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.     

Isolation and characterization of the carbon catabolite‐derepressing protein kinase Snf1 from the stress tolerant yeast Torulaspora delbrueckii

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae snf1Δ mutant strain. DNA sequence analysis revealed that the fragment contained a complete open reading frame (ORF), which shares a high similarity with the S. cerevisiae energy sensor protein kinase Snf1. The cloned TdSNF1 gene was able to restore growth of the S. cerevisiae snf1Δ mutant strain on media containing nonfermentable carbon sources. Furthermore, cells of the Tdsnf1Δ mutant were unable to proliferate under nonfermenting conditions. Finally, protein domain analysis showed that TdSnf1p contains a typical catalytic protein kinase domain (positions 41–293), which is also present in other Snf1p homologues. Within this region we identified a protein kinase ATP‐binding region (positions 48–71) and a consensus Ser/Thr protein kinase active site (positions 160–172). The GenBank Accession No. for the sequenced DNA fragment is HM131845. Copyright © 2010 John Wiley & Sons, Ltd.     

Secalin蛋白检测及其与面团黏性关系的研究

用SDS—PAGE检测了70个小麦品种是否带有黑麦Secalin蛋白，并测试了它们的面团黏性，用PCR检测了部分品种是否含有1RS染色体片断，结果表明：70％的品种为1B／1R易位系；有54．3％的品种面团发黏；带有黑麦Secalin蛋白的品种约有72．5％面团发黏，而不带有黑麦Secalin蛋白的品种只有30％面团发黏；影响面团黏性的主要因素为来源于黑麦的Secalin蛋白；PCR扩增结果表明，在我国存在不带有Sec-1基因的1B／1R小片断易位系。     

Schizosaccharomyces pombe Pmf1p is structurally and functionally related to Mmf1p of Saccharomyces cerevisiae

A novel family of small proteins, termed p14.5 or YERO57c/YJGFc, has been identified. Independent studies indicate that p14.5 family members are multifunctional proteins involved in several pathways, e.g. regulation of translation or activation of the protease mu-calpain. We have previously shown that Mmf1p, a p14.5 of the budding yeast Saccharomyces cerevisiae, is localized in the mitochondria and influences mitochondrial DNA stability. In addition, we have demonstrated that Mmf1p is functionally related to p14.5 of mammalian cells. To explore further the evolutionary conservation of the mitochondrial function(s) of the p14.5s we have extended our study to the fission yeast, Schizosaccharomyces pombe. In this organism two p14.5 homologous proteins are present: Pmf1p (pombe mitochondrial factor 1) and Hpm1p (homologous Pmf1p factor 1). We have generated a specific Pmf1p antibody, which recognizes a single band of approximately 15 kDa in total cellular extracts. Cellular fractionation experiments indicate that Pmf1p localizes in the mitochondria as well as in the cytoplasm. We also show that Pmf1p shares several properties of S. cerevisiae Mmf1p. Indeed, Pmf1p restores the wild-type phenotype when expressed in delta mmf1 S. cerevisiae cells. Deletion of the leader sequence of Pmf1p abrogates its ability to localize in mitochondria and to functionally replace Mmf1p. Thus, these data together with our previous study show that the mitochondrial function(s) of the p14.5 family members are highly conserved in eukaryotic cells.     

Isolation and sequence of the MIG1 homologue from the yeast Candida utilis

The Mig1p repressor from the food yeast Candida utilis has been isolated using a homologous PCR hybridization probe. This probe was amplified with two sets of degenerate primers designed on the basis of highly conserved motifs in the DNA-binding region (zinc-finger domain) from yeast Mig1p and fungi CreA repressors. The cloned gene was sequenced and found to encode a polypeptide of 345 amino acids which shows significant identity with other yeast and fungus repressors in the DNA-binding domain and also with the yeast Mig1 proteins in the C-terminal region (effector domain). The MIG1 repressor gene from C. utilis was able to complement functionally the mig1 mutation of S. cerevisiae. The sequence presented here has been deposited in the EMBL data library under Accession No. AJ277830.     

Structure-function analysis of Knr4/Smi1, a newly member of intrinsically disordered proteins family, indispensable in the absence of a functional PKC1-SLT2 pathway in Saccharomyces cerevisiae

The coordination between cell wall synthesis and cell growth in the yeast Saccharomyces cerevisiae implicates the PKC1-dependent MAP kinase pathway. KNR4, encoding a 505 amino acid long protein, participates in this coordination, since it displays synthetic lethality with all the members of the PKC1 pathway and shows physical interaction with Slt2/Mpk1. The recent finding that KNR4 interacts genetically or physically with more than 100 partners implicated in different cellular processes raised the question of how these interactions may occur and their physiological significance. This called for an in-depth structure-function analysis of the Knr4 protein, which is reported in the present paper. Computational analysis supported by biochemical and biophysical data characterize Knr4 as a newly identified member of the growing family of intrinsically disordered proteins. Despite disordered regions that are located at the N- and C-termini and are probably responsible for fine regulatory function; this protein contains a structured central core (amino acid residues 80-340) that is able to restore wild-type phenotypes of knr4Delta mutant in stress conditions. However, this fragment was unable to complement synthetic lethality between knr4 mutations and deletions of genes encoding protein kinases of the PKC1-dependent pathway. For these crucial events to occur, the presence of the N-terminal part of Knr4 protein is indispensable. Moreover, we demonstrate that this protein is essential for cell viability in the absence of a functional Pkc1-Slt2 pathway, since the lethality caused by KNR4 deletion in such a genetic background could not be compensated by overexpression of any gene from yeast genomic libraries.     

Identification and functional analysis of the Saccharomyces cerevisiae nicotinamidase gene, PNC1

Nicotinamidase (NAMase) from the budding yeast, Saccharomyces cerevisiae, was purified by Ni(2+) affinity chromatography and gel filtration. N-terminal microsequencing revealed sequence identity with a hypothetical polypeptide encoded by the yeast YGL037C open reading frame sharing 30% sequence identity with Escherichia coli pyrazinamidase/nicotinamidase. A yeast strain in which the NAMase gene, hereafter named PNC1, was deleted shows a decreased intracellular NAD(+) concentration, consistent with the loss of NAMase activity in the null mutant. In wild-type strains, NAMase activity is stimulated during the stationary phase of growth, by various hyperosmotic shocks or by ethanol treatment. Using a P(PNC1)::lacZ gene fusion, we have shown that this stimulation of NAMase activity results from increased levels of the protein and requires stress response elements in the 5'non-coding region of PNC1. These results suggest that NAMase helps yeast cells to adapt to various stress conditions and nutrient depletion, most likely via the activation of NAD-dependent biological processes.     

Molecular characterization of Candida boidinii MIG1 and its role in the regulation of methanol-inducible gene expression

Methanol-inducible gene promoters in methanol-utilizing yeasts are used in high-level heterologous gene expression systems. Generally, expression of methanol-inducible genes is completely repressed by the presence of glucose. In this study we identified the MIG1 gene in Candida boidinii, which encodes a homologue of the glucose repressor Mig1p of Saccharomyces cerevisiae. Disruption of the CbMIG1 gene had no growth effect on various carbon sources. Activation of the methanol-inducible AOD1 gene, which encodes alcohol oxidase, was increased in the early stage of methanol induction when cells of the CbMIG1-disrupted strain were transferred from glucose medium to methanol medium. Furthermore, CbMig1p tagged with yellow fluorescent protein was primarily localized in the nucleus of glucose-grown cells, but was diffuse in the cytosol of methanol-grown cells. This cytosolic diffusion in methanol-grown cells occurred in a CbMsn5p-dependent manner. These results suggest that CbMig1p is involved in negative regulation of methanol-inducible gene expression in C. boidinii.     

Functional characterization of human and fungal MAP kinases in Saccharomyces cerevisiae

A functional comparison of three human SAPKs with fungal Hog1p was undertaken, using Saccharomyces cerevisiae as a heterologous expression system. We characterized the role of mammalian MAP kinases in sensitivity to both osmotic and oxidative stress of a S. cerevisiae hog1 mutant. Western blot analyses indicated that S. cerevisiae can only phosphorylate mammalian MAP kinases in response to osmotic stress but not to oxidative stress, while morphogenetic defects characteristic of hog1 mutants under hyperosmotic stress are only suppressed by fungal and not mammalian Hog1p. Our data demonstrate the functional conservation of MAPKs although they also evidence differential aspects among the three human SAPKs and the fungal MAPKs.     

AFLP analysis of type strains and laboratory and industrial strains of Saccharomyces sensu stricto and its application to phenetic clustering

Using nine primer pairs, amplified fragment length polymorphism (AFLP) analysis was conducted to characterize industrial, laboratory and type strains of Saccharomyces sensu stricto. S. cerevisiae, S. bayanus, S. carlsbergensis and S. paradoxus had species-specific AFLP profiles, with some variations among the strains. Nineteen wine, ale, bakery, whisky and laboratory strains of S. cerevisiae were differentiated by two primer pairs, while out of 19 strains of sake yeast, two groups consisting of two and eight strains were not differentiated using nine primer pairs. A phenogram of 41 strains of S. cerevisiae, two strains of S. bayanus, the type strain of S. pastorianus, three strains of S. carlsbergensis, one hybrid strain of S. cerevisiae and S. bayanus and the type strain of S. paradoxus was obtained by the unweighted pair group method, using arithmetic averages (UPGMA) based on the percentage of shared AFLP fragments of each sample pair. This phenogram demonstrated clear separations of S. cerevisiae, S. bayanus, S. carlsbergensis and S. paradoxus. However, S. pastorianus ATCC 12752(T) showed the highest percentages of shared fragments with the strains of S. bayanus, and formed a cluster with them. Except for the type strain of S. pastorianus, the percentages of shared fragments showed a similar tendency with reported data of DNA relatedness. The cluster of S. cerevisiae separated into three subclusters: one consisting of sake and shochu strains and a whisky strain; another consisting of bakery, wine, ale and whisky strains; and a third consisting of laboratory strains.     

Kluyveromyces lactis SSO1 and SEB1 genes are functional in Saccharomyces cerevisiae and enhance production of secreted proteins when overexpressed

The SEB1/SBH1 and the SSO genes encode components of the protein secretory machinery functioning at the opposite ends, ER translocation and exocytosis, respectively, of the secretory pathway in Saccharomyces cerevisiae. Overexpression of these genes can rescue temperature-sensitive (ts) growth defect of many sec mutants impaired in protein secretion. Furthermore, their overexpression in wild-type yeast enhances production of secreted proteins in S. cerevisiae, which suggests that they may be rate-limiting factors in this process. Here we report isolation of Kluyveromyces lactis homologues of these genes. KlSSO1 and KlSEB1 were isolated as clones capable of rescuing growth of ts sso2-1 and seb1Delta seb2Delta sem1Delta strains, respectively, at the restrictive temperature. The encoded Kluyveromyces proteins are up to 70% identical with the S. cerevisiae homologues at the amino acid level and can functionally replace them. Interestingly, KlSSO1 and KlSEB1 show similar enhancing effect on production of a secreted protein as the SSO and SEB1 genes of S. cerevisiae when overexpressed. In accordance with the high homology level of the secretory pathway proteins in different yeast species, the polyclonal antibodies raised against S. cerevisiae Seb1p, Sso2p and Sec4p can detect homologous proteins in cell lysates of K. lactis and Pichia pastoris, the latter also in Candida utilis. The GenBank Accession Nos are AF307983 (K. lactis SSO1) and AF318314 (K. lactis SEB1).     

X–XI. Yeast mapping reports. The use of random-breakage mapping to locate the genes APN1 and YUH1 in the Saccharomyces genome,and to determine gene order near the left end of chromosome XI

We have used the previously described technique of random-breakage mapping to locate the two yeast genes APN1 and YUH1. The APN1 locus is located ～235 kb from the left telomere of chromosome XI, and shows weak (～53 cM) genetic linkage to ura1. The YUH1 locus is located ～140 kb from the right telomere of chromosome X, and genetically maps 3·6 cM distal to cdc11. In addition, we show by random-breakage mapping that TRP3 is located ～45 kb from the left telomere of chromosome XI, whereas FAS1 is ～110 kb from the same telomere. This supports a gene order on the left distal portion of chromosome XI that agrees with other physical reports but is inverted with respect to Edition 11 of the published genetic map. This report confirms that random-breakage mapping is a rapid and convenient method of locating cloned genes.     

Cloning and genetic characterization of a calcium- and phospholipid-binding protein from Saccharomyces cerevisiae that is homologous to translation elongation factor-1γ

We have isolated a gene (CAM1) from the yeast Saccharomyces cerevisiae that encodes a protein homologous to the translational cofactor elongation factor-1γ (EF-1γ) first identified in the brine shrimp Artemia salina. The predicted Cam1 amino acid sequence consists of 415 residues that share 32% identity with the Artemia protein, increasing to 72% when conservative substitutions are included. The calculated M_r of Cam1p (47 092 Da) is in close agreement with that of EF-1γ (M_r = 49 200 Da), and hydropathy plots of each protein exhibit strikingly similar profiles. Disruption of the CAM1 locus yields four viable meiotic progeny, indicating that under normal growth conditions the Cam1 protein is non-essential. Attempts to elicit a translational phenotype have been unsuccessful. Since EF-1γ participates in the regulation of a GTP-binding protein (EF-1α), double mutants with cam1 disruptions and various mutant alleles of known GTP-binding proteins were constructed and examined. No evidence was found for an interaction of CAM1 with TEF1, TEF2, SEC4, YPT1, RAS1, RAS2, CDC6, ARF1, ARF2 or CIN4. The possibility that Cam1p may play a redundant role in the regulation of protein synthesis or another GTP-dependent process is discussed.