Boron-dependent regulation of translation through AUGUAA sequence in yeast

Under high boron (B) conditions, nodulin 26-like intrinsic protein 5;1 (NIP5;1) mRNA, a boric acid channel, is destabilized to avoid excess B entry into roots of Arabidopsis thaliana. In this regulation, the minimum upstream open reading frame (uORF), AUGUAA, in its 5′-untranslated region (5′-UTR) is essential, and high B enhances ribosome stalling at AUGUAA and leads to suppression of translation and mRNA degradation. This B-dependent AUGUAA-mediated regulation occurs also in animal transient expression and reticulocyte lysate translation systems. Thus, uncovering the ubiquitousness of B-dependent unique regulation is important to reveal the evolution of translational regulation. In the present study, we examined the regulation in Saccharomyces cerevisiae. Reporter assay showed that in yeast, carrying ATGTAA in 5′-UTR of NIP5;1 upstream of the reporter gene, the relative reporter activities were reduced significantly under high B conditions compared with control, whereas deletion of ATGTAA abolished such responses. This suggests that AUGUAA mediates B-dependent regulation of translation in Saccharomyces cerevisiae. Moreover, the deletion of ATGTAA resulted in up to 10-fold increase in general reporter activities indicating the suppression effect of AUGUAA on translation of the main ORF. Interestingly, mRNA level of the reporter gene was not affected by B in both yeast cells with and without AUGUAA. This finding reveals that in yeast, unlike the case in plants, mRNA degradation is not associated with AUGUAA regulation. Together, results suggest that B-dependent AUGUAA-mediated translational regulation is common among eukaryotes.     

Mutations in the N‐terminal region of the Schizosaccharomyces pombe glutathione transporter pgt1+ allows functional expression in Saccharomyces cerevisiae

Pgt1p encodes a glutathione transporter in Schizosaccharomyces pombe, orthologous to the Saccharomyces cerevisiae glutathione transporter, Hgt1p. Despite high similarity to Hgt1p, Pgt1p failed to display functionality during heterologous expression in S. cerevisiae. In the present study we employed a genetic strategy to investigate the reason behind the non‐functionality of pgt1⁺ in S. cerevisiae. Functional mutants were isolated after in vitro mutagenesis. Several mutants were obtained and four mutants analysed. Among these, three yielded different point mutations in the N‐terminal region (301–350 bp) of the transporter before the first transmembrane domain, while one mutant contained a deletion of 42 nucleotides within the same region. The mutant pgt1⁺ proteins not only expressed and localized correctly, but displayed high‐affinity glutathione transport capabilities in S. cerevisae. Comparison of wild‐type pgt1⁺ with the functional mutants revealed that a loss in protein expression was responsible for lack of functionality of wild‐type pgt1⁺ in S. cerevisiae. The mRNA levels in wild‐type and mutants were comparable, suggesting that the block was in translation. The formation of a strong stem–loop structure appeared to be responsible for inefficient translation in pgt1⁺ and disruption of these structures in the mutants was probably permitting translation. This was confirmed by making silent mutations in this region of wild‐type pgt1⁺, which led to their functionality in S. cerevisiae. This genetic strategy to relieve functional blocks in expression should greatly facilitate the study of these and other transporters from more intractable genetic organisms in a heterologous expression system. Copyright © 2012 John Wiley & Sons, Ltd.     

Molecular,functional and evolutionary characterization of the gene encoding HMG-CoA reductase in the fission yeast,Schizosaccharomyces pombe

The synthesis of mevalonate, a molecule required for both sterol and isoprene biosynthesis in eukaryotes, is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG-CoA reductase, hmg1+, from the fission yeast Schizosaccharomyces pombe (Accession Number L76979). Specifically, hmg1+ was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMG-CoA reductase. Gene disruption analysis showed that hmg1+ was an essential gene. This result provided evidence that, unlike Saccharomyces cerevisiae, S. pombe contained only a single functional HMG-CoA reductase gene. The presence of a single HMG-CoA reductase gene was confirmed by genomic hybridization analysis. As observed for the S. cerevisiae HMG1p, the hmg1+ protein induced membrane proliferations known as karmellae. A previously undescribed ‘feed-forward’ regulation was observed in which elevated levels of HMG-CoA synthase, the enzyme catalysing the synthesis of the HMG-CoA reductase substrate, induced elevated levels of hmg1+ protein in the cell and conferred partial resistance to lovastatin. The amino acid sequences of yeast and human HMG-CoA reductase were highly divergent in the membrane domains, but were extensively conserved in the catalytic domains. We tested whether the gene duplication that produced the two functional genes in S. cerevisiae occurred before or after S. pombe and S. cerevisiae diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specifying post-divergence duplication had significantly higher likelihood. Moreover, phylogenetic analyses of available HMG-CoA reductase sequences also suggested that the lineages of S. pombe and S. cerevisiae diverged approximately 420 million years ago but that the duplication event that produced two HMG-CoA reductase genes in the budding yeast occurred only approximately 56 million years ago. To date, S. pombe is the only unicellular eukaryote that has been found to contain a single HMG-CoA reductase gene. Consequently, S. pombe may provide important opportunities to study aspects of the regulation of sterol biosynthesis that have been difficult to address in other organisms and serve as a test organism to identify novel therapies for modulating cholesterol synthesis.     

A quantitative model for mRNA translation in Saccharomyces cerevisiae

Messenger RNA (mRNA) translation is an essential step in eukaryotic gene expression that contributes to the regulation of this process. We describe a deterministic model based on ordinary differential equations that describe mRNA translation in Saccharomyces cerevisiae. This model, which was parameterized using published data, was developed to examine the kinetic behaviour of translation initiation factors in response to amino acid availability. The model predicts that the abundance of the eIF1–eIF3–eIF5 complex increases under amino acid starvation conditions, suggesting a possible auxiliary role for these factors in modulating translation initiation in addition to the known mechanisms involving eIF2. Our analyses of the robustness of the mRNA translation model suggest that individual cells within a randomly generated population are sensitive to external perturbations (such as changes in amino acid availability) through Gcn2 signalling. However, the model predicts that individual cells exhibit robustness against internal perturbations (such as changes in the abundance of translation initiation factors and kinetic parameters). Gcn2 appears to enhance this robustness within the system. These findings suggest a trade‐off between the robustness and performance of this biological network. The model also predicts that individual cells exhibit considerable heterogeneity with respect to their absolute translation rates, due to random internal perturbations. Therefore, averaging the kinetic behaviour of cell populations probably obscures the dynamic robustness of individual cells. This highlights the importance of single‐cell measurements for evaluating network properties. Copyright © 2010 John Wiley & Sons, Ltd.     

Sequence and analysis of a 33 kb fragment from the right arm of chromosome XV of the yeast Saccharomyces cerevisiae

We have determined the nucleotide sequence of a cosmid (pEOA423) from chromosome XV of Saccharomyces cerevisiae. Analysis of the 33,173 bp sequence reveals the presence of 20 putative open reading frames (ORFs). Five of them correspond to previously known genes (MGM1, STE4, CDC44, STE13, RPB8). The previously published nucleotide sequences are in perfect agreement with our sequence except for STE4 and MGM1. In the latter case, 59 amino acids were truncated from the published protein at its N-terminal end due to a frameshift. The putative translation products of six other ORFs exhibit significant homology with protein sequences in public databases: O50 03 and O50 17 products are homologs of the ANC1 and MIP1 proteins of S. cerevisiae, respectively; O50 05 product is similar to that of a protein of unknown function from Myxococcus xanthus; O50 12 product is probably a new ATP/ADP carrier; O50 13 product shows homology with group II tRNA synthetases; and the O50 16 product exhibits strong similarity with the N-terminal domain of the NifU proteins from several prokaryotes. The remaining nine ORFs show no significant similarity. Among these, two contiguous ORFs (O50 19 and O50 20) are very similar to each other, suggesting an ancient tandem duplication. The 33,173 bp sequence has been submitted to EMBL (Accession Number: X92441).     

Cloning and sequence of a 3·835 kbp DNA fragment containing the HIS4 gene and a fragment of a PEX5-like gene from Candida albicans

We have isolated the Candida albicans HIS4 (CaHIS4) gene by complementation of a his4-34 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 2514 bp, whose translation product shares a global identity of 44% and 55% to the His4 protein homologs of S. cerevisiae and Kluyveromyces lactis, respectively. Analysis of CaHIS4 sequence suggests that, similarly to S. cerevisiae HIS4, it codes for a polypeptide having three separate enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase) which reside in different domains of the protein. A C. albicans his4 strain is complemented with this gene when using a C. albicans-S. cerevisiae-Escherichia coli shuttle vector, thus enabling the construction of a host system for C. albicans genetic manipulation. In addition, upstream of the sequenced CaHIS4 sequence, we have found the 3′-terminal half of a gene encoding a PEX5-like protein. The EMBL/DDJB/GenBank Accession Number of this sequence is AJ003115. © 1998 John Wiley & Sons, Ltd.     

The Hansenula polymorpha PDD1 gene product,essential for the selective degradation of peroxisomes,is a homologue of Saccharomyces cerevisiae Vps34p

Via functional complementation we have isolated the Hansenula polymorpha PDD1 gene essential for selective, macroautophagic peroxisome degradation. HpPDD1 encodes a 116 kDa protein with high similarity (42% identity) to Saccharomyces cerevisiae Vps34p, which has been implicated in vacuolar protein sorting and endocytosis. Western blotting experiments revealed that HpPDD1 is expressed constitutively. In a H. polymorpha pdd1 disruption strain peroxisome degradation is fully impaired. Sequestered peroxisomes, typical for the first stage of peroxisome degradation in H. polymorpha, were never observed, suggesting that HpPdd1p plays a role in the tagging of redundant peroxisomes and/or sequestration of these organelles from the cytosol. Possibly, HpPdd1p is the functional homologue of ScVps34p, because—like S. cerevisiae vps34 mutants—H. polymorpha pdd1 mutants are temperature‐sensitive for growth and are impaired in the sorting of vacuolar carboxypeptidase Y. Moreover, HpPdd1p is associated to membranes, as was also observed for ScVps34p. Copyright © 1999 John Wiley & Sons, Ltd.     

Translational comparison of HPV58 long and short L1 mRNAs in yeast (Saccharomyces cerevisiae) cell-free system

Recently, we developed a yeast cell-free system for translation of human papillomavirus (HPV) 58 short L1 mRNA and VLP assembly in vitro. In the present study, we examined the translation efficiency of HPV58 long and short L1 mRNAs in our established system. Production of HPV 58 long and short L1 proteins was significantly dependent on the amounts of the L1 mRNAs used and the time length of translation reaction. Compared with the long L1 mRNA, the short L1 mRNA consistently exhibited higher translation efficiency in all the experiments conducted. Supplementation of exogenous amino acids significantly enhanced translation of the long L1 mRNA in this system. Furthermore, a single amino acid leucine can be rate-limiting for translation of the long L1 mRNA in this system. Electron microscopy demonstrated that HPV 58 virus-like particles (VLPs) were assembled from both long and short L1 capsid proteins in the yeast cell-free system.     

The Saccharomyces cerevisiae poly (A) binding protein (Pab1): Master regulator of mRNA metabolism and cell physiology

Pab1, the major poly (A) binding protein of the yeast Saccharomyces cerevisiae, is involved in many intracellular functions associated with mRNA metabolism, such as mRNA nuclear export, deadenylation, translation initiation and termination. Pab1 consists of four RNA recognition motifs (RRM), a proline-rich domain (P) and a carboxy-terminal (C) domain. Due to its modular structure, Pab1 can simultaneously interact with poly (A) tails and different proteins that regulate mRNA turnover and translation. Furthermore, Pab1 also influences cell physiology under stressful conditions by affecting the formation of quinary assemblies and stress granules, as well as by stabilizing specific mRNAs to allow translation re-initiation after stress. The main goal of this review is to correlate the structural complexity of this protein with the multiplicity of its functions.     

Bax induces activation of the unfolded protein response by inducing HAC1 mRNA splicing in Saccharomyces cerevisiae

Bax, a multidomain pro‐apoptotic Bcl‐2 protein, localizes to the endoplasmic reticulum (ER), where it regulates ER stress‐induced apoptosis. Adaptation to ER stress depends on the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). This study examined the death‐inducing activity of Bax and its ability to induce UPR signalling pathways in yeast. We observed that inhibition of global translation in yeast cells expressing Bax correlated with Bax‐induced cell death. Using a lacZ reporter containing several UPR cis‐activating regulatory elements, we also found that Bax directly activated the UPR. Furthermore, this correlated with the splicing of HAC1 mRNA, a gene involved in UPR activation. Bax induced expression of representative UPR target genes such as KAR2, DER1 and GCN4. Finally, we found that Ire1p function is critical for Bax‐induced cell death. Copyright © 2012 John Wiley & Sons, Ltd.     

Influence of sulfur fertilization on S-containing, phenolic, and carbohydrate metabolites in rosy garlic (Allium roseum L.): a wild edible species in North Africa

Allium roseum L., a North African endemic species, is a rich source of many important nutrients and bioactive compounds responsible for many promising beneficial health physiological effects. The influence of sulfur fertilization (S fertilization) on the flavor, total polyphenols, and carbohydrates content in A. roseum was studied, using three sulfur concentrations (0.01, 1.50 and 4.50 mmol L^?1) under controlled conditions. S fertilization showed a significant increase in the allicin concentration of A. roseum bulbs with an average of 0.859–2.285 g kg^?1 FW for bulbs grown at 0.01 and 1.50 mmol L^?1 SO₄ ^2?, respectively. The same trend was observed for total polyphenol content. On the contrary, the highest level of S decreased the content of reduced carbohydrates. These results provide evidence that the concentrations of allicin and polyphenols in A. roseum are increased by S fertilization, potentially amplifying its beneficial impacts on health.