Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione-GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.     

Differential regulation of the N-methyl transferases responsible for phosphatidylcholine synthesis in Saccharomyces cerevisiae

The enzymes catalyzing the conversion of phosphatidylethanolamine to phosphatidylcholine were assayed by measuring the incorporation of label from [¹⁴C-CH₃]-S-adenosyl-methionine into the endogenous phospholipids of particulate, cell-free preparations from S. cerevisiae grown in the presence of N-methylethanolamine, N,N-dimethylethanolamine, or choline. The results indicate that each base in the growth medium results in reduced levels of all the N-methyltransferase activity involved in the formation of the phosphatidyl ester of the given base. By following the conversion of exogenous [³²P]-phosphatidyldimethylethanolamine to [³²P]-phosphatidylcholine it has been shown that the activity of the third methyl transfer is 90% lower in particles prepared from choline grown cells than in particles prepared from cells grown without choline. The results suggest that there are at least two enzymes involved in the conversion of phosphatidylethanolamine to phosphatidylcholine and that their levels can be regulated individually.Supplementing the growth medium with any of the three methylated aminoethanols results in markedly increased cellular levels of their corresponding phosphatidyl esters and decreased levels of the precursor phosphatidyl esters. The fatty acid composition of phosphatidylcholine also changes when the medium is supplemented with choline suggesting that the proportions of the molecular species of this phosphatide depends on whether synthesis is via methylation of phosphatidylethanolamino or from the supplemented aminoethanol.     

Structural and functional analysis of Saccharomyces cerevisiae Mob1

The Mob proteins function as activator subunits for the Dbf2/Dbf20 family of protein kinases. Human and Xenopus Mob1 protein structures corresponding to the most conserved C-terminal core, but lacking the variable N-terminal region, have been reported and provide a framework for understanding the mechanism of Dbf2/Dbf20 regulation. Here, we report the 2.0 A X-ray crystal structure of Saccharomyces cerevisiae Mob1 containing both the conserved C-terminal core and the variable N-terminal region. Within the N-terminal region, three novel structural elements are observed; namely, an alpha-helix denoted H0, a strand-like element denoted S0 and a short beta strand denoted S-1. Helix H0 associates in an intermolecular manner with a second Mob1 molecule to form a Mob1 homodimer. Strand S0 binds to the core domain in an intramolecular manner across a putative Dbf2 binding site mapped by Mob1 temperature-sensitive alleles and NMR binding experiments. In vivo functional analysis demonstrates that Mob1 mutants that target helix H0 or its reciprocal binding site are biologically compromised. The N-terminal region of Mob1 thus contains structural elements that are functionally important.     

Structural analysis of Saccharomyces cerevisiae myo-inositol phosphate synthase

The New York Structural Genomics Research Consortium has targeted highly conserved but uncharacterized enzyme families for structure determination. As part of this effort, the 2.65-Å crystal structure has been determined for Saccharomyces cerevisiae myo-inositol 1-phosphate synthase (MIP), an essential enzyme that catalyzes critical steps in inositol biosynthesis. The structure determination of four independent monomers in the asymmetric unit (240 kDa) reveals atomic details and residue composition for the partially closed NAD-containing active sites in apo-configuration. The structure further reveals extensive interactions involved in tetrameric assembly of the enzyme complex.     

Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae

Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme, and its deficiency in humans causes the accumulation of 2,8-dihydroxyadenine. It is the sole catalyst for adenine recycling in most eukaryotes. The most commonly expressed APRTase has subunits of approximately 187 amino acids, but the only crystal structure is from Leishmania donovani, which expresses a long form of the enzyme with 237 residues. Saccharomyces cerevisiae APRTase was selected as a representative of the short APRTases, and the structure of the apo-enzyme and sulfate bound forms were solved to 1.5 and 1.75 A, respectively. Yeast APRTase is a dimeric molecule, and each subunit is composed of a central five-stranded beta-sheet surrounded by five alpha-helices, a structural theme found in all known purine phosphoribosyltransferases. The structures reveal several important features of APRTase function: (i) sulfate ions bound at the 5'-phosphate and pyrophosphate binding sites; (ii) a nonproline cis peptide bond (Glu67-Ser68) at the pyrophosphate binding site in both apo-enzyme and sulfate-bound forms; and (iii) a catalytic loop that is open and ordered in the apo-enzyme but open and disordered in the sulfate-bound form. Alignment of conserved amino acids in short-APRTases from 33 species reveals 13 invariant and 15 highly conserved residues present in hinges, catalytic site loops, and the catalytic pocket. Mutagenesis of conserved residues in the catalytic loop, subunit interface, and phosphoribosylpyrophosphate binding site indicates critical roles for the tip of the catalytic loop (Glu106) and a catalytic site residue Arg69, respectively. Mutation of one loop residue (Tyr103Phe) increases k(cat) by 4-fold, implicating altered dynamics for the catalytic site loop.     

Functional production of mammalian concentrative nucleoside transporters in Saccharomyces cerevisiae

The transport of nucleosides and nucleobases in the yeast Saccharomyces cerevisiae is reviewed and the use of this organism to study recombinant mammalian concentrative nucleoside transport (CNT) proteins is described. A selection strategy based on the ability of an expressed nucleoside transporter cDNA to mediate thymidine uptake by yeast under a selective condition that depletes endogenous thymidylate was used to assess the transport capacity of heterologous transporter proteins. The pyrimidine-nucleoside selective concentrative transporters from human (hCNT1) and rat (rCNT1) complemented the imposed thymidylate depletion in S. cerevisiae, as did N-terminally truncated versions of hCNT1 and rCNT1 lacking up to 31 amino acids. Transporter-mediated rescue of S. cerevisiae by both nucleoside transporters was inhibited by cytidine, uridine and adenosine, but not by guanosine or inosine. This work represents the development of a new model system for the functional production of recombinant nucleoside transporters of the CNT family of membrane proteins.     

Functional production of mammalian concentrative nucleoside transporters in Saccharomyces cerevisiae

The transport of nucleosides and nucleobases in the yeast Saccharomyces cerevisiae is reviewed and the use of this organism to study recombinant mammalian concentrative nucleoside transport (CNT) proteins is described. A selection strategy based on the ability of an expressed nucleoside transporter cDNA to mediate thymidine uptake by yeast under a selective condition that depletes endogenous thymidylate was used to assess the transport capacity of heterologous transporter proteins. The pyrimidine-nucleoside selective concentrative transporters from human (hCNT1) and rat (rCNT1) complemented the imposed thymidylate depletion in S. cerevisiae, as did N-terminally truncated versions of hCNT1 and rCNT1 lacking up to 31 amino acids. Transporter-mediated rescue of S. cerevisiae by both nucleoside transporters was inhibited by cytidine, uridine and adenosine, but not by guanosine or inosine. This work represents the development of a new model system for the functional production of recombinant nucleoside transporters of the CNT family of membrane proteins.     

Saccharomyces cerevisiae cells have three Omega class glutathione S-transferases acting as 1-Cys thiol transferases

The Saccharomyces cerevisiae genome encodes three proteins that display similarities with human GSTOs (Omega class glutathione S-transferases) hGSTO1-1 and hGSTO2-2. The three yeast proteins have been named Gto1, Gto2 and Gto3, and their purified recombinant forms are active as thiol transferases (glutaredoxins) against HED (beta-hydroxyethyl disulphide), as dehydroascorbate reductases and as dimethylarsinic acid reductases, while they are not active against the standard GST substrate CDNB (1-chloro-2,4-dinitrobenzene). Their glutaredoxin activity is also detectable in yeast cell extracts. The enzyme activity characteristics of the Gto proteins contrast with those of another yeast GST, Gtt1. The latter is active against CDNB and also displays glutathione peroxidase activity against organic hydroperoxides such as cumene hydroperoxide, but is not active as a thiol transferase. Analysis of point mutants derived from wild-type Gto2 indicates that, among the three cysteine residues of the molecule, only the residue at position 46 is required for the glutaredoxin activity. This indicates that the thiol transferase acts through a monothiol mechanism. Replacing the active site of the yeast monothiol glutaredoxin Grx5 with the proposed Gto2 active site containing Cys46 allows Grx5 to retain some activity against HED. Therefore the residues adjacent to the respective active cysteine residues in Gto2 and Grx5 are important determinants for the thiol transferase activity against small disulphide-containing molecules.     

Structural homology among calf thymus alpha-polymerase polypeptides.

A sample of highly purified calf thymus alpha-polymerase contained an abundant 118,000 Mr polypeptide as well as five lower molecular weight polypeptides in the range of 54,000- to 64,000-Mr. This 118,000-Mr polypeptide was capable of DNA polymerase activity, as revealed by in situ assay after SDS-polyacrylamide gel electrophoresis. Tryptic peptide mapping indicated that the 118,000-Mr polypeptide shared extensive primary structure homology with 57,000-, 58,000- and 64,000-Mr polypeptides and some limited homology with 54,000- and 56,000-Mr polypeptides. This is the first evidence that lower and higher Mr polypeptides of purified calf thymus alpha-polymerase share sequence homology; these results are interpreted in the context of a model that predicts the existence of a common precursor with molecular weight greater than 140,000.     

Structural characterization of Saccharomyces cerevisiae prion-like protein Ure2

Sacchromyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a soluble protein that can assemble into fibers that are similar to the fibers observed in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatography, sedimentation velocity, and electron microscopy demonstrates that the soluble form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concentration-dependent. By the use of limited proteolysis, intrinsic fluorescence, and circular dichroism measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p molecules. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insoluble high molecular weight oligomers. These changes essentially affect the COOH-terminal part of the molecule. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.     

Minimal extent of homology required for completion of meiotic recombination in Saccharomyces cerevisiae

The minimal length of contiguous homology required for successful completion of meiotic recombination was investigated by using heterologous insertions to delimit homologous segments of chromosome III in the yeast Saccharomyces cerevisiae. Constructs created in vitro by insertion of selectable markers into the LEU2 locus were transplaced into haploid strains, which were then mated to create diploids containing pairs of insertion heterologies at various distances. Analysis of the meiotic products from these diploids revealed a gradient in the frequency of both reciprocal and nonreciprocal recombination declining monotonically from the 5′ end of LEU2. Both types of event were found to be restricted by the presence of the insertion heterologies. The spo 13 single division meiosis was exploited to develop a plating assay in which LEU2 diploid spores containing reciprocally recombinant strands derived from events occurring completely within the interval flanked by the insertion heterologies were selected by random spore methods. Reciprocal recombination frequencies measured with this assay decreased linearly with extent, extrapolating to a minimal homology requirement of 150–250 nucleotides. When homology was most severely restricted, unexpected flanking marker configurations among reciprocal recombinants within LEU2 demonstrated the occurrence of complex recombination events. In addition to detecting reciprocal recombinants, the system is capable of measuring the probability that a non-reciprocal recombination event will have one endpoint between the heterologous inserts and the other lying outside the interval. The minimal length of homology required for this aspect of recombination was found to be 25–60 nucleotides. © 1993 Wiley-Liss, Inc.     

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

Na+/H+ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1-3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.     

Expression of microbial virulence proteins in Saccharomyces cerevisiae models mammalian infection

Bacterial virulence proteins that are translocated into eukaryotic cells were expressed in Saccharomyces cerevisiae to model human infection. The subcellular localization patterns of these proteins in yeast paralleled those previously observed during mammalian infection, including localization to the nucleus and plasma membrane. Localization of Salmonella SspA in yeast provided the first evidence that SspA interacts with actin in living cells. In many cases, expression of the bacterial virulence proteins conferred genetically exploitable growth phenotypes. In this way, Yersinia YopE toxicity was demonstrated to be linked to its Rho GTPase activating protein activity. YopE blocked polarization of the yeast cytoskeleton and cell cycle progression, while SspA altered polarity and inhibited depolymerization of the actin cytoskeleton. These activities are consistent with previously proposed or demonstrated effects on higher eukaryotes and provide new insights into the roles of these proteins in pathogenesis: SspA in directing formation of membrane ruffles and YopE in arresting cell division. Thus, study of bacterial virulence proteins in yeast is a powerful system to determine functions of these proteins, probe eukaryotic cellular processes and model mammalian infection.     

Molecular cloning of mouse p47, a second group mammalian RuvB DNA helicase-like protein: homology with those from human and Saccharomyces cerevisiae

A 47k protein (p47) in a high-salt buffer extract of a rat liver nuclear matrix fraction was purified by means of a wheat germ agglutinin affinity column, reversed phase HPLC, and SDS-PAGE, and partial amino acid sequences were analyzed. Based on these sequences, the mouse cDNA of the protein was cloned and sequenced, and its amino acid sequence was deduced. Mouse p47 consists of 463 amino acid residues with a molecular weight of 51,112. The amino acid sequences of human and Saccharomyces cerevisiae p47s were also deduced from the nucleotide sequences of "expressed sequence tag" fragments and genomic DNA, respectively. These sequences contain helicase motifs and show homology to bacterial RuvB DNA helicases acting in homologous recombination. They also show homology with the putative mammalian helicases p50/TIP49 and RUVBL1. Comparison of the amino acid sequences of p47 group proteins and those of p50/TIP49 group proteins revealed the p47 group proteins to comprise a group distinct from the p50/TIP49 proteins. Ultracentrifugation and gel filtration analyses showed that p47 in the rat liver cytosol fraction exists as large complexes of 697k.     

Structural modeling and electrostatic properties of aspartate transcarbamylase from Saccharomyces cerevisiae

In Saccharomyces cerevisiae the first two reactions of the pyrimidine pathway are catalyzed by a multifunctional protein which possesses carbamylphosphate synthetase and aspartate transcarbamylase activities. Genetic and proteolysis studies suggested that the ATCase activity is carried out by an independently folded domain. In order to provide structural information for ongoing mutagenesis studies, a model of the three-dimensional structure of this domain was generated on the basis of the known X-ray structure of the related catalytic subunit from E. coli ATCase. First, a model of the catalytic monomer was built and refined by energy minimization. In this structure, the conserved residues between the two proteins were found to constitute the hydrophobic core whereas almost all the mutated residues are located at the surface. Then, a trimeric structure was generated in order to build the active site as it lies at the interface between adjacent chains in the E. coli catalytic trimer. After docking a bisubstrate analog into the active site, the whole structure was energy minimized to regularize the interactions at the contact areas between subunits. The resulting model is very similar to that obtained for the E. coli catalytic trimer by X-ray crystallography, with a remarkable conservation of the structure of the active site and its vicinity. Most of the interdomain and intersubunit interactions that are essential for the stability of the E. coli catalytic trimer are maintained in the yeast enzyme even though there is only 42% identity between the two sequences. Free energy calculations indicate that the trimeric assembly is more stable than the monomeric form. Moreover an insertion of four amino acids is localized in a loop which, in E. coli ATCase, is at the surface of the protein. This insertion exposes hydrophobic residues to the solvent. Interestingly, such an insertion is present in all the eukaryotic ATCase genes sequenced so far, suggesting that this region is interacting with another domain of the multifunctional protein. © 1994 Wiley-Liss, Inc.