Molecular biology of translation in yeast

The combination of genetic, molecular and biochemical approaches have made the yeastSaccharomyces cerevisiae a convenient organism to study translation. The sequence similarity of translation factors from yeast and other organisms suggests a high degree of conservation in the translational machineries. This view is also strengthened by a functional analogy of some proteins implicated in translation. Beautiful genetic experiments have confirmed existing models and added new insights in the mechanism of translation. This review summarizes recent experiments using yeast as a model system for the analysis of this complex process.     

Saturation mutagenesis of a +1 programmed frameshift-inducing mRNA sequence derived from a yeast retrotransposon

Errors during the process of translating mRNA information into protein products occur infrequently. Frameshift errors occur less frequently than other types of errors, suggesting that the translational machinery has more robust mechanisms for precluding that kind of error. Despite these mechanisms, mRNA sequences have evolved that increase the frequency up to 10,000-fold. These sequences, termed programmed frameshift sites, usually consist of a heptameric nucleotide sequence, at which the change in frames occurs along with additional sequences that stimulate the efficiency of frameshifting. One such stimulatory site derived from the Ty3 retrotransposon of the yeast Saccharomyces cerevisiae (the Ty3 stimulator) comprises a 14 nucleotide sequence with partial complementarity to a Helix 18 of the 18S rRNA, a component of the ribosome's accuracy center. A model for the function of the Ty3 stimulator predicts that it base pairs with Helix 18, reducing the efficiency with which the ribosome rejects erroneous out of frame decoding. We have tested this model by making a saturating set of single-base mutations of the Ty3 stimulator. The phenotypes of these mutations are inconsistent with the Helix 18 base-pairing model. We discuss the phenotypes of these mutations in light of structural data on the path of the mRNA on the ribosome, suggesting that the true target of the Ty3 stimulator may be rRNA and ribosomal protein elements of the ribosomal entry tunnel, as well as unknown constituents of the solvent face of the 40S subunit.     

Identification of rate-limiting steps in yeast heme biosynthesis

The heme biosynthesis pathway in the yeast Saccharomyces cerevisiae is a highly regulated system, but the mechanisms accounting for this regulation remain unknown. In an attempt to identify rate-limiting steps in heme synthesis, which may constitute potential regulatory points, we constructed yeast strains overproducing two enzymes of the pathway: the porphobilinogen synthase (PBG-S) and deaminase (PBG-D). Biochemical analysis of the enzyme-overproducing strains revealed intracellular porphobilinogen and porphyrin accumulation. These results indicate that both enzymes play a rate-limiting role in yeast heme biosynthesis.     

Systems Biology in Aging: Linking the Old and the Young

Aging can be defined as a process of progressive decline in the physiological capacity of an organism, manifested by accumulated alteration and destabilization at the whole system level. Systems biology approaches offer a promising new perspective to examine the old problem of aging. We begin this review by introducing the concepts of systems biology, and then illustrate the application of systems biology approaches to aging research, from gene expression profiling to network analysis. We then introduce the network that can be constructed using known lifespan and aging regulators, and conclude with a look forward to the future of systems biology in aging research. In summary, systems biology is not only a young field that may help us understand aging at a higher level, but also an important platform that can link different levels of knowledge on aging, moving us closer to a more comprehensive control of systematic decline during aging.     

RNA isolation from yeast using silica matrices.

RNA isolation from yeast is complicated by the need to initially break the cell wall. While this can be accomplished by glass bead disruption or enzyme treatment, these approaches result in DNA contamination and/or the need for incubation periods. We have developed a protocol for the isolation of RNA samples from yeast that minimizes degradation by RNases and incorporates two purification steps: acid phenol extraction and binding to a silica matrix. The procedure requires no precipitation steps, facilitating automation, and can be completed in less than 90 min. The RNA quality is ideal for microarray analysis.     

High yield electroextraction of proteins from yeast by a flow process

High yields of intracellular enzymes from yeast can be obtained by application of a series of electric field pulses with a flow process. Up to 80-90% of the total activity can be liberated without any further or previous treatment of cells. The method is based on electroinduced changes in the cell envelope leading to a leakage of part of the intracellular proteins without formation of debris and permits treatment of large volumes. Field parameters require a limited electrical power. Treatment of at least 20% wet weight suspensions is possible. The optimal field conditions must be adjusted to the suspension concentration. Maximal yield is obtained within 4h at 30 degrees C for enzymes from Saccharomyces cerevisiae such as hexokinase, 3-phosphoglycerate kinase, and glyceraldehyde-3-phosphate dehydrogenase. The extraction of beta-D-galactosidase from Kluyveromyces lactis lasts 10h but can be accelerated by adding dithiothreitol in the postpulse medium. The specific activities of the electroextracted enzymes are higher than those obtained by mechanical disintegration or enzymatic lysis.     

Systems biology of lipid metabolism: From yeast to human

Lipid metabolism is highly relevant as it plays a central role in a number of human diseases. Due to the highly interactive structure of lipid metabolism and its regulation, it is necessary to apply a holistic approach, and systems biology is therefore well suited for integrated analysis of lipid metabolism. In this paper it is demonstrated that the yeast Saccharomyces cerevisiae serves as an excellent model organism for studying the regulation of lipid metabolism in eukaryotes as most of the regulatory structures in this part of the metabolism are conserved between yeast and mammals. Hereby yeast systems biology can assist to improve our understanding of how lipid metabolism is regulated.     

Integration of omics sciences to advance biology and medicine

In the past two decades, our ability to study cellular and molecular systems has been transformed through the development of omics sciences. While unlimited potential lies within massive omics datasets, the success of omics sciences to further our understanding of human disease and/or translating these findings to clinical utility remains elusive due to a number of factors. A significant limiting factor is the integration of different omics datasets (i.e., integromics) for extraction of biological and clinical insights. To this end, the National Cancer Institute (NCI) and the National Heart, Lung and Blood Institute (NHLBI) organized a joint workshop in June 2012 with the focus on integration issues related to multi-omics technologies that needed to be resolved in order to realize the full utility of integrating omics datasets by providing a glimpse into the disease as an integrated “system”. The overarching goals were to (1) identify challenges and roadblocks in omics integration, and (2) facilitate the full maturation of ‘integromics’ in biology and medicine. Participants reached a consensus on the most significant barriers for integrating omics sciences and provided recommendations on viable approaches to overcome each of these barriers within the areas of technology, bioinformatics and clinical medicine.     

Biogenesis of yeast mitochondrial cytochrome c: a unique relationship to the TOM machinery

The import of cytochrome c into the mitochondrial intermembrane space is not understood at a mechanistic level. While the precursor apocytochrome c can insert into protein-free lipid bilayers, the purified translocase of the outer membrane (TOM) complex supports the translocation of apocytochrome c into proteoliposomes. We report an in organello analysis of cytochrome c import into yeast mitochondria from wild-type cells and different mutants cells, each defective in one of the seven Tom proteins. The import of cytochrome c is not affected by removal of the receptor Tom20 or Tom70. Moreover, neither the transfer protein Tom5 nor the assembly factors Tom6 and Tom7 are needed for import of cytochrome c. When the general import pore (GIP)-protein Tom40 is blocked, the import of cytochrome c is moderately affected. Mitochondria lacking the central receptor and organizing protein Tom22 contain greatly reduced levels of cytochrome c. We conclude that up to two components of the TOM complex, Tom22 and possibly the GIP, are involved in the biogenesis of cytochrome c.     

Destabilization and recovery of a yeast prion after mild heat shock

Yeast prion [PSI⁺] is a self-perpetuating amyloid of the translational termination factor Sup35. Although [PSI⁺] propagation is modulated by heat shock proteins (Hsps), high temperature was previously reported to have little or no effect on [PSI⁺]. Our results show that short-term exposure of exponentially growing yeast culture to mild heat shock, followed by immediate resumption of growth, leads to [PSI⁺] destabilization, sometimes persisting for several cell divisions after heat shock. Prion loss occurring in the first division after heat shock is preferentially detected in a daughter cell, indicating the impairment of prion segregation that results in asymmetric prion distribution between a mother cell and a bud. Longer heat shock or prolonged incubation in the absence of nutrients after heat shock led to [PSI⁺] recovery. Both prion destabilization and recovery during heat shock depend on protein synthesis. Maximal prion destabilization coincides with maximal imbalance between Hsp104 and other Hsps such as Hsp70-Ssa. Deletions of individual SSA genes increase prion destabilization and/or counteract recovery. The dynamics of prion aggregation during destabilization and recovery are consistent with the notion that efficient prion fragmentation and segregation require a proper balance between Hsp104 and other (e.g., Hsp70-Ssa) chaperones. In contrast to heat shock, [PSI⁺] destabilization by osmotic stressors does not always depend on cell proliferation and/or protein synthesis, indicating that different stresses may impact the prion via different mechanisms. Our data demonstrate that heat stress causes asymmetric prion distribution in a cell division and confirm that the effects of Hsps on prions are physiologically relevant.     

Stress and prions: lessons from the yeast model

Yeast self-perpetuating amyloids (prions) provide a convenient model for studying the cellular control of highly ordered aggregates involved in mammalian protein assembly disorders. The very ability of an amyloid to propagate a prion state in yeast is determined by its interactions with the stress-inducible chaperone Hsp104. Prion formation and propagation are also influenced by other stress-related chaperones (Hsp70 and Hsp40), and by alterations of the protein trafficking and degradation networks. Some stress conditions induce prion formation or loss. It is proposed that prions arise as byproducts of the reversible assembly of highly ordered complexes, protecting certain proteins during unfavorable conditions.     

Influence of compartmental localization on the function of yeast NADP+-specific isocitrate dehydrogenases

Three differentially compartmentalized isozymes of isocitrate dehydrogenase (mitochondrial IDP1, cytosolic IDP2, and peroxisomal IDP3) in the yeast Saccharomyces cerevisiae catalyze the NADP(+)-dependent oxidative decarboxylation of isocitrate to form alpha-ketoglutarate. These enzymes are highly homologous but exhibit some significant differences in physical and kinetic properties. To examine the impact of these differences on physiological function, we exchanged promoters and altered organellar targeting information to obtain expression of IDP2 and IDP3 in mitochondria and of IDP1 and IDP3 in the cytosol. Physiological function was assessed as complementation by mislocalized isozymes of defined growth defects of isocitrate dehydrogenase mutant strains. These studies revealed that the IDP isozymes are functionally interchangeable for glutamate synthesis, although mitochondrial localization has a positive impact on this function during fermentative growth. However, IDP2, whether located in mitochondria or in the cytosol, provided the highest level of defense against endogenous or exogenous oxidative stress.     

Recycling of the yeast a-factor receptor

The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway and a ligand-dependent uptake pathway. Whereas the constitutive pathway leads to degradation of the receptor in the vacuole, the present work finds that receptor internalized via the ligand-dependent pathway recycles. With the a-factor ligand continuously present in the culture medium, trafficking of the receptor achieves an equilibrium in which continuing uptake to endosomal compartments is balanced by its recycling return to the plasma membrane. Withdrawal of ligand from the medium leads to a net return of the internalized receptor back to the plasma membrane. Although recycling is demonstrated for receptors that lack the signal for constitutive endocytosis, evidence is provided indicating a participation of recycling in wild-type Ste3p trafficking as well: a-factor treatment both slows wild-type receptor turnover and results in receptor redistribution to intracellular endosomal compartments. Apparently, a-factor acts as a switch, diverting receptor from vacuole-directed endocytosis and degradation, to recycling. A model is presented for how the two Ste3p endocytic modes may collaborate to generate the polarized receptor distribution characteristic of mating cells.     

Replicative aging of the yeast does not require DNA replication

Glucose addition to a stationary culture of wild-type Saccharomyces cerevisiae BY4742 cells with zero activity of MDR pumps resuspended in a fresh medium causes pump resynthesis (measured as pump-effected diS-C3(3) efflux). In a stationary culture in its original growth medium, this glucose-induced pump resynthesis fails to occur due to depletion of essential nutrients or to extracellular metabolites produced by cells during growth. Direct pump inactivation by metabolites is excluded since exponential cells with high MDR pump activity cultured in a medium with high concentration of extracellular metabolites retain this activity for at least 2 h. The metabolites also do not affect pump synthesis on the level of gene expression as addition of concentrated growth medium or an amino acid mixture to stationary cells in spent growth medium restores glucose-induced pump synthesis. The block of MDR pump synthesis is therefore due to the lack of essential nutrients in spent medium.     

Global gene expression in recombinant and non-recombinant yeast Saccharomyces cerevisiae in three different metabolic states

Global gene expression of two strains of Saccharomyces cerevisiae, one recombinant (P+), accumulating large amounts of an intracellular protein Superoxide Dismutase (SOD) and one non-recombinant (P−) which does not contain the recombinant plasmid, were compared in batch culture during diauxic growth when cells were growing exponentially on glucose, when they were growing exponentially on ethanol, and in the early stationary phase when glycerol was being utilized.When comparing the gene expression for P− (and P+) during growth on ethanol to that on glucose (Eth/Gluc), overexpression is related to an increase in consumption of glycerol, activation of the TCA cycle, degradation of glycogen and metabolism of ethanol. Furthermore, 97.6% of genes (80 genes) involved in the central metabolic pathway are overexpressed. This is similar to that observed by DeRisi et al. [DeRisi, J.L., Iyer, V.R. & Brown, P.O. 1997. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278:680–686.] but very different from was observed for Metabolic Flux Analysis (MFA), where the specific growth rate is lowered to ca. 40%, the fluxes in the TCA cycle are reduced to ca. 40% (to 30% in P+), glycolysis is reduced to virtually 0 and protein synthesis to ca. 50% (to 40% in P+). Clearly it is not possible to correlate in a simple or direct way, quantitative mRNA expression levels with cell function which is shown by the Metabolic Flux Analysis (MFA).When comparing the two strains in the 3 growth stages, 4 genes were found to be under or overexpressed in all cases. The products of all of these genes are expressed at the plasma membrane or cell wall of the yeast. While comparing the strains (P+/P−) when growing on glucose, ethanol and in the early stationary phase, many of the genes of the central metabolic pathways are underexpressed in P+, which is similar to the behaviour of the metabolic fluxes of both strains (MFA). Comparing the gene expression for P− (and to some extent P+) during the early stationary phase to growth on ethanol (Stat/Eth), underexpression is generalized. This shows that the switch in metabolism between ethanol and early stationary phases has an almost instantaneous effect on gene expression but a much more retarded effect on metabolic fluxes and that the “early stationary” phase represents a “late ethanol” phase from the metabolic analysis point of view since ethanol is still present and being consumed although at a much slower rate.     

Oxygen stress: a regulator of apoptosis in yeast.

Oxygen radicals are important components of metazoan apoptosis. We have found that apoptosis can be induced in the yeast Saccharomyces cerevisiae by depletion of glutathione or by low external doses of H2O2. Cycloheximide prevents apoptotic death revealing active participation of the cell. Yeast can also be triggered into apoptosis by a mutation in CDC48 or by expression of mammalian bax. In both cases, we show oxygen radicals to accumulate in the cell, whereas radical depletion or hypoxia prevents apoptosis. These results suggest that the generation of oxygen radicals is a key event in the ancestral apoptotic pathway and offer an explanation for the mechanism of bax-induced apoptosis in the absence of any established apoptotic gene in yeast.     

Isomaltulose production via yeast surface display of sucrose isomerase from Enterobacter sp. FMB-1 on Saccharomyces cerevisiae

The gene encoding sucrose isomerase from Enterobacter sp. FMB-1 species (ESI) was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a glycosylphosphatidylinositol (GPI) anchor attachment signal sequence. Fluorescence activated cell sorting (FACS) analysis and immunofluorescence microscopy confirmed the localization of ESI on the yeast cell surface. The displayed ESI (dESI) was stable at a broad range of temperatures (35-55 °C) and pHs (pH 5-7) with optimal temperature and pH at 45 °C and pH 7.0, respectively. In addition, the thermostability of the dESI was significantly enhanced compared with the recombinant ESI expressed in Escherichia coli. Biotransformation of sucrose to isomaltulose was observed in various ranges of substrate concentrations (50-250 mM) with a 6.4-7.4% conversion yield. It suggested that the bioconversion of sucrose to isomaltulose can be successfully performed by the dESI on the surface of host S. cerevisiae.