粟酒裂殖酵母中Mef2在线粒体中功能的研究
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摘要
由核编码基因控制的线粒体翻译对线粒体中电子传递链复合物的合成是必不可少的。旨在揭示粟酒裂殖酵母中Mef2蛋白的主要功能。利用同源重组的方法构建Δmef2突变体,观察Δmef2在以甘油为唯一碳源的非发酵培养基的生长表型;生物信息学分析结果显示Mef2的N端含有一段由31个氨基酸组成的线粒体定位序列(MTS),为进一步确定Mef2蛋白的定位,在Mef2的C端添加一个GFP荧光标记,观察GFP绿色荧光的位置。接着采用Northern blotting检测mef2的缺失对线粒体基因组编码m RNAs的影响。最后,运用Western blotting检测mef2的缺失对线粒体基因组编码的蛋白的影响。研究结果表明,Δmef2菌株在非发酵培养基上表现出生长缺陷,是线粒体呼吸缺陷型菌;GFP绿色荧光定位实验证实了Mef2定位于线粒体中;Northern blotting实验结果显示mef2的缺失不影响线粒体基因组编码m RNAs的转录;Western blotting检测结果显示mef2的缺失导致Cox1、Cox3、Atp6和Cob1蛋白的表达量降低。综上所述,Mef2是一个与线粒体功能密切相关的蛋白,并且参与了线粒体编码蛋白Cox1、Cox3、Atp6和Cob1的翻译。
Mitochondrial translation,essential for synthesis of the electron transport chain complexes in the mitochondria,is governedby nuclear encoded gene. This paper aims to reveal the main function of Mef2 protein in Schizosaccharomyces pombe. First,we constructed thestrain of mef2 gene deletion by homologous recombination,and observed its growth phenotype in non-fermentative medium with glycerol asthe sole carbon source. Second,the bioinformatics analysis demonstrated that the N-terminal of Mef2 contained a mitochondrial localizationsequence(MTS)consisting of 31 amino acids. To further determine the localization of Mef2 protein,a GFP fluorescent label was addedto the C-terminus of Mef2 to observe GFP green fluorescence. Third,Northern blotting was used to detect the effect of mef2 deletion on thelevel of mitochondrial genome encoding m RNAs. Finally,Western blotting was employed to detect the influence of the deletion of mef2 on theexpression of mitochondrial genome-encoded protein. The results showed that Δmef2 strain caused growth defects on non-fermentation medium,which was one with mitochondrial respiratory defect. GFP green fluorescence localization experiments confirmed that Mef2 was localized inmitochondria. Northern blotting revealed that the deletion of mef2 did not affect the transcription of m RNAs encoded by the mitochondrialgenome. Western blotting showed that the deletion of mef2 resulted in decrease in the expression of Cox1,Cox3,Atp6,and Cob1 proteins. Insummary,Mef2 is a protein that is closely related to mitochondrial function and is involved in the translation of mitochondrial encoded proteinsCox1,Cox3,Atp6,and Cob1.
Mitochondrial translation,essential for synthesis of the electron transport chain complexes in the mitochondria,is governedby nuclear encoded gene. This paper aims to reveal the main function of Mef2 protein in Schizosaccharomyces pombe. First,we constructed thestrain of mef2 gene deletion by homologous recombination,and observed its growth phenotype in non-fermentative medium with glycerol asthe sole carbon source. Second,the bioinformatics analysis demonstrated that the N-terminal of Mef2 contained a mitochondrial localizationsequence(MTS)consisting of 31 amino acids. To further determine the localization of Mef2 protein,a GFP fluorescent label was addedto the C-terminus of Mef2 to observe GFP green fluorescence. Third,Northern blotting was used to detect the effect of mef2 deletion on thelevel of mitochondrial genome encoding m RNAs. Finally,Western blotting was employed to detect the influence of the deletion of mef2 on theexpression of mitochondrial genome-encoded protein. The results showed that Δmef2 strain caused growth defects on non-fermentation medium,which was one with mitochondrial respiratory defect. GFP green fluorescence localization experiments confirmed that Mef2 was localized inmitochondria. Northern blotting revealed that the deletion of mef2 did not affect the transcription of m RNAs encoded by the mitochondrialgenome. Western blotting showed that the deletion of mef2 resulted in decrease in the expression of Cox1,Cox3,Atp6,and Cob1 proteins. Insummary,Mef2 is a protein that is closely related to mitochondrial function and is involved in the translation of mitochondrial encoded proteinsCox1,Cox3,Atp6,and Cob1.
引文
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[16]Valente L,Tiranti V,Marsano RM,et al.Infantile encephalopathyand defective mitochondrial DNA translation in patientswith mutations of mitochondrial elongation factors EFG1 andEFTu[J].American Journal of Human Genetics,2007,80(1):44.
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[18]Greenberg RA,Sobhian B,Pathania S,et al.Multifactorialcontributions to an acute DNA damage response by BRCA1/BARD1-containing complexes[J].Genes&Development,2006,20(1):34.
[19]Chalfie M,Tu Y,Euskirchen G,et al.Green fluorescent protein asa marker for gene expression[J].Science,1994,263(5148):802.
[20]Christian B,Haque E,Spremulli L.Ribosome shifting or splitting:It is all up to the EF-G[J].Molecular Cell,2009,35(4):400-402.
[21]Maheshwari KK,Marzuki S.Defective assembly of themitochondrial ribosomes in yeast cells grown in the presence ofmitochondrial protein synthesis inhibitors[J].Biochimica etBiophysica Acta,1985,824(4):273-283.
[22]Callegari S,Gregory PA,Sykes MJ,et al.Polymorphisms inthe mitochondrial ribosome recycling factor EF-G2mt/MEF2compromise cell respiratory function and increase atorvastatintoxicity[J].PLo S Genetics,2011,8(6):e1002755.
[2]Lemaire C,Dujardin G.Preparation of respiratory chaincomplexes from Saccharomyces cerevisiae wild-type and mutantmitochondria[M].Humana Press,2008:65-81.
[3]H?jlund K,Wrzesinski K,Larsen PM,et al.Proteome analysisreveals phosphorylation of ATP synthase beta-subunit in humanskeletal muscle and proteins with potential roles in type 2diabetes[J].Journal of Biological Chemistry,2003,278(12):10436-10442.
[4]Moss DA,Bendall DS.Cyclic electron transport in chloroplasts.The Q-cycle and the site of action of antimycin[J].Biochimicaet Biophysica Acta(BBA)-Bioenergetics,1984,767(3):389-395.
[5]Sullivan LB,GUI DY,Hosios AM,et al.Supporting aspartatebiosynthesis is an essential function of respiration in proliferatingcells[J].Cell,2015,162(3):552-63.
[6]Zhao X,León IR,Bak S,et al.Phosphoproteome analysis offunctional mitochondria isolated from resting human muscle revealsextensive phosphorylation of inner membrane protein complexes andenzymes[J].Molecular&Cellular Proteomics,2011,10(1):M110.000299.
[7]马丽晶,徐勉.线粒体基因突变所致糖尿病发病机制及治疗进展[J].医学综述,2010,16(2):275-277.
[8]Scipioni L,Thompson W,Sijbrandij S,et al.An essential role ofthe mitochondrial electron transport chain in cell proliferation is toenable aspartate synthesis[J].Cell,2015,162(3):540-551.
[9]Neufeldcohen A,Robles MS,Aviram R,et al.Circadian control ofoscillations in mitochondrial rate-limiting enzymes and nutrientutilization by PERIOD proteins[J].Proceedings of the NationalAcademy of Sciences of the United States of America,2016,113(12):201519650.
[10]Kai SD,Fritz S,Fuchs F,et al.Genetic basis of mitochondrialfunction and morphology in Saccharomyces cerevisiae[J].Molecular Biology of the Cell,2002,13(3):847-853.
[11]Maheshwari KK,Marzuki S.Defective assembly of themitochondrial ribosomes in yeast cells grown in the presence ofmitochondrial protein synthesis inhibitors[J].Biochimica etBiophysica Acta,1985,824(4):273-283.
[12]Merz S,Westermann B.Genome-wide deletion mutant analysisreveals genes required for respiratory growth,mitochondrial genomemaintenance and mitochondrial protein synthesis in Saccharomyces cerevisiae[J].Genome Biology,2009,10(9):1-18.
[13]Rasmussen SW.A 37.5 kb region of yeast chromosome X includesthe SME1,MEF2,GSH1 and CSD3 genes,a TCP-1-related gene,an open reading frame similar to the DAL80 gene,and a t RNA(Arg)[J].Yeast,1995,11(9):873-883.
[14]Hammarsund M,Wilson W,Corcoran M,et al.Identification andcharacterization of two novel human mitochondrial elongation factorgenes,h EFG2,and h EFG1,phylogenetically conserved throughevolution[J].Human Genetics,2001,109(5):542-550.
[15]Tsuboi M,Morita H,Nozaki Y,et al.EF-G2mt is an exclusiverecycling factor in mammalian mitochondrial proteinsynthesis[J].Molecular Cell,2010,82(9):825-31.
[16]Valente L,Tiranti V,Marsano RM,et al.Infantile encephalopathyand defective mitochondrial DNA translation in patientswith mutations of mitochondrial elongation factors EFG1 andEFTu[J].American Journal of Human Genetics,2007,80(1):44.
[17]Sasarman F,Antonicka H,Shoubridge EA.The A3243G t RNALeu(UUR)MELAS mutation causes amino acid misincorporation anda combined respiratory chain assembly defect partially suppressedby overexpression of EFTu and EFG2[J].Human MolecularGenetics,2008,17(23):3697-3707.
[18]Greenberg RA,Sobhian B,Pathania S,et al.Multifactorialcontributions to an acute DNA damage response by BRCA1/BARD1-containing complexes[J].Genes&Development,2006,20(1):34.
[19]Chalfie M,Tu Y,Euskirchen G,et al.Green fluorescent protein asa marker for gene expression[J].Science,1994,263(5148):802.
[20]Christian B,Haque E,Spremulli L.Ribosome shifting or splitting:It is all up to the EF-G[J].Molecular Cell,2009,35(4):400-402.
[21]Maheshwari KK,Marzuki S.Defective assembly of themitochondrial ribosomes in yeast cells grown in the presence ofmitochondrial protein synthesis inhibitors[J].Biochimica etBiophysica Acta,1985,824(4):273-283.
[22]Callegari S,Gregory PA,Sykes MJ,et al.Polymorphisms inthe mitochondrial ribosome recycling factor EF-G2mt/MEF2compromise cell respiratory function and increase atorvastatintoxicity[J].PLo S Genetics,2011,8(6):e1002755.