Isolation and Characterization of Pep5, a Gene Essential for Vacuolar Biogenesis in Saccharomyces Cerevisiae

pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37 degrees. The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursors forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein.     

PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors.

The proteinase A structural gene of Saccharomyces cerevisiae was cloned by using an immunological screening procedure that allows detection of yeast cells which are aberrantly secreting vacuolar proteins (J. H. Rothman, C. P. Hunter, L. A. Valls, and T. H. Stevens, Proc. Natl. Acad. Sci. USA, 83:3248-3252, 1986). A second cloned gene was obtained on a multicopy plasmid by complementation of a pep4-3 mutation. The nucleotide sequences of these two genes were determined independently and were found to be identical. The predicted amino acid sequence of the cloned gene suggests that proteinase A is synthesized as a 405-amino-acid precursor which is proteolytically converted to the 329-amino-acid mature enzyme. Proteinase A shows substantial homology to mammalian aspartyl proteases, such as pepsin, renin, and cathepsin D. The similarities may reflect not only analogous functions but also similar processing and intracellular targeting mechanisms for the two proteins. The cloned proteinase A structural gene, even when it is carried on a single-copy plasmid, complements the deficiency in several vacuolar hydrolase activities that is observed in a pep4 mutant. A strain carrying a deletion in the genomic copy of the gene fails to complement a pep4 mutant of the opposite mating type. Genetic linkage data demonstrate that integrated copies of the cloned proteinase A structural gene map to the PEP4 locus. Thus, the PEP4 gene encodes a vacuolar aspartyl protease, proteinase A, that is required for the in vivo processing of a number of vacuolar zymogens.     

Biogenesis of the yeast vacuole (lysosome). Mutation in the active site of the vacuolar serine proteinase yscB abolishes proteolytic maturation of its 73-kDa precursor to the 41.5-kDa pro-enzyme and a newly detected 41-kDa peptide.

The codon of the catalytic serine in the active site of the vacuolar serine proteinase yscB (PrB) was changed to alanine, yielding the mutant gene prb1-Ala519. Following replacement of the wild-type PRB1 allele with prb1-Ala519, only a 73-kDa molecule was detected by immunoprecipitation with PrB-specific antiserum. The size of the mutant molecule corresponds to the unprocessed cytoplasmic precursor (pre-super-pro-PrB), as detected in sec61 mutants, when translocation into the endoplasmic reticulum is blocked. However, the mutant molecule is completely translocated into the secretory pathway, as indicated by protection from proteinase K digestion in spheroplast lysates in the absence of detergent. When N-glycosylation was inhibited in prb1-Ala519 mutant cells by tunicamycin, a smaller molecule of about 71 kDa appeared consistent with single N-glycosylation and signal-sequence cleavage of the translocated mutant PrB molecule in the endoplasmic reticulum. Thus, the active-site mutation prevents the wild-type processing of the N-glycosylated 73-kDa precursor of PrB to the 41.5 kDa pro-PrB in the endoplasmic reticulum. In order to characterize the processing of wild-type super-pro-PrB in more detail, we generated antibodies against the non-enzymatic superpeptide domain of the 73-kDa precursor expressed in Escherichia coli. We find that, in addition to pro-PrB, a distinct protein (superpeptide) with a mobility of about 41 kDa in SDS/PAGE is generated in the endoplasmic reticulum. Pulse-chase experiments indicate rapid degradation of the 41-kDa superpeptide in wild-type cells. Correspondingly, the superpeptide was virtually undetectable by immunoblotting wild-type cell extracts. In contrast, no degradation of radioactively labeled 41-kDa superpeptide was observed within 60 min in mutant strains deficient in the vacuolar proteinase yscA (PrA), in which maturation of vacuolar pro-PrB to active PrB is blocked. Accordingly, superpeptide antigenic material was readily detected by immunoblotting cell extracts and enriched in vacuolar preparations of PrA deficient mutant cells. These results indicate that the superpeptide and pro-PrB travel to the vacuole, where the superpeptide is rapidly degraded upon pro-PrB activation to PrB. Using purified vacuoles, rapid degradation of the superpeptide was reconstituted in vitro by addition of either mature PrA or mature PrB. However, the PrA-triggered in vitro degradation of the superpeptide required PrB activity, as this process was inhibited in the presence of the PrB inhibitor chymostatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

The pep4 gene encoding proteinase A is involved in dimorphism and pathogenesis of Ustilago maydis

Vacuole proteases have important functions in different physiological processes in fungi. Taking this aspect into consideration, and as a continuation of our studies on the analysis of the proteolytic system of Ustilago maydis, a phytopathogenic member of the Basidiomycota, we have analysed the role of the pep4 gene encoding the vacuolar acid proteinase PrA in the pathogenesis and morphogenesis of the fungus. After confirmation of the location of the protease in the vacuole using fluorescent probes, we obtained deletion mutants of the gene in sexually compatible strains of U. maydis (FB1 and FB2), and analysed their phenotypes. It was observed that the yeast to mycelium dimorphic transition induced by a pH change in the medium, or the use of a fatty acid as sole carbon source, was severely reduced in Δpep4 mutants. In addition, the virulence of the mutants in maize seedlings was reduced, as revealed by the lower proportion of plants infected and the reduction in size of the tumours induced by the pathogen, when compared with wild‐type strains. All of these phenotypic alterations were reversed by complementation of the mutant strains with the wild‐type gene. These results provide evidence of the importance of the pep4 gene for the morphogenesis and virulence of U. maydis.     

Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information.

An inactive precursor form of proteinase A (PrA) transits through the early secretory pathway before final vacuolar delivery. We used gene fusions between the gene coding for PrA (PEP4) and the gene coding for the secretory enzyme invertase (SUC2) to identify vacuolar protein-sorting information in the PrA precursor. We found that the 76-amino-acid preprosegment of PrA contains at least two sorting signals: an amino-terminal signal peptide that is cleaved from the protein at the level of the endoplasmic reticulum followed by the prosegment which functions as a vacuolar protein-sorting signal. PrA-invertase hybrid proteins that carried this sequence information were accurately sorted to the yeast vacuole as determined by cell fractionation and immunolocalization studies. Hybrid proteins lacking all or a portion of the PrA prosegment were secreted from the cell. Our gene fusion data together with an analysis of the wild-type PrA protein indicated that N-linked carbohydrate modifications are not required for vacuolar sorting of this protein. Furthermore, results obtained with a set of deletion mutations constructed in the PrA prosegment indicated that this sequence also contributes to proper folding of this polypeptide into a stable transit-competent molecule.     

Analysis of a processing system for proteases using yeast cell surface engineering: conversion of precursor of proteinase A to active proteinase A

The display of a protease, carboxypeptidase Y (CPY) or procarboxypeptidase Y (proCPY), which is the vacuolar protease, on the yeast-cell surface was successfully performed using yeast-cell-surface engineering for the first time. Through that we could confirm the processing of vacuolar proteases containing proteinase A (PrA) and proteinase B (PrB) which are related to the maturation of proCPY, using a novel cell-surface engineering technique. Various protease-knockout strains of Saccharomyces cerevisiae with the CPY-displaying system were constructed to evaluate the operation of the activation process of CPY. The display of CPY (CPY-agg, which is a fusion protein of CPY with C-terminal half of α-agglutinin) on the cell surface was confirmed by immunofluorescence staining. The activity of the CPY-agg was determined after the conversion of proCPY to active CPY by treatment of whole cells with proteinase K. In the proCPY-displaying CPY-knockout strain and PrB-knockout strain, CPY was displayed as an active (mature) form, but in the proCPY-displaying PrA-knockout strain, CPY was present as an inactive form (proCPY). These facts indicate that PrA had been already activated before its transport to the vacuole and that active mature PrA might convert proCPY to CPY before the transport of proCPY to the vacuole. From these results, it was suggested that by using the yeast-cell-surface engineering at the location of the initial step, the autocatalytic activation from proPrA to PrA might occur before the vacuolar branch separates from the main secretory pathway.     

Expression of GAI gene and disruption of PEP4 gene in an industrial brewer's yeast strain

Aims:  Construction of an industrial brewer's yeast strain, which could improve foam stability and reduce calorific values of beer.
Methods and Results:  An industrial brewer's yeast strain (Ts-10) was constructed by integrating glucoamylase encoding gene GAI amplified from Saccharomycopsis fibuligera by PCR into the locus of proteinase A (PrA) gene ( PEP4 ). The resulting recombinant strain identified by PCR could grow on YNB minimal medium plate with starch as sole carbon source. Its highest GAI activity was 91·69 U ml⁻¹, but it had no PrA activity. The real extract was reduced by 21·07% and the main residual maltotriose content was reduced by 14% in wort fermented with the recombinants strain. Its foam retention in beer was higher 39 s and the contents of potential off-flavour compounds, such as diacetyl, pentanedione and acetaldehyde were lowered by 16%, 13% and 14%, respectively, as compared with the industrial brewer's yeast YSF-5.
Conclusions:  An industrial brewer's yeast strain was constructed by introducing GAI gene and disrupting PEP4 gene.
Significance and Impact of the Study:  The recombinant strain (Ts-10) had better foam performance and mouthfeel in addition to low-calories values. It was free of heterologous DNA sequences and drug-resistance genes and could be safely used in beer production.     

Modeling the growth and proteinase A production in continuous cultures of recombinant Saccharomyces cerevisiae

Overexpression of the homologous protein proteinase A (PrA) in Saccharomyces cerevisiae has been achieved by inserting the PrA gene (PEP4) with its own promoter on a 2mu multicopy plasmid. With this system the specific PrA production rate was found to be described well by a linear function of the oxidative glucose metabolism, the reductive glucose metabolism, and the oxidative ethanol metabolism, with a significant lower yield resulting from the reductive glucose metabolism compared with the oxidative glucose metabolism. To describe the experimental data, a simple mathematical model has been set up. The model is based on an assumption of a limited respiratory capacity as suggested by Sonnleitner and K?ppeli but extended to describe production of an extracellular protein. The model predicts correctly the critical dilution rate to be between 0.15 and 0.16 h(-1), the decrease in the biomass yield above the critical dilution rate, and the production of proteinase A at different dilution rates. Both the experimental data and model simulations suggest that the optimum operating conditions for protein production is just at the critical dilution rate. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 447-454, 1997.     

Serine proteinase over-expression in relation to deltamethrin resistance in Culex pipiens pallens

Two serine proteinase genes were isolated from Culex pipiens pallens as significantly up-regulated genes in a deltamethrin-resistant strain through a combination of suppression substractive hybridization and gene expression profiling by macroarrays. These two genes were found to be expressed at least threefold higher in the resistant strain than in the susceptible one. By using rapid amplification of cDNA ends to screen the constructed cDNA library, we cloned these two sequences. There were 909 bp with an open reading frame of 786 bp in the sequence of trypsin cDNA (GenBank/NCBI AF468495), the deduced protein had 261 amino acids, which was most similar to the trypsin gene of Anopheles gambiae. There were 992 bp with an open reading frame of 816 bp in the chymotrypsin cDNA (GenBank/NCBI AY034060), and its deduced amino acid sequence had 271 amino acids, which was most similar to the chymotrypsin-like protein from Aedes aegypti. The two genes were stably expressed in mosquito C6/36 cells, and the expected 29 and 30 kDa bands were shown with Western blot, respectively. In these cells, after deltamethrin treatment, they had protective effects on the viability. The results indicate that trypsin and chymotrypsin were more highly expressed in the deltamethrin-resistant strain, and was related to insecticide resistance in mosquitoes, Cx. pipiens pallens.     

Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases.

vps35 mutants of Saccharomyces cerevisiae exhibit severe defects in the localization of carboxypeptidase Y, a soluble vacuolar hydrolase. We have cloned the wild-type VPS35 gene by complementation of the vacuolar protein sorting defect exhibited by the vps35-17 mutant. Sequence analysis revealed an open reading frame predicted to encode a protein of 937 amino acids that lacks any obvious hydrophobic domains. Subcellular fractionation studies indicated that 80% of Vps35p peripherally associates with a membranous particulate cell fraction. The association of Vps35p with this fraction appears to be saturable; when overproduced, the vast majority of Vps35p remains in a soluble fraction. Disruption of the VPS35 gene demonstrated that it is not essential for yeast cell growth. However, the null allele of VPS35 results in a differential defect in the sorting of vacuolar carboxypeptidase Y (CPY), proteinase A (PrA), proteinase B (PrB), and alkaline phosphatase (ALP). proCPY was quantitatively missorted and secreted by delta vps35 cells, whereas almost all of proPrA, proPrB, and proALP were retained within the cell and converted to their mature forms, indicating delivery to the vacuole. Based on these observations, we propose that alternative pathways exist for the sorting and/or delivery of proteins to the vacuole.     

马铃薯腐烂茎线虫L 型半胱氨酸蛋白酶新基因(Dd-cpl-1) 的克隆与序列分析

L型半胱氨酸蛋白酶基因 (Cathepsin L-like cysteine proteinase gene) 为与植物寄生线虫寄生能力相关的多功能基因。运用RT-PCR和RACE的方法从马铃薯腐烂茎线虫Ditylenchus destructor中克隆出1个L型半胱氨酸蛋白酶新基因Dd-cpl-1 (GenBank登录号为GQ180107)。该基因Dd-cpl-1 cDNA全长序列含有1个1 131 bp的开放性阅读框 (ORF),编码376个氨基酸残基,其5′末端及3′末端分别含有29 bp和159 bp的非编码区 (UTR)。Dd-cpl-1内含子外显子结构分析结果表明,其基因组序列包含7个内含子,且各内含子两端剪接位点序列遵守GT/AG规则。Dd-cpl-1基因推定的蛋白Dd-CPL-1与松材线虫L型半胱氨酸蛋白酶高度同源,一致性达到77％。以不同物种中L 型半胱氨酸蛋白酶氨基酸序列进行比对分析,推测推定的蛋白 Dd-CPL-1含有L型半胱氨酸蛋白酶基因家族高度保守的催化三联体 (Cys183,His322 和Asn343) 以及ERFNIN基系和GNFD基系。半胱氨酸蛋白酶系统发育分析表明,Dd-cpl-1 属于由L型半胱氨酸蛋白酶组成的进化分支。Dd-cpl-1的这些序列特征进一步表明其为L型半胱氨酸蛋白酶基因。这是首次在马铃薯腐烂茎线虫中克隆到的L型半胱氨酸蛋白酶,为今后在蛋白水平对其进行进一步的功能分析提供基础。     

Autoactivation of proteinase A initiates activation of yeast vacuolar zymogens.

The Saccharomyces cerevisiae PEP4 gene encodes proteinase A, an aspartyl protease. pep4 mutants are defective in the activation of many vacuolar hydrolases, including proteinase B. We have expressed a pep4 mutation which directs the accumulation of pro-proteinase A with a defective active site. Co-expression with PEP4 leads to normal processing, i.e. the mutant zymogen is functional as a substrate for the maturation reaction in trans. We conclude that wild-type pro-proteinase A has the ability to mediate its own activation. Elimination of the co-expressed PEP4 gene did not effectively stop the processing of the mutant zymogen, owing to a strong, proteinase-B-dependent, phenotypic lag. In a proteinase-B-negative strain, processing of pro-proteinase A led to an active form of a higher molecular mass than the normal mature form.     

Cloning and partial sequencing of the proteinase gene complex from Lactococcus lactis subsp. lactis UC317.

The proteinase genes from Lactococcus lactis subsp. lactis UC317 were identified on a plasmid, pCI310, which is a deletion derivative of a cointegrate between pCI301, the 75 kb Lac Prt plasmid from UC317 and the 38.5 kb cryptic plasmid from that strain. The prt genes were cloned using a replacement cloning strategy whereby fragments from pCI310 were exchanged with the equivalent fragments in pNZ521, which contains the cloned proteinase genes from L. lactis subsp. lactis SK112. This generated two plasmids which encoded a cell-envelope-associated and a secreted proteinase, respectively. Specific regions of the UC317 structural prtP gene known to encode seven of the amino acids essential for substrate cleavage specificity were sequenced and compared with the known sequences of prt genes from L. lactis strains SK112, Wg2 and NCDO763. In spite of various differences that were detected in the nucleotide sequence of this region, it appears that these seven amino acids in strains UC317 and NCDO763 are identical, and represent a combination of three of the amino acids from SK112 and four from Wg2. These results indicate that the UC317 proteinase is a natural hybrid of the SK112 and Wg2 proteinases.     

Biogenesis of the yeast vacuole (lysosome). Proteinase yscB contributes molecularly and kinetically to vacuolar hydrolase-precursor maturation.

The vacuolar proteinase yscB (PrB) has been implicated in the final maturation of procarboxypeptidase yscY (pro-CpY) to the mature wild-type form CpYb of 61 kDa. In PrB-deficient mutants, only the proteinase yscA processed form CpYa of 62 kDa is found [Mechler, B., Müller, H. & Wolf, D. H. (1987) EMBO J. 6, 2157-2163]. We report now that, akin to CpY, two forms of mature proteinase yscA (PrA) can be distinguished. In PrB-deficient mutant cells, PrAa, migrating at about 43 kDa in SDS/PAGE, is found, whereas PrAb, found in wild-type cells, had the known molecular mass of 42 kDa. In the PrB-deficient strain, pro-PrA and pro-CpY matured only to the higher-molecular-mass forms, PrAa and CpYa, and the maturation of both precursors was slower than in the isogenic wild-type strain. Pulse-labeling experiments indicated that the mature forms, PrAb or CpYb, are generated directly in the PrB-containing wild-type strain in vivo. In vitro experiments showed that PrB is able to trigger maturation of its 42-kDa pro-PrB precursor to mature PrB in the absence of PrA. Mature PrB and its proteolytic activity, however, shows a higher stability in the presence of mature PrA. The data indicate a molecular and kinetic participation of proteinase yscB in vacuolar hydrolase precursor maturation.     

大肠杆菌ppsA和tktA基因的串联表达

ppsA和tktA是芳香族氨基酸生物合成中心途径的两个关键酶基因,在大肠杆菌中,ppsA基因编码磷酸烯醇式丙酮酸合成酶A(PpsA),该酶催化丙酮酸合成磷酸烯醇式丙酮酸;tktA基因编码转酮酶A,该酶在磷酸戊糖途径中生成4-磷酸赤藓糖起主要作用。采用PCR方法从大肠杆菌K-12株中扩增到ppsA和tktA,并实现了两基因的高效表达,其中ppsA活性提高了10．8倍,tktA活性提高了3．9倍,当这两个基因串联在一个质粒上导入大肠杆菌进行表达时,PpsA的活性变化较大(2．1～9．1倍),TktA的活性相对稳定(3．9～4．5倍),且这两个基因单独表达和串联表达都能使芳香族氨基酸生物合成共同途径中关键中间产物DAHP的产量提高,且串联表达比单独表达较高。     

Analysis of the prtP gene encoding porphypain, a cysteine proteinase of Porphyromonas gingivalis.

The cloning and sequencing of the gene encoding porphypain, a cysteine proteinase previously isolated from detergent extracts of the Porphyromonas gingivalis W12 cell surface, are described. The prtP gene encoded a unique protein of 1,732 amino acids, including a putative signal sequence for protein secretion. The predicted molecular mass for the mature protein was 186 kDa, which was close to the observed molecular mass of 180 kDa. There was one copy of prtP in the genomes of seven P. gingivalis strains examined. The gene was located 5' to a region with a high degree of homology to the insertion element IS1126 in P. gingivalis W12. The PrtP protein had regions of high homology to HagA, a hemagglutinin of P. gingivalis, and to several purported proteinases of P. gingivalis that have Arg-X specificity. A detailed comparison of genes encoding the latter and cpgR suggested that rgp-1, prpR1, prtR, agp, cpgR, and possibly prtH were derived from identical genetic loci. Although an rgp-1-like locus was detected in seven P. gingivalis strains by Southern blot analyses, agp and cpgR were not detected, not even in the strains from which they were originally isolated. In addition, at least 20 copies of a repeat region common to PrtP, the Rgp-1-like proteins, and HagA were observed in each of the seven genomes examined. The repeat region hybridization patterns for strains W83 and W50 were very similar, and they were identical for strains 381 and ATCC 33277, providing further evidence that these strains are closely related genetically.     

Molecular cloning of the gene encoding the mosaic neurotoxin, composed of parts of botulinum neurotoxin types C1 and D, and PCR detection of this gene from Clostridium botulinum type C organisms.

The DNA fragment common to the genes encoding botulinum neurotoxin types C1 (BN/C1) and D (BN/D) was amplified by PCR from the culture supernatant of Clostridium botulinum type C strain 6813 (C6813) that was treated with either DNase I or proteinase K but not from the supernatant that was treated with both DNase I and proteinase K, suggesting the neurotoxin gene is located on a certain bacteriophage DNA. Thus, to isolate the neurotoxin gene, we performed PCR with the culture supernatant of C6813 and seven primer pairs designed from the genes encoding BN/C1 and BN/D. The coding region in the connected sequence encodes a neurotoxin composed of 1,280 amino acids with a molecular weight of 147,817. The neurotoxin from C6813 has 95% amino acid identity to BN/C1, except for its C-terminal one-third, which is quite similar to the C-terminal one-third of BN/D (95% identity). When we performed PCRs with four primer pairs designed from the 5'-terminal two-thirds of the BN/C1 gene and two primers from the 3'-terminal one-third of the BN/D gene, DNA fragments of the expected sizes (0.5 to 1.3 kbp) could be amplified from C. botulinum type C strains 6812 and 6814. These results suggest that some strains of C. botulinum type C contain the gene encoding the mosaic neurotoxin composed of parts of BN/C1 and BN/D.     

The cloning and sequencing of the UDP-galactose 4-epimerase gene (galE) from Avibacterium paragallinarum.

The putative uridine diphosphate (UDP)-galactose 4-epimerase encoding gene, galE, was isolated from Avibacterium paragallinarum with the use of degenerate primers, colony hybridization and inverse PCR. The data revealed an open reading frame of 1017 bp encoding a protein of 338 amino acids with a molecular weight of 37 kDa and an isoelectric point of 5.5. High sequence homology was obtained with an 87, 91 and 89% sequence identity on protein level towards the galE genes from Actinobacillus pleuropneumoniae, Haemophilus influenza and Pasteurella multocida, respectively. To verify that the cloned galE gene encodes for a UDP-galactose 4-epimeras, this gene was cloned into the pYES-2 expression vector, followed by transformation in a Saccharomyces cerevisiae gal10 deletion strain. Complementation of the gal10 deletion mutant with the galE gene confirmed that this gene encodes a UDP-galactose 4-epimerase.