Preparation Of Schizosaccharomyces Pombe Ppr10-Mpa1 Complex

Mitochondria,a self-replicating organelle with a bilayer membrane structure,is the main site for intracellular synthesis of ATP.The expression of the mitochondrial genome(mtDNA)is critical for mitochondrial function in Schizosaccharomyces pombe.It primarily encodes seven key subunits of the oxidative phosphorylation complex,two ribosomal subunits,25 tRNAs required for mitochondrial protein synthesis and RNA subunit of RNase P(rnpB).The PPR protein contains 2-30 PPR motifs consisting of degenerate 35 tandem repeating amino acids that bind to RNA.Regulation of the level of translation of mitochondrial gene expression is critical.In human and mouse,plant and budding yeast species,PPR proteins are involved in the transcriptional and post-transcriptional regulation of mtDNA-encoded gene expression.PPR protein specifically binds to RNA and is closely related to RNA 5'maturation,intron cleavage,RNA editing and stability.The laboratory uses Schizosaccharomyces pombe to study the function of PPR proteins.Our laboratory reports that Ppr10 can immunoprecipitate Mpa1,Ppr10 and Mpa1 are involved in the translation of mitochondrial proteins.However,the physiological significance of Ppr10and Mpa1 forming a complex is not clear.This paper carries out research on the above issues.Firstly,the interaction of Ppr10 and Mpa1 in living cells is detected by bimolecular fluorescence complementation(BiFC)method.Western blotting and fluorescence microscopy show that Ppr10 and Mpa1 interact directly in vivo.On the other hand,the key to studying the significance of the interaction between Ppr10 and Mpa1 is to obtain the Ppr10-Mpa1 complex and Ppr10.In this study,the Ppr10-Mpa1 complex and Ppr10 are expressed using the S.pombe ESP~?expression system.To obtain the Ppr10-Mpa1 complex,ppr10 and mpa1 are cloned into the pESP2 plasmid using the ESP~?expression system to achieve co-expression of the proteins Ppr10 and Mpa1 in S.pombe.The Ppr10-Mpa1complex is purified by FastPrep-24 instrument and GST affinity chromatography column.The complex basically meet the requirement of protein purity in vitro.As a control experiment,Ppr10 is expressed in S.pombe by the same method and GST-Ppr10 is purified.This study lays the foundation for the subsequent physiological study of the interaction between Ppr10 and Mpa1 in vitro,such as the use of thermal shift assay to detect whether Mpa1 promotes the stability of Ppr10.Atp4 in Schizosaccharomyces pombe is predicted to be a constituent protein of mitochondrial ATP synthase F0,but the specific function is not clear.The phenotype study shows that the growth of the strain after the knockdown of atp4 is significantly affected.Fluorescence microscopy revealed that atp4 is localized in the mitochondria.Deletion of Atp4 results in a dramatic decrease in the expression levels of the mtDNA-encoded proteins Cob1,Cox1,Cox2 and Atp6.The above studies indicate that atp4 is important for the stability of mitochondrial protein expression.