In vitro mutagenesis of the mitochondrial leucyl tRNA synthetase ofSaccharomyces cerevisiae shows that the suppressor activity of the mutant proteins is related to the splicing function of the wild-type protein

TheNAM2 gene ofSaccharomyces cerevisiae encodes the mitochondrial leucyl tRNA synthetase (mLRS), which is necessary for the excision of the fourth intron of the mitochondrialcytb gene (bI4) and the fourth intron of the mitochondrialcoxI gene (aI4), as well as for mitochondrial protein synthesis. Some dominant mutant alleles of the gene are able to suppress mutations that inactivate the bI4 maturase, which is essential for the excision of the introns aI4 and bI4. Here we report mutagenesis studies which focus on the splicing and suppressor functions of the protein. Small deletions in the C-terminal region of the protein preferentially reduce the splicing, but not the synthetase activity; and all the C-terminal deletions tested abolish the suppressor activity. Mutations which increase the volume of the residue at position 240 in the wild-type mLRS without introducing a charge, lead to a suppressor activity. The mutant 238C, which is located in the suppressor region, has a reduced synthetase activity and no detectable splicing activity. These data show that the splicing and suppressor functions are linked and that the suppressor activity of the mutant alleles results from a modification of the wild-type splicing activity.     

Divergence of the mitochondrial leucyl tRNA synthetase genes in two closely related yeasts Saccharomyces cerevisiae and Saccharomyces douglasii: a paradigm of incipient evolution

Summary We studied the NAM2 genes of Saccharomyces douglasii and Saccharomyces cerevisiae, and showed that they are interchangeable for all the known functions of these genes, both mitochondrial protein synthesis and mitochondrial mRNA splicing. This confirms the prediction that the S. douglasii NAM2D gene encodes the mitochondrial leucyl tRNA synthetase (EC 6.1.1.4). The observation that these enzymes are interchangeable for their mRNA splicing functions, even though there are significant differences in the intron/exon structure of their mitochondrial genome, suggests that they may have a general role in yeast mitochondrial RNA splicing. A short open reading frame (ORF) precedes the synthetase-encoding ORF, and we showed that at least in S. cerevisiae this is not essential for the expression of the gene; however, it may be involved in a more subtle type of regulation. Sequence comparisons of S. douglasii and S. cerevisiae revealed a particularly interesting situation from the evolutionary point of view. It appears that the two yeasts have diverged relatively recently: there is remarkable nucleotide sequence conservation, with no deletions or insertions, but numerous (albeit non-saturating) silent substitutions resulting from transitions. This applies not only to the NAM2 coding regions, but also to two other ORFs flanking the NAM2 ORF. The regions between the ORFs (believed to be intergenic regions) are much less conserved, with several deletions and insertions. Thus S. douglasii and S. cerevisiae provide an ideal system for the study of molecular evolution, being two yeasts caught in the act of speciation.     

An extragenic suppressor ofprp24-1 defines genetic interaction betweenPRP24 andPRP21 gene products ofSaccharomyces cerevisiae

The temperature-sensitiveprp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperaturesensitive (ts)prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic toprp21-1. This suppressor,prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that ofprp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in theprp24-1 strain. Genetic analysis of the suppressor showed thatprp21-2 is not a bypass suppressor ofprp24-1. The suppression ofprp24-1 byprp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2–U6 snRNA interactions.     

The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed.

Summary We have characterized the nuclear geneNAM8 inSaccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wildtype strains, its overexpression is a prerequisite for the suppressor action. Inactivation of theNAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. TheNAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.     

Two-hybrid interaction of a human UBC9 homolog with centromere proteins ofSaccharomyces cerevisiae

Using a two-hybrid system, we cloned a human cDNA encoding a ubiquitin-conjugating enzyme (UBC), hUBC9, which interacts specifically with all three subunits of theSaccharomyces cerevisiae centromere DNA-binding core complex, CBF3. The hUBC9 protein shows highest homology to a new member of the UBC family: 54% identity toS. cerevisiae Ubc9p and 64% identity toSchizosaccharomyces pombe (Sp) hus5. Overexpression of hUBC9 partially suppresses aS. cerevisiae ubc9 temperature-sensitive mutation, indicating that theUBC9 gene family is also functionally conserved. Like hUBC9, Sphus5 also interacts specifically with all three subunits of the CBF3 complex. However,S. cerevisiae Ubc9p interacts only with the Cbf3p subunit (64 kDa) of the CBF3 complex, indicating the specificity of the interaction betweenS. cerevisiae Ubc9 and Cbf3p proteins. The function of Ubc9p in the G₂/M phase ofS. cerevisiae could be related to regulation of centromere proteins in chromosome segregation in mitosis. Therefore, the ubiquitination process and centromere function may be linked to chromosome segregation. We also provide further in vivo evidence that Mck1p, a protein kinase, is specifically associated with the centromere proteins Cbf2p and Cbf5p, which were previously shown to interact in vitro.     

TheCBP2 gene fromSaccharomyces douglasii is a functional homologue of theSaccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome

InSaccharomyces cerevisiae the only known role of theCBP2 gene is the excision of the fifth intron of the mitochondrialcyt b gene (bI5). We have cloned theCBP2 gene fromSaccharomyces douglasii (a close relative ofS. cerevisiae). A comparison of theS. douglasii andS. cerevisiae sequences shows that there are 14% nucleotide substitutions in the coding region, with transitions being three times more frequent than transversions. At the protein level sequence identity is 87%. We have demonstrated that theS. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-typeS. douglasii mitochondrial genome, but not in the presence of an intronlessS. cerevisiae mitochondrial genome. Also theS. douglasii andS. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from theS. douglasii mitochondrial genome.     

Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase

In Saccharomyces cerevisiae the utilization of lactate occurs via specific oxidation of l- and d-lactate to pyruvate catalysed by l-lactate ferricytochrome c oxidoreductase (L-LCR) (EC 1.1.2.3) encoded by the CYB2 gene, and d-lactate ferricytochrome c oxidoreductase (D-LCR) (EC 1.1.2.4), respectively. We selected several lactate^– pyruvate⁺ mutants in a cyb2 genetic background. Two of them were devoid of D -LCR activity (dld mutants, belonging to the same complementation group). The mutation mapped in the structural gene. This was demonstrated by a gene dosage effect and by the thermosensitivity of the enzyme activity of thermosensitive revertants. The DLD gene was cloned by complementation for growth on d-, l-lactate in the strain WWF18-3D, carrying both a CYB2 disruption and the dld mutation. The minimal complete complementing sequence was localized by subcloning experiments. From the sequence analysis an open reading frame (ORF) was identified that could encode a polypeptide of 576 amino-acids, corresponding to a calculated molecular weight of 64000 Da. The deduced protein sequence showed significant homology with the previously described microsomal flavoprotein l-gulono--lactone oxidase isolated from Rattus norvegicus, which catalyses the terminal step of l-ascorbic acid biosynthesis. These results are discussed together with the role of L-LCR and D-LCR in lactate metabolism of S. cerevisiae.     

酿酒酵母表达和纯化功能性的铁载体合成蛋白PchE

【目的】利用酿酒酵母表达系统,通过乙醇脱氢酶启动子异源表达细菌源的铁载体合成蛋白PchE,并与来源于枯草芽孢杆菌的泛酰化酶Sfp同宿主共表达,探索真核表达体系表达具有生化活性的细菌源蛋白。【方法】从大肠杆菌BAP1染色体上扩增sfp基因,将pchE基因及串联的pchE与sfp基因分别构建到酵母-大肠杆菌穿梭质粒pXW55中,各自转化酿酒酵母BJ5464-npg A表达,经过亲和层析和离子交换层析纯化蛋白,利用HPLC检测细菌源与酵母源表达的PchE在体外重构生化反应中的催化活性。【结果】利用酿酒酵母表达系统可以获得高纯度的原核蛋白PchE。真菌源的泛酰化基因NpgA和细菌源的Sfp,均可泛酰化修饰PchE,合成中间产物HPT-Cys。【结论】在酿酒酵母Saccharomyces cerevisiae BJ5464-npgA表达系统中,首次证明真菌源的泛酰化基因NpgA和细菌源的Sfp,均可泛酰化修饰细菌源的非核糖体肽合酶。比较酵母和细菌宿主的目标蛋白表达,证明酵母表达的巨大蛋白PchE的纯度更高,非特异性条带减少,推测酵母宿主可能更适合表达纯化功能性的巨型蛋白质。     

ACR1, a gene encoding a protein related to mitochondrial carriers, is essential for acetyl-CoA synthetase activity in Saccharomyces cerevisiae

The utilization of ethanol via acetate by the yeast Saccharomyces cerevisiae requires the presence of the enzyme acetyl-coenzyme A synthetase (acetyl-CoA synthetase), which catalyzes the activation of acetate to acetyl-coenzyme A (acetyl-CoA). We have isolated a mutant, termed acr1, defective for this activity by screening for mutants unable to utilize ethanol as a sole carbon source. Genetic and biochemical characterization show that, in this mutant, the structural gene for acetyl-CoA synthetase is not affected. Cloning and sequencing demonstrated that the ACR1 gene encodes a protein of 321 amino acids with a molecular mass of 35 370 Da. Computer analysis suggested that the ACR1 gene product (ACR1) is an integral membrane protein related to the family of mitochondrial carriers. The expression of the gene is induced by growing yeast cells in media containing ethanol or acetate as sole carbon sources and is repressed by glucose. ACR1 is essential for the utilization of ethanol and acetate since a mutant carrying a disruption in this gene is unable to grow on these compounds.     

CDC7 protein kinase activity is required for mitosis and meiosis in Saccharomyces cerevisiae

Summary The product of the CDC7 gene of Saccharomyces cerevisiae has multiple cellular functions, being needed for the initiation of DNA synthesis during mitosis as well as for synaptonemal complex formation and commitment to recombination during meiosis. The CDC7 protein has protein kinase activity and contains the conserved residues characteristic of the protein kinase catalytic domain. To determine which of the cellular functions of CDC7 require this protein kinase activity, we have mutated some of the conserved residues within the CDC7 catalytic domain and have examined the ability of the mutant proteins to support mitosis and meiosis. The results indicate that the protein kinase activity of the CDC7 gene product is essential for its function in both mitosis and meiosis and that this activity is potentially regulated by phosphorylation of the CDC7 protein.     

Chitin synthetase activity is bound to chitosomes anf to the plasma membrane in protoplasts of Saccharomyces cerevisiae

The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacoular proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40–110 nm; bouyant density (d) = 1.146 g/cm³). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm³) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with ³H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100–250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.     

酿酒酵母全基因组范围内筛选MTM1基因的抑制基因

MTM1 基因对于维持锰超氧化物歧化酶的活性和线粒体正常功能十分重要,MTM1 基因的缺失会严重影响酵母锰超氧化物歧化酶活性,并损伤线粒体功能,因此在非发酵培养基上不能生长．利用MTM1 基因缺失的突变体在非发酵培养基上的生长缺陷,转入酵母基因组文库筛选MTM1 抑制基因,发现MTM1基因缺失造成的损伤一旦形成不可逆转,重新引入MTM1 基因也无法挽救,直接筛选无法得到抑制基因．为了避免MTM1缺失造成的不可逆损伤,在野生型酵母中先转入带有MTM1 基因的质粒,再敲除染色体上的MTM1 基因,随后转入基因组文库,再利用药物5-氟乳清酸(5-FOA)迫使细胞丢失表达MTM1基因的外源质粒,再筛选能在非发酵培养基上生长的转化子,通过这种方法筛选发现,POR2等5个基因的过表达可以挽救MTM1 基因缺失造成的非发酵培养基上的生长缺陷,为深入了解MTM1基因的功能提供了线索,对筛选其他造成不可逆损伤的突变基因的抑制基因提供了一条可行的研究思路．     

Man8GlcNAc2糖基化的酿酒酵母菌株的构建

酿酒酵母糖蛋白的N-糖基化经过高尔基体的修饰后形成聚合度约150-200的甘露寡糖,高尔基体N-糖基化的糖基转移酶Mnn1p和Och1p在甘露寡糖的形成过程中起关键作用。通过同源重组置换敲除了酵母中的MNN1和OCH1基因阻断高尔基体N-糖基化修饰,分离纯化了mnn1 och1突变株中的N-糖蛋白,糖酰胺酶PNGaseF酶解释放的N-糖链经过2-氨基吡啶衍生后,利用HPLC和MALDITOF/MS结合的方法分析了突变株糖蛋白上的N-糖链。结果显示mnn1 och1突变株中的糖蛋白的N-糖链为结构单一的糖链,分子量为1794.66,推测为Man₈GlcNAc₂。     

Inactivation of the yeast Sen1 protein affects the localization of nucleolar proteins

A mutation in the Saccharomyces cerevisiae SEN1 gene causes accumulation of end-matured, intron-containing pre-tRNAs. Cells containing the thermosensitive sen1-1 mutation exhibit reduced tRNA splicing endonuclease activity. However, Sen1p is not the catalytic subunit of this enzyme. We have used Sen1p-specific antibodies for cell fractionation studies and immunofluorescent microscopy and determined that Sentp is a low abundance protein of about 239 kDa. It localizes to the nucleus with a granular distribution. We verified that a region in SEN1 containing a putative nuclear localization signal sequence (NLS) is necessary for nuclear targeting. Furthermore, we found that inactivation of Sen1p by temperature shift of a strain carrying sen1-1 leads to mislocalization of two nucleolar proteins, Nopt and Ssb1 Possible mechanisms are discussed for several related nuclear functions of Sen1p, including tRNA splicing and the maintenance of a normal crescent-shaped nucleolus.     

The glucose repression and RAS-cAMP signal transduction pathways of Saccharomyces cerevisiae each affect RNA processing and the synthesis of a reporter protein

Previously we reported that mutations in the Saccharomyces cerevisiae REG1 gene encoding a negative regulator of glucose-repressible genes, suppress the RNA processing defects and temperature-sensitive growth of rna1-1 and prp cells. This result and the fact that growth on non-glucose carbon sources also suppresses rna1-1 led us to propose that RNA processing and export of RNA from the nucleus are responsive to carbon source regulation. To understand how carbon source affects these processes, we used p70, an antigen regulated by REGI and by glucose availability, as a reporter. We found that the response of p70 to glucose availability is mediated by both the SNFI-SSN6-dependent glucose repression and the RAS-cAMP pathways. These results led us to test whether the RAS-cAMP pathway interacts with RNA1. We found that suppression of rnal-1 appears to be mediated, at least in part, by the RAS-cAMP pathway.     

Structure of the HOM2 gene of Saccharomyces cerevisiae and regulation of its expression

Summary In Saccharomyces cerevisiae the HOM2 gene encodes aspartic semi-aldehyde dehydrogenase (ASA DH). The synthesis of this enzyme had been shown to be derepressed by growth in the presence of high concentrations of methionine. In the present work we have cloned and sequenced the HOM2 gene and found that the promoter region of this gene bears one copy of the consensus sequence for general control of amino acid synthesis. This prompted us to study the regulation of the expression of the HOM2 gene. We have found that ASA DH is the first reported enzyme of the related threonine and methionine pathway to be regulated by the general control of amino acid synthesis.     

Characterization of the cyrl-2 UGA mutation in Saccharomyces cerevisiae

Summary cyrl-2 is a temperature-sensitive mutation of the yeast adenylate cyclase structural gene, CYR1. The cyrl-2 mutation has been suggested to be a UGA mutation since a UGA suppressor SUP201 has been isolated as a suppressor of the cyrl-2 mutation. Construction of chimeric genes restricted the region containing the cyrl-2 mutation, and the cyrl-2 UGA mutation was identified at codon 1282, which lies upstream of the region coding for the catalytic domain of adenylate cyclase. Alterations in the region upstream of the cyrl-2 mutation site result in null mutations. The complete open reading frame of the cyrl-2 gene expressed under the control of the GAL1 promoter complemented cyrl-dl in a galactose-dependent manner. These results suggest that at the permissive temperature weak readthrough occurs at the cyrl-2 mutation site to produce low levels of active adenylate cyclase. An endogenous suppressor in yeast cells is assumed to be responsible for this readthrough.     

The archaebacterial membrane protein bacterio-opsin is expressed and N-terminally processed in the yeast Saccharomyces cerevisiae

The bop gene codes for the membrane protein bacterio-opsin (BO), which on binding all-trans-retinal, constitutes the light-driven proton pump bacteriorhodopsin (BR) in the archaebacterium Halobacterium salinarium . This gene was cloned in a yeast multi-copy vector and expressed in Saccharomyces cerevisiae under the control of the constitutive ADH1 promoter. Both the authentic gene and a modified form lacking the precursor sequence were expressed in yeast. Both proteins are incorporated into the membrane in S. cerevisiae. The presequence is thus not required for membrane targeting and insertion of the archaebacterial protein in budding yeast, or in the fission yeast Schizosaccharomyces pombe, as has been shown previously. However, in contrast to S. pombe transformants, which take on a reddish colour when all-trans-retinal is added to the culture medium as a result of the in vivo regeneration of the pigment, S. cerevisiae cells expressing BO do not take on a red colour. The precursor of BO is processed to a protein identical in size to the mature BO found in the purple membrane of Halobacterium. The efficiency of processing in S. cerevisiae is dependent on growth phase, as well as on the composition of the medium and on the strain used. The efficiency of processing of BR is reduced in S. pombe and in a retinal-deficient strain of H. salinarium, when retinal is present in the medium.

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Use of synthetic lethal mutants to clone and characterize a novel CTP synthetase gene in Saccharomyces cerevisiae

In the pyrimidine biosynthetic pathway, CTP synthetase catalyses the conversion of uridine 5-triphosphate (UTP) to cytidine 5-triphosphate (CTP). In the yeast Saccharomyces cerevisiae, the URA7 gene encoding this enzyme was previously shown to be nonessential for cell viability. The present paper describes the selection of synthetic lethal mutants in the CTP biosynthetic pathway that led us to clone a second gene, named URA8, which also encodes a CTP synthetase. Comparison of the predicted amino acid sequences of the products of URA7 and URA8 shows 78% identity. Deletion of the URA8 gene is viable in a haploid strain but simultaneous presence of null alleles both URA7 and URA8 is lethal. Based on the codon bias values for the two genes and the intracellular concentrations of CTP in strains deleted for one of the two genes, relative to the wild-type level, URA7 appears to be the major gene for CTP biosynthesis. Nevertheless, URA8 alone also allows yeast growth, at least under standard laboratory conditions.