions of some WW domains suppress growth defects of arp2 and end3 cells. The defects in endocytosis, actin cytoskeleton, and morphology of arp2 are also suppressed. (ii) Rsp5p and Sla2p colocalize in abnormal F-actin-containing clumps in arp2 and pan1 mutants. Immunoprecipitation experiments confirmed that Rsp5p and Act1p colocalize in pan1 mutants. (iii) Rsp5p and Sla2p coimmunoprecipitate and partially colocalize to punctate structures in wild-type cells. These studies provide the first evidence for an interaction of an actin cytoskeleton protein with Rsp5p. (iv) rsp5-w1 mutants are resistant to latrunculin A, a drug that sequesters actin monomers and depolymerizes actin filaments, consistent with the fact that Rsp5p is involved in actin cytoskeleton dynamics.

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The Cloning and Mapping of ADR6, a Gene Required for Sporulation and for Expression of the Alcohol Dehydrogenase II Isozyme From Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/116.4.531 ◽

1987 ◽

Vol 116 (4) ◽

pp. 531-540

Author(s):

Aileen K W Taguchi ◽

Elton T Young

Keyword(s):

Saccharomyces Cerevisiae ◽

Alcohol Dehydrogenase ◽

Single Gene ◽

Carbon Sources ◽

Transcription Unit ◽

Yeast Cells ◽

Recessive Mutation ◽

Yeast Saccharomyces Cerevisiae ◽

Upstream Activating Sequence ◽

A Site

ABSTRACT The alcohol dehydrogenase II (ADH2) gene of the yeast, Saccharomyces cerevisiae, is not transcribed during growth on fermentable carbon sources such as glucose. Growth of yeast cells in a medium containing only nonfermentable carbon sources leads to a marked increase or derepression of ADH2 expression. The recessive mutation, adr6-1, leads to an inability to fully derepress ADH2 expression and to an inability to sporulate. The ADR6 gene product appears to act directly or indirectly on ADH2 sequences 3' to or including the presumptive TATAA box. The upstream activating sequence (UAS) located 5' to the TATAA box is not required for the Adr6- phenotype. Here, we describe the isolation of a recombinant plasmid containing the wild-type ADR6 gene. ADR6 codes for a 4.4-kb RNA which is present during growth both on glucose and on nonfermentable carbon sources. Disruption of the ADR6 transcription unit led to viable cells with decreased ADHII activity and an inability to sporulate. This indicates that both phenotypes result from mutations within a single gene and that the adr6-1 allele was representative of mutations at this locus. The ADR6 gene mapped to the left arm of chromosome XVI at a site 18 centimorgans from the centromere.

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Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.442-451.1989 ◽

1989 ◽

Vol 9 (2) ◽

pp. 442-451

Author(s):

M Nishizawa ◽

R Araki ◽

Y Teranishi

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Pyruvate Kinase ◽

Transcriptional Activation ◽

Regulatory Elements ◽

Noncoding Region ◽

Yeast Cells ◽

Kinase Gene ◽

Yeast Saccharomyces Cerevisiae ◽

Upstream Activating Sequence

To clarify carbon source-dependent control of the glycolytic pathway in the yeast Saccharomyces cerevisiae, we have initiated a study of transcriptional regulation of the pyruvate kinase gene (PYK). By deletion analysis of the 5'-noncoding region of the PYK gene, we have identified an upstream activating sequence (UASPYK1) located between 634 and 653 nucleotides upstream of the initiating ATG codon. The promoter activity of the PYK 5'-noncoding region was abolished when the sequence containing the UASPYK1 was deleted from the region. Synthetic UASPYK1 (26mer), in either orientation, was able to restore the transcriptional activity of UAS-depleted mutants when placed upstream of the TATA sequence located at -199 (ATG as +1). While the UASPYK1 was required for basal to intermediate levels of transcriptional activation, a sequence between -714 and -811 was found to be necessary for full activation. On the other hand, a sequence between -344 and -468 was found to be responsible for transcriptional repression of the PYK gene when yeast cells were grown on nonfermentable carbon sources. This upstream repressible sequence also repressed transcription, although to a lesser extent, when glucose was present in the medium. The possible mechanism for carbon source-dependent regulation of PYK expression through these cis-acting regulatory elements is discussed.

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INTERCONVERSION OF YEAST MATING TYPES II. RESTORATION OF MATING ABILITY TO STERILE MUTANTS IN HOMOTHALLIC AND HETEROTHALLIC STRAINS

Genetics ◽

10.1093/genetics/85.3.373 ◽

1977 ◽

Vol 85 (3) ◽

pp. 373A-393

Author(s):

James B Hicks ◽

Ira Herskowitz

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Yeast Cells ◽

Mating Types ◽

Yeast Saccharomyces Cerevisiae ◽

Type Locus ◽

Mating Ability ◽

Mating Type Locus ◽

Regulatory Information ◽

Ho Gene

ABSTRACT The two mating types of the yeast Saccharomyces cerevisiae can be interconverted in both homothallic and heterothallic strains. Previous work indicates that all yeast cells contain the information to be both a and α and that the HO gene (in homothallic strains) promotes a change in mating type by causing a change at the mating type locus itself. In both heterothallic and homothallic strains, a defective α mating type locus can be converted to a functional a locus and subsequently to a functional α locus. In contrast, action of the HO gene does not restore mating ability to a strain defective in another gene for mating which is not at the mating type locus. These observations indicate that a yeast cell contains an additional copy (or copies) of α information, and lead to the "cassette" model for mating type interconversion. In this model, HM a and hmα loci are blocs of unexpressed α regulatory information, and HMα and hm a loci are blocs of unexpressed a regulatory information. These blocs are silent because they lack an essential site for expression, and become active upon insertion of this information (or a copy of the information) into the mating type locus by action of the HO gene.

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The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.5010-5019.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 5010-5019 ◽

Cited By ~ 1

Author(s):

J Heitman ◽

A Koller ◽

J Kunz ◽

R Henriquez ◽

A Schmidt ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Cyclosporin A ◽

Secretory Pathway ◽

Yeast Cells ◽

Amino Acid Transporters ◽

Yeast Saccharomyces Cerevisiae ◽

Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

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GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats

Molecular and Cellular Biology ◽

10.1128/mcb.11.10.5101-5112.1991 ◽

1991 ◽

Vol 11 (10) ◽

pp. 5101-5112

Author(s):

J S Flick ◽

M Johnston

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Interactions ◽

Tandem Repeats ◽

Glucose Repression ◽

Molecular Data ◽

Primary Response ◽

Yeast Cells ◽

Cell Fractionation ◽

Protein Protein Interactions ◽

Yeast Saccharomyces Cerevisiae

Growth of the yeast Saccharomyces cerevisiae on glucose leads to repression of transcription of many genes required for alternative carbohydrate metabolism. The GRR1 gene appears to be of central importance to the glucose repression mechanism, because mutations in GRR1 result in a pleiotropic loss of glucose repression (R. Bailey and A. Woodword, Mol. Gen. Genet. 193:507-512, 1984). We have isolated the GRR1 gene and determined that null mutants are viable and display a number of growth defects in addition to the loss of glucose repression. Surprisingly, grr1 mutations convert SUC2, normally a glucose-repressed gene, into a glucose-induced gene. GRR1 encodes a protein of 1,151 amino acids that is expressed constitutively at low levels in yeast cells. GRR1 protein contains 12 tandem repeats of a sequence similar to leucine-rich motifs found in other proteins that may mediate protein-protein interactions. Indeed, cell fractionation studies are consistent with this view, suggesting that GRR1 protein is tightly associated with a particulate protein fraction in yeast extracts. The combined genetic and molecular data are consistent with the idea that GRR1 protein is a primary response element in the glucose repression pathway and is required for the generation or interpretation of the signal that induces glucose repression.

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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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Evolution of ethylene by Saccharomyces cerevisiae as influenced by the carbon source for growth and the presence of air

Canadian Journal of Microbiology ◽

10.1139/m78-107 ◽

1978 ◽

Vol 24 (6) ◽

pp. 637-642 ◽

Cited By ~ 6

Author(s):

K. C. Thomas ◽

Mary Spencer

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Ethylene Production ◽

Atp Synthesis ◽

Yeast Cells ◽

Yeast Culture ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Absolute Requirement ◽

Wild Type Yeast

Effects of the carbon source and oxygen on ethylene production by the yeast Saccharomyces cerevisiae have been studied. The amounts of ethylene evolved by the yeast culture were less than those detected in the blank (an equal volume of uninoculated medium), suggesting a net absorption of ethylene by the yeast cells. Addition of glucose to the lactate-grown yeast culture induced ethylene production. This glucose-induced stimulation of ethylene production was inhibited to a great extent by cycloheximide. Results suggested that the yeast cells in the presence of glucose synthesized an ethylene precursor and passed it into the medium. The conversion of this precursor to ethylene might be stimulated by oxygen. The fact that ethylene was produced by the yeast growing anaerobically and also by respiration-deficient mutants isolated from the wild-type yeast suggested that mitochondrial ATP synthesis was not an absolute requirement for ethylene biogenesis.

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