Study On Rescuing Of Deletion Of Yeast ATP23 By COX1 Site-directed Mutation

Mitochondrial ATP synthase is a key enzyme catalyzing the synthesis of ATP in eukaryotic cell.Many nuclear genes encoded proteins are involved in regulation of its biogenesis and assembly.The previous study in our lab found that Atp23 p was a metalloprotease that was necessary for the maturation of subunit 6 of mitochondrial ATP synthase and for assembly of mitochondrial ATP synthase in Saccharomyces cerevisiae.When yeast ATP23 is absent,the newly synthesized subunit 6 cannot be changed into mature Atp6 p,and the mitochondrial ATP synthase cannot be assembled.However,it remains unkown whether there are genes functional complementary to Atp23 p to rescue ATP23 deletion.Therefore,it is of great significance exploring the genes functional complementary to Atp23 p to find methods of rescuing ATP23 deletion,which may also play important roles in understanding the functional relationship among mitochondrial proteins.Moreover,protein sequences of Atp23 p are highly conserved from yeast to human.If mutation of ATP23 leads to human disease,exploration of ATP23 complementary genes might provide new ideas and methods for the research and therapy of the related mitochondrial diseases.In order to search for genes functional complementary to yeast ATP23,we screened and purified the revertants of ATP23 null mutant,and found that the mitochondrial ATP synthase in these revertants could be partially assembled.Further analysis showed that the revertible function of the revertants was caused by the T1121 G mutation of coding sequence of mitochondrial DNA COX1 which led to the amino acid mutation of Val374 Gly.Next,this research constructed exogenous COX1-HA hybrid plasmid containing T1121 G mutation and transformed it into ATP23 null mutant,and found that the mitochondrial ATP synthase of the transformants could be partially assembled.This result further verified that COX1 T1121 G mutation could partially restore deficiency of mitochondrial ATP synthase because of ATP23 deletion.By alignment Cox1 p protein sequences from difference species,it was found that the Valine at p.Val374 site was highly conserved from yeast to human,and no significant effect on the tertiary structure of Cox1 p protein was found by analzing with the protein structure prediction software.By Co-immunoprecipitation(Co-IP)experiment,the result showed that Atp23 p and Cox1 p did not form protein complex to achieve the functional complementation.In addition,Atp6 p precursor was stable in revertants,but it degraded in ATP23 null mutant.Our further study revealed that the stability of the Atp6 p precursor in the revertants increased,and the increased stability of Atp6 p was not caused by increased proteins expression level of Nam1 p which played an important role in Atp6p's stability or of molecular chaperone Atp10 p which could assist Atp6 p to assemble into mitochondrial ATP synthase.Therefore,this study revealed that the coding sequence T1121 G mutation(resulting in the coding amino acid Val374 Gly mutation)of yeast COX1 gene which coded mitochondrial respiratory chain cytochrome C oxidase subunit I could partially restore unassembly of mitochondrial ATP synthase in ATP23 null mutant by increasing the stability of Atp6 p.Therefore,this study found the method to partially rescue yeast ATP23 deltion mutation and revealed the functional links between the mitochondrial ATP synthase and the cytochrome C oxidase complex on the mitochondrial respiratory chain,which provided a deeper understanding of relationship among protein complex of the yeast mitochondrial respiratory chain.