粟酒裂殖酵母Atp10在线粒体中的功能研究
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摘要
线粒体是真核细胞中的动力工厂,细胞生命活动所需能量大多来自于线粒体氧化磷酸化.粟酒裂殖酵母是真核生物研究的模式生物.本研究借助基因敲除方法获得atp10基因缺失菌株Δatp10,在以甘油和半乳糖为唯一碳源的非发酵型培养基中,Δatp10的生长出现缺陷.利用生物信息学分析,Atp10在N端含有32个氨基酸残基组成的线粒体定位序列,GFP绿色荧光观察Atp10蛋白定位在线粒体中;Western blotting检测显示,Atp10缺失导致线粒体相关蛋白的表达几乎丧失,影响线粒体呼吸链复合体的组装.综上所述,Atp10是一个与线粒体功能密切相关的蛋白,是线粒体呼吸链正常发挥功能所必须的蛋白.
Mitochondria are power plants in eukaryotic cells. Most of the energy required for cell life activities comes from oxidative phosphorylation of mitochondria. First,we constructed the strain of atp10 gene deletion by homologous recombination and observed its growth phenotype in non-fermentation medium with glycerol and galactose as the sole carbon source. Then,bioinformatics analyses revealed that Atp10 contains a 32-amino-acids mitochondrial localization sequence(MTS)at the N-terminus. The green fluorescence confirmed that Atp10 is localized in mitochondria. Western blotting assay showed that the deletion of Atp10 resulted in the decrease of the expression of mitochondrial-related proteins and affected the assembly of mitochondrial respiratory chain complex. In short,Atp10 is a protein closely related to mitochondrial function,which is essential for the normal functioning of mitochondrial respiratory chain.
Mitochondria are power plants in eukaryotic cells. Most of the energy required for cell life activities comes from oxidative phosphorylation of mitochondria. First,we constructed the strain of atp10 gene deletion by homologous recombination and observed its growth phenotype in non-fermentation medium with glycerol and galactose as the sole carbon source. Then,bioinformatics analyses revealed that Atp10 contains a 32-amino-acids mitochondrial localization sequence(MTS)at the N-terminus. The green fluorescence confirmed that Atp10 is localized in mitochondria. Western blotting assay showed that the deletion of Atp10 resulted in the decrease of the expression of mitochondrial-related proteins and affected the assembly of mitochondrial respiratory chain complex. In short,Atp10 is a protein closely related to mitochondrial function,which is essential for the normal functioning of mitochondrial respiratory chain.
引文
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[19] 黄敏.基因敲除技术及其应用[J].广东轻工职业技术学院学报,2009,8(4):12-14.
[20] PAUL M F,BARRIENTOS A,TZAGOLOFF A.A single amino acid change in subunit 6 of theyeast mitochondrial ATPase suppresses a nullmutation in ATP10[J].The journal of biological chemistry,2000,275(38):29238-29243.
[21] DE J P,VAN DIJKEN J P,PRONK J T.Metabolic fluxes in chemostat cultures of Schizosaccharomyces pombe grown on mixtures of glucose and ethanol[J].Microbiology,1996,142(6):1399-1407.
[2] LURIN C1,ANDRéS C,AUBOURG S,et al.Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis[J].Plant cell,2004,16(8):2089-2103.
[3] PAPA S,SCACCO S,SCHLIEBS M,et al.Mitochondrial diseases and aging[J].Molecular aspects of medicine,1996,17(6):513-563.
[4] RAULE N,SEVINI F,SANTORO A,et al.Association studies on human mitochondrial DNA:methodological aspects and results in the most common age-related diseases[J].Mitochondrion,2007,7(1/2):29-38.
[5] DOWLING D K.Evolutionary perspectives on the links between mitochondrial genotype and disease phenotype.[J].Biochimica Et Biophysica Acta,2014,1840(4):1393-1403.
[6] SCHON E A,DIMAURO S,HIRANO M.Human mitochondrial DNA:roles of inherited andsomatic mutations[J].Nature reviews genetics,2012,13(12):878-890.
[7] NEWSHOLME P,GAUDEL C,KRAUSE M.Mitochondria and diabetes:an intriguing pathogenetic role[J].Advances in experimental medicine and biology,2012(942):235-247.
[8] JAMES A M,COLLINS Y,LOGAN A,et al.Mitochondrial oxidative stress and the metabolic syndrome[J].Trends in endocrinology and metabolism,2012,23(9):429-434.
[9] HATAKEYAMA H,GOTO Y I.Respiratory chain complex disorganization impairs mitochondrial and cellular integrity:phenotypic variation in cytochrome c oxidase deficiency[J].American journal of pathology,2017,187(1):110-121.
[10] MAYR J A,HAACK T B,FREISINGER P,et al.Spectrum of combined respiratory chain de fects[J].Journal of inherited metabolic disease,2015,38(4):629-640.
[11] ROLOFF G A,HENRY M F.Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria[J].Molecular biology of the cell,2015,26(16):2885-2894.
[12] CROSS R L.The mechanism and regulation of ATP synthesis by F1-ATPases[J].Annu Rev Biochem,1981,50:681-714.
[13] WALKER J E,FEARNLEY I M,GAY N J,et al.Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria[J].Journal of molecular biology,1985,184(4):677-701.
[14] FUTAI M,KANAZAWA H.Structure and function of proton-translocating adenosine triphosphatase(F0F1):biochemical and molecular biological approaches[J].Microbiological reviews,1983,47(3):285-312.
[15] TZAGOLOFF A,MEAGHER P.Assembly of the mitochondrial membrane system.V.properties of a dispersed preparation of the rutamycin-sensitive adenosine triphosphatase of yeast mitochondria[J].The journal of biological chemistry,1971,246(23):7328-7336.
[16] ALEXANDER T,ANTONI B,WALTER N,et al.Atp10p assists assembly of Atp6p into the Fo unit of the yeast mitochondrial ATPase[J].The journal of biological chemistry,2004,279(19):19775-19780.
[17] CLAROS M G,VINCENS P.Computational method to predict mitochondrially imported proteins and their targeting sequences[J].European journal of biochemistry,1996,241(3):779-786.
[18] 岳强,周惠.粟酒裂殖酵母——一种良好的真核模式生物[J].韶关学院学报,2003,24(3):100-102.
[19] 黄敏.基因敲除技术及其应用[J].广东轻工职业技术学院学报,2009,8(4):12-14.
[20] PAUL M F,BARRIENTOS A,TZAGOLOFF A.A single amino acid change in subunit 6 of theyeast mitochondrial ATPase suppresses a nullmutation in ATP10[J].The journal of biological chemistry,2000,275(38):29238-29243.
[21] DE J P,VAN DIJKEN J P,PRONK J T.Metabolic fluxes in chemostat cultures of Schizosaccharomyces pombe grown on mixtures of glucose and ethanol[J].Microbiology,1996,142(6):1399-1407.