scholarly journals Mutational analysis of the Saccharomyces cerevisiae ABD1 gene: cap methyltransferase activity is essential for cell growth.

1996 ◽  
Vol 16 (2) ◽  
pp. 475-480 ◽  
Author(s):  
X Mao ◽  
B Schwer ◽  
S Shuman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Yeast Cells ◽  
Terminal Deletion ◽  
Methyltransferase Activity

RNA (guanine-7-)-methyltransferase is the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA. The Saccharomyces cerevisiae enzyme is a 436-amino-acid protein encoded by the essential ABD1 gene. In this study, deletion and point mutations in ABD1 were tested for the ability to support growth of an abd1 null strain. Elimination of 109 amino acids from the N terminus had no effect on cell viability, whereas a more extensive N-terminal deletion of 155 residues was lethal, as was a C-terminal deletion of 55 amino acids. Alanine substitution mutations were introduced at eight conserved residues within a 206-amino-acid region of similarity between ABD1 and the methyltransferase domain of the vaccinia virus capping enzyme. ABD1 alleles H253A (encoding a substitution of alanine for histidine at position 253), T282A, E287A, E361A, and Y362A were viable, whereas G174A, D178A, and Y254A were either lethal or severely defective for growth. Alanine-substituted and amino-truncated ABD1 proteins were expressed in bacteria, purified, and tested for cap methyltransferase activity in vitro. Mutations that were viable in yeast cells had either no effect or only a moderate effect on the specific methyltransferase activity of the mutated ABD1 protein, whereas mutations that were deleterious in vivo yielded proteins that were catalytically defective in vitro. These findings substantiate for the first time the long-held presumption that cap methylation is an essential function in eukaryotic cells.

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (8) ◽  
pp. 5010-5019 ◽  
Author(s):  
J Heitman ◽  
A Koller ◽  
J Kunz ◽  
R Henriquez ◽  
A Schmidt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Cyclosporin A ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Amino Acid Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

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 The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (8) ◽  
pp. 5010-5019 ◽  
Author(s):  
J Heitman ◽  
A Koller ◽  
J Kunz ◽  
R Henriquez ◽  
A Schmidt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Cyclosporin A ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Amino Acid Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

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 A Saccharomyces cerevisiae mitochondrial transcription factor, sc-mtTFB, shares features with sigma factors but is functionally distinct.

1995 ◽  
Vol 15 (4) ◽  
pp. 2101-2108 ◽  
Author(s):  
G S Shadel ◽  
D A Clayton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Amino Acid ◽  
Mutational Analysis ◽  
Dna Supercoiling ◽  
Normal Function ◽  
Sigma Factors ◽  
Promoter Recognition

In Saccharomyces cerevisiae mitochondria, sc-mtTFB is a 341-amino-acid transcription factor required for initiation of transcription from mitochondrial DNA promoters. Specific transcription in vitro requires only sc-mtTFB and the bacteriophage-related core sc-mtRNA polymerase. Mutational analysis of sc-mtTFB has defined two regions of the protein that are important for normal function both in vivo and in vitro. These regions overlap portions of the protein that exhibit similarity to conserved region 2 of bacterial sigma factors. One mutation in this region of sc-mtTFB (tyrosine 108 to arginine [Y108R]) has a defective phenotype that matches that observed for mutations in the corresponding residue of Bacillus subtilis sigma A and sigma E proteins. However, mutations in the sigma 2.4-like region, including a 5-amino-acid deletion corresponding to crucial promoter-contacting amino acids of sigma factors, did not eliminate the ability of sc-mtTFB to initiate transcription specifically in vitro. This suggests a mechanism of promoter recognition for sc-mtRNA polymerase different from that used by bacterial RNA polymerases. Two mutations in a basic region of sc-mtTFB resulted in defective proteins that were virtually dependent on supercoiled DNA templates in vitro. These mutations may have disrupted a DNA-unwinding function of sc-mtTFB that is only manifested in vitro and is partially rescued by DNA supercoiling.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

Effect of Cycloheximide on In Vitro and In Vivo Protein Synthesis by Certain Tissues of Rats and Pigeons with Special Reference to Muscle

1973 ◽  
Vol 51 (12) ◽  
pp. 933-941 ◽  
Author(s):  
Njanoor Narayanan ◽  
Jacob Eapen
Keyword(s):  
Amino Acids ◽  
Body Weight ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Incorporation ◽  
Contrast Administration ◽  
Acid Incorporation ◽  
Tissue Homogenates

The effect of cycloheximide in vitro and in vivo on the incorporation of labelled amino acids into protein by muscles, liver, kidneys, and brain of rats and pigeons was studied. In vitro incorporation of amino acids into protein by muscle microsomes, myofibrils, and myofibrillar ribosomes was not affected by cycloheximide. In contrast, administration of the antibiotic into intact animals at a concentration of 1 mg/kg body weight resulted in considerable inhibition of amino acid incorporation into protein by muscles, liver, kidneys, and brain. This inhibition was observed in all the subcellular fractions of these tissues during a period of 10–40 min after the administration of the precursor. Tissue homogenates derived from in vivo cycloheximide-treated animals did not show significant alteration in in vitro amino acid incorporation with the exception of brain, which showed a small but significant enhancement.

scholarly journals Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

2006 ◽  
Vol 6 (2) ◽  
pp. 328-336 ◽  
Author(s):  
Kariona A. Grabińska ◽  
Paula Magnelli ◽  
Phillips W. Robbins
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chain Length ◽  
Attachment Site ◽  
Chitin Synthase ◽  
Yeast Cells ◽  
Average Chain Length ◽  
Calcofluor White ◽  
Protein Factor

ABSTRACT Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a ∼60% decrease in CSIII activity, which is correlated with a ∼30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro.

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