MicroReview Control of translation initiation in Saccharomyces cerevisiae

The first observations regarding the control of translation initiation in the yeast Saccharomyces cerevisiae were made by Fred Sherman and his colleagues in 1971. Elegant genetic studies of the CYC1 gene resulted in the formulation of 'Sherman's Rules' for translation initiation as follows: (i) AUG is the only initiator codon. (ii) the most proximal AUG from the 5' end of a message will serve as the start site of translation; and (iii) if the upstream AUG codon is mutated then initiation begins at the next available AUG in the message. Hidden within these rules is the mechanism of eukaryotic translation initiation, as these very same rules were later shown to apply to higher eukaryotic organisms and were formulated into the scanning model. However, only in the past five years has yeast been taken seriously as an organism for studying the mechanism of eukaryotic translation initiation. The basis for this is that the yeast genes for at least four mammalian translation initiation factor homologues have been identified and the number is growing. Similar factors suggest similar mechanisms for translation initiation between yeast and mammals. For some translation initiation factors, the genetics of yeast has provided new insights into their function. A mechanism for regulating translation initiation in mammalian cells is now evident in yeast. It seems clear that the molecular genetics of yeast coupled with the available in vitro translation system will provide a wealth of information in the future regarding translational control and regulatory mechanisms. The purpose of this review is to summarize what is known about translational control in S. cerevisiae.     

Efficiency of translation initiation by non-AUG codons in Saccharomyces cerevisiae.

The quantitative levels of initiation of protein synthesis at codons other than AUG were determined with a CYC7-lacZ fused gene in the yeast Saccharomyces cerevisiae. AUG was the only codon which efficiently initiated translation, although some non-AUG codons allowed initiation at very low efficiency, below 1% of the normal level. Since translation initiates at codons other than AUG in at least two wild-type genes from eucaryotes, other factors presumably play a role in enhancing the activity of non-AUG codons.     

Phosphatidylinositol 4-phosphate is required for translation initiation in Saccharomyces cerevisiae

The small natural product wortmannin inhibits protein synthesis by modulating several phosphatidylinositol (PI) metabolic pathways. A primary target of wortmannin in yeast is the plasma membrane-associated PI 4-kinase (PI4K) Stt4p, which is required for actin cytoskeleton organization. Here we show that wortmannin treatment or inactivation of Stt4p, but not disorganization of the actin cytoskeleton per se, leads to a rapid attenuation of translation initiation. Interestingly, inactivation of Pik1p, a wortmannin-insensitive, functionally distinct PI4K, implicated in the regulation of Golgi functions and secretion, also results in severe translation initiation defects with a marked increase of the phosphorylation of the translation initiation factor eIF2alpha. Because wortmannin largely phenocopies the effects of rapamycin (e.g. it triggers nuclear accumulation of Gln3p), it likely also inhibits the PI kinase-related, target of rapamycin (TOR) kinases. Importantly, however, neither inactivation of Stt4p nor Pik1p significantly affects TOR-controlled readouts other than translation initiation, indicating that these PI4Ks do not simply function upstream of TOR. Together, our results reveal the existence of a novel translation initiation control mechanism in yeast that is tightly coupled to the synthesis of distinct PI4P pools.     

Suppressors of translation initiation defect in hem12 locus of Saccharomyces cerevisiae

A system for the positive selection of transational initiation suppressors in S. cerevisiae has been developed. A mutant with an ATA initiation codon in the HEM12 gene, encoding uroporphyrinogen decarboxylase, was used to select cis- and trans-acting suppressors. These suppressors partially restore growth on nonfermentable carbon sources, such as glycerol, but still allow the accumulation of porphyrins. All extragenic suppressors are mapped to the SUI1 locus, encoding initiation factor eIF1. The effect of the hem12 mutation is also partially reversed by the known SUI3 suppressor encoding the beta subunit of eIF2. In contrast, the sui2 suppressor encoding the a subunit of eIF2 does not affect the hem12 phenotype. The intragenic suppressors are able to restore the translation of hem12 due to the generation of additional, in frame AUG codons upstream of the hem12-14 mutation. Mutational analysis of the HEM12 leader sequence was also performed to determine the role of small open reading frames (uORFs) present upstream of the HEM12 ORF. Studies on the expression of integrated hem12-1/4-lacZ fusion, devoid of all upstream ATGs, indicate a lack of regulatory effect of uORFs on HEM12 translation.     

Immature small ribosomal subunits can engage in translation initiation in Saccharomyces cerevisiae

It is generally assumed that, in Saccharomyces cerevisiae, immature 40S ribosomal subunits are not competent for translation initiation. Here, we show by different approaches that, in wild‐type conditions, a portion of pre‐40S particles (pre‐SSU) associate with translating ribosomal complexes. When cytoplasmic 20S pre‐rRNA processing is impaired, as in Rio1p‐ or Nob1p‐depleted cells, a large part of pre‐SSUs is associated with translating ribosomes complexes. Loading of pre‐40S particles onto mRNAs presumably uses the canonical pathway as translation‐initiation factors interact with 20S pre‐rRNA. However, translation initiation is not required for 40S ribosomal subunit maturation. We also provide evidence suggesting that cytoplasmic 20S pre‐rRNAs that associate with translating complexes are turned over by the no go decay (NGD) pathway, a process known to degrade mRNAs on which ribosomes are stalled. We propose that the cytoplasmic fate of 20S pre‐rRNA is determined by the balance between pre‐SSU processing kinetics and sensing of ribosome‐like particles loaded onto mRNAs by the NGD machinery, which acts as an ultimate ribosome quality check point.     

Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae

We have devised an approach for analyzing shotgun proteomics datasets based on the normalized spectral abundance factor that can be used for quantitative proteomics analysis. Three biological replicates of samples enriched for plasma membranes were isolated from S. cerevisiae grown in 14N-rich media and 15N-minimal media and analyzed via quantitative multidimensional protein identification technology. The natural log transformation of NSAF values from S. cerevisiae cells grown in 14N YPD media and 15N-minimal media had a normal distribution. The t-test analysis demonstrated 221 of 1316 proteins were significantly overexpressed in one or the other growth conditions with a p value <0.05. Notably, amino acid transporters were among the 14 membrane proteins that were significantly upregulated in cells grown in minimal media, and we functionally validated these increases in protein expression with radioisotope uptake assays for selected proteins.     

A proteome analysis of the cadmium response in Saccharomyces cerevisiae

Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood. We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins. A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal. Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium.     

Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis

The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique. Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures. Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity. Whereas mitochondrial preparations isolated by means of differential centrifugation include a considerable degree of non-mitochondrial proteins (16%), this contamination could be effectually removed by the inclusion of a ZE-FFE purification step (2%). This higher degree of purity led to the identification of many more proteins from ZE-FFE purified mitochondrial protein extracts (n = 129), compared to mitochondrial protein extracts isolated by differential centrifugation (n = 80). Moreover, a marked decrease of degraded proteins was found in the ZE-FFE purified mitochondrial protein extracts. It is noteworthy that even at a low 2-DE resolution level, a four-fold higher number (17 versus 4) of presumably low abundance proteins could be identified in the ZE-FFE purified mitochondrial protein extracts. Therefore these results represent a feasible approach for an in-depth proteome analysis of mitochondria and possibly other organelles.     

Depletion and deletion analyses of eucaryotic translation initiation factor 1A in Saccharomyces cerevisiae

Translation initiation factor eIF1A is a highly conserved, small, acidic protein that is required for cell growth in yeast. Biochemical studies in vitro implicate eIF1A in dissociating ribosomes, promoting methionyl-tRNA(i) binding to 40S ribosomal subunits, scanning of mRNAs and recognizing the AUG initiation codon. To elucidate the pleiotropic functions of eIF1A in vivo, the factor was depleted by placing its gene behind the repressible GAL1 promoter. After Saccharomyces cerevisiae cells were shifted to glucose medium, depletion of eIF1A was seen after 3-4 generations, corresponding with cessation of cell growth. Polysome profiles of the depleted strain showed ribosome run-off from mRNAs, indicating that eIF1A is involved in the initiation phase of translation. A decrease in free 40S ribosomes and an apparent increase in free 60S ribosomes were attributed to the formation of 40S subunit dimers. The result suggests that one of the functions of eIF1A is to prevent formation of 40S dimers. Mutant forms of eIF1A lacking either the positively charged N-terminal region or the negatively charged C-terminal region were constructed and tested for their ability to confer cell growth as the sole source of eIF1A. Either deletion supports cell growth, albeit at a slower rate, and causes a reduction in polysomes, although eIF1A lacking the N-terminal region is more deleterious. Therefore the charged terminal regions contribute to, but are not absolutely essential for, eIF1A function.     

RNA-binding activity of translation initiation factor eIF4G1 from Saccharomyces cerevisiae

We identified and mapped RNA-binding sites of yeast Saccharomyces cerevisiae translation initiation factor eIF4G1 and examined their importance for eIF4G1 function in vitro and in vivo. Yeast eIF4G1 binds to single-stranded RNA with three different sites, the regions of amino acids 1-82 (N terminus), 492-539 (middle), and 883-952 (C terminus). The middle and C-terminal RNA-binding sites represent RS (arginine and serine)-rich domains; the N-terminal site is asparagine-, glutamine- and glycine-rich. The three RNA-binding sites have similar affinity for single-stranded RNA, whereas the affinity for single-stranded RNA full-length eIF4G1 is about 100-fold higher (approximate K(d) of 5 x 10(-8) M). Replacement of the arginine residues in the middle RS site by alanine residues abolishes its RNA-binding activity. Deletion of individual RNA-binding sites shows that eIF4G1 molecules lacking one binding site are still active in supporting growth of yeast cells and translation in vitro, whereas eIF4G1 molecules lacking two or all three RNA-binding sites are strongly impaired or inactive. These data suggest that RNA-binding activity is required for eIF4G1 function.     

Dynamics of histone acetylation in Saccharomyces cerevisiae

Rates of turnover for the posttranslational acetylation of core histones were measured in logarithmically growing yeast cells by radioactive acetate labeling to near steady-state conditions. On average, acetylation half-lives were approximately 15 min for histone H4, 10 min for histone H3, 4 min for histone H2B, and 5 min for histone H2A. These rates were much faster than the several hours that have previously been reported for the rate of general histone acetylation and deacetylation in yeast. The current estimates are in line with changes in histone acetylation detected directly at specific chromatin locations and the speed of changes in gene expression that can be observed. These results emphasize that histone acetylation within chromatin is subject to constant flux. Detailed analysis revealed that the turnover rates for acetylation of histone H3 are the same from mono- through penta-acetylated forms. A large fraction of acetylated histone H3, including possibly all tetra- and penta-acetylated forms, appears subject to acetylation turnover. In contrast, the rate of acetylation turnover for mono- and di-acetylated forms of histones H4 and H2B, and the fraction subject to acetylation turnover, was lower than for multi-acetylated forms of these histones. This difference may reflect the difference in location of these histones within the nucleosome, a difference in the spectrum of histone-specific acetylating and deacetylating enzymes, and a difference in the role of acetylation in different histones.     

Intracellular translation initiation factor levels in Saccharomyces cerevisiae and their role in cap-complex function

Knowledge of the balance of activities of eukaryotic initiation factors (eIFs) is critical to our understanding of the mechanisms underlying translational control. We have therefore estimated the intracellular levels of 11 eIFs in logarithmically growing cells of Saccharomyces cerevisiae using polyclonal antibodies raised in rabbits against recombinant proteins. Those factors involved in 43S complex formation occur at levels comparable (i.e. within a 0.5- to 2.0-fold range) to those published for ribosomes. In contrast, the subunits of the cap-binding complex eIF4F showed considerable variation in their abundance. The helicase eIF4A was the most abundant eIF of the yeast cell, followed by eIF4E at multiple copies per ribosome, and eIF4B at approximately one copy per ribosome. The adaptor protein eIF4G was the least abundant of the eIF4 factors, with a copy number per cell that is substoichiometric to the ribosome and similar to the abundance of mRNA. The observed excess of eIF4E over its functional partner eIF4G is not strictly required during exponential growth: at eIF4E levels artificially reduced to 30% of those in wild-type yeast, growth rates and the capacity for general protein synthesis are only minimally affected. This demonstrates that eIF4E does not exercise a higher level of rate control over translation than other eIFs. However, other features of the yeast life cycle, such as the control of cell size, are more sensitive to changes in eIF4E abundance. Overall, these data constitute an important basis for developing a quantitative model of the workings of the eukaryotic translation apparatus.     

Starved Saccharomyces cerevisiae cells have the capacity to support internal initiation of translation.

Internal initiation of translation, whereby ribosomes are directed to internal AUG codon independently of the 5' end of the mRNA, has been observed rarely in higher eucaryotes and has not been demonstrated in living yeast. We report here that starved yeast cells are capable of initiating translation of a dicistronic message internally. The studied element that functions as an internal ribosome entry site (IRES) is hardly functional or not functional at all in logarithmically growing cells. Moreover, during the logarithmic growth phase, this element seems to inhibit translation reinitiation when placed as an intercistronic spacer or to inhibit translation when placed in the 5'-untranslated region of a monocistronic message. Inhibition of translation is likely due to the putative strong secondary structure of the IRES that interferes with the cap-dependent scanning process. When cells exit the logarithmic growth phase, or when artificially starved for carbon source, translation of the IRES-containing messages is substantially induced. Our findings imply that the capacity to translate internally is a characteristic of starved rather than vegetatively growing yeast cells.     

Eap1p, a novel eukaryotic translation initiation factor 4E-associated protein in Saccharomyces cerevisiae

Ribosome binding to eukaryotic mRNA is a multistep process which is mediated by the cap structure [m(7)G(5')ppp(5')N, where N is any nucleotide] present at the 5' termini of all cellular (with the exception of organellar) mRNAs. The heterotrimeric complex, eukaryotic initiation factor 4F (eIF4F), interacts directly with the cap structure via the eIF4E subunit and functions to assemble a ribosomal initiation complex on the mRNA. In mammalian cells, eIF4E activity is regulated in part by three related translational repressors (4E-BPs), which bind to eIF4E directly and preclude the assembly of eIF4F. No structural counterpart to 4E-BPs exists in the budding yeast, Saccharomyces cerevisiae. However, a functional homolog (named p20) has been described which blocks cap-dependent translation by a mechanism analogous to that of 4E-BPs. We report here on the characterization of a novel yeast eIF4E-associated protein (Eap1p) which can also regulate translation through binding to eIF4E. Eap1p shares limited homology to p20 in a region which contains the canonical eIF4E-binding motif. Deletion of this domain or point mutation abolishes the interaction of Eap1p with eIF4E. Eap1p competes with eIF4G (the large subunit of the cap-binding complex, eIF4F) and p20 for binding to eIF4E in vivo and inhibits cap-dependent translation in vitro. Targeted disruption of the EAP1 gene results in a temperature-sensitive phenotype and also confers partial resistance to growth inhibition by rapamycin. These data indicate that Eap1p plays a role in cell growth and implicates this protein in the TOR signaling cascade of S. cerevisiae.     

Bioinformatics analysis reveals biophysical and evolutionary insights into the 3-nitrotyrosine post-translational modification in the human proteome

Protein 3-nitrotyrosine is a post-translational modification that commonly arises from the nitration of tyrosine residues. This modification has been detected under a wide range of pathological conditions and has been shown to alter protein function. Whether 3-nitrotyrosine is important in normal cellular processes or is likely to affect specific biological pathways remains unclear. Using GPS-YNO2, a recently described 3-nitrotyrosine prediction algorithm, a set of predictions for nitrated residues in the human proteome was generated. In total, 9.27 per cent of the proteome was predicted to be nitratable (27 922/301 091). By matching the predictions against a set of curated and experimentally validated 3-nitrotyrosine sites in human proteins, it was found that GPS-YNO2 is able to predict 73.1 per cent (404/553) of these sites. Furthermore, of these sites, 42 have been shown to be nitrated endogenously, with 85.7 per cent (36/42) of these predicted to be nitrated. This demonstrates the feasibility of using the predicted dataset for a whole proteome analysis. A comprehensive bioinformatics analysis was subsequently performed on predicted and all experimentally validated nitrated tyrosine. This found mild but specific biophysical constraints that affect the susceptibility of tyrosine to nitration, and these may play a role in increasing the likelihood of 3-nitrotyrosine to affect processes, including phosphorylation and DNA binding. Furthermore, examining the evolutionary conservation of predicted 3-nitrotyrosine showed that, relative to non-nitrated tyrosine residues, 3-nitrotyrosine residues are generally less conserved. This suggests that, at least in the majority of cases, 3-nitrotyrosine is likely to have a deleterious effect on protein function and less likely to be important in normal cellular function.