Search

Resveratrol (RSV) is a naturally occurring polyphenolic compound endowed with interesting biological properties/functions amongst which are its activity as an antioxidant and as Sirtuin activating compound towards SIRT1 in mammals. Sirtuins comprise a family of NAD+-dependent protein deacetylases that are involved in many physiological and pathological processes including aging and age-related diseases. These enzymes are conserved across species and SIRT1 is the closest mammalian orthologue of Sir2 of Saccharomyces cerevisiae. In the field of aging researches, it is well known that Sir2 is a positive regulator of replicative lifespan and, in this context, the RSV effects have been already examined. Here, we analyzed RSV effects during chronological aging, in which Sir2 acts as a negative regulator of chronological lifespan (CLS). Chronological aging refers to quiescent cells in stationary phase; these cells display a survival-based metabolism characterized by an increase in oxidative stress. We found that RSV supplementation at the onset of chronological aging, namely at the diauxic shift, increases oxidative stress and significantly reduces CLS. CLS reduction is dependent on Sir2 presence both in expired medium and in extreme Calorie Restriction. In addition, all data point to an enhancement of Sir2 activity, in particular Sir2-mediated deacetylation of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (Pck1). This leads to a reduction in the amount of the acetylated active form of Pck1, whose enzymatic activity is essential for gluconeogenesis and CLS extension., Graphical abstract fx1, Highlights • RSV supplementation at the diauxic shift strongly reduces chronological lifespan. • RSV supplementation increases oxidative stress during chronological aging. • RSV supplementation decreases trehalose stores during chronological aging. • RSV supplementation determines a decrease in the acetylated active form of Pck1. • Sir2 is required for the pro-aging effects elicited by RSV supplementation.

Nicotinamide, nicotinic acid and nicotinamide riboside are vitamin B3 precursors of NAD+ in the human diet. NAD+ has a fundamental importance for cellular biology, that derives from its essential role as a cofactor of various metabolic redox reactions, as well as an obligate co-substrate for NAD+-consuming enzymes which are involved in many fundamental cellular processes including aging/longevity. During aging, a systemic decrease in NAD+ levels takes place, exposing the organism to the risk of a progressive inefficiency of those processes in which NAD+ is required and, consequently, contributing to the age-associated physiological/functional decline. In this context, dietary supplementation with NAD+ precursors is considered a promising strategy to prevent NAD+ decrease and attenuate in such a way several metabolic defects common to the aging process. The metabolism of NAD+ precursors and its impact on cell longevity have benefited greatly from studies performed in the yeast Saccharomyces cerevisiae, which is one of the most established model systems used to study the aging processes of both proliferating (replicative aging) and non-proliferating cells (chronological aging). In this review we summarize important aspects of the role played by nicotinamide, nicotinic acid and nicotinamide riboside in NAD+ metabolism and how each of these NAD+ precursors contribute to the different aspects that influence both replicative and chronological aging. Taken as a whole, the findings provided by the studies carried out in S. cerevisiae are informative for the understanding of the complex dynamic flexibility of NAD+ metabolism, which is essential for the maintenance of cellular fitness and for the development of dietary supplements based on NAD+ precursors.

Yeast chronological aging is regarded as a model for aging of mammalian post-mitotic cells. It refers to changes occurring in stationary phase cells over a relatively long period of time. How long these cells can survive in such a non-dividing state defines the chronological lifespan. Several factors influence cell survival including two well known normal by-products of yeast glucose fermentation such as ethanol and acetic acid. In fact, the presence in the growth medium of these C2 compounds has been shown to limit the chronological lifespan. In the chronological aging paradigm, a pro-aging role has also emerged for the deacetylase Sir2, the founding member of the Sirtuin family, whose loss of function increases the depletion of extracellular ethanol by an unknown mechanism. Here, we show that lack of Sir2 strongly influences carbon metabolism. In particular, we point out a more efficient acetate utilization which in turn may have a stimulatory effect on ethanol catabolism. This correlates with an enhanced glyoxylate/gluconeogenic flux which is fuelled by the acetyl-CoA produced from the acetate activation. Thus, when growth relies on a respiratory metabolism such as that on ethanol or acetate, SIR2 inactivation favors growth. Moreover, in the chronological aging paradigm, the increase in the acetate metabolism implies that sir2Δ cells avoid acetic acid accumulation in the medium and deplete ethanol faster; consequently pro-aging extracellular signals are reduced. In addition, an enhanced gluconeogenesis allows replenishment of intracellular glucose stores which may be useful for better long-term cell survival.

Nicotinamide (NAM), a form of vitamin B3, is a byproduct and noncompetitive inhibitor of the deacetylation reaction catalyzed by Sirtuins. These represent a family of evolutionarily conserved NAD+-dependent deacetylases that are well-known critical regulators of metabolism and aging and whose founding member is Sir2 of Saccharomyces cerevisiae. Here, we investigated the effects of NAM supplementation in the context of yeast chronological aging, the established model for studying aging of postmitotic quiescent mammalian cells. Our data show that NAM supplementation at the diauxic shift results in a phenocopy of chronologically aging sir2Δ cells. In fact, NAM-supplemented cells display the same chronological lifespan extension both in expired medium and extreme Calorie Restriction. Furthermore, NAM allows the cells to push their metabolism toward the same outcomes of sir2Δ cells by elevating the level of the acetylated Pck1. Both these cells have the same metabolic changes that concern not only anabolic pathways such as an increased gluconeogenesis but also respiratory activity in terms both of respiratory rate and state of respiration. In particular, they have a higher respiratory reserve capacity and a lower non-phosphorylating respiration that in concert with a low burden of superoxide anions can affect positively chronological aging.

Black band disease (BBD) is a widespread coral pathology caused by a microbial consortium dominated by cyanobacteria, which is significantly contributing to the loss of coral cover and diversity worldwide. Since the effects of the BBD pathogens on the physiology and cellular stress response of coral polyps appear almost unknown, the expression of some molecular biomarkers, such as Hsp70, Hsp60, HO-1, and MnSOD, was analyzed in the apparently healthy tissues of Goniopora columna located at different distances from the infection and during two disease development stages. All the biomarkers displayed different levels of expression between healthy and diseased colonies. In the healthy corals, low basal levels were found stable over time in different parts of the same colony. On the contrary, in the diseased colonies, a strong up-regulation of all the biomarkers was observed in all the tissues surrounding the infection, which suffered an oxidative stress probably generated by the alternation, at the progression front of the disease, of conditions of oxygen supersaturation and hypoxia/anoxia, and by the production of the cyanotoxin microcystin by the BBD cyanobacteria. Furthermore, in the infected colonies, the expression of all the biomarkers appeared significantly affected by the development stage of the disease. In conclusion, our approach may constitute a useful diagnostic tool, since the cellular stress response of corals is activated before the pathogens colonize the tissues, and expands the current knowledge of the mechanisms controlling the host responses to infection in corals.

Saccharomyces cerevisiae grows fast on glucose, while growth slows down on ethanol as cells move from glucose fermentation to oxidative metabolism. The type of carbon source influences both the specific growth rate and cell cycle progression, as well as cell size. Yeast cells grown on glucose have a larger size than cells grown on ethanol. Here, we analysed the behaviour of a sfp1 null mutant during balanced and transitory states of growth in batch in response to changes in the growth medium carbon sources. In a screening for mutants affected in cell size at Start, SFP1 has been identified as a gene whose deletion caused one of the smallest whi phenotype. Findings presented in this work indicate that in the sfp1 null mutant the reduction in cell size is not only a consequence of the reduced growth rate but it is tightly linked to the cellular metabolism. The SFP1 gene product is required to sustain the increase of both rRNA and protein content that in wild-type cells takes place in respiro-fermentative growth conditions, while it seems dispensable for growth on non-fermentable carbon sources. It follows that sfp1 cells growing on ethanol have a larger size than cells growing on glucose and, noticeably, the former enter the S phase with a critical cell size higher than the latter. These features, combined with the role of Sfp1p as a transcriptional factor, suggest that Sfp1p could be an important element in the control of the cell size modulated by nutrients. Copyright © 2005 John Wiley & Sons, Ltd.

Gas1p is a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae and is a representative of Family GH72 of glycosidases/transglycosidases, which also includes proteins from human fungal pathogens. Gas1p, Phr1-2p from Candida albicans and Gel1p from Aspergillus fumigatus have been shown to be beta-(1,3)-glucanosyltransferases required for proper cell wall assembly and morphogenesis. Gas1p is organized into three modules: a catalytic domain; a cys-rich domain; and a highly O-glycosylated serine-rich region. In order to provide an experimental system for the biochemical and structural analysis of Gas1p, we expressed soluble forms in the methylotrophic yeast Pichia pastoris. Here we report that 48 h after induction with methanol, soluble Gas1p was produced at a yield of approximately 10 mg x L(-1) of medium, and this value was unaffected by the further removal of the serine-rich region or by fusion to a 6 x His tag. Purified soluble Gas1 protein showed beta-(1,3)-glucanosyltransferase activity that was abolished by replacement of the putative catalytic residues, E161 and E262, with glutamine. Spectral studies confirmed that the recombinant soluble Gas1 protein assumed a stable conformation in P. pastoris. Interestingly, thermal denaturation studies demonstrated that Gas1p is highly resistant to heat denaturation, and a complete refolding of the protein following heat treatment was observed. We also showed that Gas1p contains five intrachain disulphide bonds. The effects of the C74S, C103S and C265S substitutions in the membrane-bound Gas1p were analyzed in S. cerevisiae. The Gas1-C74S protein was totally unable to complement the phenotype of the gas1 null mutant. We found that C74 is an essential residue for the proper folding and maturation of Gas1p.

UBP10 codes for a deubiquitinating enzyme of Saccharomyces cerevisiae whose loss of function determines slow growth rate and partial impairment of silencing at telomeres and HM loci. A genome-wide analysis performed on a ubp10 disruptant revealed alterations in expression of subtelomeric genes together with a broad change in the whole transcriptional profile, closely parallel to that induced by oxidative stress. This response was accompanied by intracellular accumulation of reactive oxygen species as well as by DNA fragmentation and phosphatidylserine externalization, two markers of apoptosis. SIR4 inactivation mitigated the wide transcriptome remodeling of the ubp10 null mutant affecting particularly the stress transcriptional profile. Moreover, the ubp10sir4 disruptant did not display apoptotic markers. These results argue in favor of an involvement of deubiquitination in transcriptional control and suggest a linkage between oxidative stress and apoptotic pathway in budding yeast.

The existence of a compensatory mechanism in response to cell wall damage has been proposed in yeast cells. The increase of chitin accumulation is part of this response. In order to study the mechanism of the stress-related chitin synthesis, we tested chitin synthase I (CSI), CSII, and CSIII in vitro activities in the cell-wall-defective mutant gas1 Δ. CSI activity increased twofold with respect to the control, a finding in agreement with an increase in the expression of the CHS1 gene. However, deletion of the CHS1 gene did not affect the phenotype of the gas1 Δ mutant and only slightly reduced the chitin content. Interestingly, in chs1 gas1 double mutants the lysed-bud phenotype, typical of chs1 null mutant, was suppressed, although in gas1 cells there was no reduction in chitinase activity. CHS3 expression was not affected in the gas1 mutant. Deletion of the CHS3 gene severely compromised the phenotype of gas1 cells, despite the fact that CSIII activity, assayed in membrane fractions, did not change. Furthermore, in chs3 gas1 cells the chitin level was about 10% that of gas1 cells. Thus, CSIII is the enzyme responsible for the hyperaccumulation of chitin in response to cell wall stress. However, the level of enzyme or the in vitro CSIII activity does not change. This result suggests that an interaction with a regulatory molecule or a posttranslational modification, which is not preserved during membrane fractionation, could be essential in vivo for the stress-induced synthesis of chitin.

A novel 1,3-beta-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus was recently characterized. This enzyme splits internally a 1,3-beta-glucan molecule and transfers the newly generated reducing end to the non-reducing end of another 1, 3-beta-glucan molecule forming a 1,3-beta linkage, resulting in the elongation of 1,3-beta-glucan chains. The GEL1 gene encoding this enzyme was cloned and sequenced. The predicted amino acid sequence of Gel1p was homologous to several yeast protein families encoded by GAS of Saccharomyces cerevisiae, PHR of Candida albicans, and EPD of Candida maltosa. Although the expression of these genes is required for correct morphogenesis in yeast, the biochemical function of the encoded proteins was unknown. The biochemical assays performed on purified recombinant Gas1p, Phr1p, and Phr2p showed that these proteins have a 1,3-beta-glucanosyltransferase activity similar to that of Gel1p. Biochemical data and sequence analysis have shown that Gel1p is attached to the membrane through a glycosylphosphatidylinositol in a similar manner as the yeast homologous proteins. The activity has been also detected in membrane preparations, showing that this 1,3-beta-glucanosyltransferase is indeed active in vivo. Our results show that transglycosidases anchored to the plasma membrane via glycosylphosphatidylinositols can play an active role in fungal cell wall synthesis.

The yeast cell wall, which for years has been regarded as a static cellular component, has been revealed to be dynamic in its structure and composition and complex in its enzymatic activity. The S. cerevisiae cell wall is composed of beta-1,3/beta-1,6-glucans, mannoproteins, and chitin, which are assembled into an extracellular matrix essential for maintenance of cell integrity. Gas1p, a glycoprotein anchored to the outer leaflet of the plasma membrane through a glycosylphosphatidylinositol, plays a key role in cell wall assembly. Loss of Gas1p leads to several morphogenetic defects and to a decrease in the amount of cross-links between the cell wall glucans. These defects in turn trigger a compensatory response that guarantees cell viability. Several Gas1p homologs have been isolated from Candida species and S. pombe. The Gas1p family also includes two plant proteins with endo-beta-1,3-glucanase activity. Sequence comparisons reveal that Gas1p family proteins have a modular organization of domains. The genetic and molecular analyses reviewed here suggest that Gas1p could play a role as a polymer cross-linker, presumably by catalyzing a transglycosylation reaction.

Analysis of Candida albicans cells using antibodies directed against Gas1p/Ggp1p, Saccharomyces cerevisiae homolog of Phr1p, revealed that Phr1p is a glycoprotein of about 88 kDa whose accumulation increases with the rise of external pH. This polypeptide is present both in the yeast form and during germ tube induction. In the Phr1 − cells at pH 8 the solubility of glucans in alkali is greatly affected. In the parental strain the alkali-soluble/-insoluble glucan ratio shows a 50% decrease at pH 8 with respect to pH 4.5, whereas in the null mutant it is unchanged, indicating the lack of a polymer cross-linker activity induced by the rise of pH. The mutant has a sixfold increase in chitin level and is hypersensitive to calcofluor. Consistently with a role of chitin in strengthening the cell wall, Phr1 − cells are more sensitive to nikkomycin Z than the parental strain.

During growth on fermentable substrates, such as glucose, pyruvate, which is the end-product of glycolysis, can be used to generate acetyl-CoA in the cytosol via acetaldehyde and acetate, or in mitochondria by direct oxidative decarboxylation. In the latter case, the mitochondrial pyruvate carrier (MPC) is responsible for pyruvate transport into mitochondrial matrix space. During chronological aging, yeast cells which lack the major structural subunit Mpc1 display a reduced lifespan accompanied by an age-dependent loss of autophagy. Here, we show that the impairment of pyruvate import into mitochondria linked to Mpc1 loss is compensated by a flux redirection of TCA cycle intermediates through the malic enzyme-dependent alternative route. In such a way, the TCA cycle operates in a “branched” fashion to generate pyruvate and is depleted of intermediates. Mutant cells cope with this depletion by increasing the activity of glyoxylate cycle and of the pathway which provides the nucleocytosolic acetyl-CoA. Moreover, cellular respiration decreases and ROS accumulate in the mitochondria which, in turn, undergo severe damage. These acquired traits in concert with the reduced autophagy restrict cell survival of the mpc1∆ mutant during chronological aging. Conversely, the activation of the carnitine shuttle by supplying acetyl-CoA to the mitochondria is sufficient to abrogate the short-lived phenotype of the mutant.

The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells. In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells.

InSaccharomyces cerevisiae, the chronological lifespan (CLS) is defined as the length of time that a population of nondividing cells can survive in stationary phase. In this phase, cells remain metabolically active, albeit at reduced levels, and responsive to environmental signals, thus simulating the postmitotic quiescent state of mammalian cells. Many studies on the main nutrient signaling pathways have uncovered the strong influence of growth conditions, including the composition of culture media, on CLS. In this context, two byproducts of yeast glucose fermentation, ethanol and acetic acid, have been proposed as extrinsic proaging factors. Here, we report that ethanol and acetic acid, at physiological levels released in the exhausted medium, both contribute to chronological aging. Moreover, this combined proaging effect is not due to a toxic environment created by their presence but is mainly mediated by the metabolic pathways required for their utilization as carbon/energy sources. In addition, measurements of key enzymatic activities of the glyoxylate cycle and gluconeogenesis, together with respiration assays performed in extreme calorie restriction, point to a long-term quiescent program favoured by glyoxylate/gluconeogenesis flux contrary to a proaging one based on the oxidative metabolism of ethanol/acetate via TCA and mitochondrial respiration.

The GGP1/GAS1/CWH52 gene of Saccharomyces cerevisiae encodes a major exocellular 115 kDa glycoprotein (gp115) anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI). The function of gp115 is still unknown but the analysis of null mutants suggests a possible role in the control of morphogenesis. PHR1 gene isolated from Candida alibicans is homologous to the GGP1 gene. In this report we have analysed the ability of PHR1 to complement a ggp1 delta mutation in S. cerevisiae. The expression of PHR1 controlled by its natural promoter or by the GGP1 promoter has been studied. In both cases we have observed a complete complementation of the mutant phenotype. Moreover, immunological analysis has revealed that PHR1 in budding yeast gives rise to a 75-80 kDa protein anchored to the membrane through a GPI, indicating that the signal for GPI attachment present in the C. albicans gene product is functional in S. cerevisiae.

ACH1 encodes a mitochondrial enzyme of Saccharomyces cerevisiae endowed with CoA-transferase activity. It catalyzes the CoASH transfer from succinyl-CoA to acetate generating acetyl-CoA. It is known that ACH1 inactivation results in growth defects on media containing acetate as a sole carbon and energy source which are particularly severe at low pH. Here, we show that chronological aging ach1Δ cells which accumulate a high amount of extracellular acetic acid display a reduced chronological lifespan. The faster drop of cell survival is completely abrogated by alleviating the acid stress either by a calorie restricted regimen that prevents acetic acid production or by transferring chronologically aging mutant cells to water. Moreover, the short-lived phenotype of ach1Δ cells is accompanied by reactive oxygen species accumulation, severe mitochondrial damage, and an early insurgence of apoptosis. A similar pattern of endogenous severe oxidative stress is observed when ach1Δ cells are cultured using acetic acid as a carbon source under acidic conditions. On the whole, our data provide further evidence of the role of acetic acid as cell-extrinsic mediator of cell death during chronological aging and highlight a primary role of Ach1 enzymatic activity in acetic acid detoxification which is important for mitochondrial functionality.

The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality.

Carbonylation of proteins is an irreversible oxidative damage that increases during both chronological and replicative yeast aging. In the latter, a spatial protein quality control system that relies on Sir2 is responsible for the asymmetrical damage segregation in the mother cells. Proper localization of Sir2 on chromatin depends on the deubiquitinating enzyme Ubp10, whose loss of function deeply affects the recombination and gene-silencing activities specific to Sir2. Here, we have analyzed the effects of SIR2 and UBP10 inactivations on carbonylated protein patterns obtained in two aging models such as stationary phase cells and size-selected old mother ones. In line with the endogenous situation of higher oxidative stress resulting from UBP10 inactivation, an increase of protein carbonylation has been found in the ubp10Delta stationary phase cells compared with sir2Delta ones. Moreover, Calorie Restriction had a salutary effect for both mutants by reducing carbonylated proteins accumulation. Remarkably, in the replicative aging model, whereas SIR2 inactivation resulted in a failure to establish damage asymmetry, the Sir2-dependent damage inheritance is maintained in the ubp10Delta mutant which copes with the increased oxidative damage by retaining it in the mother cells. This indicates that both Ubp10 and a correct association of Sir2 with the silenced chromatin are not necessary in such a process but also suggests that additional Sir2 activities on non-chromatin substrates are involved in the establishment of damage asymmetry.

Saccharomyces cerevisiae SFP1 promotes transcription of a large cluster of genes involved in ribosome biogenesis. During growth in shake flasks, a mutant deleted for SFP1 shows a small size phenotype and a reduced growth rate. We characterized the behaviour of an sfp1Δ mutant compared to an isogenic reference strain growing in chemostat cultures at the same specific growth rate. By studying glucose (anaerobic)- and ethanol (aerobic)-limited cultures we focused specifically on nutrient-dependent effects. Major differences in the genome-wide transcriptional profiles were observed during glucose-limited growth. In particular, Sfp1 appeared to be involved in the control of ribosome biogenesis but not of ribosomal protein gene expression. Flow cytometric analyses revealed size defects for the mutant under both growth conditions. Our results suggest that Sfp1 plays a role in transcriptional and cell size control, operating at two different levels of the regulatory network linking growth, metabolism and cell size. © 2008 SGM.

on host physiology The organisers would like to thank Novozymes Delta Ltd who generously supported the meeting. Meeting

The aim of the study was the identification, cloning and disruption of the GAS1 homologue of Pichia pastoris. Gas1p is a glycoprotein anchored to the outer layer of the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. Gas1p is a β-1,3-glucanosyltransglycosylase (EC 2.4.1.-). This cross-linking enzyme highly affects the structure and permeability of the yeast cell wall. The gene coding for the GAS1 homologue of P. pastoris was cloned by PCR, and its functionality was proven in a Saccharomyces cerevisiae GAS1 null mutant. Based on the nucleotide sequence information of the P. pastoris GAS1 homologue, a disruption cassette was constructed for the knockout of the GAS1 in P. pastoris. The morphology of ΔGAS1 P. pastoris was identical to that of S. cerevisiae GAS1 mutants. Finally, the impact of GAS1 disruption on secretion of three recombinant model proteins in P. pastoris, human trypsinogen, human serum albumin and Rhizopus oryzae lipase, was evaluated. While the disruption had no effect on the secretion of trypsinogen and albumin, the amount of lipase released from the cells was doubled. © 2006 Federation of European Microbiological Societies.

We have isolated a Schizosaccharomyces pombe cDNA encoding a small heat-shock protein, designated Hsp9. The deduced amino acid sequence shares significant homology with the Saccharomyces cerevisiae Hsp12 gene product. Northern blot analysis identified a 600-base transcript which is expressed at a low level in S. pombe exponentially growing cells, but is strongly induced by heat-shock and upon entry into the stationary phase. An increase in the transcript level is also observed in response to glucose deprivation.

The three-dimensional (3D) structure of the catalytic domain of Gas1p, a protein belonging to the only family of beta-(1,3)-glucan transferases so far identified in yeasts and some pathogenic fungi (family GH-72), has been predicted by combining results derived from threading methods, multiple sequence alignments and secondary-structure predictions. The 3D model has allowed the identification of several residues that are predicted to play a crucial role in structural integrity, substrate recognition and catalysis. In particular, the model of the catalytic domain can be useful for designing site-directed mutagenesis experiments and for developing inhibitors of Gas1p enzymatic activity.

New secretory signals and strategies can be attempted to improve the secretion of heterologous proteins of biotechnological interest which encounter difficulties being exported in yeast. The GGPI gene of Saccharomyces cerevisiae codes for a 125 kDa glycoprotein transported through the secretory pathway and anchored to the plasma membrane by means of a glycosylphosphatidylinositol. The regions coding for the secretory signal or also for the first 46 amino acids were tested for efficiency in secretion by fusion to the lacZ gene of Escherichia coli resulting in the synthesis of the endoplasmic reticulum-targeted 1-22- and 1-68-GgpIp/beta-gal hybrids. A cytoplasmic form was also examined. The 1-22 beta gal is partially transported to the cell surface and in the medium in an unglycosylated form. The 1-68 beta gal is completely retained in the intracellular membranes and is N-glycosylated in the GgpIp moiety. The amount of hybrid protein produced is similar and independent from its targeted site, suggesting that translocation through endoplasmic reticulum is not a limiting step, whereas the amount of active enzyme is from 50 to 80% lower for the endoplasmic reticulum forms compared with the cytoplasmic form. BiP/Kar2p putative precursor is accumulated in cells expressing the endoplasmic reticulum-targeted forms but not in those producing the cytosolic beta-galactosidase or over-expressing an endogenous secretory protein. Thus, glycosylation and abnormal folding rather than over-expression are among the factors responsible for the decreased activity and exit of beta-galactosidase from the endoplasmic reticulum and for induction of BiP. The results obtained indicate that the sole secretory signal of GgpIp is suitable to drive secretion of foreign products with complex folding and point to the importance of the endoplasmic reticulum quality control in the secretion of heterologous proteins in yeast.

A cDNA encoding glyceraldehyde-3-phosphate dehydrogenase (GPD) was isolated from the fission yeast Schizosaccharomyces pombe. The nucleotide and the predicted amino acid sequences were determined. A very high degree of homology with different GPD sequences present in the SWISS-PROT data bank was observed in accordance with the evolutionary conservation of this enzyme. Northern analysis and the comparison with other fungal GPD sequences suggest that the cDNA corresponds to an actively expressed gene encoding a functional GPD protein. Finally, codon usage analysis revealed the same codon bias observed for three highly expressed genes in S. pombe

gp115 is a N- and O-glycosylated protein of Saccharomyces cerevisiae. It is also modified by addition of glycosylphosphatidylinositol, which anchors the protein to the plasma membrane. The gene encoding gp115 (GGP1) has been cloned by a two-step procedure. By an immunoscreening of a yeast genomic DNA library in the expression vector lambda gt11, a 3'-terminal 0.9-kilobase portion of the gene has been isolated and then used as a molecular probe to screen a yeast genomic DNA library in YEp24. In this way, the whole GGP1 gene has been cloned. Its identity with the gp115 gene has been confirmed by gene disruption, which has also indicated that the function of gp115 is not essential for cell viability. The features of the sequence are also entirely consistent with it corresponding to the gp115 gene. The nucleotide sequence of GGP1 predicts a 60-kDa polypeptide, in agreement with the molecular mass of the gp115 precursor detected in sec53 mutant cells at restrictive temperature. Two hydrophobic sequences, one NH2- and the other COOH-terminal were found. The former has the features of the cleavable signal sequence, which allows the entry of proteins in the secretory pathway. The latter could be the signal sequence that has to be removed during the addition of glycosylphosphatidylinositol. The predicted amino acid sequence of gp115 shows 10 sequons for N-glycosylation and a high proportion of serine-threonine residues (22%) that could provide several sites for O-glycosylation. The unusual concentration of 27 serines in the COOH-terminal portion of the protein shares homology with a similar polyserine repeat of the serine repeat antigen (SERA protein) of Plasmodium falciparum. A two-dimensional analysis of the "in vitro" translational product of the GGP1 mRNA has been carried out, allowing the identification of the "in vivo" gp115 precursor in a two-dimensional gel.

The glycoprotein gp115 (Mr = 115,000, pI 4.8-5) is localized in the plasma membrane of Saccharomyces cerevisiae cells and maximally expressed during G1 phase. To gain insight on the mechanism regulating its synthesis, we have examined various conditions of cell proliferation arrest. We used pulse-labeling experiments with [35S]methionine and two-dimensional gel electrophoresis analysis, which allow the detection of the well characterized 100-kDa precursor of gp115 (p100). In the cAMP-requiring mutant cyr1, p100 synthesis is active during exponential growth, shut off by cAMP removal, and induced when growth is restored by cAMP readdition. The inhibition of p100 synthesis also occurs in TS1 mutant cells (ras1ras2-ts1) shifted from 24 to 37 degrees C. During nitrogen starvation of rca1 cells, a mutant permeable to cAMP, p100 synthesis is also inhibited. cAMP complements the effect of ammonium deprivation, promoting p100 synthesis, even when added to cells which have already entered G0. Experiments with the bcy1 and cyr1bcy1 mutants have indicated the involvement of the cAMP-dependent protein kinases in the control of p100 synthesis. Moreover, the synthesis of p100 was unaffected in A364A cells, terminally arrested at START B by alpha-factor. These results indicate that the switch operating on p100 synthesis is localized in early G1 (START A) and is one of the multiple events controlled by the cAMP pathway.

In Saccharomyces cerevisiae cells (strain A364A) during a shift-down from glucose to raffinose, a rapid reduction in the rate of RNA accumulation was observed whereas the rate of protein accumulation was unaffected for at least 2 h. Following the transition the percentage of unbudded cells slightly increased and the cell volume distribution showed a newly formed subpopulation of smaller cells. To study the effects of the shift-down on the protein synthesis pattern, total [35S]-methionine pulse-labeled extracts were fractionated by high-resolution two-dimensional gel electrophoresis. The synthesis of two classes of proteins (I and II) was modulated during the transitory state of growth: one positively, the other negatively. Two polypeptides of 57 kDa showed the most dramatic increase in synthesis during the shift-down. Also a heat-shock protein (HSP 256) appeared to be positively correlated to the shift-down transition.

The cell cycle modulated protein gp115 (115 kDa, isoelectric point about 4.8–5) of Saccharomyces cerevisiae undergoes various post-translational modifications. It is N-glycosylated during its maturation along the secretory pathway where an intermediary precursor of 100 kDa (p100), dynamically related to the mature gp115 protein, is detected at the level of endoplasmic reticulum. Moreover, we have shown by the use of metabolic labeling with [35S]methionine, [3H]palmitic acid and myo-[3H]inositol combined with high resolution two-dimensional gel electrophoresis and immunoprecipitation with a specific antiserum, that gp115 is one of the major palmitate- and inositol-containing proteins in yeast. These results, and the susceptibility of gp115 to phosphatidylinositol-specific phospholipase C treatment strongly indicate that gp115 contains the glycosylphosphatidylinositol (GPI) structure as membrane anchor domain. The two-dimensional analysis of the palmitate- and inositol-labeled proteins has also allowed the characterization of other polypeptides which possibly contain a GPI structure.

The effects of temperature (in the range 15-36 degrees C) on growth and the nuclear and budding cycle have been studied in populations of the yeast Saccharomyces cerevisiae exponentially growing in batch on yeast nitrogen base (YNB) glucose medium. The maximal rate of exponential growth is achieved at 30 degrees C, and a transition point is apparent at about 20 degrees C. At all tested temperatures DNA replication begins when cells are still unbudded and both the budded period and the postreplicative period have the same temperature dependence. A temperature compensatory mechanism seems to operate in S phase, during which duration remains relatively constant, in the range 21-36 degrees C, while duration of G2+ M phases shows a much more pronounced temperature dependence. The results are discussed in terms of a cell-cycle model for budding yeast.

Immunoblot analysis with anti-phosphotyrosine antibodies of total extracts from exponentially growing yeast cells reveals a unique cross-reactive polypeptide of about 75 Kd (p75). The specificity of the immunodecorations has been checked by experiments of competition with phosphoaminoacids. A common behaviour has been observed for the 75 kd band and the 170 kd band corresponding to the platelet-derived growth factor receptor from Swiss 3T3 cells, which it has been known to be autophosphorylated on tyrosine upon ligand binding and used as a control throughout this work. We have found that p75 is associated to detergent insoluble cytoplasmic matrices. The stability of p75 detection by antibodies following treatments that specifically hydrolyze phosphohistidine and its susceptibility to potato acid phosphatase treatment provide further evidences that the epitope recognized by these antibodies in the yeast p75 polypeptide is indeed phosphotyrosine.

The molecular events of start, the regulatory step that commits yeast cells to DNA replication, have recently begun to be investigated. One of the gene products required for completion of start has been found to have a significant structural homology with oncogenes endowed with protein kinase activity. Our experiments provide data on the biosynthetic pathway of a previously identified labile protein (p100, molecular weight 100,000, isoelectric point of approximately 4.8-5) involved in cell cycle progression at start, which appears to be specifically made during the release from cell cycle arrest of a temperature-sensitive mutant (cdc25) of Saccharomyces cerevisiae. On two-dimensional gel, p100 migrates very close to another 100-kDa labile protein (p100*) which behaves as a cell cycle modulated protein with reduced synthesis in G1. Pulse and chase labeling of protein with [35S]methionine suggests that both p100 and p100* are processed to a protein (p115) of slightly higher molecular weight (Mr = 115,000). Peptide mapping analysis indicates that p100 and p100 yield identical maps and that both p100 and p100* are very much similar to p115. p115 is a glycosylated protein as shown by a labeling experiment with [3H]glucosamine and by the fact that the synthesis of both p100 and p115 is inhibited if cells are cultured in the presence of tunicamycin. A protein having the same heterogeneous aspect of migration on sodium dodecyl sulfate-polyacrylamide gel and the same apparent molecular weight and isoelectric point of p115 is abundantly present in a preparation of membranes from S. cerevisiae and the isolated radioactive p115 comigrates with it. Taken together these results favor the idea that terminal glycosylation of both p100 and p100* gives rise to the fully glycosylated p115 protein which appears to be a membrane-associated protein.

The plasma membrane H + -ATPases from fungi and yeasts have similar catalytic and molecular properties. A structural comparison has been performed using immunoblot analysis with polyclonal antibodies directed toward the 102 kDa polypeptide of the plasma membrane H + -ATPase from Neurospora crassa . A strong cross-reactivity is observed between the fungal H + -ATPase and the enzyme from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe . Structural homologies are indicated also by the analysis of the cross-reactive peptides originated by proteolytic digestion of Neurospora and S.cerevisiae purified enzymes. Neither enzyme from these two sources appears to be glycosylated by a highly sensitive concanavalin A affinity assay on blotted proteins. A glycoprotein of M r 115000 and p I 4.8–5, which comigrates with a cell cycle-modulated protein on 2D gel, is present in partially purified preparations of plasma membrane H + -ATPase of S.cerevisiae and it is shown to be structurally unrelated to H + -ATPase.