Study On The Effect Of Ppr10 Protein Of Schizosaccharomyces Pombe On The Translation Of MtDNA-encoded Protein

Mitochondria are the major sites of ATP production in eukaryotes and the mitochondrial genome?mtDNA?-encoded proteins are important subunits of the mitochondrial respiratory chain complex.PPR protein refers to a protein containing two or more tandem repeats of the PPR motif,while PPR motif consists of 35 amino acid residues.The Ppr10 protein is critical to mitochondrial translation in Schizosaccharomyces pombe.In previous studies,we found deletion of ppr10 in the strains which contain mitochondrial introns?yHL6381?not only affected the level of RNA encoded by mitochondrial genome,but also impaired the synthesis of proteins encoded by mitochondrial genome in varying degrees.Therefore,this article mainly explores how Ppr10 protein is involved in mitochondrial genome translation.P3 is a strain that does not contain mitochondrial introns,and its mtDNA-encoded mRNAs do not require intron splicing.P3 strains can help us to explain how Ppr10 protein participates in the translation of mitochondrial genome.The mtDNA of S.pombe contains only three introns.Two of the type I introns in cox1 are cox1 I 1b and cox1 I 2b and one type II intron in cob1 is cob1 I 1,cox1 I 1b encodes DNA endonuclease and mature enzyme and cox1 I 2b encodes DNA endonuclease,the protein encoded by cob1 I 1 has reverse transcriptase activity and mature enzyme activity and participates in self-splicing.We constructed?ppr10/P3 strains.The dot-loop experiment shows that the growth of?ppr10/P3 strains was inhibited in non-fermented medium,which indicated that Ppr10 was still closely related to the function of mitochondria in the absence of mitochondrial introns.The results of qRT-PCR and Northern blot showed that the mRNAs levels of cox1 and cob1 in?ppr10/P3 strains were not affected compared with P3 strains.By RT-PCR experiments,the deletion of ppr10 in P3 strains affects the cleavage of cox1,cob1 mRNAs.On the other hand,we found that the deletion of ppr10 affected the synthesis of the mtDNA-encoded protein through[³⁵S]-Protein Labeling of mtDNA-encoded proteins in vivo.Western blotting results showed that Cob1,Cox1,Cox2,Cox3 and Atp6 were almost undetected and the homeostasis of Cox4 also decreased significantly.Therefore,the deletion of ppr10 affects the synthesis of mitochondrial respiratory chain subunits and consequently the function of mitochondrial.Ppr10 is an activator of the entire mitochondrial protein translation machinery.Deletion of the ppr10 gene affected the translation of mitochondrial proteins and therefore does not normally encode the mature enzymes required for splicing of mitochondrial mRNAs.The function of Ppr10 in mitochondrial translation was further verified.