Two isoforms of Saccharomyces cerevisiae glutaredoxin 2 are expressed in vivo and localize to different subcellular compartments

Glutaredoxin (Grx)2 from Saccharomyces cerevisiae is a member of the two-cysteine (dithiol) subfamily of Grxs involved in the defence against oxidative stress in yeast. Recombinant yeast Grx2p, expressed in Escherichia coli, behaves as a ‘classical’ Grx that efficiently catalyses the reduction of hydroxyethyl disulphide by GSH. Grx2p also catalyses the reduction of GSSG by dihydrolipoamide with even higher efficiency. Western blot analysis of S. cerevisiae crude extracts identifies two isoforms of Grx2p of 15.9 and 11.9kDa respectively. The levels of these two isoforms reach a peak during the exponential phase of growth in normal yeast extract/peptone/dextrose ('YPD') medium, with the long form predominating over the short one. From immunochemical analysis of subcellular fractions, it is shown that both isoforms are present in mitochondria, but only the short one is detected in the cytosolic fraction. On the other hand, only the long form is prominent in microsomes. Mitochondrial isoforms should represent the processed and unprocessed products of an open reading frame (YDR513W), with a putative start codon 99bp upstream of the GRX2 start codon described thus far. These results indicate that GRX2 contains two in-frame start codons, and that translation from the first AUG results in a product that is targeted to mitochondria. The cytosolic form would result either by initiation from the second AUG, or by differential processing of one single translation product.

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Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2091-2103.1993 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2091-2103

Author(s):

S Türkel ◽

P J Farabaugh

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Activation ◽

Binding Sites ◽

Transcriptional Repression ◽

Physiological Control ◽

Reading Frame ◽

Negative Region ◽

Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

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Saccharomyces cerevisiae ubiquitin-like protein Rub1 conjugates to cullin proteins Rtt101 and Cul3 in vivo

Biochemical Journal ◽

10.1042/bj20030755 ◽

2004 ◽

Vol 377 (2) ◽

pp. 459-467 ◽

Cited By ~ 17

Author(s):

Jose M. LAPLAZA ◽

Magnolia BOSTICK ◽

Derek T. SCHOLES ◽

M. Joan CURCIO ◽

Judy CALLIS

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein A ◽

Fold Increase ◽

Lysine Residue ◽

Reading Frame ◽

E3 Ligases ◽

C Terminus ◽

Dependent Modification ◽

F Box Protein

In Saccharomyces cerevisiae, the ubiquitin-like protein Rub1p (related to ubiquitin 1 protein) covalently attaches to the cullin protein Cdc53p (cell division cycle 53 protein), a subunit of a class of ubiquitin E3 ligases named SCF (Skp1–Cdc53–F-box protein) complex. We identified Rtt101p (regulator of Ty transposition 101 protein, where Ty stands for transposon of yeast), initially found during a screen for proteins to confer retrotransposition suppression, and Cul3p (cullin 3 protein), a protein encoded by the previously uncharacterized open reading frame YGR003w, as two new in vivo targets for Rub1p conjugation. These proteins show significant identity with Cdc53p and, therefore, are cullin proteins. Modification of Cul3p is eliminated by deletion of the Rub1p pathway through disruption of either RUB1 or its activating enzyme ENR2/ULA1. The same disruptions in the Rub pathway decreased the percentage of total Rtt101p that is modified from approx. 60 to 30%. This suggests that Rtt101p has an additional RUB1- and ENR2-independent modification. All modified forms of Rtt101p and Cul3p were lost when a single lysine residue in a conserved region near the C-terminus was replaced by an arginine residue. These results suggest that this lysine residue is the site of Rub1p-dependent and -independent modifications in Rtt101p and of Rub1p-dependent modification in Cul3p. An rtt101Δ strain was hypersensitive to thiabendazole, isopropyl (N-3-chlorophenyl) carbamate and methyl methanesulphonate, but rub1Δ strains were not. Whereas rtt101Δ strains exhibited a 14-fold increase in Ty1 transposition, isogenic rub1Δ strains did not show statistically significant increases. Rtt101K791Rp, which cannot be modified, complemented for Rtt101p function in a transposition assay. Altogether, these results suggest that neither the RUB1-dependent nor the RUB1-independent form of Rtt101p is required for Rtt101p function. The identification of additional Rub1p targets in S. cerevisiae suggests an expanded role for Rub in this organism.

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cis- and trans-acting suppressors of a translation initiation defect at the cyc1 locus of Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/132.1.97 ◽

1992 ◽

Vol 132 (1) ◽

pp. 97-112 ◽

Cited By ~ 3

Author(s):

I Pinto ◽

J G Na ◽

F Sherman ◽

M Hampsey

Keyword(s):

Saccharomyces Cerevisiae ◽

Cytochrome C ◽

Point Mutations ◽

Start Codon ◽

Reading Frame ◽

Codon Recognition ◽

Short Open Reading Frame ◽

Initiator Codon ◽

Extragenic Suppressors ◽

Cis And Trans

Abstract The cyc1-362 mutant of Saccharomyces cerevisiae is deficient in iso-1-cytochrome c as a consequence of an aberrant ATG codon that initiates a short open reading frame (uORF) in the cyc1 transcribed leader region. We have isolated and characterized functional revertants of cyc1-362 in an effort to define cis- and trans-acting factors that can suppress the effect of the uORF. Genetic and DNA sequence analyses have defined three classes of revertants: (i) those that acquired point mutations in the upstream ATG (uATG), restoring iso-1-cytochrome c to its normal level; (ii) substitution of the normal A residue at position -1 relative to the uATG by either C or T, enhancing iso-1-cytochrome c production from approximately 2% to 6% (C) or 10% (T) of normal, indicating that the nucleotide immediately preceding the initiator codon can affect the efficiency of AUG start codon recognition and that purines are preferred over pyrimidines at this site; and (iii) extragenic suppressors that enhance iso-1-cytochrome c expression to 10-40% of normal while retaining the uATG. These suppressors are represented by five different genes, designated sua1-sua4 and sua6. In contrast to the previously described sua7 and sua8 suppressors, they do not compensate for the uATG by affecting cyc1 transcription start site selection. Potential suppressor mechanisms are discussed.

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A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth.

Molecular and Cellular Biology ◽

10.1128/mcb.8.8.3094 ◽

1988 ◽

Vol 8 (8) ◽

pp. 3094-3103 ◽

Cited By ~ 55

Author(s):

C K Shih ◽

R Wagner ◽

S Feinstein ◽

C Kanik-Ennulat ◽

N Neff

Keyword(s):

Saccharomyces Cerevisiae ◽

Atp Synthase ◽

Resistance Mutation ◽

Amino Acid Sequences ◽

Wild Type ◽

Reading Frame ◽

F0f1 Atp Synthase ◽

General Inhibitor

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.

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Glycerophosphocholine-dependent Growth Requires Gde1p (YPL110c) and Git1p in Saccharomyces cerevisiae

Journal of Biological Chemistry ◽

10.1074/jbc.m507051200 ◽

2005 ◽

Vol 280 (43) ◽

pp. 36110-36117 ◽

Cited By ~ 58

Author(s):

Edward Fisher ◽

Claudia Almaguer ◽

Roman Holic ◽

Peter Griac ◽

Jana Patton-Vogt

Keyword(s):

Saccharomyces Cerevisiae ◽

Wild Type Strain ◽

Open Reading Frame ◽

Reading Frame ◽

Intracellular Fraction ◽

Dependent Growth ◽

Phosphate Source ◽

Glycerophosphocholine Phosphodiesterase ◽

In Cells

Glycerophosphocholine is formed via the deacylation of the phospholipid phosphatidylcholine. The protein encoded by Saccharomyces cerevisiae open reading frame YPL110c effects glycerophosphocholine metabolism in vivo, most likely by acting as a glycerophosphocholine phosphodiesterase. Deletion of YPL110c causes an accumulation of glycerophosphocholine in cells prelabeled with [14C]choline. Correspondingly, overexpression of YPL110c results in reduced intracellular glycerophosphocholine in cells prelabeled with [14C]choline. Glycerophospho[3H]choline supplied in the growth medium accumulates to a much greater extent in the intracellular fraction of a YPL110Δ strain than in a wild type strain. Furthermore, glycerophospho[3H]choline accumulation requires the transporter encoded by GIT1, a known glycerophosphoinositol transporter. Growth on glycerophosphocholine as the sole phosphate source requires YPL110c and the Git1p permease. In contrast to glycerophosphocholine, glycerophosphoinositol metabolism is unaffected by deletion of YPL110c. The open reading frame YPL110c has been termed GDE1.

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Proline utilization in Saccharomyces cerevisiae: sequence, regulation, and mitochondrial localization of the PUT1 gene product

Molecular and Cellular Biology ◽

10.1128/mcb.7.12.4431-4440.1987 ◽

1987 ◽

Vol 7 (12) ◽

pp. 4431-4440

Author(s):

S S Wang ◽

M C Brandriss

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Initiation ◽

Initiation Site ◽

Translation Initiation Site ◽

Lacz Gene ◽

Oxygen Regulation ◽

Reading Frame ◽

Proline Utilization ◽

Beta Galactosidase

The PUT1 gene of Saccharomyces cerevisiae, believed to encode proline oxidase, has been completely sequenced and contains an open reading frame capable of encoding a polypeptide of 476 amino acids in length. The amino terminus of the protein deduced from the DNA sequence has a characteristic mitochondrial import signal; two PUT1-lacZ gene fusions were constructed that produced mitochondrially localized beta-galactosidase in vivo. The transcription initiation and termination sites of the PUT1 mRNA were determined. By using a PUT1-lacZ gene fusion that makes a cytoplasmic beta-galactosidase, the regulation of the PUT1 gene was studied. PUT1 is inducible by proline, responds only slightly to carbon catabolite repression, and is not regulated by the cytochrome activator proteins HAP1 and HAP2. The PUT1 gene is under oxygen regulation; expression in anaerobically grown cells is 10-fold lower than in aerobically grown cells. Oxygen regulation is abolished when cells are respiratory deficient. PUT1 expression in a [rho-] strain grown either aerobically or anaerobically is as high as that seen in a [rho+] strain grown aerobically. Studies on PUT1 promoter deletions define a region between positions -458 and -293 from the translation initiation site that is important for full expression of the PUT1 gene and required for oxygen regulation.

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Differential Processing of Leading- and Lagging-Strand Ends at Saccharomyces cerevisiae Telomeres Revealed by the Absence of Rad27p Nuclease

Genetics ◽

10.1093/genetics/162.4.1583 ◽

2002 ◽

Vol 162 (4) ◽

pp. 1583-1594

Author(s):

Julie Parenteau ◽

Raymund J Wellinger

Keyword(s):

Saccharomyces Cerevisiae ◽

Double Mutant ◽

Telomerase Activity ◽

Differential Processing ◽

Okazaki Fragment Processing ◽

Mutant Strains ◽

Leading Strand ◽

Replication Intermediates ◽

Lagging Strand

Abstract Saccharomyces cerevisiae strains lacking the Rad27p nuclease, a homolog of the mammalian FEN-1 protein, display an accumulation of extensive single-stranded G-tails at telomeres. Furthermore, the lengths of telomeric repeats become very heterogeneous. These phenotypes could be the result of aberrant Okazaki fragment processing of the C-rich strand, elongation of the G-rich strand by telomerase, or an abnormally high activity of the nucleolytic activities required to process leading-strand ends. To distinguish among these possibilities, we analyzed strains carrying a deletion of the RAD27 gene and also lacking genes required for in vivo telomerase activity. The results show that double-mutant strains died more rapidly than strains lacking only telomerase components. Furthermore, in such strains there is a significant reduction in the signals for G-tails as compared to those detected in rad27Δ cells. The results from studies of the replication intermediates of a linear plasmid in rad27Δ cells are consistent with the idea that only one end of the plasmid acquires extensive G-tails, presumably the end made by lagging-strand synthesis. These data further support the notion that chromosome ends have differential requirements for end processing, depending on whether the ends were replicated by leading- or lagging-strand synthesis.

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Dbp6p Is an Essential Putative ATP-Dependent RNA Helicase Required for 60S-Ribosomal-Subunit Assembly inSaccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.18.4.1855 ◽

1998 ◽

Vol 18 (4) ◽

pp. 1855-1865 ◽

Cited By ~ 58

Author(s):

Dieter Kressler ◽

Jesús de la Cruz ◽

Manuel Rojo ◽

Patrick Linder

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Subunit ◽

Rna Helicase ◽

Open Reading Frame ◽

Northern Hybridization ◽

Ribosomal Subunits ◽

Steady State Analysis ◽

Reading Frame ◽

Dead Box

A previously uncharacterizedSaccharomyces cerevisiaeopen reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network. YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore namedDBP6(DEAD-box protein 6). Dbp6p is essential for cell viability. In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs. Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus. We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.

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A DNA Helicase Required for Maintenance of the Functional Mitochondrial Genome in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.20.5.1816-1824.2000 ◽

2000 ◽

Vol 20 (5) ◽

pp. 1816-1824 ◽

Cited By ~ 37

Author(s):

Tiina Sedman ◽

Silja Kuusk ◽

Sirje Kivi ◽

Juhan Sedman

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitochondrial Genome ◽

Signal Sequence ◽

Carbon Sources ◽

Point Mutations ◽

Nuclear Genome ◽

Dna Helicase ◽

Dependent Manner ◽

Reading Frame

ABSTRACT A novel DNA helicase, a homolog of several prokaryotic helicases, including Escherichia coli Rep and UvrD proteins, is encoded by the Saccharomyces cerevisiae nuclear genome open reading frame YOL095c on the chromosome XV. Our data demonstrate that the helicase is localized in the yeast mitochondria and is loosely associated with the mitochondrial inner membrane during biochemical fractionation. The sequence of the C-terminal end of the 80-kDa helicase protein is similar to a typical N-terminal mitochondrial targeting signal; deletions and point mutations in this region abolish transport of the protein into mitochondria. The C-terminal signal sequence of the helicase targets a heterologous carrier protein into mitochondria in vivo. The purified recombinant protein can unwind duplex DNA molecules in an ATP-dependent manner. The helicase is required for the maintenance of the functional ([rho +]) mitochondrial genome on both fermentable and nonfermentable carbon sources. However, the helicase is not essential for the maintenance of several defective ([rho −]) mitochondrial genomes. We also demonstrate that the helicase is not required for transcription in mitochondria.

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Forespore-Specific Expression of Bacillus subtilis yqfS, Which Encodes Type IV Apurinic/Apyrimidinic Endonuclease, a Component of the Base Excision Repair Pathway

Journal of Bacteriology ◽

10.1128/jb.185.1.340-348.2003 ◽

2003 ◽

Vol 185 (1) ◽

pp. 340-348 ◽

Cited By ~ 17

Author(s):

Norma Urtiz-Estrada ◽

José M. Salas-Pacheco ◽

Ronald E. Yasbin ◽

Mario Pedraza-Reyes

Keyword(s):

Bacillus Subtilis ◽

Excision Repair ◽

Transcriptional Start Site ◽

Northern Blotting ◽

Start Codon ◽

Start Site ◽

Specific Expression ◽

Reading Frame ◽

Type Iv

ABSTRACT The temporal and spatial expression of the yqfS gene of Bacillus subtilis, which encodes a type IV apurinic/apyrimidinic endonuclease, was studied. A reporter gene fusion to the yqfS opening reading frame revealed that this gene is not transcribed during vegetative growth but is transcribed during the last steps of the sporulation process and is localized to the developing forespore compartment. In agreement with these results, yqfS mRNAs were mainly detected by both Northern blotting and reverse transcription-PCR, during the last steps of sporulation. The expression pattern of the yqfS-lacZ fusion suggested that yqfS may be an additional member of the EσG regulon. A primer extension product mapped the transcriptional start site of yqfS, 54 to 55 bp upstream of translation start codon of yqfS. Such an extension product was obtained from RNA samples of sporulating cells but not from those of vegetatively growing cells. Inspection of the nucleotide sequence lying upstream of the in vivo-mapped transcriptional yqfS start site revealed the presence of a sequence with good homology to promoters preceding genes of the σG regulon. Although yqfS expression was temporally regulated, neither oxidative damage (after either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment induced the transcription of this gene.

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