Research Of Kub3 In Regulating The Assembly Of Yeast Mitochondrial ATP Synthase And Embryonic Lung Development Of Mouse

Kub3 was originally reported to be a Ku70-binding protein.Kub3 and its homologous proteins exist in yeast,Arabidopsis thaliana,zebrafish,mouse,and humans,and is conserved from yeast to humans.Previous studies have found that Atp23 p,yeast homologous protein of mammalian Kub3,was located in the inner membrane of mitochondria and had dual functions including processing of yeast Atp6 p precursor and assisting assembly of yeast mitochondrial ATP synthase.The metalloprotease motif(HEXXH)of Atp23 p is responsible for its function of processing Atp6 p precursor.However,the regions of Atp23 p responsible for assisting assembly of mitochondrial ATP synthase remains unknown.Moreover,nuclear-encoded Atp10 p was previously shown to regulate assisting yeast Atp6 p assembly to mitochondrial ATP synthase,and extra expression of Atp23 p could partially rescue atp10 null mutant,which indicated that Atp23 p and Atp10 p could coordinate to regulate assembly of yeast mitochondrial ATP synthase,but the mechanism of this synergistic regulation remains to be further studied.In addition,the metalloprotease motif also exists in mammalian Kub3 protein sequences,whereas,subunit6 in mammalian cells was not synthesized as a precursor.Does Kub3 in mammalian cells have functions different from those of Atp23 p in yeast? The physiological functions of Kub3 in mammalian cells remain to be elucidated.Firstly,the regions of Atp23 p,yeast homologous protein of Kub3,responsible for the function of assisting assembly of yeast mitochondrial ATP synthase,and the mechanism of Atp23 p and Atp10 p coordinating to regulate the assembly of mitochondrial ATP synthase were investigated.Four conserved sequence regions were selected by alignmenting Atp23 p protein sequences in different fungi and different atp23 deletion mutants were constructed and characterized.The results showed that the residues 112-115(LRDK)of Atp23 p were required for its function in assisting assembly of the synthase.Rescue experiments by transforming atp10 null mutant with ATP23 mutant plasmids(without processing function or without assembly function)showed that both functions of Atp23 p,including function in processing of Atp6 p precursor and function in assisting assembly of the ATP synthase,were required for the rescue of atp10 null mutant.Further research indicated that the stability of subunit 6 increased in atp10 null mutant strain upon overexpressing Atp23 p.Co-IP(Coimmunoprecipitation)and BN-PAGE(Blue-native polyacrylamide gel electrophoresis)experiments indicated that Atp23 p and Atp10 p were physically associated with each other and formed protein complex.Furthermore,atp10 null mutant showed leaky growth on respiratory substrates after 72 hours,and showed partial mitochondrial ATP synthase assembly and increased expression of Atp23 p.These results indicated that overexpression of Atp23 p could partially rescue atp10 null mutant by increasing the stability of Atp6 p,and Atp23 p and Atp10 p jointly regulate mitochondrial ATP synthase assembly in vivo by forming a protein complex.In atp10 null mutant,ATP10 deficiency could be partially complemented with increased expression of Atp23 p and lead to partial assembly of mitochondrial ATPase.The physiological roles of Kub3 in mammalian cells were further explored.By generating Kub3 gene knockout(KO)mice,it showed that KO mice died between E18.5and before birth or died within 6 hours after birth.Tissue distribution assay by Western-blot showed that Kub3 was expressed in most key organs in mice,especially with high expression in lung.Compared with lungs of WT at E18.5,Kub3-KO lungs displayed less amounts of alveoli,collapse of alveolar space and increased thickness of mesenchymal septa,which suggested that Kub3 deletion results in abnormal lung development at E18.5.Moreover,it was found that the abnormalities of KO lungs were obvious from the late microtubule stage(E17.5),suggesting that lung defects of KO were evident from late embryonic lung development.Cell proliferation and apoptosis in E18.5 embryonic lungs were analyzed by PCNA and TUNEL immunofluorescence imaging and Western-blotting experiments.The results showed that no differences in cell differentiation and apopotosis were detected between Kub3-KO lungs with WT lungs at E18.5.However,the differentiation of alveolar epithelial cells was impaired in KO lungs at E18.5 by detecting the marker proteins of type I epithelial cells and type II epithelial cells.Further PAS(Periodic acid Schiff)staining and transmission electron microscopy analysis showed an excessive accumulation of glycogen and reduction of lamellarbodys in E18.5 KO lungs,which indicated that Kub3 deletion leads to aberration of maturation of type II epithelial cells.The expression of marker proteins of the critical regulatory signaling pathways,including WNT,SHH(Sonic Hedgehog),BMP(Bone Morphogenesis Protein),Notch,and FGF(Fibroblast Growth Factor)signaling,were further investigated,and the results showed that the expression of marker proteins of Wnt,Shh,Bmp and Notch signaling had no obvious changes between KO and WT lungs at E18.5,whereas Kub3 deficiency causes abnormal FGF signaling at E18.5,which showed increased Fgf10 expression and RASMAPK signaling in Kub3-KO lung at E18.5.Taking all the data together,this study demonstrated that the residues 112-115(LRDK)of Atp23 p were required for its function in assisting assembly of yeast mitochondrial synthase.Meanwhile,Atp23 p and Atp10 p coordinate the assembly of yeast mitochondrial ATP synthase by forming a protein complex and promoting partial mitochondrial ATP synthase assembly by increasing Atp23 p expression in the absence of Atp10 p,which advanced understanding of the functions of yeast Atp23 p and the mechanism of assembly of yeast mitochondrial ATP synthase.Moreover,by generating Kub3 gene knockout mice and revealed that Kub3 gene knockout mice died between E18.5 and before birth or died within 6 hours after birth,and found that Kub3-KO results in abnormal lung development at E18.5.Further studies showed that the abnormal lung development results from aberrant differentiation of alveolar epithelial cells and immaturation of type II epithelial cells due to disturbed FGF signaling pathway.These results uncovered an essential role of Kub3 in mice prenatal lung development and improved the function research of Kub3,which advances our knowledge of regulatory factors in embryonic lung development and provides a new thought for exploring the mechanism of disease related to perinatal lung development.