Isolation of mutant Saccharomyces cerevisiae strains that survive without sphingolipids

1990 ◽  
Vol 10 (5) ◽  
pp. 2176-2181
Author(s):  
R C Dickson ◽  
G B Wells ◽  
A Schmidt ◽  
R L Lester
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Lipids ◽  
Molecular Genetic ◽  
Suppressor Gene ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Chain Base ◽  
Long Chain Base ◽  
Long Chain

Sphingolipids comprise a large, widespread family of complex eucaryotic-membrane constituents of poorly defined function. The yeast Saccharomyces cerevisiae is particularly suited for studies of sphingolipid function because it contains a small number of sphingolipids and is amenable to molecular genetic analysis. Moreover, it is the only eucaryote in which mutants blocked in sphingolipid biosynthesis have been isolated. Beginning with a nonreverting sphingolipid-defective strain that requires the addition of the long-chain-base component of sphingolipids to the culture medium for growth, we isolated two strains carrying secondary, suppressor mutations that permit survival in the absence of exogenous long-chain base. Remarkably, the suppressor strains made little if any sphingolipid. A study of how the suppressor gene products compensate for the lack of sphingolipids may reveal the function(s) of these membrane lipids in yeast cells.

scholarly journals Isolation of mutant Saccharomyces cerevisiae strains that survive without sphingolipids.

1990 ◽  
Vol 10 (5) ◽  
pp. 2176-2181 ◽  
Author(s):  
R C Dickson ◽  
G B Wells ◽  
A Schmidt ◽  
R L Lester
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Lipids ◽  
Molecular Genetic ◽  
Suppressor Gene ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Chain Base ◽  
Long Chain Base ◽  
Long Chain

Sphingolipids comprise a large, widespread family of complex eucaryotic-membrane constituents of poorly defined function. The yeast Saccharomyces cerevisiae is particularly suited for studies of sphingolipid function because it contains a small number of sphingolipids and is amenable to molecular genetic analysis. Moreover, it is the only eucaryote in which mutants blocked in sphingolipid biosynthesis have been isolated. Beginning with a nonreverting sphingolipid-defective strain that requires the addition of the long-chain-base component of sphingolipids to the culture medium for growth, we isolated two strains carrying secondary, suppressor mutations that permit survival in the absence of exogenous long-chain base. Remarkably, the suppressor strains made little if any sphingolipid. A study of how the suppressor gene products compensate for the lack of sphingolipids may reveal the function(s) of these membrane lipids in yeast cells.

scholarly journals Induction of the synthesis of an additional family of long-chain dolichols in the yeast Saccharomyces cerevisiae. Effect of starvation and ageing.

2002 ◽  
Vol 49 (3) ◽  
pp. 781-787 ◽  
Author(s):  
Anna Szkopinska ◽  
Ewa Swiezewska ◽  
Joanna Rytka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stationary Phase ◽  
Yeast Cells ◽  
Logarithmic Phase ◽  
Cellular Membranes ◽  
Yeast Saccharomyces Cerevisiae ◽  
Chemical Changes ◽  
Physico Chemical ◽  
Long Chain

The yeast Saccharomyces cerevisiae strain W303 synthesizes in the early logarithmic phase of growth dolichols of 14-18 isoprene residues. The analysis of the polyisoprenoids present in the stationary phase revealed an additional family which proved to be also dolichols but of 19-24 isoprene residues, constituting 39% of the total dolichols. The transfer of early logarithmic phase cells to a starvation medium lacking glucose or nitrogen resulted in the synthesis of the longer chain dolichols. The additional family of dolichols represented 13.8% and 10.3% of total dolichols in the glucose and nitrogen deficient media, respectively. The level of dolichols in yeast cells increased with the age of the cultures. Since both families of dolichols are present in stationary phase cells we postulate that the longer chain dolichols may be responsible for the physico-chemical changes in cellular membranes allowing yeast cells to adapt to nutrient deficient conditions to maintain long-term viability.

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.

scholarly journals Sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae: genetics, physiology, and a method for their selection.

1992 ◽  
Vol 174 (8) ◽  
pp. 2565-2574 ◽  
Author(s):  
W J Pinto ◽  
B Srinivasan ◽  
S Shepherd ◽  
A Schmidt ◽  
R C Dickson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chain Base ◽  
Long Chain Base ◽  
Long Chain

Isolation and characterization of a cDNA encoding a Δ8 sphingolipid desaturase from Aquilegia vulgaris

2002 ◽  
Vol 30 (6) ◽  
pp. 1073-1075 ◽  
Author(s):  
L. V. Michaelson ◽  
A. J. Longman ◽  
O. Sayanova ◽  
A. K. Stobart ◽  
J. A. Napier
Keyword(s):  
Functional Characterization ◽  
Yeast Cells ◽  
Sphingoid Bases ◽  
Yeast Saccharomyces Cerevisiae ◽  
Isolation And Characterization ◽  
Long Chain Base ◽  
In The Wild ◽  
Cis And Trans ◽  
Long Chain

We have isolated a cDNA encoding the Δ8 sphingolipid desaturase from the plant Aquilegia vulgaris L. via a PCR-based strategy using primers designed to target the conserved histidine box regions of microsomal desaturases. The function of the cDNA was confirmed by expression in the yeast, Saccharomyces cerevisiae. Analysis of the long-chain sphingoid bases as their dinitrophenyl derivatives by reverse-phase HPLC demonstrated the accumulation of cis- and trans-desaturated sphingoid bases which were not present in the wild-type yeast cells. The Δ8 desaturated products co-eluted with known Δ8-desaturated phytosphingenine and the molecular mass of these products was confirmed by liquid chromatography-MS. The Δ8 long-chain base desaturase was also able to desaturate dihydrosphingosine substrates. This is the first report of the functional characterization of an A. vulgaris gene product.

scholarly journals 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.

scholarly journals Ceramide/Long-Chain Base Phosphate Rheostat in Saccharomyces cerevisiae: Regulation of Ceramide Synthesis by Elo3p and Cka2p

2003 ◽  
Vol 2 (2) ◽  
pp. 284-294 ◽  
Author(s):  
Scott D. Kobayashi ◽  
Marek M. Nagiec
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Wild Type ◽  
Α Subunit ◽  
Chain Base ◽  
Long Chain Base ◽  
Ceramide Synthesis ◽  
Long Chain ◽  
Ck2 Kinase

ABSTRACT Sphingolipid precursors, namely, ceramide and long-chain base phosphates (LCBPs), are important growth regulators with often opposite effects on mammalian cells. A set of enzymes that regulate the levels of these precursors, referred to as a ceramide/LCBP rheostat, is conserved in all eukaryotes. In order to gain further insight into the function of the rheostat in Saccharomyces cerevisiae, we searched for mutants that are synthetically lethal with a deletion of the LCB3 gene encoding LCBP phosphatase. In addition to acquiring expected mutants lacking the LCBP lyase, the screen revealed elo3 (sur4) mutants that were defective in fatty acid elongation and cka2 mutants lacking the α′ subunit of the protein kinase CK2 (casein kinase). Both mutations affected the in vivo activity of the acyl coenzyme A (acyl-CoA)-dependent and fumonisin B1-sensitive ceramide synthase (CS). The Elo3 protein is necessary for synthesis of C26-CoA, which in wild-type yeast is a source of C26 fatty acyls found in the ceramide moieties of all sphingolipids. In the in vitro assay, CS had a strong preference for acyl-CoAs containing longer acyl chains. This finding suggests that a block in the formation of C26-CoA in yeast may cause a reduction in the conversion of LCBs into ceramides and lead to an overaccumulation of LCBPs that is lethal in strains lacking the Lcb3 phosphatase. In fact, elo3 mutants were found to accumulate high levels of LCBs and LCBPs. The cka2 mutants, on the other hand, exhibited only 25 to 30% of the in vitro CS activity found in wild-type membranes, indicating that the α′ subunit of CK2 kinase is necessary for full activation of CS. The cka2 mutants also accumulated high levels of LCBs and had elevated levels of LCBPs. In addition, both the elo3 and cka2 mutants showed increased sensitivity to the CS inhibitors australifungin and fumonisin B1. Together, our data demonstrate that the levels of LCBPs in yeast are regulated by the rate of ceramide synthesis, which depends on CK2 kinase activity and is also strongly affected by the supply of C26-CoA. This is the first evidence indicating the involvement of protein kinase in the regulation of de novo sphingolipid synthesis in any organism.

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