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PubMed1995

Characterization of yeast translation initiation factor 1A and cloning of its essential gene.

Translation initiation factor eIF1A is required in vitro for maximal rates of protein synthesis in mammalian systems. It functions primarily by dissociating ribosomes and stabilizing 40 S preinitiation complexes. To better elucidate its precise role in promoting the translation initiation process, the yeast form of eIF1A has been identified in Saccharomyces cerevisiae and purified to homogeneity on the basis of its cross-reaction with antibodies prepared against mammalian eIF1A. The apparent mass of yeast eIF1A (22 kDa) resembles that of the mammalian homolog (20 kDa), and the yeast factor is active in stimulating methionyl-puromycin synthesis in an assay composed of mammalian components. The gene encoding yeast eIF1A, named TIF11, was cloned and shown to be single copy. TIF11 encodes a protein comprising 153 amino acids (17.4 kDa); the deduced amino acid sequence exhibits 65% identity with the sequence of human eIF1A. Both human and yeast eIF1A contain clusters of positive residues at the N terminus and negative residues at the C terminus. Deletion/disruption of TIF11 demonstrates that eIF1A is essential for cell growth. Expression of human eIF1A cDNA rescues the growth defect of TIF11-disrupted cells, indicating that the structure/function of yeast and mammalian eIF1A is highly conserved.

Amino Acid Sequence↗
PubMed1995

Identification of strong modifications in cation selectivity in an Arabidopsis inward rectifying potassium channel by mutant selection in yeast.

The Arabidopsis thaliana cDNA, KAT1, encodes a hyperpolarization-activated K+ channel. In the present study, we utilized a combination of random site-directed mutagenesis, genetic screening in a potassium uptake-deficient yeast strain, and electrophysiological analysis in Xenopus oocytes to identify strong modifications in cation selectivity of the inward rectifying K+ channel KAT1. Threonine at position 256 was replaced by 11 other amino acid residues. Six of these mutated KAT1 cDNAs complemented a K+ uptake-deficient yeast strain at low concentrations of potassium. Among these, two mutants (T256D and T256G) showed a sensitivity of yeast growth toward high ammonium concentrations and a dramatic increase in current amplitudes of rubidium and ammonium ions relative to K+ by 39-72-fold. These single site mutations gave rise to Rb+- and NH4(+)-selective channels with Rb+ and NH4+ currents that were approximately 10-13-fold greater in amplitude than K+ currents, whereas the NH4+ to K+ current amplitude ratio of wild type KAT1 was 0.28. This strong conversion in cation specificity without loss of general selectivity exceeds those reported for other mutations in the pore domain of voltage-dependent K+ channels. Yeast growth was greatly impaired by sodium in two other mutants at this site (T256E and T256Q), which were blocked by millimolar sodium (K1/2 = 1.1 mM for T256E), although the wild type channel was not blocked by 110 mM sodium. Interestingly, the ability of yeast to grow in the presence of toxic cations correlated to biophysical properties of KAT1 mutants, illustrating the potential for qualitative K+ channel mutant selection in yeast. These data suggest that the size of the side chain of the amino acid at position 256 in KAT1 is important for enabling cation permeation and that this site plays a crucial role in determining the cation selectivity of hyperpolarization-activated potassium channels.

Amino Acid Sequence↗
PubMed1995

Prediction and identification of new natural substrates of the yeast mitochondrial intermediate peptidase.

Most mitochondrial precursor proteins are processed to the mature form in one step by mitochondrial processing peptidase (MPP), while a subset of precursors destined for the matrix or the inner membrane are cleaved sequentially by MPP and mitochondrial intermediate peptidase (MIP). We showed previously that yeast MIP (YMIP) is required for mitochondrial function in Saccharomyces cerevisiae. To further define the role played by two-step processing in mitochondrial biogenesis, we have now characterized the natural substrates of YMIP. A total of 133 known yeast mitochondrial precursors were collected from the literature and analyzed for the presence of the motif RX(decreases)(F/L/I)XX(T/S/G)XXXX(decreases), typical of precursors cleaved by MPP and MIP. We found characteristic MIP cleavage sites in two distinct sets of proteins: respiratory components, including subunits of the electron transport chain and tricarboxylic acid cycle enzymes, and components of the mitochondrial genetic machinery, including ribosomal proteins, translation factors, and proteins required for mitochondrial DNA metabolism. Representative precursors from both sets were cleaved to predominantly mature form by mitochondrial matrix or intact mitochondria from wild-type yeast. In contrast, intermediate-size forms were accumulated upon incubation of the precursors with matrix from mip1 delta yeast or intact mitochondria from mip1ts yeast, indicating that YMIP is necessary for maturation of these proteins. Consistent with the fact that some of these substrates are essential for the maintenance of mitochondrial protein synthesis and mitochondrial DNA replication, mip1 delta yeast undergoes loss of functional mitochondrial genomes.

Amino Acid Sequence↗
PubMed1996

Binding of centrins and yeast calmodulin to synthetic peptides corresponding to binding sites in the spindle pole body components Kar1p and Spc110p.

Centrins contain four potential Ca2+ binding sites, known as EF-hands, and have essential functions in centrosome duplication and filament contraction. Here we report that centrins from yeast, green algae, and humans bound with high affinity to a peptide of the yeast centrosomal component Kar1p. Interestingly, centrin binding was regulated by physiological relevant changes in [Ca2+], and this Ca2+ dependence was influenced by acidic amino acids within the Kar1p peptide, which also prevented efficient binding of the related yeast calmodulin. However, a hybrid protein with the third and fourth EF-hands from the yeast centrin Cdc31p and the amino-terminal half from yeast calmodulin behaved more like Cdc31p, indicating that the carboxyl-terminal half of Cdc31p influences binding specificity. Besides Kar1p, centrins bound to a yeast calmodulin binding site, explaining the dosage-dependent suppression of a calmodulin mutant by CDC31. Consistent with an essential role of Ca2+ for centrin functions, mutations in the first or the fourth EF-hands of Cdc31p, impairing the Ca2+-induced conformational change of Cdc31p, resulted in nonfunctional proteins in vivo. Our results suggest that centrins are involved in Ca2+ signaling, likely by influencing the properties of target proteins in response to changes in [Ca2+].

Amino Acid Sequence↗
PubMed1997

Casein kinase II catalyzes tyrosine phosphorylation of the yeast nucleolar immunophilin Fpr3.

In the yeast Saccharomyces cerevisiae, the nucleolar immunophilin, Fpr3, is phosphorylated at tyrosine and dephosphorylated by the phosphotyrosine-specific phosphoprotein phosphatase, Ptp1. In Ptp1-deficient cells, Fpr3 contains phospho-Tyr at a single site (Tyr184), but also contains phospho-Ser and phospho-Thr. Ser186 (adjacent to Tyr184) is situated within a canonical site for phosphorylation by casein kinase II (CKII). Yeast cell lysates contain an activity that binds to Fpr3 in vitro and phosphorylates Fpr3 at Ser, Thr, and Tyr; this activity was found to be dependent on expression of functional yeast CKII. Moreover, purified Fpr3 was phosphorylated on Tyr184 in vitro by either purified yeast CKII or purified, bacterially-expressed human CKII. Likewise, phosphorylation of Fpr3 at tyrosine in vivo was markedly enhanced in yeast cells overexpressing a heterologous (Drosophila) CKII, but was undetectable in yeast cells carrying only a temperature-sensitive allele of the endogenous CKII, even when the cells were grown at a permissive temperature. Phosphorylation of Fpr3 at Tyr184 by CKII in vitro lagged behind phosphorylation of Fpr3 at Ser, and was accelerated by pre-phosphorylation of Fpr3 at Ser using CKII. Furthermore, synthetic peptides corresponding to the sequence surrounding Tyr184 that contained P-Ser (or Glu) at position 186 were much more efficient substrates for CKII phosphorylation of Tyr184 than a synthetic peptide containing Ala at position 186. These findings indicate that CKII phosphorylates Fpr3 at tyrosine and serine both in vivo and in vitro and thus possesses dual specificity. These results also indicate that Tyr184 is phosphorylated by CKII via a two-step process, in which phosphorylation at the +2 position provides a negatively-charged specificity determinant that allows subsequent phosphorylation of Tyr184.

Amino Acid Isomerases↗
PubMed1997

Identification of the region in yeast S-II that defines species specificity in its interaction with RNA polymerase II.

Yeast S-II was found to stimulate yeast RNA polymerase II only and not mouse RNA polymerase II. To identify the molecular region of S-II that defines species specificity, we constructed six hybrid S-II molecules consisting of three regions from yeast and/or Ehrlich cell S-II and examined their activity in terms of RNA polymerase II specificity and suppression of 6-azauracil sensitivity in the yeast S-II null mutant. We found that the region 132-270 (amino acid positions) of yeast S-II is indispensable for specific interaction with yeast RNA polymerase II in vitro and for suppression of 6-azauracil sensitivity in vivo. The corresponding region of Ehrlich cell S-II, the region 132-262, was also shown to be essential for its interaction with mouse RNA polymerase II. This region is known to be less conserved than the N- and C-terminal regions in the S-II family suggesting that it is important in the interaction with transcription machinery proteins in a tissue and/or species-specific manner.

Amino Acid Sequence↗
PubMed1997

Functional complementation of the yeast divalent cation transporter family SMF by NRAMP2, a member of the mammalian natural resistance-associated macrophage protein family.

The mammalian NRAMP gene family has two members, NRAMP1 and NRAMP2 that encode integral membrane proteins. Nramp1 is expressed exclusively in macrophages where it is found in the phagosomal membrane, and NRAMP1 mutations cause susceptibility to infection by abrogating the capacity of macrophages to control intracellular microbial replication. Nramp2 is highly similar to Nramp1, but is expressed in several tissues and cell types. The Nramp protein family is remarkably conserved throughout evolution, and recent data suggest that the mammalian Nramp2 and the yeast homologues Smf1 and Smf2 transport divalent cations. We tested whether structural similarity between the mammalian Nramp and the yeast Smf proteins results in functional complementation in yeast. Wild-type and mutant variants of the Nramp1 and Nramp2 proteins were expressed in a yeast mutant bearing null alleles at the SMF1 and SMF2 loci, and complementation of the phenotypes of this yeast mutant was investigated. Nramp2, but not Nramp1, was found to complement hypersensitivity to EGTA of the smf1/smf2 mutant under oxidative stress conditions (methyl viologen). We also observed that the smf1/smf2 double mutant is hypersensitive to growth at alkaline pH (pH 7.9) and that Nramp2 could complement this phenotype as well. Complementation by Nramp2 was specific and required a functional protein as independent mutations in residues highly conserved in all members of the Nramp family abrogated Nramp2 complementation. Since Mn2+ was the only divalent cation capable of completely suppressing both the EGTA and pH phenotypes, our results suggest that Nramp2 can transport Mn2+ in yeast.

Amino Acid Sequence↗
PubMed1997

Expression of the human aryl hydrocarbon receptor complex in yeast. Activation of transcription by indole compounds.

The human aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator protein (ARNT) were coexpressed in the yeast Saccharomyces cerevisiae to create a system for the study of this heterodimeric transcription factor. Specific transcriptional activation mediated by AHR/ARNT heterodimer, which is a functional indicator of receptor expression, was assessed by beta-galactosidase activity produced from a reporter plasmid. Yeast expressing AHR and ARNT displayed constitutive transcriptional activity that was not augmented by addition of AHR agonists in strains that required exogenous tryptophan for viability. In contrast, strains with an intact pathway for tryptophan biosynthesis responded to AHR agonists and had lower levels of background beta-galactosidase activity. Hexachlorobenzene, benzo(a)pyrene, and beta-naphthoflavone were effective AHR agonists in the yeast system, and had EC50 values of 200, 40, and 20 nM, respectively, for beta-galactosidase activity induction. Tryptophan, indole, indole acetic acid, and tryptamine activated transcription in yeast coexpressing AHR and ARNT (EC50 values approximately 300 microM). Indole-3-carbinol was an exceptionally potent AHR agonist (EC50 approximately 10 microM) in yeast. This yeast system is useful for the study of AHR/ARNT protein complexes, and may be generally applicable to the investigation of other multiprotein complexes.

Antioxidants↗
PubMed1998

Cloning and characterization of shk2, a gene encoding a novel p21-activated protein kinase from fission yeast.

We describe the characterization of a novel gene, shk2, encoding a second p21(cdc42/rac)-activated protein kinase (PAK) homolog in fission yeast. Like other known PAKs, Shk2 binds to Cdc42 in vivo and in vitro. While overexpression of either shk2 or cdc42 alone does not impair growth of wild type fission yeast cells, cooverexpression of the two genes is toxic and leads to highly aberrant cell morphology, providing evidence for functional interaction between Cdc42 and Shk2 proteins in vivo. Fission yeast shk2 null mutants are viable and exhibit no obvious phenotypic defects. Overexpression of shk2 restores viability and normal morphology but not full mating competence to fission yeast cells carrying a shk1 null mutation. Additional genetic data suggest that Shk2, like Cdc42 and Shk1, participates in Ras-dependent morphological control and mating response pathways in fission yeast. We also show that overexpression of byr2, a gene encoding a Ste11/MAPK kinase kinase homolog, suppresses the mating defect of cells partially defective for Shk1 function, providing evidence of a link between PAKs and mitogen-activated protein kinase signaling in fission yeast. Taken together, our results suggest that Shk2 is partially overlapping in function with Shk1, with Shk1 being the dominant protein in function.

Amino Acid Sequence↗
PubMed1998

The respiratory chain in yeast behaves as a single functional unit.

Inhibitor titrations using antimycin have been used to study the pool behavior of ubiquinone and cytochrome c in the respiratory chain of the yeast Saccharomyces cerevisiae. If present in a homogeneous pool, these carriers should be able to diffuse freely through or along the membrane respectively and accept and subsequently donate electrons to an infinite number of the respective respiratory complex. However, we show that under physiological conditions neither ubiquinone nor cytochrome c exhibits pool behavior, implying that the respiratory chain in yeast is one functional unit. Pool behavior can be introduced for both small carriers by adding chaotropic agents to the reaction medium. We conclude that these agents disrupt the interaction between the respiratory complexes, thereby causing them to become randomly arranged in the membrane. In such a situation, ubiquinone and cytochrome c become mobile carriers, shuttling between the large respiratory complexes. Furthermore, we conclude from the respiratory activities found for different substrates that the respiratory units in yeast vary in composition with respect to the ubiquinone reducing enzyme. All units contain the cytochrome chain, supplemented with either succinate dehydrogenase or the internal or the external NADH dehydrogenase. This implies that when only one substrate is available, only a certain fraction of the cytochrome chain is used in respiration. The molecular organization of the respiratory chain in yeast is compared with that of higher eukaryotes and to the electron transfer systems of photosynthetic membranes. Differences between the organization of the respiratory chain of yeast and that of higher eukaryotes are discussed in terms of the ability of yeast to radically alter its metabolism in response to change of the available carbon source.

Antimycin A↗
PubMed1999

Yeast and rat Coq3 and Escherichia coli UbiG polypeptides catalyze both O-methyltransferase steps in coenzyme Q biosynthesis.

Ubiquinone (coenzyme Q or Q) is a lipid that functions in the electron transport chain in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes. Q-deficient mutants of Saccharomyces cerevisiae harbor defects in one of eight COQ genes (coq1-coq8) and are unable to grow on nonfermentable carbon sources. The biosynthesis of Q involves two separate O-methylation steps. In yeast, the first O-methylation utilizes 3, 4-dihydroxy-5-hexaprenylbenzoic acid as a substrate and is thought to be catalyzed by Coq3p, a 32.7-kDa protein that is 40% identical to the Escherichia coli O-methyltransferase, UbiG. In this study, farnesylated analogs corresponding to the second O-methylation step, demethyl-Q(3) and Q(3), have been chemically synthesized and used to study Q biosynthesis in yeast mitochondria in vitro. Both yeast and rat Coq3p recognize the demethyl-Q(3) precursor as a substrate. In addition, E. coli UbiGp was purified and found to catalyze both O-methylation steps. Futhermore, antibodies to yeast Coq3p were used to determine that the Coq3 polypeptide is peripherally associated with the matrix-side of the inner membrane of yeast mitochondria. The results indicate that one O-methyltransferase catalyzes both steps in Q biosynthesis in eukaryotes and prokaryotes and that Q biosynthesis is carried out within the matrix compartment of yeast mitochondria.

Animals↗
PubMed1999

Alternative mechanisms of vacuolar acidification in H(+)-ATPase-deficient yeast.

Acidification of the endosomal/lysosomal pathway by the vacuolar-type proton translocating ATPase (V-ATPase) is necessary for a variety of essential eukaryotic cellular functions. Nevertheless, yeasts lacking V-ATPase activity (Deltavma) are viable when grown at low pH, suggesting alternative methods of organellar acidification. This was confirmed by directly measuring the vacuolar pH by ratio fluorescence imaging. When Deltavma yeasts were cultured and tested in the acidic conditions required for growth of V-ATPase-deficient mutants, the vacuolar pH was 5.9. Fluid-phase pinocytosis of acidic extracellular medium cannot account for these observations, because the V-ATPase-independent vacuolar acidification was unaffected in mutants deficient in endocytosis. Similarly, internalization of the plasmalemmal H(+)-ATPase (Pma1p) was ruled out, because overexpression of Pma1p failed to complement the Deltavma phenotype and did not potentiate the vacuolar acidification. To test whether weak electrolytes present in the culture medium could ferry acid equivalents to the vacuole, wild-type and the Deltavma yeasts were subjected to sudden changes in extracellular pH. In both cell types, the vacuoles rapidly alkalinized when external pH was raised from 5.5 (the approximate pH of the culture medium) to 7.5 and re-acidified when the yeasts were returned to a medium of pH 5.5. Importantly, these rapid pH changes were only observed when NH(4)(+), routinely added as a nitrogen source, was present. The NH(4)(+)-dependent acidification was not due to efflux of NH(3) from the vacuole, as cells equilibrated to pH 7.5 in the absence of weak electrolytes rapidly acidified when challenged with an acidic medium containing NH(4)(+). These findings suggest that although NH(3) can act as a cell-permeant proton scavenger, NH(4)(+) may function as a protonophore, facilitating equilibration of the pH across the plasma and vacuolar membranes of yeast. The high concentration of NH(4)(+) frequently added as a nitrogen source to yeast culture media together with effective NH(4)(+) transporters thereby facilitate vacuolar acidification when cells are suspended in acidic solutions.

Cell Membrane↗
PubMed1999

Translational repression by human 4E-BP1 in yeast specifically requires human eIF4E as target.

4E-binding proteins (4E-BPs) are believed to have important regulatory functions in controlling the rate of translation initiation in mammalian cells. They do so by binding to the mRNA cap-binding protein, eIF4E, thereby inhibiting formation of the cap-binding complex, a process essential for cap-dependent translation initiation. We have reproduced the translation-repressive function of human 4E-BP1 in yeast and find its activity to be dependent on substitution of human eIF4E for its yeast counterpart. Translation initiation and growth are inhibited when human 4E-BP1 is expressed in a strain with the human eIF4E substitution, but not in an unmodified strain. We have compared the relative affinities of human 4E-BP1 for human and yeast eIF4E, both in vitro using an m7GTP cap-binding assay and in vivo using a yeast two-hybrid assay, and find that the affinity of human 4E-BP1 for human eIF4E is markedly greater than for yeast eIF4E. Thus yeast eIF4E lacks structural features required for binding to human 4E-BP1. These results therefore demonstrate that the features of eIF4E required for binding to 4E-BP1 are distinct from those required for cap-complex assembly.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein.

Our studies of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are aimed at clarifying the cofactor dependence and catalytic mechanism and obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease). We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine beta-synthase. The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine beta-synthase. The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism. The reaction of the enzyme with L-serine in the absence of L-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm. The formation of this intermediate from the product, L-cystathionine, demonstrates the partial reversibility of the reaction. Our results establish the overall catalytic mechanism of yeast cystathionine beta-synthase and provide a useful system for future studies of structure and function. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase.

Animals↗
PubMed2005

Mammalian prion protein suppresses Bax-induced cell death in yeast.

Several lines of evidence suggest that PrP(C), the non-infectious form of the prion protein, may function to protect neurons and other cells from stress or toxicity. In this paper, we report on the use of the yeast Saccharomyces cerevisiae as a model system to assay the cytoprotective activity of PrP(C). The mammalian pro-apoptotic protein, Bax, confers a lethal phenotype when expressed in yeast. Since overexpression of PrP(C) has been found to prevent Bax-mediated cell death in cultured human neurons, we explored whether PrP could also suppress Bax-induced cell death in yeast. We utilized a form of mouse PrP containing a modified signal peptide that we had previously shown is efficiently targeted to the secretory pathway in yeast. We found that this PrP potently suppressed the death of yeast cells expressing mammalian Bax under control of a galactose-inducible promoter. In contrast, cytosolic PrP-(23-231) failed to rescue growth of Bax-expressing yeast, indicating that protective activity requires targeting of PrP to the secretory pathway. Deletion of the octapeptide repeat region did not affect the rescuing activity of PrP, but deletion of a charged region encompassing residues 23-31 partially eliminated activity. We also tested several PrP mutants associated with human familial prion diseases and found that only a mutant containing nine extra octapeptide repeats failed to suppress Bax-induced cell death. These findings establish a simple and genetically tractable system for assaying a putative biological activity of PrP(C).

Animals↗
PubMed2000

A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast.

The mammalian inducer of apoptosis Bax is lethal when expressed in yeast and plant cells. To identify potential inhibitors of Bax in plants we transformed yeast cells expressing Bax with a tomato cDNA library and we selected for cells surviving after the induction of Bax. This genetic screen allows for the identification of plant genes, which inhibit either directly or indirectly the lethal phenotype of Bax. Using this method a number of cDNA clones were isolated, the more potent of which encodes a protein homologous to the class theta glutathione S-transferases. This Bax-inhibiting (BI) protein was expressed in Escherichia coli and found to possess glutathione S-transferase (GST) and weak glutathione peroxidase (GPX) activity. Expression of Bax in yeast decreases the intracellular levels of total glutathione, causes a substantial reduction of total cellular phospholipids, diminishes the mitochondrial membrane potential, and alters the intracellular redox potential. Co-expression of the BI-GST/GPX protein brought the total glutathione levels back to normal and re-established the mitochondrial membrane potential but had no effect on the phospholipid alterations. Moreover, expression of BI-GST/GPX in yeast was found to significantly enhance resistance to H(2)O(2)-induced stress. These results underline the relationship between oxidative stress and Bax-induced death in yeast cells and demonstrate that the yeast-based genetic strategy described here is a powerful tool for the isolation of novel antioxidant and antiapoptotic genes.

Amino Acid Sequence↗
PubMed2000

Yeast glyoxalase I is a monomeric enzyme with two active sites.

The tertiary structure of the monomeric yeast glyoxalase I has been modeled based on the crystal structure of the dimeric human glyoxalase I and a sequence alignment of the two enzymes. The model suggests that yeast glyoxalase I has two active sites contained in a single polypeptide. To investigate this, a recombinant expression clone of yeast glyoxalase I was constructed for overproduction of the enzyme in Escherichia coli. Each putative active site was inactivated by site-directed mutagenesis. According to the alignment, glutamate 163 and glutamate 318 in yeast glyoxalase I correspond to glutamate 172 in human glyoxalase I, a Zn(II) ligand and proposed general base in the catalytic mechanism. The residues were each replaced by glutamine and a double mutant containing both mutations was also constructed. Steady-state kinetics and metal analyses of the recombinant enzymes corroborate that yeast glyoxalase I has two functional active sites. The activities of the catalytic sites seem to be somewhat different. The metal ions bound in the active sites are probably one Fe(II) and one Zn(II), but Mn(II) may replace Zn(II). Yeast glyoxalase I appears to be one of the few enzymes that are present as a single polypeptide with two active sites that catalyze the same reaction.

Base Sequence↗
PubMed2000

An oxysterol-derived positive signal for 3-hydroxy- 3-methylglutaryl-CoA reductase degradation in yeast.

Sterol synthesis by the mevalonate pathway is modulated, in part, through feedback-regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In mammals, both a non-sterol isoprenoid signal derived from farnesyl diphosphate (FPP) and a sterol-derived signal appear to act together to positively regulate the rate of HMGR degradation. Although the nature and number of sterol-derived signals are not clear, there is growing evidence that oxysterols can serve in this capacity. In yeast, a similar non-sterol isoprenoid signal generated from FPP acts to positively regulate HMGR degradation, but the existence of any sterol-derived signal has thus far not been revealed. We now demonstrate, through the use of genetic and pharmacological manipulation of oxidosqualene-lanosterol cyclase, that an oxysterol-derived signal positively regulated HMGR degradation in yeast. The oxysterol-derived signal acted by specifically modulating HMGR stability, not endoplasmic reticulum-associated degradation in general. Direct biochemical labeling of mevalonate pathway products confirmed that oxysterols were produced endogenously in yeast and that their levels varied appropriately in response to genetic or pharmacological manipulations that altered HMGR stability. Genetic manipulation of oxidosqualene-lanosterol cyclase did result in the buildup of detectable levels of 24,25-oxidolanosterol by gas chromatography, gas chromatography-mass spectroscopy, and NMR analyses, whereas no detectable amounts were observed in wild-type cells or cells with squalene epoxidase down-regulated. In contrast to mammalian cells, the yeast oxysterol-derived signal was not required for HMGR degradation in yeast. Rather, the function of this second signal was to enhance the ability of the FPP-derived signal to promote HMGR degradation. Thus, although differences do exist, both yeast and mammalian cells employ a similar strategy of multi-input regulation of HMGR degradation.

Down-Regulation↗