Functional Study Of Fzo1 Protein In Schizosaccharomyces Pombe

Schizosaccharomyces pombe was a single cell organism,which could be easily manipulated by genes.Schizosaccharomyces pombe was a unique yeast,which shared many characteristics with higher eukaryotes,making it an informative and accurate model system for eukaryotic molecular biology research.Mitochondria are semi-autonomous organelles that contain their own genome(mt DNA)and encode a small number of mitochondrial proteins.Mitochondria are essential in a variety of metabolic processes,including oxidative phosphorylation,amino acid metabolism,and the formation of iron-sulfur clusters.Mitochondria are highly dynamic organelles that constantly fuse and divide.Mitochondrial fusion and fission are needed by the cell so that the mitochondria can best adapt to the metabolic needs of the cell.Meanwhile,mitochondrial dynamics is also considered to resist aging and constitute the mechanism of organelle quality control.FZO1 protein plays the most critical role in the mitochondrial fusion process of Budding yeast.while homologous protein Fzo1 in S.pombe is rarely studied.Therefore,this paper focuses on the further study of the function of Fzo1 protein,which is divided into two parts: On the one hand,to study the effect of Fzo1 protein on mitochondrial function,We first expressed the exogenous p YJ19-fzo1-GFP recombinant plasmid in fission yeast cells,and the Fzo1 protein was clearly localized in mitochondria by fluorescence co-localization experiments,We then found that the fzo1 knockout did not grow properly in glycerol-based carbon media,suggesting that the ?fzo1 strain is a respiratory-deficient strain.The combination of full-length point-in-time phenotyping experiments and growth curve measurements showed that the ?fzo1 strain grew more slowly than the wild type in late culture,suggesting that the fzo1 knockout affected the normal growth lifespan of the yeast.The expression levels of the mitochondrial respiratory chain proteins of the ?fzo1 strain were significantly reduced by measuring the expression levels of Cob1,Cox1,Cox3 and Atp6 proteins,while the those mitochondrial respiratory chain protein RNA level was not affected;On the other hand,HA tags were added to the C-terminal of Fzo1 protein through homologous recombination,Fzo1 protein expression levels were then detected by immunoblotting at different time points,and the results showed that Fzo1 protein expression was down-regulated in yeast at late growth stages.The late degradation of Fzo1 protein was not found in yeast cells treated with proteasome inhibitors and in the ?rsv2/Fzo1-HA strain,and the down-regulation of Fzo1 protein was determined to be via the ubiquitin-proteasome pathway.To further investigate the specific ubiquitination pathway involved in the downregulation of Fzo1 protein,We examined Fzo1 protein expression in 10 E2 ubiquitin-conjugating enzyme knockout strains and found that Fzo1 protein was not degraded late in the ?ubc8/Fzo1-HA strain,suggesting that Ubc8 E2 ubiquitin-conjugating enzyme is involved in the ubiquitin-proteasome degradation pathway of Fzo1 protein,and we found that Fzo1 protein was not degraded upon overexpression of Fzo1 protein and had no effect on Ubc8 protein expression was not affected.In this paper,the results of the functional study of Fzo1 protein are divided into two main parts.Firstly,we found that Fzo1 protein is important for normal mitochondrial respiration,and secondly,we found that the expression of Fzo1 protein is down-regulated in yeast at late growth stage,and the down-regulation of Fzo1 protein expression is through the ubiquitin-proteasome system,Ubc8 E2ubiquitin-binding enzyme is involved in the degradation of Fzo1 protein.These results provide a theoretical basis for further understanding the mitochondrial fusion protein Fzo1 in S.pombe.