Suppression of ρ 0 lethality by mitochondrial ATP synthase F1 mutations in Kluyveromyces lactis occurs in the absence of F0

Specific mgi mutations in the α, β or γ subunits of the mitochondrial F₁-ATPase have previously been found to suppress ρ⁰ lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F₁ is dependent on the F₀ sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, ρ⁰ mutants can be isolated, upon treatment with ethidium bromide, that lack six major F₀ subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of ρ⁰ mutants indicates that an F₁-complex carrying a mgi mutation can assemble in the absence of F₀ subunits and that suppression of ρ⁰ lethality is an intrinsic property of the altered F₁ particle. Received: 7 April 1998 / Accepted: 10 June 1998     

线粒体H~ -ATPase超分子结构及其催化机制的研究现状

质子泵H~ -ATPase广泛存在于线粒体,叶绿体,异养菌和光合细菌中,是生物体能量转换的核心酶,有极为重要的生理作用。近几年来,对H~ -ATPase的结构、功能和调控机制的研究进展很快,对复合体的组装有了进一步的认识。对H~ -ATPase的主要亚基已经完成序列测定及分析,对各亚基生理功能也进行了较为深入的研究。生物化学及分子生物学工作揭示:生物体内以多种方式对编码H~ -ATPase主要亚基的基因表达和该酶的活力进行调控。其中,对于线粒体H~ -ATPasc的研究显得尤为突出。本文综述了线粒体H~ -ATPase的结构、功能、和调节方面的研究现状,并进一步提出了一些值得深入探讨的问题。     

线粒体H^＋-ATPase超分子结构及其催化机制的研究现状

质子泵H^＋-ATPase广泛存在于线粒体,叶绿体,异养菌和光合细菌中,是生物体能量转换的核心酶,有极为重要的生理作用。近几年来,对H^＋-ATPase的结构、功能和调控机制的研究进展很快,对复合体的组装有了进一步的认识。对H^＋-ATPase的主要亚基已经完成序列测定及分析,对各亚基生理功能也进行了较为深入的研究。生物化学及分子生物学工作揭示：生物体内以多种方式对编码H^＋-ATPase主要亚基的基因表达和该酶的活力进行调控。其中,对于线粒体H^＋-ATPasc的研究显得尤为突出。本文综述了线粒体H^＋-ATPase的结构、功能、和调节方面的研究现状,并进一步提出了一些值得深入探讨的问题。     

牛凝乳酶原基因的合成及其在乳酸克鲁维酵母中的表达

凝乳酶在奶酪加工中应用广泛,为获得高活性的凝乳酶制剂,采用乳酸克鲁维酵母为宿主,首次对经密码子优化的牛凝乳酶原基因进行表达。利用DNAWorks3.0软件辅助设计,用两步PCR法合成了小牛凝乳酶原基因(GenBank Accession No.AA30448)。将该基因插入酵母表达载体pKLAC1,构建了重组载体pKLAC1-Prochy,并用电脉冲法将线性化的重组质粒转化到乳酸克鲁维酵母GG799中。通过含1%酪蛋白的YEPD平板活性筛选,PCR鉴定,最后获得了一株多拷贝整合的基因工程菌chy1。该菌株可分泌表达牛凝乳酶原,经SDS-PAGE分析,证明重组牛凝乳酶原的分子量约为41kDa,符合预期大小,酸化处理后为36kDa,证明可以正确自我剪切。液体培养96h后,酶活最高达到99.67SU/mL。分别以半乳糖和葡萄糖为碳源的条件下表达,其酶活性差异不大,说明在发酵期间,可以不经过半乳糖诱导即可产生高水平的牛凝乳酶原产物。该工程菌的获得为进一步优化产酶条件及放大工艺提供了条件,并为凝乳酶的工业化生产奠定了基础。     

乙醇对F_1-ATP酶和H~+-ATP酶的抑制与其核苷酸结合位点状态的关系

 本文利用动力学方法研究了乙醇对F_1-ATP酶和H~(+)-ATP酶复合体的抑制与其结合核苷酸位点状态的关系,结果表明天然情况下乙醇对F_1呈现反竞争性抑制类型,对H~(+)-ATP酶呈现非竞争性抑制类型,且乙醇对F_1和H~(+)-ATP酶的抑制与核苷酸结合位点的构象密切相关。游离状态下和膜结合状态下的F_1在部分结合的核苷酸被洗脱前后动力学行为的不同,反映了二种状态下的F_1具有不同的构象,且F_0和膜脂对F_1起着一定的调控作用。     

生物膜能量偶联ATP酶的研究牛心线粒体F_1冷盐解离后各部分的亚基分析

纯化的牛心线粒体Ｆ１ＡＴＰ酶（Ｆ_１）在有１ｍｏｌ／ＬＫＣｌ的介质中,０℃保温１ｈ,酶活性降到接近于零,经脱盐并在室温（２０—２５℃）保温,可恢复约６０％的酶活性。电泳结果表明,冷盐处理１ｈ的样品解来成了四个亚部分,这四个部分的亚基组成分别是Ⅰ,α,γ,δ,ε,Ⅱ,β,δ,ε,Ⅲ,β,ε,Ⅳ,β。经冷盐处理１ｈ和５ｈ的Ｆ_１进行ＨＰＬＣ分析的结果有显著的差别。     

The KlPGS1 gene encoding phosphatidylglycerolphosphate synthase in Kluyveromyces lactis is essential and assigned to chromosome I

The phosphatidylglycerolphosphate synthase (CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase, EC 2.7.8.5) is an essential enzyme in biosynthesis of cardiolipin. In this work we report the isolation, heterological cloning, molecular characterization and physical mapping of the Saccharomyces cerevisiae PEL1/PGS1 homologue from Kluyveromyces lactis. The pel1 mutant strain of S. cerevisiae was used to isolate this homologue by screening a K. lactis genomic library. The novel cloned gene was named KlPGS1. Its coding region was found to consist of 1623 bp. The corresponding protein exhibits 55% amino acid identity to its S. cerevisiae counterpart. The presence of the mitochondrial presequence indicates its mitochondrial localization. Sporulation and ascus dissection of diploids heterozygous for single-copy disruption of KlPGS1 revealed that the KlPGS1 gene, is essential in K. lactis. Using a DIG-dUTP-labeled DNA probe-originated from the KlPGS1 gene and Southern hybridization of contour-clamped homogeneous electric field (CHEF)-separated K. lactis chromosomal DNA, the KlPGS1 gene was assigned to chromosome I. The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the Accession No. AY176328.     

Potential relationship between glutathione metabolism and flocculation in the yeast Kluyveromyces lactis

Reduced glutathione (GSH) is involved in biochemical and physiological processes in cells. Flocculation is an important mechanism in microorganisms. The present study concerned the potential relationship between GSH metabolism and flocculation. Two yeast strains, a flocculent (Kluyveromyces lactis 5c) and a nonflocculent (Kluyveromyces lactis 5a) strain, were used. The level of intracellular GSH measured during the growth period was significantly higher in the nonflocculent than in the flocculent strain; in contrast, the flocculent strain exhibited brighter staining of vacuoles than the nonflocculent strain when observed using epifluorescence microscopy. Compounds acting either on flocculation (EDTA, galactose) or on GSH metabolism (buthionine sulfoximine, and N-acetylcysteine) were tested on the flocculent strain during the growth period. Both EDTA and galactose fully inhibited flocculation and induced GSH overproduction of 58% and 153%, respectively. Buthionine sulfoximine decreased GSH level by 76% but had no effect on flocculation; N-acetylcysteine increased the GSH level and flocculation by 106% and 41%, respectively. Combination of EDTA and N-acetylcysteine produced similar effects than with each of them. Combination of galactose and N-acetylcysteine increased the GSH level but decreased flocculation. These results demonstrated that GSH homeostasis is linked to the flocculation mechanism. A hypothesis related to stress is given.     

Significance of the KlLAC1 gene in glucosylceramide production by Kluyveromyces lactis

Each of the 12 genes involved in the synthesis of glucosylceramide was overexpressed in cells of Kluyveromyces lactis to construct a strain accumulating a high quantity of glucosylceramide. Glucosylceramide was doubled by the KlLAC1 gene, which encodes ceramide synthase, and not by 11 other genes, including the KlLAG1 gene, a homologue of KlLAC1 . Disruption of the KlLAC1 gene reduced the content below the detection level. Heterologous expression of the KlLAC1 gene in the cells of Saccharomyces cerevisiae caused the accumulation of ceramide, composed of C₁₈ fatty acid. The KlLAC1 protein preferred long-chain (C₁₈) fatty acids to very-long-chain (C₂₆) fatty acids for condensation with sphingoid bases and seemed to supply a ceramide moiety as the substrate for the formation of glucosylceramide. When the amino acid sequences of ceramide synthase derived from eight yeast species were compared, LAC1 proteins from five species producing glucosylceramide were clearly discriminated from those of the other three species and all LAG1 proteins. The LAC1 protein of K. lactis is the enzyme that plays a crucial role in the synthesis of glucosylceramide.     

Mannoproteins from the cell wall of Kluyveromyces lactis

Abstract Wall mannoproteins from Kluyveromyces lactis have been solubilised by treatment of cell walls with sodium dodecyl sulphate (SDS) or zymolyase. While the former reagent liberates a large number of molecular species, zymolyase preferentially releases a high-molecular-weight material that is sensitive to endo- β - N -acetylglucosaminidase H, and a 29-kDa molecule that reacts with the antiserum raised against a similar species from walls of Saccharomyces cerevisiae . In contrast with observations on isolated walls of S. cerevisiae , dithiothreitol pretreatment of K. lactis walls does not enhance the effect of zymolyase upon mannoprotein release. However, the action of thiol agents is still necessary to obtain protoplasts by zymolyase digestion from K. lactis whole cells.     

Enhanced secretion of heterologous proteins in Kluyveromyces lactis by overexpression of the GDP-mannose pyrophosphorylase, KlPsa1p

GDP-mannose is the mannosyl donor for the glycosylation reactions and is synthesized by GDP-mannose pyrophosphorylase from GTP and d-mannose-1-phosphate; in Saccharomyces cerevisiae this enzyme is encoded by the PSA1/VIG9/SRB1 gene. We isolated the Kluyveromyces lactis KlPSA1 gene by complementing the osmotic growth defects of S. cerevisiae srb1/psa1 mutants. KlPsa1p displayed a high degree of similarity with other GDP-mannose pyrophosphorylases and was demonstrated to be the functional homologue of S. cerevisiae Psa1p. Phenotypic analysis of a K. lactis strain overexpressing the KlPSA1 gene revealed changes in the cell wall assembly. Increasing the KlPSA1 copy number restored the defects in O-glycosylation, but not those in N-glycosylation, that occur in K. lactis cells depleted for the hexokinase Rag5p. Overexpression of GDP-mannose pyrophosphorylase also enhanced heterologous protein secretion in K. lactis as assayed by using the recombinant human serum albumin and the glucoamylase from Arxula adeninivorans.     

α-Lipoic acid protects mitochondrial enzymes and attenuates lipopolysaccharide-induced hypothermia in mice

Hypothermia is a key symptom of sepsis, but the mechanism(s) leading to hypothermia during sepsis is largely unknown and thus no effective therapy is available for hypothermia. Therefore, it is important to investigate the mechanism and develop effective therapeutic methods. Lipopolysaccharide (LPS)-induced hypothermia accompanied by excess nitric oxide (NO) production leads to a reduction in energy production in wild-type mice. However, mice lacking inducible nitric oxide synthase did not suffer from LPS-induced hypothermia, suggesting that hypothermia is associated with excess NO production during sepsis. This observation is supported by the treatment of wild-type mice with α-lipoic acid (LA) in that it effectively attenuates LPS-induced hypothermia with decreased NO production. We also found that LA partially restored ATP production, and activities of the mitochondrial enzymes involved in energy metabolism, which were inhibited during sepsis. These data suggest that hypothermia is related to mitochondrial dysfunction, which is probably compromised by excess NO production and that LA administration attenuates hypothermia mainly by protecting mitochondrial enzymes from NO damage.     

Identification and characterization of a novel glucose-phosphorylating enzyme in Kluyveromyces lactis

Recent data suggest that hexokinase KlHxk1 (Rag5) represents the only glucose-phosphorylating enzyme of Kluyveromyces lactis, which also is required for glucose signalling. Long-term growth studies of a K. lactis rag5 mutant, however, reveal slow growth on glucose, but no growth on fructose. Isolation of the permissive glucose-phosphorylating enzyme, mass spectrometric tryptic peptide analysis and determination of basic kinetic data identify a novel glucokinase (KlGlk1) encoded by ORF KLLA0C01,155g. In accordance with the growth characteristics of the rag5 mutant, KlGlk1 phosphorylates glucose, but fails to act on fructose as a sugar substrate. Multiple sequence alignment indicates the presence of at least one glucokinase gene in all sequenced yeast genomes.     

Galactose transport in Kluyveromyces lactis: major role of the glucose permease Hgt1

In Kluyveromyces lactis, galactose transport has been thought to be mediated by the lactose permease encoded by LAC12. In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1, previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12, and the double mutant hgt1lac12, suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium.