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Glucose triggers transcriptional and post-transcriptional mechanisms that increase the amount and the activity of Saccharomyces cerevisiae plasma membrane H+-ATPase. In a previous study, we found that a mutation in the Rsp5 ubiquitin-protein ligase enzyme affected the post-transcriptional activation of the enzyme by glucose. Mutations at the RSP5 locus alter the glucose-triggered K(m) decrease. In a genetic screening for multicopy suppressors of the rsp5 mutation, we identified the WSC2/YNL283c gene. Deletion of the WSC2 gene disturbs ATPase activation by glucose, abolishing the K(m) decrease that occurs during this process. Wsc2 is a component of the PKC1-MPK1 mitogen-activated protein kinase (MAPK) signaling pathway that controls the cell wall integrity. Deletion of the MPK1/SLT2 gene disturbs the glucose-triggered K(m) decrease in ATPase. (C) 2000 Elsevier Science B.V., Spanish Grant DGICYT-PB97-0054 supported this study.

The proper spatial and temporal coordination of mitosis and cytokinesis is essential for maintaining genomic integrity. We describe the identification and characterization of the Saccharomyces cerevisiae IBD1 gene, which encodes a novel protein that regulates the proper nuclear division and bud separation. IBD1 was identified by the limited homology to byr4, a dosage-dependent regulator of cytokinesis in Schizosaccharomyces pombe. IBD1 is not an essential gene, and the knock-out cells show no growth defects except for the reduced mating efficiency [1]. However, upon ectopic expression from an inducible promoter, IBD1 is lethal to the cell and leads to abnormal nuclear division and bud separation. In detail, approximately 90% of the IBD1 overexpressing cells arrest at large bud stages with dividing or divided nuclei. In some IBD1 overexpressing cells, spindle elongation and chromosome separation occur within the mother cell, leading to anucleated and binucleate daughter cells. The anucleated cell can not bud, but the binucleate cell proceeds through another cell cycle(s) to produce a cell with multiple nuclei and multiple buds. Observations of the F-actin and chitin rings in the IBD1 overexpressing cells reveal that these cells lose the polarity for bud site selection and growth or attain the hyper-polarity for growth. Consistent with the phenotypes, the IBD1 overexpressing cells contain a broad range of DNA content, from 2 to 4 N or more. A functional Ibd1p-GFP fusion protein localizes to a single dot at the nuclear DNA boundary in the divided nuclei or to double dots in dividing nuclei, suggesting its localization on the spindle pole body (SPB). The cross-species expressions of IBD1 in S. pombe and byr4 in S. cerevisiae cause defects in shape, implicating the presence of a conserved mechanism for the control of cytokinesis in eukaryotes. We propose that Ibd1p is an SPB associated protein that links proper nuclear division to cytokinesis and bud separation.

Potassium efflux in yeast induced by several cationic compounds showed different characteristics. All of the observed efflux required glucose as substrate at the concentrations used. For most of them, the phenomenon required binding of the cationic compound to the cell surface and increased with the negative cell surface charge, and for all the compounds tested, it depended on a metabolizable substrate. Efflux induced with terbium chloride appeared more likely due to the function of a K+/H+ antiporter. With DEAE-dextran and dihydrostreptomycin, potassium efflux was dependent on the cell potassium content and was also sensitive to osmotic changes of the medium. DEAE-dextran-provoked efflux was not due to cell disruption. Dihydrostreptomycin seemed to activate a potassium efflux system which could not be studied in isolation, but its inhibition of potassium uptake may also be involved. Except for cells treated with ethidium bromide, no appreciable cell disruption was observed. The potassium efflux observed appears to be a membrane phenomenon reversible after washing with magnesium chloride.

The green fluorescent protein (GFP) was used as a marker to study the intracellular transport of vacuolar and secretory proteins in yeast. Therefore, the following gene constructs were expressed in Saccharomyces cerevisiae under control of the GAL1 promoter: GFP N-terminally fused to the yeast secretory invertase (INV-GFP), the plant vacuolar chitinase (CHN-GFP) and its secretory derivative (CHNΔVTP-GFP), which did not contain the vacuolar targeting peptide (VTP), both chitinase forms (CHN and CHNΔVTP), GFP without any targeting information and two secretory GFP variants with and without the VTP of chitinase (N-GFP-V and N-GFP). Whereas chitinase without VTP is accumulated in the culture medium the other gene products are retained inside the cell up to 48 h of induction. Independently of a known VTP they are transported to the vacuole, so far as they contain a signal peptide for entering the endoplasmic reticulum. This was demonstrated by confocal laser scanning microscopy, immunocytochemical analysis and subcellular fractionation experiments as well. The transport of the GFP fusion proteins is temporary delayed by a transient accumulation in electron-dense structures very likely derived from the ER, because they also contain the ER chaperone Kar2p/Bip. Our results demonstrate that GFP directs secretory proteins without VTP to the yeast vacuole, possibly by the recognition of an unknown vacuolar signal and demonstrates, therefore, a first limitation for the application of GFP as a marker for the secretory pathway in yeast.

The in vivo activation of Saccharomyces cerevisiae plasma membrane H+-ATPase by ethanol was observed during ethanol-stressed cultivation or following the rapid incubation of cells with ethanol (6% (v/v)). Ethanol activated both the basal and the glucose-activated forms of the enzyme being the H+-ATPase fully activated by glucose (5% (w/v)) still additionally activable by ethanol. The kinetic parameters of ethanol-activated and non-activated H+-ATPase were calculated based directly on Michaëlis–Menten equation (with MgATP concentrations in the range 0.16–8.18 mM and 7.5 mM of free Mg2+); the rectangular hyperbolic function was solved using iterative procedures. Ethanol-induced stimulation of plasma membrane H+-ATPase activity was associated to the increase of Vmax whereas the Km for MgATP increased. Results obtained with mutants constructed and used in previous studies envisaging the analysis of the molecular mechanisms underlying plasma membrane ATPase activation by glucose, external acidification and nitrogen starvation, suggested that the carboxyl-terminus (C-terminus) regulatory domain may also be involved in the in vivo activation by ethanol.

Saccharomyces cerevisiae cells stored oxovanadium (IV) ions in a dimeric form. In the late stationary phase Saccharomyces cerevisiae cells grown in rich medium containing concentrations of oxovanadium (V), orthovanadate from 12 to 18 mM, causing growth stasis, a dimeric oxovanadium (IV) species was identified by EPR spectroscopy. The EPR spectrum exhibited at 110 K the low-field forbidden deltaMs = +/-2 transition at g around 4 and the half-field deltaMs = +/-1 15-lines feature at g around 2 out of the presence of a triplet state by the coupling of the oxovanadium (IV) ions in a dimeric form. Hyperfine splitting of 75.2 x 10(-4) cm(-1) and an interionic distance of about 4.4 angstroms was calculated. The dimeric species was localized in the cellular cytoplasmic space.

Saccharomyces cerevisiae YGR262c gene, whose disruption causes severely defective growth, encodes a putative protein kinase shorter than any other protein kinase biochemically characterized to date and lacking some of the conserved features of these enzymes. Here we show that the product of the YGR262c gene, piD261, expressed in E. coli with a C-terminal (His)6 tag, is a bona fide Ser/Thr protein kinase as judged from its capability to autophosphorylate and to phosphorylate casein and osteopontin in the presence of [γ-32P]ATP. In contrast, no phosphorylation of histones, myelin basic protein, phosvitin, bovine serum albumin and poly(Glu/Tyr)4:1 could be detected. Mn2+ or, less effectively, Co2+ are required for piD261 catalytic activity, which is conversely undetectable in the presence of Mg2+, a behaviour unique among Ser/Thr protein kinases.

The NHA1 gene (2958 nt) encoding a putative Na+/H+antiporter (986 aa) in Saccharomyces cerevisiae was cloned by selection based on increased NaCl tolerance. The putative protein is highly similar to sodium/proton antiporters from Schizosaccharomyces pombe (gene sod2), and Zygosaccharomyces rouxii (gene Z-SOD2). Overexpression of the NHA1 gene results in higher and partially pH-dependent tolerance to sodium and lithium; its disruption leads to an increased sensitivity towards these ions.

To determine the topology of subunit VIII of the yeast ubiquinol-cytochrome c oxidoreductase in the mitochondrial inner membrane, an epitope has been introduced in the N-terminal half of this protein. Previous topology studies had shown that at least the C-terminus faces the intermembrane space [Hemrika and Berden (1990) Eur. J. Biochem. 192, 761–765]. Based on sensitivity of the protein to proteinase K digestion we now suggest that the N-terminus of subunit VIII is similarly oriented, implying that this subunit does not span the membrane. Despite this, however, subunit VIII cannot be extracted from the membrane even after treatment with 0.1 M Na 2 CO 3 at pH 11.5, showing that the protein is integrally embedded in the membrane. A similar behaviour was displayed by another low molecular weight protein of the complex, subunit VII, which faces the matrix side. A model for the topology of these subunits in the membrane is discussed with respect to the structure of the complex and their involvement in quinone binding.

Na+ efflux and Na+ tolerance depend on a putative P-type ATPase encoded by the gene ENA1(PMR2) in Saccharomyces cerevisiae and on a putative Na+/H+ antiporter encoded by the gene sod2 in Schizosaccharomyces pombe. This report shows that a sod2::ura4 mutant of S. pombe transformed with the ENA1 gene of S. cerevisiae expressed the ENA1 protein, and recovered Na+ efflux and Na+ tolerance. The efflux of Na+ in the wild strain of S. pombe was sensitive to the transmembrane Na+ and H+ gradients, whereas in the sod2::ura4 mutant transformed with ENA1 it was independent of these gradients. The data give further support to the notion that ENA1 and sod2 encode Na+ transporters and not regulators of the process of Na+ export; they show also the physiological consequences of exporting Na+ through an Na+-ATPase or an Na+/H+ antiporter.

The objective of this work was to evaluate the quantitative characteristics of carcass, and qualitative of the loin and shoulder of kids ¾ Boer + ¼ Saanen and Saanen and to evaluate the feed intake, dry matter nutrients digestibility, nitrogen balance and to estimate nutrients total digestible of diets with inclusion of dry yeast replacing the soybean meal. Twenty-seven carcasses of 18 ¾ Boer + ¼ Saanen and nine Saanen noncastrated male goats were used with a slaughter weight of 30.1 ± 0.27 kg, to evaluate the carcass characteristics and qualitative characteristics of the loin and shoulder. The animals were distributed in a completely randomized design in factorial arrangement 2x3 (racial group x diet). The diets were made by adding dry yeast: soybean meal, soybean meal + dry yeast or dry yeast. The common ingredients were: oat hay (30% of DM), corn and mineral mix. The hot carcass weight, cold carcass weight, loss of cooling, true carcass yield and carcass compacity index were not affected (P>0.05) by treatments. otherwise, the group Saanen had higher value of carcass commercial yield, however, they had 15 days longer for reach the slaughter weight. The yield of commercial cuts were not affected (P>0.05) by treatments, but, significant interaction for shoulder yield was observed. The treatments did not influence the measures of loin, muscle:bone rate, cooking loss, slice shear force and centesimal composition. For proportion of muscle in the loin, the treatments showed interaction, verifying that the inclusion of yeast in the ration for group ¾ Boer + ¼ Saanen was higher. In shoulder the treatments did not influence the ratio of muscle, bone, humidity and ash content. However, for the group Saanen the ratio of bone and humidity content were higher and total lipids content was lower. For the digestibility were used five non-castrated male goats (31.8 ± 1.44 kg) housed in digestibility cages, in 5x5 latin square design. The rations were formulated with inclusion of dry yeast (0; 25; 50; 75; 100% of DM) replacing the soybean meal in diets for kid goats, the rations being composed of oat hay (30% of DM), ground corn, soybean meal and/or dry yeast and mineral supplement. The total feces (with canvas bags) and urine were collected for each experimental period, which constituted of 10 days of adaptation to the rations and five for collections. The dry matter intake and total carbohydrates were unchanged by the inclusion of dry yeast in rations. However, for: organic matter, crude protein, ether extract and neutral detergent fiber intake there were a quadratic effect. For the digestibility of ether extract there was negative linear effect with the inclusion of dry yeast in rations. The dry matter, organic matter, crude protein, total carbohydrates, neutral detergent fiber and total digestible nutrients digestibility showed quadratic effects. The nitrogen balance did not have any change with the inclusion of dry yeast in rations. The group ¾ Boer + ¼ Saanen had a higher precocity with the reduction of slaughter age resulting from the heterosis obtained from the crossing. Dry yeast can be included in rations for kid goats as protein source without change the feed intake of the dry matter and nitrogen balance, however, attention for maximum limits of its inclusion must be hard for not reduce the digestibility of dry matter and nutrients. O objetivo deste trabalho foi avaliar as características quantitativas da carcaça, e qualitativas do lombo e da paleta de cabritos ¾ Boer + ¼ Saanen e Saanen e avaliar a ingestão, a digestibilidade da matéria seca e dos nutrientes digestíveis, o balanço de nitrogênio e estimar os nutrientes digestíveis totais de rações contendo levedura seca em substituição ao farelo de soja. Foram utilizadas 27 carcaças de cabritos machos não castrados, confinados, sendo 18 ¾ Boer + ¼ Saanen e nove Saanen com peso de abate de 30,1 ± 0,27 kg. Os animais foram distribuídos em delineamento inteiramente casualizado em arranjo fatorial 2x3 (grupo racial x rações). As rações foram constituídas pela inclusão de levedura seca, sendo: farelo de soja, farelo de soja + levedura seca ou levedura seca, sendo comum feno de aveia (30% da MS), milho moído e mistura mineral.O peso de carcaça quente, peso de carcaça fria, perdas de peso por resfriamento, rendimento verdadeiro de carcaça e índice de compacidade de carcaça não foram influenciadas (P>0,05) pelos tratamentos. No entanto, o grupo Saanen apresentou maior rendimento comercial de carcaça, porém, 15 dias a mais de idade para atingir o peso de abate. Os rendimentos de cortes não foram influenciados (P>0,05) pelos tratamentos, mas, ocorreu interação significativa para rendimento de paleta. Os tratamentos não influenciaram nas mensurações do lombo, razão músculo: osso, perdas de peso por cocção, força de cisalhamento e composição centesimal. Para a proporção de músculo do lombo os tratamentos apresentaram interação significativa, verificando que a inclusão da levedura seca nas rações dentro do grupo ¾ Boer + ¼ Saanen foi superior. Na paleta os tratamentos não alteraram as proporções de músculo, gordura, teor de proteína e cinzas. No entanto, para o grupo Saanen a proporção de osso e teor de umidade foram maiores e menor teor de lipídios totais. Para a digestibilidade foram utilizados cinco cabritos sem raça definida (SRD), não castrados (31,8 ± 1,44 kg) alojados em gaiolas metabólicas distribuídos em delineamento quadrado latino 5x5. As rações foram compostas pela inclusão da levedura seca (0; 25; 50; 75 e 100% da MS) em substituição ao farelo de soja nas rações para cabritos, sendo compostas por feno de aveia (30% da MS), milho moído, farelo de soja e/ou levedura seca e mistura mineral. As coletas totais de fezes (com o auxílio de sacolas de lona) e urina foram realizadas a cada período experimental, sendo constituído de 10 dias para adaptação à ração e cinco dias para as coletas. As ingestões de matéria seca e carboidratos totais não foram alterados pela inclusão da levedura seca nas rações. No entanto, para a ingestão de matéria orgânica, proteína bruta, extrato etéreo e fibra em detergente neutro observou-se efeito quadrático. A digestibilidade do extrato etéreo apresentou efeito linear decrescente com a inclusão da levedura seca nas rações. A digestibilidade da matéria seca, matéria orgânica, proteína bruta, carboidratos totais, fibra em detergente neutro e nutriente digestíveis total apresentaram efeito quadrático. O balanço de nitrogênio não foi alterado em função da substituição do farelo de soja. O grupo ¾ Boer + ¼ Saanen apresentou maior precocidade com redução de idade de abate resultante da heterose obtida a partir do cruzamento. A levedura seca pode ser incluída nas rações de cabritos como fonte protéica sem alterar a ingestão de matéria seca e o balanço de nitrogênio, no entanto, deve-se ter atenção nos limites máximos de sua inclusão para não reduzir a digestibilidade da matéria seca e dos nutrientes. xiv, 54 f

The yeast succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits, Sdh1p–Sdh4p, that couples the oxidation of succinate to the transfer of electrons to ubiquinone. One of the membrane anchor subunits, Sdh4p, has an unusual 30 amino acid extension at the C-terminus that is not present in SDH anchor subunits of other organisms. We identify Lys-132 in the Sdh4p C-terminal region as necessary for enzyme stability, ubiquinone reduction, and cytochrome b 562 assembly in SDH. Five Lys-132 substituted SDH4 genes were constructed by site-directed mutagenesis and introduced into an SDH4 knockout strain. The mutants, K132E, K132G, K132Q, K132R, and K132V were characterized in vivo for respiratory growth and in vitro for ubiquinone reduction, enzyme stability, and cytochrome b 562 assembly. Only the K132R substitution, which conserves the positive charge of Lys-132, produces a wild-type enzyme. The remaining four mutants do not affect the ability of SDH to oxidize succinate in the presence of the artificial electron acceptor, phenazine methosulfate, but impair quinone reductase activity, enzyme stability, and heme insertion. Our results suggest that the presence of a positive charge on residue 132 in the C-terminus of Sdh4p is critical for establishing a stable conformation in the SDH hydrophobic domain that is compatible with ubiquinone reduction and cytochrome b 562 assembly. In addition, our data suggest that heme does not play an essential role in quinone reduction.

An exopolyphosphatase (polyPase) with a specific activity of 60 U/mg protein has been purified from the vacuolar sap of Saccharomyces cerevisiae . The molecular mass of the intact enzyme was found to be 245 kDa. It is highly specific towards high-molecular polyphosphates (polyP). The activity with polyP 9 is 24% of that with polyP 208 . The apparent K m for polyP 15 and polyP 208 hydrolysis is 93 and 2.4 μM, respectively. The enzyme is slightly active with polyP 3 and adenosine-5′-tetraphosphate, but does not hydrolyze pyrophosphate, ATP, GTP and p -nitrophenylphosphate. It is stimulated by divalent metal cations. Co 2+ , the best activator, stimulates it 6-fold. Antibodies that inhibit the cell envelope and cytosol polyPases of S. cerevisiae have no effect on the vacuolar polyPase. The vacuolar polyPase differs from other yeast polyPases in molecular mass, substrate specificity and effects of activators.

The levels of two trehalose hydrolysing enzymes, acid trehalase (AT) and neutral trehalase (NT), have been investigated in Candida utilis at different stages of growth; in complete contrast to Saccharomyces cerevisiae, significant AT activity appears to be absent at all stages of growth studied in C. utilis. In addition, presence of only very low amounts of iso-aspartyl methyl transferase (IMT) activity at the onset of stationary phase and lower survival ability in early stationary phase in contrast to that of S. cerevisiae lend support to the ideas that (a) lower degree of survival of C. utilis in the stationary phase may be a direct consequence of inability to mobilise stored trehalose due to absence of intracellular AT and reduced levels of IMT activities and (b) trehalose may have a dual role vis-à-vis stress resistance in yeasts.

Previous studies on the resistance of yeast cells to dehydration pointed towards the protective role of trehalose and the importance of the specific trehalose transporter in guaranteeing survival. The present report demonstrates that the trehalose transporter is essential during the germination process in order to translocate trehalose from the cytosol to the external environment. Diploids that lack the trehalose transporter germinate poorly and do not form 4 spore tetrads although they accumulate trehalose and show trehalase activity. Furthermore, addition of exogenous trehalose to the germination medium enhances germination and normal segregation. The ability to transport trehalose is dominant and seems to be related to a single gene.

Evidence is presented here that in Saccharomyces cerevisiae IGC 3507, grown either on glycerol, ethanol or acetate, glycerol is transported by a high affinity uptake system of the electrogenic proton symport type, with K m of 1.7±0.7 mM, V max 441±19 μmol h −1 g −1 dry weight and a stoichiometry of 1 : 1 proton per molecule of glycerol, at 30°C and pH 5.0. No competitors were found among other polyols and sugars. Glycerol maximum accumulation ratios followed p.m.f. with extracellular pH. CCCP prevented glycerol accumulation, and inhibited uptake. NaCl did not interfere with H + /glycerol kinetics and energetics. This transport system was shown to be under glucose repression and inactivation. Glucose-grown cells presented, instead, a lower affinity permease for glycerol, probably a facilitated diffusion. Growth on glucose in the presence of NaCl did not induce the high affinity carrier. The stringent control of cell physiological condition over induction suggests for glycerol proton symport rather a physiological role connected with growth under gluconeogenic conditions.

After heterologous expression in E. coli, functionally active phosphate carrier (PIC) from Saccharomyces cerevisiae mitochondria was purified and reconstituted into giant liposomes and used for patch clamp experiments. Single channel currents across excised patches revealed an anion channel function of the PIC protein. Besides the three transport modes known to date, namely phosphate/phosphate exchange, phosphate/OH− exchange and mercurial-induced unidirectional transport, this channel activity represents the fourth transport mode of the PIC. The PIC channel activity was sensitive towards phosphate as its physiological substrate. Phosphate (10 mM) blocked in a specific but reversible manner the PIC channel, suggesting a phosphate-dependent conformational change of the protein into the carrier mode. Furthermore, the current through the channel and its gating activity were affected by divalent cations. In the presence of Ca2+ and Mg2+, the channel displayed a mean conductance of 25±5 pS whereas 40±10 pS was observed in the absence of divalent cations. Also, the dwell times in either the open or closed state of the PIC channel appeared to be prolonged in the presence of Ca2+ and Mg2+. The observed PIC channel characteristics are discussed with respect to previously reported electrophysiological in situ measurements on anion channels of the inner mitochondrial membrane. Similarities of the PIC channel to the inner mitochondrial anion channel (IMAC) have been found.

The Candida albicans CAN1 gene, encoding a high-affinity permease for arginine, lysine and histidine, was tagged at its C-terminus with a c-myc epitope and introduced into strains of Saccharomyces cerevisiae lacking basic amino acid permeases. The expression levels of Ca-Can1p were influenced by the available nitrogen source, being almost negligible when cells were grown in the presence of ammonia. Ca-Can1p was shown to follow the secretory pathway in S. cerevisiae. Ca-Can1p activity was not detected in a secretion-defective sec1-1 mutant grown at a non-permissive temperature. Shr3p, an ER protein that participates in the biogenesis of amino acid permeases was also required for the functional expression of Ca-Can1p. The shr3 mutation does not affect the affinity for substrate but does decrease the number of Can1p molecules reaching the plasma membrane.

The rapid in vivo activation of Saccharomyces cerevisiae plasma membrane H + -ATPase that has been attributed to medium acidification from pH 6.5 to pH 3.5 is not caused by the low pH itself but is induced by the weak organic acid (succinic) used as the acidulant. The activation induced by 50 mM succinic acid at pH 3.5 occurred in both the presence or absence of glucose. Activation at pH 3.5 was also induced by acetic acid and it was maximal at 50 mM concentration. To investigate the role of plasma membrane ATPase activation in pH homeostasis, the internal pH (cytosolic and vacuolar) of yeast cells incubated in media at pH 6.5 or at pH 3.5, acidified either with HCl or acetic acid, were compared by using in vivo 31 P-NMR. Despite plasma membrane ATPase activation by acetic acid, the decrease in cytosolic pH caused by external acidification was much more important when the permeant acetic acid was used instead of HCl as the acidulant. The supplementation of the incubation medium at pH 3.5 with glucose led to higher cytosolic pH values, consistent with the observed in vivo activation of plasma membrane ATPase by glucose. At the external pH value of 6.5 the vacuole was maintained at a mildly acidic pH (around 6) while the cytosol was at about neutral pH; however, when cytoplasmic pH decreased due to external acidification, vacuolar pH accompanied that decrease. Vacuolar pH reached 5.4–5.5 during incubation with HCl and dropped sharply to values below 4.4 in cells incubated with acetic acid. These results indicate that the vacuole also plays a role in homeostasis of the intracellular pH.

Funiculosin is a well-known inhibitor of the mitochondrial respiratory chain, probably acting at the ubiquinone reducing site or center i of QH2 cytochrome c reductase. We report here the isolation, mapping and RNA sequence analysis of yeast apo-cytochrome b mutants resistant to this inhibitor. Funiculosin-resistance was found to be conferred, in 4 independent isolates, upon replacement of a leucine residue by phenylalanine in position 198 of the cytochrome b polypeptide chain.

The activity of the plasma membrane ATPase in growing yeast is increased by low pH and by glucose, conditions which result in a higher demand for proton pumping. The amount of enzyme is not significatively modified under these conditions. The amount of ATPase is only slightly increased by introducing extra copies of its gene in autonomous plasmids. In addition, the expression of the ATPase gene in a multi-copy plasmid causes a reduction of the copy number of the plasmid and slows growth. Therefore, overexpression of the ATPase is detrimental for the cell, justifying a regulatory mechanism based on increasing the catalytic activity and not the amount of enzyme.

The sequence of the gene encoding pyruvate kinase from Saccharomyces cerevisiae was re-determined because of failures with oligonucleotide-directed mutagenesis experiments involving a region thought to contain a string of five contiguous non-preferred codons. This region was found to be difficult to sequence and was shown to have three extra bases when compared with the published sequence [(1983) J. Biol. Chem. 258, 2193–2201]. The revised sequence demonstrates that the yeast pyruvate kinase gene does not have a cluster of non-preferred codons, and that it therefore is not an example of the class of genes which possibly exhibit translational control by the presence of non-preferred codons.

We describe here the nuclear gene for a yeast protein showing unexpectedly high homology with mammalian aldo/keto reductases as well as with ϱ-crystallin, one of the prominent proteins of the frog eye lens. Although it could be proven that the gene occurs as a single copy in the haploid yeast genome, replacement of the intact by a disrupted, nonfunctional allele led to no obvious phenotype, indicating that the gene is dispensable. The gene was assigned to chromosome XV. It is transcribed in vivo into an mRNA of about 1300 bases with a coding capacity for a protein of 312 amino acids (estimated Mr 35000).

A ferritin-like molecule was purified from iron-loaded cells of Saccharomyces cerevisiae, but its iron content was very low and was not representative of the cellular iron content. A study of the intracellular distribution of iron has shown that the vacuoles are involved in the storage of iron in the yeast cell. Moreover, it seems that this vacuolar iron can be further utilised by the cells for iron-requiring processes such as mitochondriogenesis.

Yeast mitochondrial adenylate kinase (high molecular mass form, gene locus: AKY2) is encoded on chromosome IV of the same DNA strand as histone H2A-1. The nontranslated intergenic region spans 560 bp, the nontranscribed spacer can be estimated to comprise at most 300 bp. The TATA-box sequence is contained in a striking environment consisting of 20 alternating pyrimidines and purines. The AKY2 transcript is made constitutively: (i) the cellular mRNA concentration does not vary significantly with either growth conditions or elapse of the cell cycle; (ii) β-galactosidase activity is about constant in yeast cells grown on various carbon sources after transformation with AKY2-promoter/lacZ fusions; (iii) primer elongation analysis shows that utilization of 5 initiation sites is qualitatively and quantitatively independent of the growth conditions and the carbon source used; (iv) Western blot analysis and adenylate kinase activity measurements indicate the absence of post-transciptional controls as well.

A series of mitochondrially inherited mutants of yeast has been analysed, which were previously identified as showing resistance to the antibiotics venturicidin or ossamycin and whose mutations showed tight linkage to oligomycin-resistance alleles affecting subunit 9 of the mitochondrial ATP synthase. DNA sequence analysis of the oli1 gene of these mutants has been used to define the nature of amino acid substitution in the subunit 9 protein. In the case of the two venturicidin-resistant mutants, mutations affect amino acids on the N-terminal stem of the protein, namely G1y25 → Ser (venR ossS oliR) and Ala 27 → Gly (venR ossS oliS). The mutations found in the two ossamyein-resistant mutants affect amino acids on the C-terminal stem of the protein, namely Leu53 → Phe (vanS ossR oliR) and Leu57 → Phe (venS OSSR oliS). These results allow us to further develop a fine structure map of domains within the subunit 9 protein involved in antibiotic interaction.

Proteins of the small and large subunits of mitochondrial ribosomes from the yeast Saccharomyces cerevisiae were isolated and characterized by two-dimensional gel electrophoresis. Ribosomal proteins of the large subunit were separated by reverse-phase HPLC and up to 37 amino acid residues of the N-terminal sequences of L3, L4, L9 and L31 were determined. No significant homology to ribosomal protein sequences so far determined from other organisms was found.

Strains of Saccharomyces cerevisiae have been constructed that possess temperature-sensitive defects in tRNA precursor (pre-tRNA) splicing and which also lack the processing endonuclease that acts at the 3′-terminus of 5 S rRNA and 35 S rRNA precursors (pre-rRNAs). The unspliced pre-tRNAs accumulated by such strains at the nonpermissive temperature are identical in structure to those accumulated by pre-tRNA splicing-defective strains with a functional pre-5 S RNA processing enzyme. The pre-RNA processing activity is therefore not obligatorily involved in maturation of several yeast tRNAs. However, gels of the pulse-labelled RNAs of RNA82+ and rna82.1 strains provide evidence that this enzyme acts upon a few small unstable transcripts that are not 5 S RNA forms. The most prominent of these transcripts on gels was, in wild-type strains, an RNA 145 ± 2 nucleotides in length.

Methods for isolation of the ADP/ATP carrier (AAC) from yeast (Saccharomyces cerevisiae) are described which allow separation of the carrier from the initially copurified porin which poses a specific problem in yeast. The procedure varies according to whether one wishes to obtain a stable CAT-AAC complex, the free and active AAC for reconstitution, or the SDS-denatured pure AAC peptide. CNBr cleavage of AAC enabled us to differentiate clearly between isogenes AAC-1 and AAC-2 recently found in yeast, due to the exclusive occurrence of a methionine (M-115) residue at the end of the first domain in AAC-2. Thus the AAC isolated from wild-type yeast is primarily or exclusively AAC-2. The isolated AAC is active in ADP/ATP exchange in reconstituted liposomes with a Vmax of 1100 mumol/min per g protein and Km = 15 microM for ADP, and a Vmax of 900 mumol/min per g protein and Km = 9 microM for ATP.

Ca2+-dependent chromatography of soluble cytosolic proteins on calmodulin-Sepharose gave a fraction that exhibited Ca2+- and calmodulin-dependent phosphorylation of several polypeptides, including 60, 56 and 45 kDa species. At 0.2 microM beef calmodulin the phosphorylation was optimal at 3 microM free Ca2+, and at 80 microM free Ca2+ it was half-maximal at about 0.1 microM beef calmodulin. It is concluded that the fraction contains calmodulin-dependent protein kinase(s) which is (are) autophosphorylated or associated with substrates.

The galactose transport system of Saccharomyces cerevisiae consists of one component which shows a Km value of approx. 4mM in growing cells. A rapid and irreversible inactivation of this transport is detected on impairment of protein synthesis. This inactivation shows the following characteristics: (i) it is due to changes in the Km and Vmax of the transport system; (ii) it follows first-order kinetics; (iii) it is an energy-dependent process and is stimulated by the presence of an exogenous carbon source; (iv) fermentable sub-dependent process and is stimulated by the presence of an exogenous carbon source; (iv) fermentable substrates stimulate inactivation more efficiently than non-fermentable substrates.

Direct fusions have been constructed between each of subunits 8 and 9 from mitochondrial ATPase of Saccharomyces cerevisiae, proteins normally encoded inside mitochondria, and the cleavable N-terminal transit peptide from the nuclearly encoded precursor to subunit 9 of Neurospora crassa mitochondrial ATPase. The subunit 8 construct was imported efficiently into isolated yeast mitochondria and was processed at or very near the fusion point. When expressed in vivo from its artificial nuclear gene, this cytoplasmically synthesized form of subunit 8 restored the growth defects of aap1 mutants unable to produce subunit 8 inside the mitochondria. The subunit 9 construct was, however, unable to be imported into isolated mitochondria and could not, following nuclear expression in vivo, complement growth defects in mitochondrial oli1 mutants. This behaviour is contrasted with the previously demonstrated import competence of another yeast subunit 9 fusion, bearing the first five residues of mature N. crassa subunit 9 interposed between its own transit peptide and the yeast subunit 9 moiety.

We describe the use of classical and molecular genetic techniques to investigate the folding, stability, and enzymatic requirements of iso-l-cytochrome c from the yeast Saccharomyces cerevisiae. Interpretation of the defects associated with an extensive series of altered forms of iso-l-cytochrome c was facilitated by the recently resolved three dimensional structure of iso-l-cytochrome c [(1987) J. Mol. Biol. 199, 295–314], and by comparison with the phylogenetic series of eukaryotic cytochromes c. Residue replacements that abolish iso-l-cytochrome c function appear to do so by affecting either heme attachment or protein stability; no replacements that abolish electron transfer function without affecting protein structure were uncovered. Most nonfunctional forms retained at least partial covalent attachment to the heme moiety; heme attachment was abolished only by replacements of Cys19 and Cys22, which are required for thioether linkage, and His23, a heme ligand. Replacements were uncovered that retain function at varying levels, including replacements at evolutionarily conserved positions, some of which were structurally and functionally indistinguishable from wild type iso-l-cytochrome c.

A specific substrate, M 13 DNA:RNA-[32P]DNA, was synthesized to investigate the mode of cleavage of enzymes with RNase H activity. RNase H(70) from Saccharomyces cerevisiae hydrolyzes the phosphodiester bond at the RNA-DNA junction of this substrate, thereby producing a 5'-monophosphate-terminated polydeoxyribonucleotide and 3'-hydroxyl-terminated oligoribonucleotides.

The ADP/ATP carrier from yeast was photoaffinity-labeled in mitochondria with 2-azido-[alpha-32P]ATP in a binding-center-specific, i.e. carboxyatractylate-sensitive, manner. After isolation, fragmentation possibilities unique for the yeast AAC-2 could be exploited to assign the insertion to a narrow range of the sequence. The CNBr fragment 115-210 contained all the incorporated label which corresponds to the second domain within the triple-domain primary structure of the AAC. With hydroxylamine cleavage directed to the Asn 171-Gly 172 site, all the label was found in the C-terminal 16 kDa fragment. Thus the 2-azido-ATP incorporation is clearly delimited to the 172-210 segment. 8-Azido-[alpha-32P]ATP could be site-specifically incorporated only in isolated AAC since it has a much lower affinity for AAC than 2-azido-ATP. The label was also exclusively found in the 172-210 region. With both forms no incorporation into the C-terminal region was found, as claimed for bovine AAC. The labeled segment contains Lys 179 and 182 which are homologous to bovine Lys 162 and 165 and which have been proposed to be in the translocation path.

The ARO1 gene of Saccharomyces cerevisiae encodes the arom multifunctional enzyme. Specific inhibitors of amino acid biosynthesis have been used to obtain evidence that expression of a cloned ARO1 gene is regulated in response to amino acid limitation. Northern blot analysis and sequence studies indicate that ARO1 is regulated by the well characterised S. cerevisiae ‘general control’ mechanism. This provides a very economical means of simultaneously tailoring the synthesis of five shikimate pathway enzymes to the needs of the cell.

We have shown that OSCP, a subunit of yeast mitochondrial ATP synthase, can be incorporated into the intact enzyme as a fusion protein representing OSCP fused at its C-terminus to the green fluorescent protein (GFP) of Aequorea victoria. The relevant fusion OSCP-GFP-h6 additionally contains a hexahistidine tag at the C-terminus. Expression of OSCP-GFP-h6 in yeast cells lacking endogenous OSCP led to the efficient restoration of growth of cells on the non-fermentable substrate, ethanol. Confocal laser scanning microscopy revealed fluorescence due to GFP in mitochondria of cells expressing OSCP-GFP-h6. Use of immobilised metal ion affinity chromatography enabled the recovery of assembled ATP synthase complexes which contained OSCP-GFP-h6 identified by its mobility on SDS-PAGE and immunoreactivity to anti-OSCP and anti-GFP antibodies. The successful isolation of the assembled multisubunit ATP synthase containing GFP fused to one of the essential subunits of the complex widely expands the potential applications of GFP. In principle, these include the spatial and temporal monitoring of ATP synthase complexes in vivo, and the exploration of interactions involving ATP synthase subunits by fluorescence resonance energy transfer (FRET).

The function of the amino- and carboxyl-terminal domains of the yeast plasma membrane H+-ATPase have been investigated by constructing deletions in vitro and selectively expressing the mutant enzymes in vivo. The first 27 amino acids are dispensable but deletion of a further 33 amino acids greatly decreases the appearance of the enzyme in the plasma membrane. Membrane localization is also prevented by carboxyl-terminal deletions which include the last hydrophobic stretch, but the last 46 amino acids of the ATPase are not required. Removal of the last 11 amino acids produces an enzyme in glucose-starved cells with the kinetic parameters of the wild-type ATPase activated by glucose fermentation. This region seems to constitute a regulatory domain.

The storage disaccharide, trehalose, is accumulated in yeast during a temperature shift from 30 to 45 degrees C. The response peaks at 90 min and is transient since levels of trehalose decline rapidly in cells returned to 30 degrees C. Storage of trehalose is inhibited when cells are incubated in the presence of acridine orange or ethidium bromide prior to and during temperature shift, suggesting a requirement for de novo RNA synthesis. Accumulation of trehalose occurs when cells are exposed to either ethanol, copper sulphate or hydrogen peroxide at 30 degrees C, indicating that the phenomenon may be a general response to physiological stress. Parallels are drawn between the trehalose accumulation response and the heat shock response in yeast.

Several nonhistone chromatin proteins (NHPs) have been isolated from Saccharomyces cerevisiae nuclei. They have molecular masses and amino acid compositions typical of the high mobility group (HMG) proteins from higher eukaryotic cells. Polyclonal antisera raised against two of the NHPs have been used in immunoblots of proteins from subcellular fractions of yeast to show that the NHPs are indeed nuclear. In addition, the amino-terminal amino acid sequences of several of the NHPs were determined. Importantly, the amino-terminal sequence of one of the proteins, NHP6, has significant (60%) identity with a stretch of amino acids in calf thymus HMG1.

The metabolism of glutathione (GSH), a marker of oxidative stress and trehalose, a rather general physiological stress marker, was examined in exponentially growing Saccharomyces cerevisiae cells after treatment with 4-hydroxynonenal (HNE). GSH was entirely depleted within a 2 h incubation with 250 μM HNE. After removal of the aldehyde it was replenished by de novo synthesis leading to an overshooting GSH level, which later decreased to the basal level. In addition, trehalose was elevated 4-fold in HNE-treated yeast cells compared to control cells. We conclude that increased GSH levels upon HNE treatment are a general phenomenon of eukaryotic cells to ensure protection and survival during further harsh conditions. Furthermore, we have discovered a new indication for the stress marker trehalose in S. cerevisiae.© 1997 Federation of European Biochemical Societies.

A yeast gene termed YKR coding for a putative protein kinase was isolated by using the cloned cDNA for rabbit protein kinase C as a hybridization probe. The encoded protein (YKR), composed of 380 amino acid residues, shows extensive sequence homology to serine/threonine-specific protein kinases from various species in the approx. 320 C-terminal amino acid residues, strongly suggesting that YKR is endowed with a protein kinase activity. The observed homologies to the cdc25 suppressing protein kinase from yeast, the catalytic subunit of mammalian cAMP-dependent protein kinase, and mammalian protein kinase C were 76, 48 and 37%, respectively. Gene replacement experiments showed that YKR itself is not essential for cell proliferation.

The alpha-agglutinin responsible for mating type-specific agglutination of S. cerevisiae alpha-cells has been purified to homogeneity. The glycoprotein released from the cell surface under mild conditions has a relative molecular mass of 200 to 300 kDa as determined by SDS-gel electrophoresis. The protein moiety corresponds to 68.2 kDa. With an oligonucleotide corresponding to the N-terminal amino acid sequence, the alpha-agglutinin gene has been cloned and sequenced. From the DNA sequence, a protein of 631 amino acids with 12 potential N-glycosylation sites is predicted. The carboxy terminal one-third of the protein is not required for agglutination activity.

Newly synthesized proteins that reach the last compartment of the Golgi complex can be sorted into pathways leading either to the cell surface or to the vacuole. It now appears that there are at least two routes from the Golgi to the vacuole: the ‘CPY pathway’, which involves transit through an endosomal/prevacuolar compartment (PVC), and a recently discovered ‘ALP pathway’, which bypasses the PVC, but may involve other as yet unidentified intermediate compartments. No cytosolic signal has been identified that directs the entry of membrane proteins into the CPY pathway. In contrast, the transport of ALP through the ALP pathway is saturable and signal mediated. Much recent work has focused on the identification of proteins that regulate trafficking to the vacuole. A number of genes have been identified that are specific for either the CPY or ALP sorting pathways, while other genes affect both types of transport and may therefore act at or after a point of convergence. Progress has also been made in further elucidating the members of the SNARE complexes that act in Golgi-to-PVC transport as well as those that mediate fusion with the vacuole.

Mutation of conserved Phe-82 of yeast iso-1 cytochrome c to Tyr, Gly, Ser, Leu, or Ile affects binding to and reaction with cytochrome-c oxidase from beef heart. The observed changes of binding and kinetic constants reflect mutation-induced rearrangements in the heme vicinity brought about by the replacement of Phe-82. Such conformational rearrangements are also revealed by altered circular dichroism spectra of the oxidase-bound mutant cytochromes c. Variations in Km for cytochrome c oxidation do not parallel variations in Kd, the dissociation constant for binding of cytochrome c to the oxidase. This observation does not support an enzymatic mechanism in which the rate of cytochrome c oxidation is governed by product dissociation.

A complete malolactic fermentation was achieved using Saccharomyces cerevisiae strains coexpressing the genes mleS and mae1 coding for the Lactococcus lactis malolactic enzyme and the Schizosaccharomyces pombe malate permease under the control of yeast promoters. The expression level of mae1 greatly influences the kinetics of the reaction by controlling the rate of malate uptake meanwhile a high expression level of mleS induces a partial consumption of malate derived from glucose by the malolactic enzyme. A strain expressing several copies of mae1 and one copy of mleS degrades 3 g/l of malate almost exclusively through the malolactic pathway in 4 days under enological conditions, without metabolic side effects.

Yeast Saccharomyces cerevisiae OCH1 gene encodes the mannosyltransferase that is essential for the outer chain elongation of N-linked oligosaccharides. Mannosyltransferase activity of OCH1 gene product (Och1p) was measured on HPLC by using pyridylaminated Man8GlcNAc2 (Man8GlcNAc2-PA) as an acceptor and the reaction product was observed at the retention time corresponding to Man9GlcNAc2-PA. 1H-NMR and fast atom bombardment mass spectrometry (FAB-MS) fragmentation analysis of Man9GlcNAc2-PA showed that the additional mannose was attached with an alpha-1,6 linkage at the site where mannose outer chain elongation initiates. Substrate specificity of Och1p was investigated by using various high mannose-type oligosaccharides as acceptors. Man8GlcNAc2 was the best acceptor for Och1p. The loss of one or two alpha-1,2-mannoses from Man8GlcNAc2 reduced the mannosyltransferase activity and the Man5GlcNAc2 completely lacking alpha-1,2-mannose residues did not serve as an acceptor. Man8GlcNAcOH that involves an open sugar ring by reduction of reducing terminal GlcNAc residue did not serve as an acceptor for Och1p. The loss of three mannoses at the alpha-1,6-branch also reduced the Och1p activity. These results suggest that Och1p is an initiation specific alpha-1,6-mannosyltransferase that requires the intact structure of Man8GlcNAc for efficient mannose outer chain initiation.

cGMP is characterized as undetectable in yeast [(1986) Yeast Cell Biology, UCLA Symp. Mol. Cell Biol. (Hicks, J. ed.) p. 495], though in many organisms it contributes specifically to the regulation of metabolism. Here, we detected cGMP, using radioactive labeling and RIA techniques, after extraction of the cells with 1 mol/1 HClO4 at 37 degrees C. The cGMP 0.015-fold cAMP, about 3-times higher with exponentially growing cells than with pressed baker's yeast, and depends on glucose and O2 supply. The PDE inhibitors DMX and IBMX induce in growing cells an additional increase of the cGMP level, without similar effects on cAMP.

31P saturation transfer techniques have been used to measure phosphate kinetics in the yeast Saccharomyces cerevisiae. The phosphate consumption rate observed in acetate grown mid-log cells was combined with measurements of O2 consumption to yield P/O ratios of 2.2 and 2.9, for cells respiring on glucose and ethanol, respectively. However, no phosphate consumption activity was observed in saturation transfer experiments on anaerobic glucose fed cells. The phosphate consumption rates measured by saturation transfer in cells respiring on glucose and ethanol was attributed to the unidirectional rates of mitochondria! ATP synthesis.

Site-directed mutants of yeast ATPase were previously studied after introduction of mutant alleles into a yeast strain where these alleles were constitutively expressed while the expression of the wild-type chromosomal ATPase gene was turned off. As a functional H+ pump is essential, strong selective pressure leads to the accumulation of revertants during growth of cells harboring variants with low activity. Thus, constitutive expression of the mutant gene can select phenotypes which reflect events such as gene conversion or reversion. We have therefore re-evaluated the phenotypes of non-dominant lethal alleles in an alternative set of conditional expression systems. We show that eight of 11 previously described site-directed mutations behave as recessive lethal alleles. Copyright (C) 1999 Elsevier Science B.V., This study was supported by Spanish Grant DGICYT-PB97-0054.