scholarly journals Point Mutations Identify a Conserved Region of the Saccharomyces cerevisiae AFR1 Gene That Is Essential for Both the Pheromone Signaling and Morphogenesis Functions

Genetics ◽  
2000 ◽  
Vol 155 (1) ◽  
pp. 43-55
Author(s):  
Cordell R DeMattei ◽  
Colleen P Davis ◽  
James B Konopka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Distinct Point ◽  
Sequence Similarity ◽  
Point Mutations ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Pheromone Signaling ◽  
Essential Region ◽  
The Relationship ◽  
Mutant Genes

Abstract Mating pheromone receptors activate a G protein signal pathway that leads to the conjugation of the yeast Saccharomyces cerevisiae. This pathway also induces the production of Afr1p, a protein that negatively regulates pheromone receptor signaling and is required to form pointed projections of new growth that become the site of cell fusion during mating. Afr1p lacks strong similarity to any well-characterized proteins to help predict how it acts. Therefore, we investigated the relationship between the different functions of Afr1p by isolating and characterizing seven mutants that were defective in regulating pheromone signaling. The AFR1 mutants were also defective when expressed as fusions to STE2, the α-factor receptor, indicating that the mutant Afr1 proteins are defective in function and not in co-localizing with receptors. The mutant genes contained four distinct point mutations that all occurred between codons 254 and 263, identifying a region that is critical for AFR1 function. Consistent with this, we found that the corresponding region is very highly conserved in the Afr1p homologs from the yeasts S. uvarum and S. douglasii. In contrast, there were no detectable effects on pheromone signaling caused by deletion or overexpression of YER158c, an open reading frame with overall sequence similarity to Afr1p that lacks this essential region. Interestingly, all of the AFR1 mutants showed a defect in their ability to form mating projections that was proportional to their defect in regulating pheromone signaling. This suggests that both functions may be due to the same action of Afr1p. Thus, these studies identify a specific region of Afr1p that is critical for its function in both signaling and morphogenesis.

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

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.

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

Genetics ◽  
1992 ◽  
Vol 132 (1) ◽  
pp. 97-112 ◽  
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.

scholarly journals Genome-Scale Analysis Reveals Sst2 as the Principal Regulator of Mating Pheromone Signaling in the Yeast Saccharomyces cerevisiae

2006 ◽  
Vol 5 (2) ◽  
pp. 330-346 ◽  
Author(s):  
Scott A. Chasse ◽  
Paul Flanary ◽  
Stephen C. Parnell ◽  
Nan Hao ◽  
Jiyoung Y. Cha ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transition State ◽  
G Protein ◽  
Common Property ◽  
Rgs Proteins ◽  
Homologous Proteins ◽  
Yeast Saccharomyces Cerevisiae ◽  
Α Subunit ◽  
Pheromone Signaling

ABSTRACT A common property of G protein-coupled receptors is that they become less responsive with prolonged stimulation. Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity and do so by stabilizing the transition state conformation of the G protein α subunit. In the yeast Saccharomyces cerevisiae there are four RGS-homologous proteins (Sst2, Rgs2, Rax1, and Mdm1) and two Gα proteins (Gpa1 and Gpa2). We show that Sst2 is the only RGS protein that binds selectively to the transition state conformation of Gpa1. The other RGS proteins also bind Gpa1 and modulate pheromone signaling, but to a lesser extent and in a manner clearly distinct from Sst2. To identify other candidate pathway regulators, we compared pheromone responses in 4,349 gene deletion mutants representing nearly all nonessential genes in yeast. A number of mutants produced an increase (sst2, bar1, asc1, and ygl024w) or decrease (cla4) in pheromone sensitivity or resulted in pheromone-independent signaling (sst2, pbs2, gas1, and ygl024w). These findings suggest that Sst2 is the principal regulator of Gpa1-mediated signaling in vivo but that other proteins also contribute in distinct ways to pathway regulation.

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

Isolation and characterization of STI1, a stress-inducible gene from Saccharomyces cerevisiae

1989 ◽  
Vol 9 (9) ◽  
pp. 3638-3646
Author(s):  
C M Nicolet ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Saccharomyces Cerevisiae ◽  
Two Dimensional Gel Electrophoresis ◽  
Reading Frame ◽  
Hsp70 Family ◽  
Isolation And Characterization ◽  
Isoelectric Points ◽  
Amino Acid Analog ◽  
Inducible Gene

We have isolated a gene from the yeast Saccharomyces cerevisiae that encodes a 2.0-kilobase heat-inducible mRNA. This gene, which we have designated STI1, for stress inducible, was also induced by the amino acid analog canavanine and showed a slight increase in expression as cells moved into stationary phase. The STI1 gene encodes a 66-kilodalton protein, as determined from the sequence of the longest open reading frame. The putative STI1 protein, as identified by two-dimensional gel electrophoresis, migrated in the region of 73 to 75 kilodaltons as a series of four isoforms with different isoelectric points. STI1 is not homologous to the other conserved HSP70 family members in yeasts, despite similarities in size and regulation. Cells carrying a disruption mutation of the STI1 gene grew normally at 30 degrees C but showed impaired growth at higher and lower temperatures. Overexpression of the STI1 gene resulted in substantial trans-activation of SSA4 promoter-reporter gene fusions, indicating that STI1 may play a role in mediating the heat shock response of some HSP70 genes.

scholarly journals Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

1994 ◽  
Vol 5 (9) ◽  
pp. 933-942 ◽  
Author(s):  
H Fang ◽  
N Green
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Water Soluble ◽  
Growth Defect ◽  
Sequence Analyses ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Transmembrane Glycoprotein ◽  
Genetic Assay ◽  
Er Membrane

The sec71-1 and sec72-1 mutations were identified by a genetic assay that monitored membrane protein integration into the endoplasmic reticulum (ER) membrane of the yeast Saccharomyces cerevisiae. The mutations inhibited integration of various chimeric membrane proteins and translocation of a subset of water soluble proteins. In this paper we show that SEC71 encodes the 31.5-kDa transmembrane glycoprotein (p31.5) and SEC72 encodes the 23-kDa protein (p23) of the Sec63p-BiP complex. SEC71 is therefore identical to SEC66 (HSS1), which was previously shown to encode p31.5. DNA sequence analyses reveal that sec71-1 cells contain a nonsense mutation that removes approximately two-thirds of the cytoplasmic C-terminal domain of p31.5. The sec72-1 mutation shifts the reading frame of the gene encoding p23. Unexpectedly, the sec71-1 mutant lacks p31.5 and p23. Neither mutation is lethal, although sec71-1 cells exhibit a growth defect at 37 degrees C. These results show that p31.5 and p23 are important for the trafficking of a subset of proteins to the ER membrane.

scholarly journals Differential Regulation of Two Ca2+ Influx Systems by Pheromone Signaling in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1527-1538
Author(s):  
Eric M Muller ◽  
Emily G Locke ◽  
Kyle W Cunningham
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Differential Regulation ◽  
Calcium Signals ◽  
Yeast Saccharomyces Cerevisiae ◽  
Constitutive Activation ◽  
Cycle Arrest ◽  
Pheromone Signaling ◽  
Rich Media ◽  
Calcineurin Activity

Abstract The budding yeast Saccharomyces cerevisiae generates calcium signals during the response to mating pheromones that promote survival of unmated cells. A Ca2+ channel composed of Cch1p and Mid1p was previously shown to be necessary for the production of these calcium signals. However, we find that the Cch1p-Mid1p high-affinity Ca2+ influx system (HACS) contributes very little to signaling or survival after treatment with α-factor in rich media. HACS activity was much greater after calcineurin inactivation or inhibition, suggesting the Cch1p-Mid1p Ca2+ channel is subject to direct or indirect regulation by calcineurin. Instead a distinct low-affinity Ca2+ influx system (LACS) was stimulated by pheromone signaling in rich medium. LACS activity was insensitive to calcineurin activity, independent of Cch1p and Mid1p, and sufficient to elevate cytosolic free Ca2+ concentrations ([Ca2+]c) in spite of its 16-fold lower affinity for Ca2+. Overexpression of Ste12p or constitutive activation of this transcription factor in dig1 dig2 double mutants had no effect on LACS activity but stimulated HACS activity when calcineurin was also inactivated. Ste12p activation had no effect on Cch1p or Mid1p abundance, suggesting the involvement of another target of Ste12p in HACS stimulation. LACS activation required treatment with mating pheromone even in dig1 dig2 double mutants and also required FAR1, SPA2, and BNI1, which are necessary for proper cell cycle arrest and polarized morphogenesis. These results show that distinct branches of the pheromone-signaling pathway independently regulate HACS and LACS activities, either of which can promote survival during long-term responses.

scholarly journals Deletion of a single-copy tRNA affects microtubule function in Saccharomyces cerevisiae.

Genetics ◽  
1993 ◽  
Vol 135 (4) ◽  
pp. 955-962 ◽  
Author(s):  
R A Reijo ◽  
D S Cho ◽  
T C Huffaker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Copy ◽  
Open Reading Frame ◽  
Northern Analysis ◽  
Trna Genes ◽  
Heat Sensitivity ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Sensitive Allele

Abstract rts1-1 was identified as an extragenic suppressor of tub2-104, a cold-sensitive allele of the sole gene encoding beta-tubulin in the yeast, Saccharomyces cerevisiae. In addition, rts1-1 cells are heat sensitive and resistant to the microtubule-destabilizing drug, benomyl. The rts1-1 mutation is a deletion of approximately 5 kb of genomic DNA on chromosome X that includes one open reading frame and three tRNA genes. Dissection of this region shows that heat sensitivity is due to deletion of the open reading frame (HIT1). Suppression and benomyl resistance are caused by deletion of the gene encoding a tRNA(Arg)AGG (HSX1). Northern analysis of rts1-1 cells indicates that HSX1 is the only gene encoding this tRNA. Deletion of HSX1 does not suppress the tub2-104 mutation by misreading at the AGG codons in TUB2. It also does not suppress by interfering with the protein arginylation that targets certain proteins for degradation. These results leave open the prospect that this tRNA(Arg)AGG plays a novel role in the cell.

scholarly journals Repair of specific base pair mismatches formed during meiotic recombination in the yeast Saccharomyces cerevisiae

1991 ◽  
Vol 11 (2) ◽  
pp. 737-745
Author(s):  
P Detloff ◽  
J Sieber ◽  
T D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Homologous Chromosomes ◽  
Base Pair Mismatch ◽  
Dna Strands ◽  
Postmeiotic Segregation ◽  
Mutant Genes ◽  
His4 Gene

Heteroduplexes formed between DNA strands derived from different homologous chromosomes are an intermediate in meiotic crossing over in the yeast Saccharomyces cerevisiae and other eucaryotes. A heteroduplex formed between wild-type and mutant genes will contain a base pair mismatch; failure to repair this mismatch will lead to postmeiotic segregation (PMS). By analyzing the frequency of PMS for various mutant alleles in the yeast HIS4 gene, we showed that C/C mismatches were inefficiently repaired relative to all other point mismatches. These other mismatches (G/G, G/A, T/T, A/A, T/C, C/A, A/A, and T/G) were repaired with approximately the same efficiency. We found that in spores with unrepaired mismatches in heteroduplexes, the nontranscribed strand of the HIS4 gene was more frequently donated than the transcribed strand. In addition, the direction of repair for certain mismatches was nonrandom.

Heat shock-regulated production of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae

1983 ◽  
Vol 3 (9) ◽  
pp. 1625-1633
Author(s):  
D B Finkelstein ◽  
S Strausberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Plasmid Vector ◽  
Lacz Gene ◽  
Multicopy Plasmid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Inducible Synthesis ◽  
Beta Galactosidase

The HSP90 gene of the yeast Saccharomyces cerevisiae encodes a heat shock-inducible protein with an Mr of 90,000 (hsp90) and unknown function. We fused DNA fragments of a known sequence (namely, either end of a 1.4-kilobase EcoRI fragment which contains the S. cerevisiae TRP1 gene) to an EcoRI site within the coding sequence of the HSP90 gene. When these fusions are introduced into S. cerevisiae they direct the synthesis of unique truncated hsp90 proteins. By determining the size and charge of these proteins we were able to deduce the translational reading frame at the (EcoRI) fusion site. This information allowed us to design and construct a well-defined in-frame fusion between the S. cerevisiae HSP90 gene and the Escherichia coli lacZ gene. When this fused gene is introduced into S. cerevisiae on a multicopy plasmid vector, it directs the heat shock-inducible synthesis of a fused protein, which is an enzymatically active beta-galactosidase. Thus, for the first time, it is possible to quantitate the heat shock response in a eucaryotic organism with a simple enzyme assay.

Sign in / Sign up

Export Citation Format

Share Document