Enhanced stability in vivo of a thermodynamically stable mutant form of yeast iso-1-cytochrome c

Previous work has established that the N57I amino acid replacement in iso-1-cytochrome c from the yeast Saccharomyces cerevisiae causes an unprecedented increase in thermodynamic stability of the protein in vitro, whereas the N57G replacement diminishes stability. Spectrophotometric measurements of intact cells revealed that the N57I iso-l-cytochrome c is present at higher than normal levels in vivo. Although iso-1-cytochrome c turnover is negligible during aerobic growth, transfer of fully derepressed, aerobically grown cells to anaerobic growth conditions leads to reduction in the levels of all of the cytochromes. Pulsechase experiments carried out under these anaerobic conditions demonstrated that the N57I iso-l-cytochrome c has a longer half-life than the normal protein. This is the first report of enhanced stability in vivo of a mutant form of a protein that has an enhanced thermodynamic stability in vitro. Although the N57I protein concentration is higher than the normal level, reduced growth in lactate medium indicated that the specific activity of this iso-l-cytochrome c in vivo is diminished relative to wild-type. On the other hand, the level of the thermodynamically labile N57G iso-1-cytochrome c was below normal. The in vivo levels of the N57I and N57G iso-l-cytochrome c suggest that proteins in the mitochondrial intermembrane space can be subjected to degradation, and that this degradation may play a role in controlling their normal levels.     

Characterization of a staurosporine- and temperature-sensitive mutant, stt1, of Saccharomyces cerevisiae: STT1 is allelic to PKC1

Summary Staurosporine is an antibiotic that specifically inhibits protein kinase C. Fourteen staurosporine- and temperature-sensitive (stt) mutants of Saccharomyces cerevisiae were isolated and characterized. These mutants were divided into ten complementation groups, and characterized for their cross-sensitivity to K-252a, neomycin, or CaCl₂, The STT1 gene was cloned and sequenced. The nucleotide sequence of the STT1 gene revealed that STT1 is the same gene as PKC1. The STT1 gene conferred resistance to staurosporine on wild-type cells, when present on a high copy number plasmid. STT1/stt1::HIS3 diploid cells were more sensitive to staurosporine than STT1/STT1 diploid cells. Analysis of temperature-sensitive stt1 mutants showed that the STT1 gene product functioned in S or G2/M phase. These results suggest that a protein kinase (the STT1 gene product) is one of the essential targets of staurosporine in yeast cells.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome c₁ and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c₁ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c₁ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c₁ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

Multiphasic oxidation-reduction of cytochrome b in the succinate-cytochrome c reductase

The triphasic course previously reported for the reduction of cytochrome b in the succinate-cytochrome c reductase by either succinate or duroquinol has been shown to be dependent on the redox state of the enzyme preparation. Prior reduction with increasing concentrations of ascorbate leads to partial reduction of cytochrome c₁, and a gradual decrease in the magnitude of the oxidation phase of cytochrome b. At an ascorbate concentration sufficient to reduce cytochrome c₁ almost completely, the reduction of cytochrome b by either succinate or duroquinol becomes monophasic. Owing to the presence of a trace amount of cytochrome oxidase in the reductase preparation employed, the addition of cytochrome c makes electron flow from substrate to oxygen possible. Under such circumstances, the addition of a limited amount of either succinate or duroquinol leads to a multiphasic reduction and oxidation of cytochrome b. After the initial three phases as described previously, cytochrome b becomes oxidized before cytochrome c₁ when the limited amount of added substrate is being used up. However, at the end of the reaction when cytochrome c_a is being rapidly oxidized, cytochrome b becomes again reduced. The above observations support a cyclic scheme of electron flow in which the reduction of cytochrome b proceeds by two different routes and its oxidation controlled by the redox state of a component of the respiratory chain.     

Cloning and characterisation of the cytochrome c gene of Aspergillus nidulans

The cytochrome c gene (cycA) of the filamentous fungus Aspergillus nidulans has been isolated and sequenced. The gene is present in a single copy per haploid genome and encodes a polypeptide of 112 amino acid residues. The nucleotide sequence of the A. nidulans cycA gene shows 87% identity to the DNA sequence of the Neurospora crassa cytochrome c gene, and approximately 72% identity to the sequence of the Saccharomyces cerevisiae iso-1-cytochrome c gene (CYC1). The S. cerevisiae CYC1 gene was used as a heterologous probe to isolate the homologous gene in A. nidulans. The A. nidulans cytochrome c sequence contains two small introns. One of these is highly conserved in terms of position, but the other has not been reported in any of the cytochrome c genes so far sequenced. Expression of the cycA gene is not affected by glucose repression, but has been shown to be induced approximatly tenfold in the presence of oxygen and three- to fourfold under heatshock conditions.     

Interaction between uranium and the cytochrome c 3 of Desulfovibrio desulfuricans strain G20

Cytochrome c₃ of Desulfovibrio desulfuricans strain G20 is an electron carrier for uranium (VI) reduction. When D. desulfuricans G20 was grown in medium containing a non-lethal concentration of uranyl acetate (1 mM), the rate at which the cells reduced U(VI) was decreased compared to cells grown in the absence of uranium. Western analysis did not detect cytochrome c₃ in periplasmic extracts from cells grown in the presence of uranium. The expression of this predominant tetraheme cytochrome was not detectably altered by uranium during growth of the cells as monitored through a translational fusion of the gene encoding cytochrome c₃ (cycA) to lacZ. Instead, cytochrome c₃ protein was found tightly associated with insoluble U(IV), uraninite, after the periplasmic contents of cells were harvested by a pH shift. The association of cytochrome c₃ with U(IV) was interpreted to be non-specific, since pure cytochrome c₃ adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe₂O₃), and commercially available U(IV) oxide.An erratum to this article can be found at     

The reactivity of cytochrome c with soft ligands

The spectral changes caused by binding soft ligands to the cytochrome c iron and their correlation to ligand affinities support the hypothesis that the iron—methionine sulfur bond of this heme protein is enhanced by delocalization of the metal l₂, electrons into the empty 3d orbitals of the ligand atom. These findings also explain the unique spectrum of cytochrome c in the far red.     

Nuclear and mitochondrial revertants of a yeast mitochondrial tRNA mutant

Summary We isolated revertants capable of respiration from the respiratory deficient yeast mutant, FF1210-6C/ 170, which displays greatly decreased mitochondrial protein synthesis due to a single base substitution at the penultimate base of the tRNA^Asp gene on mitochondrial (mt) DNA. Three classical types of revertant were identified: (1) same-site revertants; (2) intragenic revertants which restore the base pairing in the acceptor stem of the mitochondrial tRNA^Asp; and (3) extragenic suppressors located in nuclear DNA. In addition a fourth type of revertant was identified in which the mutant tRNA^Asp is amplified due to the maintenance of both the original mutant mtDNA and a modified form of the mutant mtDNA in which only a small region around the tRNA^Asp gene is retained and amplified. The latter form resembles the mtDNA in vegetative petite (rho ^-) strains which normally segregates rapidly from the wild-type mtDNA. Each revertant type was characterized genetically and by both DNA sequence analysis of the mitochondrial tRNA^Asp gene and analysis of the quantity and size of RNA containing the tRNA^Asp sequence. These results indicate that the mitochondrial tRNA^Asp of the mutant retains a low level of activity and that the presence of the terminal base pair in tRNA^Asp is a determinant of both tRNA^Asp function and the maintenance of wild-type levels of tRNA^Asp.     

Dominant mutant alleles of yeast protein kinase geneCDC15 suppress thelte1 defect in termination of M phase and genetically interact withCDC14

LTE1 encodes a homolog of GDP-GTP exchange factors for the Ras superfamily and is required at low temperatures for cell cycle progression at the stage of the termination of M phase inSaccharomyces cerevisiae. We isolated extragenic suppressors which suppress the cold sensitivity oflte1 cells and confer a temperature-sensitive phenotype on cells. Cells mutant for the suppressor alone were arrested at telophase at non-permissive temperatures and the terminal phenotype was almost identical to that oflte1 cells at non-permissive temperatures. Genetic analysis revealed that the suppressor is allelic toCDC15, which encodes a protein kinase. Thecdc15 mutations thus isolated were recessive with regard to the temperature-sensitive phenotype and were dominant with respect to suppression oflte1. We isolatedCDC14 as a low-copy-number suppressor ofcdc15-rlt1.CDC14 encodes a phosphotyrosine phosphatase (PTPase) and is essential for termination of M phase. An extra copy ofCDC14 suppressed the temperature sensitivity ofcdc15-rlt1 cells, but not that ofcdc15-1 cells. In addition, some residues that are essential for the Cdc14 PTPase activity were found to be non-essential for the suppression. These results strongly indicate that Cdc14 possesses dual functions; PTPase activity is needed for one function but not for the other. We postulate that the cooperative action of Cdc14 and Cdc15 plays an essential role in the termination of M phase.     

Aspirin induces apoptosis through mitochondrial cytochrome c release

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin-induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT-4, Raji and HL-60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAD·fmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin-induced apoptosis involves caspase activation through cytochrome c release.     

水稻OsMBF1c基因的克隆及表达分析

多蛋白桥联因子1(multi protein bridging factor 1, MBF1)在植物应对逆境胁迫中起着重要的作用,而对于水稻中MBF1是否参与重金属胁迫响应机制目前尚未见相关报道。为了揭示水稻MBF1家族与重金属胁迫的相关性及其潜在作用机制,该研究利用PCR技术克隆水稻OsMBF1c基因的全长编码序列,通过生物信息学对基因功能进行分析和预测,并通过实时荧光定量PCR(RT-qPCR)分析其在镉(Cd)胁迫下的表达特征。结果表明:(1)OsMBF1c的全长编码序列为468 bp,共编码155个氨基酸,相对分子量为16.154 kDa。(2)OsMBF1c与大麦TdMBF1a.1亲缘关系最近,具有光、厌氧等环境因子诱导相关的顺式调节元件。(3)重金属Cd可诱导OsMBF1c表达且在时间上和组织中的表达水平具有特异性,100 μmol·L^-1 Cd 处理1 h 后,地上部分OsMBF1c表达量明显上调,为对照组的7倍; 100 μmol·L^-1 Cd 胁迫处理6 h后,根部OsMBF1c表达量上调为对照组的3倍。该研究结果进一步完善了非生物胁迫下MBF1家族的生物学功能研究。     

The MSS51 gene product is required for the translation of the COX1 mRNA in yeast mitochondria

Summary The MSS51 gene product has been previously shown to be involved in the splicing of the mitochondrial pre-mRNA of cytochrome oxidase subunit I (COX1). We show here that it is specifically required for the translation of the COX1 mRNA. Furthermore, the paromomycin-resistance mutation (P _inf454 ^supR ) which affects the 15 S mitoribosomal RNA, interferes, directly or indirectly, with the action of the MSS51 gene product. Possible roles of the MSS51 protein on the excision of COX1 introns are discussed.     

Plasmid stability in recombinant Saccharomyces cerevisiae

Because of many advantages, the yeast Saccharomyces cerevisiae is increasingly being employed for expression of recombinant proteins. Usually, hybrid plasmids (shuttle vectors) are employed as carriers to introduce the foreign DNA into the yeast host. Unfortunately, the transformed host often suffers from some kind of instability, tending to lose or alter the foreign plasmid. Construction of stable plasmids, and maintenance of stable expression during extended culture, are some of the major challenges facing commercial production of recombinant proteins. This review examines the factors that affect plasmid stability at the gene, cell, and engineering levels. Strategies for overcoming plasmid loss, and the models for predicting plasmid instability, are discussed. The focus is on S. cerevisiae, but where relevant, examples from the better studied Escherichia coli system are discussed. Compared to free suspension culture, immobilization of cells is particularly effective in improving plasmid retention, hence, immobilized systems are examined in some detail. Immobilized cell systems combine high cell concentrations with enhanced productivity of the recombinant product, thereby offering a potentially attractive production method, particularly when nonselective media are used. Understanding of the stabilizing mechanisms is a prerequisite to any substantial commercial exploitation and improvement of immobilized cell systems.     

Oenothera mitochondrial orf454, a gene involved in cytochrome c biogenesis corresponds to orf169 and orf322 of Marchantia

We have characterized a mitochondrial gene in Oenothera, designated orf454, capable of encoding a component of the cytochrome c biogenesis system. This open reading frame is interrupted by an intron of 941 nucleotides showing high similarity to a group II intron residing in the rpl2 gene. RNA editing, which is observed at 18 cytidine positions within the orf454 reading frame, improves the similarity to protein-coding sequences in bacteria and higher plants and removes the last 16 amino acids. orf454 also shows high sequence similarity to two overlapping reading frames (orf169 and orf322) of Marchantia mitochondria. These ORFs belong to an operon-like cluster of genes in the liverwort that is not conserved in Oenothera mitochondria. However, in bacteria these reading frames are organized like the Marchantia gene cluster. It has been shown by genetical analysis in Rhodobacter capsulatus that these genes are essential for cytochrome c biogenesis. Genes of bacterial operons — ccl1 in Rhodobacter and yejR and nrfE in Escherichia coli — show high sequence similarity to the mitochondrial reading frames orf577 and orf454 of Oenothera. orf454, which we describe here, is homologous to the C-terminal region of these bacterial genes, while the previously described orf577 is homologous to the N-terminal region.