Eng1p, an Endo-1,3-β-Glucanase Localized at the Daughter Side of the Septum, Is Involved in Cell Separation in Saccharomyces cerevisiae

ABSTRACT ENG1 (YNR067c), a gene encoding a new endo-1,3-β-glucanase, was cloned by screening a genomic library with a DNA probe obtained by PCR with synthetic oligonucleotides designed according to conserved regions found between yeast exo-1,3-β-glucanases (Exg1p, Exg2p, and Ssg1p). Eng1p shows strong sequence similarity to the product of the Saccharomyces cerevisiae ACF2 gene, involved in actin assembly “in vitro,” and to proteins present in other yeast and fungal species. It is also related to plant glucan-binding elicitor proteins, which trigger the onset of a defense response upon fungal infection. Eng1p and Acf2p/Eng2p are glucan-hydrolyzing proteins that specifically act on 1,3-β linkages, with an endolytic mode of action. Eng1p is an extracellular, heavily glycosylated protein, while Acf2p/Eng2p is an intracellular protein with no carbohydrate linked by N-glycosidic bonds. ENG1 transcription fluctuates periodically during the cell cycle; maximal accumulation occurs during the M/G1 transition and is dependent on the transcription factor Ace2p. Interestingly, eng1 deletion mutants show defects in cell separation, and Eng1p localizes asymmetrically to the daughter side of the septum, suggesting that this protein is involved, together with chitinase, in the dissolution of the mother-daughter septum.

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Signaling through Adenylyl Cyclase Is Essential for Hyphal Growth and Virulence in the Pathogenic FungusCandida albicans

Molecular Biology of the Cell ◽

10.1091/mbc.12.11.3631 ◽

2001 ◽

Vol 12 (11) ◽

pp. 3631-3643 ◽

Cited By ~ 274

Author(s):

Cintia R. C. Rocha ◽

Klaus Schröppel ◽

Doreen Harcus ◽

Anne Marcil ◽

Daniel Dignard ◽

...

Keyword(s):

Mouse Model ◽

Adenylyl Cyclase ◽

Sequence Similarity ◽

Hyphal Growth ◽

Antifungal Drugs ◽

Growth Defect ◽

Adenylyl Cyclases ◽

Gene Encoding ◽

Mutant Cells

The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. We have cloned theCaCDC35 gene encoding C. albicansadenylyl cyclase by functional complementation of the conditional growth defect of Saccharomyces cerevisiae cells with mutations in Ras1p and Ras2p. It has previously been shown that these Ras homologues regulate adenylyl cyclase in yeast. The C. albicans adenylyl cyclase is highly homologous to other fungal adenylyl cyclases but has less sequence similarity with the mammalian enzymes. C. albicans cells deleted for both alleles ofCaCDC35 had no detectable cAMP levels, suggesting that this gene encodes the only adenylyl cyclase in C. albicans. The homozygous mutant cells were viable but grew more slowly than wild-type cells and were unable to switch from the yeast to the hyphal form under all environmental conditions that we analyzed in vitro. Moreover, this morphogenetic switch was completely blocked in mutant cells undergoing phagocytosis by macrophages. However, morphogenetic switching was restored by exogenous cAMP. On the basis of epistasis experiments, we propose that CaCdc35p acts downstream of the Ras homologue CaRas1p. These epistasis experiments also suggest that the putative transcription factor Efg1p and components of the hyphal-inducing MAP kinase pathway depend on the function of CaCdc35p in their ability to induce morphogenetic switching. Homozygouscacdc35Δ cells were unable to establish vaginal infection in a mucosal membrane mouse model and were avirulent in a mouse model for systemic infections. These findings suggest that fungal adenylyl cyclases and other regulators of the cAMP signaling pathway may be useful targets for antifungal drugs.

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Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.11.12.5801-5812.1991 ◽

1991 ◽

Vol 11 (12) ◽

pp. 5801-5812

Author(s):

R A Preston ◽

M F Manolson ◽

K Becherer ◽

E Weidenhammer ◽

D Kirkpatrick ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Product ◽

Zinc Finger ◽

Genomic Library ◽

Sequence Similarity ◽

Carboxypeptidase Y ◽

Lag Phase ◽

Significant Similarity ◽

Reading Frame ◽

Isolation And Characterization

The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus E1a zinc finger. A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells. Cells bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely small vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37 degrees C, and were incapable of sporulating (as homozygotes). Fractionation of cells expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cells and is a vacuolar peripheral membrane protein. Sequence identity established that PEP3 and VPS18 (J. S. Robinson, T. R. Graham, and S. D. Emr, Mol. Cell. Biol. 11:5813-5824, 1991) are the same gene.

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RPC82 encodes the highly conserved, third-largest subunit of RNA polymerase C (III) from Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4433 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4433-4440 ◽

Cited By ~ 13

Author(s):

N Chiannilkulchai ◽

R Stalder ◽

M Riva ◽

C Carles ◽

M Werner ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Stop Codon ◽

Sequence Similarity ◽

Start Codon ◽

Rrna Synthesis ◽

Temperature Sensitive ◽

In Vitro Mutagenesis ◽

Multicopy Plasmid

RNA polymerase C (III) promotes the transcription of tRNA and 5S RNA genes. In Saccharomyces cerevisiae, the enzyme is composed of 15 subunits, ranging from 160 to about 10 kDa. Here we report the cloning of the gene encoding the 82-kDa subunit, RPC82. It maps as a single-copy gene on chromosome XVI. The UCR2 gene was found in the opposite orientation only 340 bp upstream of the RPC82 start codon, and the end of the SKI3 coding sequence was found only 117 bp downstream of the RPC82 stop codon. The RPC82 gene encodes a protein with a predicted M(r) of 73,984, having no strong sequence similarity to other known proteins. Disruption of the RPC82 gene was lethal. An rpc82 temperature-sensitive mutant, constructed by in vitro mutagenesis of the gene, showed a deficient rate of tRNA relative to rRNA synthesis. Of eight RNA polymerase C genes tested, only the RPC31 gene on a multicopy plasmid was capable of suppressing the rpc82(Ts) defect, suggesting an interaction between the polymerase C 82-kDa and 31-kDa subunits. A group of RNA polymerase C-specific subunits are proposed to form a substructure of the enzyme.

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Divergence of Eukaryotic Secretory Components: the Candida albicans Homolog of the Saccharomyces cerevisiae Sec20 Protein Is N Terminally Truncated, and Its Levels Determine Antifungal Drug Resistance and Growth

Journal of Bacteriology ◽

10.1128/jb.183.1.46-54.2001 ◽

2001 ◽

Vol 183 (1) ◽

pp. 46-54 ◽

Cited By ~ 10

Author(s):

Yvonne Weber ◽

Uwe J. Santore ◽

Joachim F. Ernst ◽

Rolf K. Swoboda

Keyword(s):

Saccharomyces Cerevisiae ◽

Candida Albicans ◽

Transcriptional Control ◽

Secretory Pathway ◽

Sodium Dodecyl ◽

Fungal Species ◽

Gene Encoding ◽

Vesicle Traffic ◽

Antifungal Drug Resistance ◽

And Function

ABSTRACT Sec20p is a component of the yeast Saccharomyces cerevisiae secretory pathway that does not have a close homolog in higher eukaryotic cells. To verify the function of Sec20p in other fungal species, we characterized the gene encoding a Sec20p homolog in the human fungal pathogen Candida albicans. The deduced protein has 27% identity with, but is missing about 100 N-terminal residues compared to S. cerevisiae Sec20p, which is part of the cytoplasmic tail interacting with the cytoplasmic protein Tip20p. Because a strain lacking both C. albicans SEC20alleles could not be constructed, we placed SEC20 under transcriptional control of two regulatable promoters, MET3pand PCK1p. Repression of SEC20 expression in these strains prevented (MET3p-SEC20 allele) or retarded (PCK1p-SEC20 allele) growth and led to the appearance of extensive intracellular membranes, which frequently formed stacks. Reduced SEC20 expression in the PCK1p-SEC20strain did not affect morphogenesis but led to a series of hypersensitivity phenotypes including supersensitivity to aminoglycoside antibiotics, to nystatin, to sodium dodecyl sulfate, and to cell wall inhibitors. These results demonstrate the occurrence and function of Sec20p in a fungal species other than S. cerevisiae, but the lack of the N-terminal domain and the apparent absence of a close TIP20 homolog in the C. albicans genome also indicate a considerable diversity in mechanisms of retrograde vesicle traffic in eukaryotes.

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ADP ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6690-6699.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6690-6699

Author(s):

T Stearns ◽

R A Kahn ◽

D Botstein ◽

M A Hoyt

Keyword(s):

Saccharomyces Cerevisiae ◽

Cold Sensitivity ◽

Animal Cells ◽

Gene Encoding ◽

Phenotypic Differences ◽

Adp Ribosylation ◽

Protein Factor ◽

Adp Ribosylation Factor ◽

Sublethal Concentrations

ADP ribosylation factor (ARF) is a ubiquitous 21-kDa GTP-binding protein in eucaryotes. ARF was first identified in animal cells as the protein factor required for the efficient ADP-ribosylation of the mammalian G protein Gs by cholera toxin in vitro. A gene (ARF1) encoding a protein homologous to mammalian ARF was recently cloned from Saccharomyces cerevisiae (Sewell and Kahn, Proc. Natl. Acad. Sci. USA, 85:4620-4624, 1988). We have found a second gene encoding ARF in S. cerevisiae, ARF2. The two ARF genes are within 28 centimorgans of each other on chromosome IV, and the proteins encoded by them are 96% identical. Disruption of ARF1 causes slow growth, cold sensitivity, and sensitivity to normally sublethal concentrations of fluoride ion in the medium. Disruption of ARF2 causes no detectable phenotype. Disruption of both genes is lethal; thus, ARF is essential for mitotic growth. The ARF1 and ARF2 proteins are functionally homologous, and the phenotypic differences between mutations in the two genes can be accounted for by the level of expression; ARF1 produces approximately 90% of total ARF. Among revertants of the fluoride sensitivity of an arf1 null mutation were ARF1-ARF2 fusion genes created by a gene conversion event in which the deleted ARF1 sequences were repaired by recombination with ARF2.

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Transposon-Disruption of a Maize Nuclear Gene, tha1, Encoding a Chloroplast SecA Homologue: In Vivo Role of cp-SecA in Thylakoid Protein Targeting

Genetics ◽

10.1093/genetics/145.2.467 ◽

1997 ◽

Vol 145 (2) ◽

pp. 467-478 ◽

Cited By ~ 4

Author(s):

Rodger Voelker ◽

Janet Mendel-Hartvig ◽

Alice Barkan

Keyword(s):

Protein Targeting ◽

Sequence Similarity ◽

Nuclear Gene ◽

Cytochrome F ◽

Transposon Insertion ◽

Nuclear Mutant ◽

Strong Sequence Similarity

A nuclear mutant of maize, tha1, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the tha1 locus facilitated the cloning of portions of the tha1 gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that tha1 encodes a SecA homologue (cp-SecA). The tha1-ref allele is either null or nearly so, in that tha1 mRNA is undetectable in mutant leaves and cp-SecA accumulation is reduced ≥40-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-Sed function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome f, and in protein targeting in the etioplast, a nonphotosynthetic plastid type. Our finding that the accumulation of properly targeted plastocyanin and cytochrome f in tha1-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.

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Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.264 ◽

1995 ◽

Vol 15 (1) ◽

pp. 264-271 ◽

Cited By ~ 63

Author(s):

M Wu ◽

B Repetto ◽

D M Glerum ◽

A Tzagoloff

Keyword(s):

Saccharomyces Cerevisiae ◽

Flavin Adenine Dinucleotide ◽

Genomic Library ◽

Sequence Similarity ◽

Complementation Group ◽

Flavin Mononucleotide ◽

Synthetase Activity ◽

Multicopy Plasmid ◽

Multiple Copies ◽

Extragenic Suppression

The FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178. Mutants in this group display a reduced level of flavin adenine dinucleotide (FAD) and an increased level of flavin mononucleotide (FMN) in mitochondria. The restoration of respiratory capability by FAD1 is shown to be due to extragenic suppression. FAD1 codes for an essential yeast protein, since disruption of the gene induces a lethal phenotype. The FAD1 product has been inferred to be yeast FAD synthetase, an enzyme that adenylates FMN to FAD. This conclusion is based on the following evidence. S. cerevisiae transformed with FAD1 on a multicopy plasmid displays an increase in FAD synthetase activity. This is also true when the gene is expressed in Escherichia coli. Lastly, the FAD1 product exhibits low but significant primary sequence similarity to sulfate adenyltransferase, which catalyzes a transfer reaction analogous to that of FAD synthetase. The lower mitochondrial concentration of FAD in G178 mutants is proposed to be caused by an inefficient exchange of external FAD for internal FMN. This is supported by the absence of FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial riboflavin kinase, the preceding enzyme in the biosynthetic pathway. A lesion in mitochondrial import of FAD would account for the higher concentration of mitochondrial FMN in the mutant if the transport is catalyzed by an exchange carrier. The ability of FAD1 to suppress impaired transport of FAD is explained by mislocalization of the synthetase in cells harboring multiple copies of the gene. This mechanism of suppression is supported by the presence of mitochondrial FAD synthetase activity in S. cerevisiae transformed with FAD1 on a high-copy-number plasmid but not in mitochondrial of a wild-type strain.

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Analysis of the interaction between piD261/Bud32, an evolutionarily conserved protein kinase of Saccharomyces cerevisiae, and the Grx4 glutaredoxin

Biochemical Journal ◽

10.1042/bj20030638 ◽

2004 ◽

Vol 377 (2) ◽

pp. 395-405 ◽

Cited By ~ 45

Author(s):

Raffaele LOPREIATO ◽

Sonia FACCHIN ◽

Geppo SARTORI ◽

Giorgio ARRIGONI ◽

Stefano CASONATO ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Hybrid Approach ◽

Inosine Monophosphate Dehydrogenase ◽

Gene Encoding ◽

Putative Protease ◽

Structural Homologues ◽

Novel Protein

The Saccharomyces cerevisiae piD261/Bud32 protein and its structural homologues, which are present along the Archaea–Eukarya lineage, constitute a novel protein kinase family (the piD261 family) distantly related in sequence to the eukaryotic protein kinase superfamily. It has been demonstrated that the yeast protein displays Ser/Thr phosphotransferase activity in vitro and contains all the invariant residues of the family. This novel protein kinase appears to play an important cellular role as deletion in yeast of the gene encoding piD261/Bud32 results in the alteration of fundamental processes such as cell growth and sporulation. In this work we show that the phosphotransferase activity of Bud32 is relevant to its functionality in vivo, but is not the unique role of the protein, since mutants which have lost catalytic activity but not native conformation can partially complement the disruption of the gene encoding piD261/Bud32. A two-hybrid approach has led to the identification of several proteins interacting with Bud32; in particular a glutaredoxin (Grx4), a putative glycoprotease (Ykr038/Kae1) and proteins of the Imd (inosine monophosphate dehydrogenase) family seem most plausible interactors. We further demonstrate that Grx4 directly interacts with Bud32 and that it is phosphorylated in vitro by Bud32 at Ser-134. The functional significance of the interaction between Bud32 and the putative protease Ykr038/Kae1 is supported by its evolutionary conservation.

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Mutations in RAD6, a yeast gene encoding a ubiquitin-conjugating enzyme, stimulate retrotransposition.

Molecular and Cellular Biology ◽

10.1128/mcb.10.3.1017 ◽

1990 ◽

Vol 10 (3) ◽

pp. 1017-1022 ◽

Cited By ~ 31

Author(s):

S Picologlou ◽

N Brown ◽

S W Liebman

Keyword(s):

Saccharomyces Cerevisiae ◽

Fold Increase ◽

Yeast Gene ◽

Gene Encoding ◽

Repair Gene ◽

Dna Repair Gene ◽

Ubiquitin Conjugating Enzyme ◽

First Time ◽

Conjugating Enzyme

The Saccharomyces cerevisiae DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme which polyubiquitinates histones in vitro. Here we show that mutations in rad6 increase the frequency of transposition of the retrotransposon Ty into the CAN1 and URA3 loci. Using isogenic RAD6 and rad6 strains, we measured a more than 100-fold increase in the spontaneous rate of retrotransposition due to rad6, although there was no increase in the Ty message level. This is the first time that a mutation in a host gene has been shown to result in an increased rate of retrotransposition.

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Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and analysis of the CEN1-ADE1-CDC15 region

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.410-419.1987 ◽

1987 ◽

Vol 7 (1) ◽

pp. 410-419

Author(s):

H Y Steensma ◽

J C Crowley ◽

D B Kaback

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Disruption ◽

Genomic Library ◽

Blot Hybridization ◽

Polyadenylated Rna ◽

Average Proportion ◽

One Step ◽

Bona Fide ◽

Chromosome I

To continue the systematic examination of the physical and genetic organization of an entire Saccharomyces cerevisiae chromosome, the DNA from the CEN1-ADE1-CDC15 region from chromosome I was isolated and characterized. Starting with the previously cloned ADE1 gene (J. C. Crowley and D. B. Kaback, J. Bacteriol. 159:413-417, 1984), a series of recombinant lambda bacteriophages containing 82 kilobases of contiguous DNA from chromosome I were obtained by overlap hybridization. The cloned sequences were mapped with restriction endonucleases and oriented with respect to the genetic map by determining the physical positions of the CDC15 gene and the centromeric DNA (CEN1). The CDC15 gene was located by isolating plasmids from a YCp50 S. cerevisiae genomic library that complemented the cdc15-1 mutation. S. cerevisiae sequences from these plasmids were found to be represented among those already obtained by overlap hybridization. The cdc15-1-complementing plasmids all shared only one intact transcribed region that was shown to contain the bona fide CDC15 gene by in vitro gene disruption and one-step replacement to delete the chromosomal copy of this gene. This deletion produced a recessive lethal phenotype that was also recessive to cdc15-1. CEN1 was located by finding a sequence from the appropriate part of the cloned region that stabilized the inheritance of autonomously replicating S. cerevisiae plasmid vectors. Finally, RNA blot hybridization and electron microscopy of R-loop-containing DNA were used to map transcribed regions in the 23 kilobases of DNA that went from CEN1 to CDC15. In addition to the transcribed regions corresponding to the ADE1 and ADC15 genes, this DNA contained five regions that gave rise to polyadenylated RNA, at least two regions complementary to 4S RNA species, and a Ty1 transposable element. Notably, a higher than average proportion of the DNA examined was transcribed into RNA.

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