Vacuolar and extracellular maturation of Saccharomyces cerevisiae proteinase A

The vacuolar aspartyl protease proteinase A (PrA) of Saccharomyces cerevisiae is encoded as a preproenzyme by the PEP4 gene and transported to the vacuole via the secretory route. Upon arrival of the proenzyme proPrA to the vacuole, active mature 42 kDa PrA is generated by specific proteolysis involving the vacuolar endoprotease proteinase B (PrB). Vacuolar activation of proPrA can also take place in mutants lacking PrB activity (prb1). Here an active 43 kDa species termed pseudoPrA is formed, probably by an autocatalytic process. When the PEP4 gene is overexpressed in wild-type cells, mature PrA can be found in the growth medium. We have found that prb1 strains overexpressing PEP4 can form pseudoPrA extracellularly. N-terminal amino acid sequence determination of extracellular, as well as vacuolar pseudoPrA showed that it contains nine amino acids of the propeptide, indicating a cleavage between Phe67 and Ser68 of the preproenzyme. This cleavage site is in accordance with the known substrate preference for PrA, supporting the notion that pseudoPrA is formed by autoactivation. When a multicopy PEP4 transformant of a prb1 mutant was grown in the presence of the aspartyl protease inhibitor pepstatin A, a significant level of proPrA was found in the growth medium. Our analyses show that overexpression of PEP4 leads to the secretion of proPrA to the growth medium where the zymogen is converted to pseudoPrA or mature PrA in a manner similar to the vacuolar processing reactions. Amino acid sequencing of secreted proPrA confirmed the predicted cleavage by signal peptidase between Ala22 and Lys23 of the preproenzyme.     

The yal017 gene on the left arm of chromosome I of Saccharomyces cerevisiae encodes a putative serine/threonine protein kinase

The DNA sequence of a region between the LTE1 and CYS3 genes on the left arm of chromosome I from Saccharomyces cerevisiae contains an open reading frame (ORF), YAL017, corresponding to the 5·0 kb FUN31 (F unction U nknown N ow) transcribed region. The predicted protein from this ORF contains 1358 amino acid residues with a molecular weight of 152 531, and an identifiable serine/threonine protein kinase catalytic domain. When compared with other yeast protein kinases, the Ya1017p kinase most resembles the SNF1 serine/threonine protein kinase which is involved in regulating sucrose fermentation genes. The Ya1017p kinase shows highest amino acid identities with two mammalian carcinoma-related serine/threonine protein kinases; PIM-1, which shows induced expression in T-cell lymphomas; and p78A1, whose expression is lost in human pancreatic carcinomas. Gene disruption of YAL017 indicates that it is non-essential for growth on glucose.     

Studies of yeast Kluyveromyces lactis mutations conferring super-secretion of recombinant proteins

We have isolated mutants responsible for a super-secretion phenotype in Kluyveromyces lactis using the gene coding for a Bacillus amyloliquefaciens alpha-amylase as a marker for secretion. These mutations defined two groups, dominant and recessive. The recessive mutant strain, which secreted the heterologous protein in five-fold excess compared to the wild-type strain, was used for the cloning of genes, restraining the super-secreting phenotype. In screening for genes affecting super-secreting phenotype, we found that multiple copies of 10 different independently isolated DNA sequences suppressed the super-secreting phenotype. The first among the genes characterized, named KlSEL1 ('secretion lowering') showed homology to Saccharomyces cerevisiae ORF YML013w. The KlSEL1 gene is predicted to encode a polypeptide of 620 amino acid residues containing a putative transmembrane domain and UBX domain, characteristic for the ubiquitin-regulatory proteins. We demonstrated that the disruption of the SEL1 orthologues in K. lactis and S. cerevisiae conferred the super-secreting phenotype. SEL1 isolated from S. cerevisiae suppressed the super-secretion phenotype in K. lactis klsel1 strain, likewise homologous KlSEL1. No other phenotypic features for strains lacking the SEL1 gene were noticed except for the S. cerevisiae mutant growth being notably slower than in a wt strain. No growth changes were observed in the K. lactis klsel1 mutant. The set of genes (suppressors of over-secreting phenotype) could be attractive for further analysis of gene functions, super-secreting mechanisms and construction of new strains. This collection could be useful for the expedient construction of reduced yeast genomes, optimized for heterologous protein secretion.     

Sequencing of a 4.3 kbp region of chromosome 2 of Candida albicans reveals the presence of homologues of SHE9 from Saccharomyces cerevisiae and of bacterial phosphatidylinositol-phospholipase C

The nucleotide sequence of a 4.3 kb fragment downstream of the LIG4 gene of Candida albicans has been determined. This fragment contains two entire ORFs (ORF1 and ORF2) and a truncated one (ORF3). ORF1 (1029 bp; EMBL databank, Accession No. AJ277539) encodes a putative protein of 343 amino acids with a high degree of similarity to phosphatidylinositol-specific phospholipases C (PI-PLC) of bacterial origin and, to a lesser degree, to similar proteins from trypanosome, fly and human. Isolated ORF1 confers PI-PLC activity to Escherichia coli transformants. ORF2 (1572 bp; EMBL databank, Accession No. AJ277538) predicts a protein of 524 amino acids with high similarity along most of the entire length to Ydr393w from Saccharomyces cerevisiae. This protein carries a domain with significant similarity to several cytoskeleton proteins of different origins. YDR393w (SHE9) is an orphan gene whose overexpression compromises cell growth. ORF3 appears to encode the homologue of the well-conserved proteasomal 26S regulatory subunit.     

Protease (PrA and PrB) and prolyl and arginyl aminopeptidase activities from Debaryomyces hansenii as a function of growth phase and nutrient sources

The effects of nutrient sources and growth phase of Debaryomyces hansenii on the protease (PrA and PrB) and aminopeptidase (prolyl-[PAP] and arginyl-[AAP] aminopeptidases) activities were investigated. These activities were also monitored during growth on a whole sarcoplasmic muscle protein extract (WSPE) and on an equivalent medium but free of compounds under 10 kDa (SPE>10 kDa). The levels of specific protease and aminopeptidase activities were higher when cells were grown in urea and dipeptides than when grown in either ammonium or free amino acids as nitrogen sources. The level of each aminopeptidase (PAP or AAP) activity was preferentially induced by its own substrate (ProLeu or LysAla), suggesting a role in the utilization of exogenous peptides. Higher specific activities for all proteolytic enzymes were detected when using acetate as carbon source. The time course experiments carried out on urea or sarcoplasmic protein-containing media revealed an increase in all activities during transition and advanced stages of stationary phase of growth. In muscle protein extracts, the absence of low molecular mass nutrients (SPE>10 kDa) initially induced the production of PrA, PrB, and AAP activities, possibly involved in the breakdown of muscle oligopeptides.     

Isolation and characterization of a novel yeast gene,ATH1, that is required for vacuolar acid trehalase activity

We have isolated a plasmid containing a gene, ATH1, that results in eight- to ten-fold higher acid trehalase activity in yeast cells when present in high copy. The screening procedure was based on overproduction-induced mislocalization of acid trehalase activity; overproduction of vacuolar enzymes that transit through the secretory pathway leads to secretion to the cell surface. A DNA fragment that confers cell surface expression of acid trehalase activity was cloned and sequenced. The deduced amino acid sequence displayed no homology to known proteins, indicating that we have identified a novel gene. A deletion in the genomic copy of the ATH1 gene eliminates vacuolar acid trehalase activity. These results suggest that ATH1 may be the structural gene encoding vacuolar acid trehalase or that the gene product may be an essential regulatory component involved in control of trehalase activity. The sequence has been deposited in the GenBank data library under Accession Number X84156 S. cerevisiae ATH1 gene.     

Expanding the genetic code of Saccharomyces cerevisiae with methionine analogues

We replaced the single N‐terminal methionine in heterologously expressed human Cu/Zn superoxide dismutase with the non‐canonical methionine analogues homopropargylglycine and norleucine in the yeast Saccharomyces cerevisiae. Our non‐canonical amino acid incorporation protocol involves a two‐step procedure. In the first step, the methionine auxotrophic yeast cells are accumulated in synthetic medium containing methionine while the target protein production is shut off. After a short methionine depletion phase, the cells are transferred to inducing medium that contains the methionine analogue instead of methionine and target protein expression is switched on. The initially low level incorporation of ～12% could be elevated to 40% by increasing the non‐canonical amino acid concentration in the medium by 10‐fold. With this approach we were able to produce up to 5 mg substituted protein per litre of yeast culture. Copyright © 2008 John Wiley & Sons, Ltd.     

Saccharomyces cerevisiae YCRO17c/CWH43 encodes a putative sensor/transporter protein upstream of the BCK2 branch of the PKC1-dependent cell wall integrity pathway.

The Saccharomyces cerevisiae cwh43-2 mutant, originally isolated for its Calcofluor white hypersensitivity, displays several cell wall defects similar to mutants in the PKC1-MPK1 pathway, including a growth defect and increased release of beta-1,6-glucan and beta-glucosylated proteins into the growth medium at increased temperatures. The cloning of CWH43 showed that it corresponds to YCR017c and encodes a protein with 14-16 transmembrane segments containing several putative phosphorylation and glycosylation sites. The N-terminal part of the amino acid sequence of Cwh43p shows 40% similarity with the mammalian FRAG1, a membrane protein that activates the fibroblast growth factor receptor of rat osteosarcoma (FGFR2-ROS) and with protein sequences of four uncharacterized ORFs from Caenorhabditis elegans and one from Drosophila melanogaster. The C-terminus of Cwh43p shows low similarities with a xylose permease of Bacillus megaterium and with putative sugar transporter from D. melanogaster, and has 52% similarity with a protein sequence from a Schizosaccharomyces pombe cDNA. A Cwh43-GFP fusion protein suggested a plasma membrane localization, although localization to the internal structure of the cells could not be excluded, and it concentrates to the bud tip of small budded cells and to the neck of dividing cells. Deletion of CWH43 resulted in cell wall defects less pronounced than those of the cwh43-2 mutant. This allele-specific phenotype appears to be due to a G-R substitution at position 57 in a highly conserved region of the protein. Genetic analysis places CWH43 upstream of the BCK2 branch of the PKC1 signalling pathway, since cwh43 mutations were synthetic lethal with pkc1 deletion, whereas the cwh43 defects could be rescued by overexpression of BCK2 and not by high-copy-number expression of genes encoding downstream proteins of the PKC1 pathway However, unlike BCK2, whose disruption in a cln3 mutant resulted in growth arrest in G(1), no growth defect was observed in a double cwh43 cln3 mutants. Taken together, it is proposed that CWH43 encodes a protein with putative sensor and transporter domains acting in parallel to the main PKC1-dependent cell wall integrity pathway, and that this gene has evolved into two distinct genes in higher eukaryotes.     

Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants

We have screened the EUROFAN (European Functional Analysis Network) deletion strain collection for yeast mutants defective in secretory/vacuolar pathways and/or associated biochemical modifications. We used systematic Western immunoblotting to analyse the electrophoretic pattern of several markers of the secretory/vacuolar pathways, the soluble alpha-factor, the periplasmic glycoprotein invertase, the plasma membrane GPI-anchored protein Gas1p, and two vacuolar proteins, the soluble carboxypeptidase Y and the membrane-bound alkaline phosphatase, which are targeted to the vacuole by different pathways. We also used colony immunoblotting to monitor the secretion of carboxypeptidase Y into the medium, to identify disruptants impaired in vacuolar targeting. We identified 25 mutants among the 631 deletion strains. Nine of these mutants were disrupted in genes identified in recent years on the basis of their involvement in trafficking (VPS53, VAC7, VAM6, APM3, SYS1), or glycosylation (ALG12, ALG9, OST4, ROT2). Three of these genes were identified on the basis of trafficking defects by ourselves and others within the EUROFAN project (TLG2, RCY1, MON2). The deletion of ERV29, which encodes a COPII vesicle protein, impaired carboxypeptidase Y trafficking from the endoplasmic reticulum to the Golgi apparatus. We also identified eight unknown ORFs, the deletion of which reduced Golgi glycosylation or impaired the Golgi to vacuole trafficking of carboxypeptidase Y. YJR044c, which we identified as a new VPS gene, encodes a protein with numerous homologues of unknown function in sequence databases.     

Isolation and characterization of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Torulaspora delbrueckii

A DNA fragment containing the URA3 gene from Torulaspora delbrueckii was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an ORF of 795 bp, encoding a 264 amino acid protein, which shares a high similarity to the Saccharomycetaceae Ura3 proteins. Furthermore, the cloned ORF fully complemented the ura3 mutation of S. cerevisiae, confirming that it encodes for the TdUra3 protein. The GeneBank Accession No. for TdURA3 is AF518402.     

Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

The tannase‐encoding Arxula adeninivorans gene ATAN1 was isolated from genomic DNA by PCR, using as primers oligonucleotide sequences derived from peptides obtained after tryptic digestion of the purified tannase protein. The gene harbours an ORF of 1764 bp, encoding a 587‐amino acid protein, preceded by an N‐terminal secretion sequence comprising 28 residues. The deduced amino acid sequence was similar to those of tannases from Aspergillus oryzae (50% identity), A. niger (48%) and putative tannases from A. fumigatus (52%) and A. nidulans (50%). The sequence contains the consensus pentapeptide motif (–Gly–X–Ser–X–Gly–) which forms part of the catalytic centre of serine hydrolases. Expression of ATAN1 is regulated by the carbon source. Supplementation with tannic acid or gallic acid leads to induction of ATAN1, and accumulation of the native tannase enzyme in the medium. The enzymes recovered from both wild‐type and recombinant strains were essentially indistinguishable. A molecular mass of ～320 kDa was determined, indicating that the native, glycosylated tannase consists of four identical subunits. The enzyme has a temperature optimum at 35–40 °C and a pH optimum at ～6.0. The enzyme is able to remove gallic acid from both condensed and hydrolysable tannins. The wild‐type strain LS3 secreted amounts of tannase equivalent to 100 U/l under inducing conditions, while the transformant strain, which overexpresses the ATAN1 gene from the strong, constitutively active A. adeninivorans TEF1 promoter, produced levels of up to 400 U/l when grown in glucose medium in shake flasks. Copyright © 2009 John Wiley & Sons, Ltd.     

Isolation and sequence analysis of the gene encoding triose phosphate isomerase from Zygosaccharomyces bailii.

The ZbTPI1 gene encoding triose phosphate isomerase (TIM) was cloned from a Zygosaccharomyces bailii genomic library by complementation of the Saccharomyces cerevisiae tpi1 mutant strain. The nucleotide sequence of a 1.5 kb fragment showed an open reading frame (ORF) of 746 bp, encoding a protein of 248 amino acid residues. The deduced amino acid sequence shares a high degree of homology with TIMs from other yeast species, including some highly conserved regions. The analysis of the promoter sequence of the ZbTPI1 revealed the presence of putative motifs known to have regulatory functions in S. cerevisiae. The GenBank Accession No. of ZbTPI1 is AF325852.     

Disruption and functional analysis of six ORFs on chromosome XV: YOL117w, YOL115w ( TRF4), YOL114c, YOL112w ( MSB4), YOL111c and YOL072w.

We have carried out the systematic disruption of six ORFs on chromosome XV, of Saccharomyces cerevisiae using the long flanking homology technique to replace each with the KanMX cassette; we have also constructed plasmids containing replacement cassettes and cognate clones for each ORF. Disruption of three of the ORFs-YOL117w, YOL114c, and YOL112w (also known as MSB4)-does not result in any noteworthy phenotype with respect to temperature or nutritional requirements, but yol112w mutants with an additional disruption of YNL293w, which encodes a protein similar to Yol112w, exhibit a slow growth phenotype. The protein specified by YOL114c shares similarity with the human DS-1 protein. Disruption of YOL115w confers slow growth, cold sensitivity and poor sporulation; this ORF has been described elsewhere as TRF4, which encodes a topoisomerase I-related protein. Cells with disruptions of YOL111c, whose product is weakly similar to the human ubiquitin-like protein GdX, are slightly impaired in mating. Mutants disrupted for YOL072w, the predicted product of which is unrelated to any protein of known function, grow slowly, are cold-sensitive and sporulate with reduced efficiency.     

Molecular cloning and expression of uricase gene from Arthrobacter globiformis in Escherichia coli and characterization of the gene product

Arthrobacter globiformis FERM BP-360 produces uricase (urate oxidase; EC 1.7.3.3) intracellularly. A genomic library of the bacterium, prepared in the plasmid vector pUC118, was screened with probes based on the amino acid sequence of the purified uricase. We found that a chimeric plasmid in the library, designated pUOD1, carries a 2.0-kb DNA insert from the Arthrobacter DNA that hybridizes with the probe. The DNA insert contains an ORF consisting of 302 amino acids with a calculated molecular mass of 33,858. The protein translated from the ORF displays the highest identity (67%) to uricase from a bacterium, Cellulomonas flavigena. X-ray fluorescence analysis showed that the Arthrobacter uricase contains copper ion. However, we found that the catalytic activity of uricase is inhibited by the excessive addition of copper ion. Although the production of A. globiformis uricase is induced by the addition of uric acid to the culture medium, Escherichia coli harboring pUOD1 produced 20-fold higher uricase than the original Arthrobacter strain, even without an inducer.     

XIV. Yeast sequencing reports. A 43·5 kb segment of yeast chromosome XIV,which contains MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1, predicts an adenosine deaminase gene and 14 new open reading frames

A 43,481 bp fragment from the left arm of chromosome XIV of Saccharomyces cerevisiae was sequenced. A gene for tRNA^phe and 23 non-overlapping open reading frames (ORFs) were identified, seven of which correspond to known yeast genes: MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1. One ORF may correspond to the yet unindentified yeast adenosine deaminase gene. Among the 15 other ORFs, four exhibit known signatures, which include a protein tyrosine phosphatase, a cytoskeleton-associated protein and two ATP-binding proteins, four have similarities with putative proteins of yeast or proteins from other organisms and seven exibit no significant similarity with amino acid sequences described in data banks. One ORF is identical to yeast expressed sequence tags (EST) and therefore corresponds to an expressed gene. Six ORFs present similarities to human dbESTs, thus identifying motifs conserved during evolution. Nine ORFs are putative transmembrane proteins. In addition, one overlapping and three antisense ORFs, which are not likely to be functional, were detected. The sequence has been deposited in the EMBL data bank under Accession Number Z46843.     

Cloning and characterization of the Hansenula polymorpha PEP4 gene encoding proteinase A

The Hansenula polymorpha PEP4 gene encoding proteinase A was cloned by Southern blot hybridization using the Saccharomyces cerevisiae PEP4 gene as probe and characterized by gene disruption and overexpression. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1239 nucleotides corresponding to a polypeptide of 413 amino acids, sharing about 67.2% sequence similarity with that of S. cerevisiae proteinase A. That the cloned H. polymorpha PEP4 gene encodes proteinase A was supported by a gene disruption experiment, which showed that the H. polymorpha pep4 mutant strain showed significantly reduced level of carboxypeptidase Y activity when assayed with an artificial substrate. When the PEP4 gene is overproduced in pep4 mutant strain, mature proteinase A could be found in the growth medium. N-terminal amino acid sequencing of extracellular proteinase A revealed the presence of a putative propeptide of 55 amino acids ending with a dibasic peptide (Lys-Arg), probably processed by Kex2p-like endopeptidase of H. polymorpha. The nucleotide sequence of the H. polymorpha PEP4 gene has been submitted to GenBank under Accession No. U67173.     

大豆rbcL基因克隆、序列分析及原核表达

利用叶绿体基因组保守性的特征,根据菜豆、豌豆、烟草的rbcL基因序列设计引物,从大豆叶绿体DNA中克隆rbcL基因,全长序列为1488bp,包括1449bp的开放阅读框,编码482个氨基酸。相似性比较显示,此序列与其它10个物种rbcL基因核苷酸的同源性为85．37％～95．31％,氨基酸的同源性为90．87％-96．47％。将该基因与表达载体pET-30a（＋）连接,转化大肠杆菌Rosseta感受态细胞,PCR和酶切鉴定筛选阳性克隆,阳性菌液IPTG诱导后经10％SDS—PAGE分析,结果显示,诱导表达出分子量约为60kD的特异融合蛋白。     

Disruption of six unknown open reading frames from Saccharomyces cerevisiae reveals two genes involved in vacuolar morphogenesis and one gene required for sporulation

In this report we describe the construction and basic phenotypic analysis of deletion mutants in six open reading frames (ORFs) of unknown function from the yeast Saccharomyces cerevisiae. Using the dominant kanMX marker and polymerase chain reaction (PCR) methods, deletion cassettes were constructed for five ORFs (YNL099c, YNL100w, YNL101w, YNL106c and YNL242w) located on chromosome XIV and one ORF (YOR109w) located on chromosome XV. The recovery of viable haploid deletant strains among the meiotic products of heterozygous deletants for each ORF demonstrated that none of the analysed ORFs was essential. With the exception of YNL242w, no alterations in growth characteristics or mating and sporulation efficiencies associated with deletion of the ORFs were observed. Homozygous diploid ynl242w delta cells obtained in three different genetic backgrounds were unable to sporulate, indicating that the product of this ORF is required for sporulation. Complementation of the sporulation defect by the cognate gene clone confirmed this observation. YNL106c and YOR109w are very similar and show strong sequence homology with a mammalian phosphatidylinositol-phosphate 5-phosphatase, synaptojanin, known to be involved in synaptic vesicle cycling. Strains bearing single and double deletions of YNL106c and YOR109w were seen to display abnormal vacuolar morphologies of varying degrees. Complementation tests indicated that YNL106c and YOR109w are redundant genes.