scholarly journals A Region of the Sir1 Protein Dedicated to Recognition of a Silencer and Required for Interaction with the Orc1 Protein in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 151 (1) ◽  
pp. 31-44 ◽  
Author(s):  
Kelly A Gardner ◽  
Jasper Rine ◽  
Catherine A Fox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Origin Recognition Complex ◽  
Dna Binding Domain ◽  
Missense Mutations ◽  
Macromolecular Complexes ◽  
Sequence Elements ◽  
Per Se ◽  
Amino Acid Segment

AbstractSilencing of the cryptic mating-type loci HMR and HML requires the recognition of DNA sequence elements called silencers by the Sir1p, one of four proteins dedicated to the assembly of silenced chromatin in Saccharomyces cerevisiae. The Sir1p is thought to recognize silencers indirectly through interactions with proteins that bind the silencer DNA directly, such as the origin recognition complex (ORC). Eight recessive alleles of SIR1 were discovered that encode mutant Sir1 proteins specifically defective in their ability to recognize the HMR-E silencer. The eight missense mutations all map within a 17-amino-acid segment of Sir1p, and this segment was also required for Sir1p's interaction with Orc1p. The mutant Sir1 proteins could function in silencing if tethered to a silencer directly through a heterologous DNA-binding domain. Thus the amino acids identified are required for Sir1 protein's recognition of the HMR-E silencer and interaction with Orc1p, but not for its ability to function in silencing per se. The approach used to find these mutations may be applicable to defining interaction surfaces on proteins involved in other processes that require the assembly of macromolecular complexes.

scholarly journals SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (5) ◽  
pp. 2154-2164 ◽  
Author(s):  
D J DeMarini ◽  
M Winey ◽  
D Ursic ◽  
F Webb ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Gene Product ◽  
Signal Sequence ◽  
Null Alleles ◽  
Nucleoside Triphosphate ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

scholarly journals The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

1990 ◽  
Vol 10 (10) ◽  
pp. 5128-5137 ◽  
Author(s):  
M M Witte ◽  
R C Dickson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Binding Specificity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Transcription Activator ◽  
Activator Proteins ◽  
Dna Binding Specificity

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae

1986 ◽  
Vol 6 (5) ◽  
pp. 1711-1721
Author(s):  
E M McIntosh ◽  
R H Haynes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Northern Analysis ◽  
Hindiii Site ◽  
Reading Frame ◽  
Hindiii Restriction Site ◽  
Cycle Dependent

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

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 Activation of the SPS Amino Acid-Sensing Pathway in Saccharomyces cerevisiae Correlates with the Phosphorylation State of a Sensor Component, Ptr3

2007 ◽  
Vol 28 (2) ◽  
pp. 551-563 ◽  
Author(s):  
Zhengchang Liu ◽  
Janet Thornton ◽  
Mário Spírek ◽  
Ronald A. Butow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Nuclear Translocation ◽  
Proteolytic Processing ◽  
Casein Kinase I ◽  
Positive Regulators ◽  
Amino Acid Permeases ◽  
Amino Acid Sensing ◽  
Amino Acid Sensor

ABSTRACT Cells of the budding yeast Saccharomyces cerevisiae sense extracellular amino acids and activate expression of amino acid permeases through the SPS-sensing pathway, which consists of Ssy1, an amino acid sensor on the plasma membrane, and two downstream factors, Ptr3 and Ssy5. Upon activation of SPS signaling, two transcription factors, Stp1 and Stp2, undergo Ssy5-dependent proteolytic processing that enables their nuclear translocation. Here we show that Ptr3 is a phosphoprotein whose hyperphosphorylation is increased by external amino acids and is dependent on Ssy1 but not on Ssy5. A deletion mutation in GRR1, encoding a component of the SCFGrr1 E3 ubiquitin ligase, blocks amino acid-induced hyperphosphorylation of Ptr3. We found that two casein kinase I (CKI) proteins, Yck1 and Yck2, previously identified as positive regulators of SPS signaling, are required for hyperphosphorylation of Ptr3. Loss- and gain-of-function mutations in PTR3 result in decreased and increased Ptr3 hyperphosporylation, respectively. We found that a defect in PP2A phosphatase activity leads to the hyperphosphorylation of Ptr3 and constitutive activation of SPS signaling. Two-hybrid analysis revealed interactions between the N-terminal signal transduction domain of Ssy1 with Ptr3 and Yck1. Our findings reveal that CKI and PP2A phosphatase play antagonistic roles in SPS sensing by regulating Ptr3 phosphorylation.

scholarly journals Genetic Analysis of Azole Resistance in the Darlington Strain of Candida albicans

2000 ◽  
Vol 44 (11) ◽  
pp. 2985-2990 ◽  
Author(s):  
Hiroshi Kakeya ◽  
Yoshitsugu Miyazaki ◽  
Haruko Miyazaki ◽  
Katherine Nyswaner ◽  
Brian Grimberg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Candida Albicans ◽  
Amino Acid ◽  
Genetic Analysis ◽  
Expression System ◽  
Single Copy ◽  
Gene Replacement ◽  
Azole Resistance ◽  
High Level

ABSTRACT High-level azole resistance in the Darlington strain ofCandida albicans was investigated by gene replacement inC. albicans and expression in Saccharomyces cerevisiae. We sequenced the ERG11 gene, which encodes the sterol C14α-demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C. albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S. cerevisiae expression system. Replacement of one copy of ERG11 in an azole-susceptible strain of C. albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the result of multiple mutations.

THE METABOLISM OF YEAST SPORULATION: VI. CHANGES IN AMINO ACID CONTENT DURING SPOROGENESIS

1964 ◽  
Vol 10 (5) ◽  
pp. 623-631 ◽  
Author(s):  
C. Ramirez ◽  
J. J. Miller
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Acid Content ◽  
Free Amino Acids ◽  
Free Amino ◽  
Amino Acid Content ◽  
Proline Content ◽  
Sporulation Medium ◽  
Free Proline

During 6-day exposures of cells of Saccharomyces cerevisiae to acetate sporulation medium, the content of free amino acids declined to approximately one-third of that of vegetative cells, but proline was exceptional in that it increased conspicuously in amount. The content of combined amino acids also diminished to about one-third, ammonia was evolved, and amino acids (not including proline) passed out of the cells into the medium. When dihydroxyacetone replaced acetate in the sporulation medium, the results were similar except that the decline in content of free and combined amino acids was much greater, more ammonia was evolved, and only very small amounts of amino acids could be detected in the medium. Transfer of sporulated cells to growth medium led to an increase in the pool of free amino acids, except for proline, which declined in amount.In two other species of Saccharomyces the free proline content also increased on exposure to sporulation medium, but in Schizosaccharomyces pombe and Torulopsis famata no such increase was observed.

scholarly journals Saccharomyces cerevisiae pms2 mutations are alleles of MLH1, and pms2-2 corresponds to a hereditary nonpolyposis colorectal carcinoma-causing missense mutation.

1996 ◽  
Vol 16 (6) ◽  
pp. 3008-3011 ◽  
Author(s):  
A Jeyaprakash ◽  
R Das Gupta ◽  
R Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Colorectal Carcinoma ◽  
Missense Mutation ◽  
Model System ◽  
Missense Mutations ◽  
Mlh1 Gene ◽  
Dna Mismatch ◽  
Hereditary Nonpolyposis ◽  
Hereditary Nonpolyposis Colorectal Carcinoma

A number of mutant Saccharomyces cerevisiae strains having phenotypes consistent with defects in DNA mismatch repair have been described, but not all have been extensively characterized. In this study we demonstrate that the pms2-1 and pms2-2 alleles arise from missense mutations in the MLH1 gene which inactivate MLH1. One of these alleles, pms2-2, causes the same amino acid substitution in a highly conserved region of the known MutL homologs as that caused by a proposed missense mutation observed in a Swedish hereditary nonpolyposis colorectal carcinoma kindred. This observation supports the functional significance of missense mutations found in hereditary nonpolyposis colorectal carcinoma kindreds and indicates that in some cases S. cerevisiae can serve as a useful model system for the analysis of such mutations.

scholarly journals Properties of the DNA-binding domain of the Saccharomyces cerevisiae STE12 protein.

1991 ◽  
Vol 11 (12) ◽  
pp. 5910-5918 ◽  
Author(s):  
Y L Yuan ◽  
S Fields
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Dna Binding ◽  
Binding Affinity ◽  
Binding Domain ◽  
Upstream Region ◽  
Dna Binding Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dnase I Footprinting

The STE12 protein of the yeast Saccharomyces cerevisiae binds to the pheromone response element (PRE) present in the upstream region of genes whose transcription is induced by pheromone. Using DNase I footprinting assays with bacterially made STE12 fragments, we localized the DNA-binding domain to 164 amino acids near the amino terminus. Footprinting of oligonucleotide-derived sequences containing one PRE, or two PREs in head-to-tail or tail-to-tail orientation, showed that the N-terminal 215 amino acids of STE12 has similar binding affinity to either of the dimer sites and a binding affinity 5- to 10-fold lower for the monomer site. This binding cooperativity was also evident on a fragment from the MFA2 gene, which encodes the a-factor pheromone. On this fragment, the 215-amino-acid STE12 fragment protected both a consensus PRE as well as a degenerate PRE containing an additional residue. Mutation of the degenerate site led to a 5- to 10-fold decrease in binding; mutation of the consensus site led to a 25-fold decrease in binding. The ability of PREs to function as pheromone-inducible upstream activation sequences in yeast correlated with their ability to bind the STE12 domain in vitro. The sequence of the STE12 DNA-binding domain contains similarities to the homeodomain, although it is highly diverged from other known examples of this motif. Moreover, the alignment between STE12 and the homeodomain postulates loops after both the putative helix 1 and helix 2 of the STE12 sequence.

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