Expression of a mammalian Na+/H+ antiporter in Saccharomyces cerevisiae

1999 ◽  
Vol 77 (1) ◽  
pp. 25-31 ◽  
Author(s):  
Mónica Montero-Lomelí ◽  
Anna L Okorokova Façanha.
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Subcellular Fractionation ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Coding Region ◽  
Phosphatidylcholine Liposomes ◽  
Yeast Transformants ◽  
Wild Type Yeast ◽  
Membrane Region

The basolateral Na+/H+ antiporter (NHE) from LLC-PK1 cells was expressed in Saccharomyces cerevisiae. Two different strategies were tested for expression. In the first, we used a yeast strain that contains a temperature-sensitive mutation in the SEC-6 gene, whose product is required for the fusion of secretory vesicles with the plasma membrane. This strain was transformed with a vector containing the coding region of the NHE1 isoform under control of a heat shock (HS) promoter (pYNHE1-HS). In the second strategy, we replaced the heat shock promoter from pYNHE1-HS with a galactose (GAL) promoter (pYNHEI-GAL) and transformed wild-type yeast. In both cases, Northern blots demonstrated a transcript that hybridized against a probe containing the membrane region of the exchanger. When an antibody against the last 40 amino acids of the carboxy-terminus of NHE1 was used for immuno-blots, a protein with a Mr of 73 000 was seen in total membranes from both yeast transformants. Subcellular fractionation revealed that NHE1 was expressed in the endoplasmic reticulum. In the case of the pYNHEI-GAL transformant, the 100 000 × g membrane pellet was reconstituted in phosphatidylcholine liposomes, and ethylisopropyl-amiloride-sensitive Na+/H+ exchange was observed. These results have paved the way for expression of the Na+/H+ exchanger in a genetically well-known microorganism.Key words: Na+/H+ exchanger, NHE1, expression, yeast.

Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5' coding region of the adenylate cyclase gene

1988 ◽  
Vol 8 (12) ◽  
pp. 5555-5560
Author(s):  
H Iida
Keyword(s):  
Heat Treatment ◽  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Adenylate Cyclase ◽  
Uv Light ◽  
Yeast Cells ◽  
Wild Type ◽  
Coding Region ◽  
Resistant Mutants ◽  
Type Strains

Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.

scholarly journals Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5' coding region of the adenylate cyclase gene.

1988 ◽  
Vol 8 (12) ◽  
pp. 5555-5560 ◽  
Author(s):  
H Iida
Keyword(s):  
Heat Treatment ◽  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Adenylate Cyclase ◽  
Uv Light ◽  
Yeast Cells ◽  
Wild Type ◽  
Coding Region ◽  
Resistant Mutants ◽  
Type Strains

Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.

scholarly journals Suppression of an Hsp70 Mutant Phenotype in Saccharomyces cerevisiae through Loss of Function of the Chromatin Component Sin1p/Spt2p

1998 ◽  
Vol 180 (24) ◽  
pp. 6484-6492 ◽  
Author(s):  
Bonnie K. Baxter ◽  
Elizabeth A. Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Molecular Chaperones ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Wild Type ◽  
Extragenic Suppressor ◽  
Slow Growing ◽  
Type Strains ◽  
Existing Data

ABSTRACT The Ssa subfamily of Hsp70 molecular chaperones in the budding yeast Saccharomyces cerevisiae has four members, encoded bySSA1, SSA2, SSA3, andSSA4. Deletion of the two constitutively expressed genes,SSA1 and SSA2, results in cells which are slow growing and temperature sensitive. In this study, we demonstrate that an extragenic suppressor of the temperature sensitivity of ssa1 ssa2 strains, EXA1-1, is a loss-of-function mutation in SIN1/SPT2, which encodes a nonhistone component of chromatin. Loss of function of Sin1p leads to overexpression ofSSA3 in the ssa1 ssa2 mutant background, at a level which is sufficient to mediate suppression. In a strain which is wild type for SSA genes, we detected no effect of Sin1p on Ssa3p expression except under conditions of heat shock. Existing data indicate that expression of SSA3 in thessa1 ssa2 mutant background as well as in heat-shocked wild-type strains is mediated by the heat shock transcription factor HSF. Our findings suggest that it is HSF-mediated induction of SSA3 which is modulated by Sin1p. TheEXA1-1 suppressor mutation thus improves the growth ofssa1 ssa2 strains by selectively increasing HSF-mediated expression of SSA3.

scholarly journals LEA (late embryonic abundant)-like protein Hsp 12 (heat-shock protein 12) is present in the cell wall and enhances the barotolerance of the yeast Saccharomyces cerevisiae

2004 ◽  
Vol 377 (3) ◽  
pp. 769-774 ◽  
Author(s):  
Precious MOTSHWENE ◽  
Robert KARREMAN ◽  
Gail KGARI ◽  
Wolf BRANDT ◽  
George LINDSEY
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Barometric Pressure ◽  
Yeast Cells ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rapid Changes ◽  
Wild Type Yeast

Yeast cells Saccharomyces cerevisiae, late embryogenic abundant-like stress response protein Hsp 12 (heat-shock protein 12) were found by immunocytochemistry to be located both in the cytoplasm and in the cell wall, from where they could be extracted with dilute NaOH solutions. Yeast cells with the Hsp 12 gene disrupted were unable to grow in the presence of either 12 mM caffeine or 0.43 mM Congo Red, molecules known to affect cell-wall integrity. The volume of yeast cells were less affected by rapid changes in the osmolality of the growth medium when compared with the wild-type yeast cells, suggesting a role for Hsp 12 in the flexibility of the cell wall. This was also suggested by subjecting the yeast cells to rapid changes in barometric pressure where it was found that wild-type yeast cells were more resistant to cellular breakage.

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system

1989 ◽  
Vol 9 (10) ◽  
pp. 4467-4472
Author(s):  
M Altmann ◽  
N Sonenberg ◽  
H Trachsel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Point Mutations ◽  
Translation Initiation Factor ◽  
Apparent Temperature ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Free System ◽  
Gal1 Promoter

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

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

1978 ◽  
Vol 24 (6) ◽  
pp. 637-642 ◽  
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.

scholarly journals The effect of calnexin deletion on the expression level of PDI in Saccharomyces cerevisiae under heat stress conditions

2008 ◽  
Vol 13 (1) ◽  
Author(s):  
Huili Zhang ◽  
Jianwei He ◽  
Yanyan Ji ◽  
Akio Kato ◽  
Youtao Song
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Heat Stress ◽  
Protein Expression ◽  
Molecular Chaperone ◽  
Mrna Level ◽  
Stress Conditions ◽  
Mrna Levels ◽  
Wild Type ◽  
Wild Type Yeast

AbstractWe cultured calnexin-disrupted and wild-type Saccharomyces cerevisiae strains under conditions of heat stress. The growth rate of the calnexin-disrupted yeast was almost the same as that of the wild-type yeast under those conditions. However, the induced mRNA level of the molecular chaperone PDI in the ER was clearly higher in calnexin-disrupted S. cerevisiae relative to the wild type at 37°C, despite being almost the same in the two strains under normal conditions. The western blotting analysis for PDI protein expression in the ER yielded results that show a parallel in their mRNA levels in the two strains. We suggest that PDI may interact with calnexin under heat stress conditions, and that the induction of PDI in the ER can recover part of the function of calnexin in calnexin-disrupted yeast, and result in the same growth rate as in wild-type yeast.

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae

1982 ◽  
Vol 2 (4) ◽  
pp. 437-442
Author(s):  
G R Taylor ◽  
B J Barclay ◽  
R K Storms ◽  
J D Friesen ◽  
R H Haynes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Frequency ◽  
Wild Type Strain ◽  
Biologically Active ◽  
Thymidylate Synthetase ◽  
Temperature Sensitive ◽  
Synthetase Activity ◽  
Enzymatic Conversion ◽  
Wild Type ◽  
Sensitive Mutant

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.

scholarly journals Mistranslating tRNA identifies a deleterious S213P mutation in the Saccharomyces cerevisiae eco1-1 allele

2020 ◽  
Vol 98 (5) ◽  
pp. 624-630 ◽  
Author(s):  
Yanrui Zhu ◽  
Matthew D. Berg ◽  
Phoebe Yang ◽  
Raphaël Loll-Krippleber ◽  
Grant W. Brown ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Genetic Disease ◽  
Elevated Temperatures ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Standard Genetic Code ◽  
Gene Encoding ◽  
Chromatid Cohesion ◽  
Sensitive Allele

Mistranslation occurs when an amino acid not specified by the standard genetic code is incorporated during translation. Since the ribosome does not read the amino acid, tRNA variants aminoacylated with a non-cognate amino acid or containing a non-cognate anticodon dramatically increase the frequency of mistranslation. In a systematic genetic analysis, we identified a suppression interaction between tRNASerUGG, G26A, which mistranslates proline codons by inserting serine, and eco1-1, a temperature sensitive allele of the gene encoding an acetyltransferase required for sister chromatid cohesion. The suppression was partial, with a tRNA that inserts alanine at proline codons and not apparent for a tRNA that inserts serine at arginine codons. Sequencing of the eco1-1 allele revealed a mutation that would convert the highly conserved serine 213 within β7 of the GCN5-related N-acetyltransferase core to proline. Mutation of P213 in eco1-1 back to the wild-type serine restored the function of the enzyme at elevated temperatures. Our results indicate the utility of mistranslating tRNA variants to identify functionally relevant mutations and identify eco1 as a reporter for mistranslation. We propose that mistranslation could be used as a tool to treat genetic disease.

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