The Mechanism Of Subcellular Localization In Mitochondria And Chaperon Function Of BmHSP20.8in Bombyx Mori

Heat stress proteins, which are also called heat shock proteins, are abundant and ubiquitous in almost all organisms, from bacteria to the mammal. They are either expressed under physiological conditions, or induced in response to adverse environmental stress and play an important role in protecting cells from being damaged. The studies on HSP in Bombyx mori also have many new progress after the completion of genomic sequencing, but are mainly focused on bioinformatic analysis, gene cloning and gene expression. Few research has been carried out in the function, regulation mechanism. The ORF of BmHSP20.8contained561bp, encoding a protein of186amino acid residues with a predicted molecular weight of20.8kDa. Prediction of subcellular localization indicated that BmHSP20.8might be distributed in mitochondria with51%possibility, in the other position with13%possibility and secreted to the outside of the cell with29%possibility. In this study, we mainly studied the subcellular localization mechanism and molecular chaperone activity of BmHSP20.8.In order to identify the subcellular localization of BmHSP20.8, we constructed three recombinant vectors, plEX-1-BmHSP20.8, pIEX-1-BmHSP20.8-EGFP and pIEX-1-EGFP-BmHSP20.8. The recombinant vectors were transfected into BmN cells, respectively, and then, cytoplasmic and mitochondrial proteins were extracted at72hours after transfection. Western blot showed BmHSP20.8was only existed in mitochondria. Futhermore, the fusion protein of BmHSP20.8and EGFP was only detected in cytoplasm when BmHSP20.8was located in N-terminal of the fusion protein, and was detected in both cytoplasm and mitochondria when BmHSP20.8was located in C-terminal. These results show that the mitochondrial localization signal domain of BmHSP20.8is located in the C terminus of BmHSP20.8. In order to find the mitochondrial localization signal area of BmHSP20.8, we cloned four truncated recombinant vectors and transfected these vectors to BmN cells, respectively. The western blot analysis of the cytoplasmic and mitochondrial proteins showed that the mitochondrial localization signal domain of BmHSP20.8is located between amino acids143to186in this protein.Molecular chaperone activity is one of the important functions of HSP. In order to detect whether BmHSP20.8has molecular chaperone activity, we constructed pETduet-HIS-SUMO-BmHSP20.8vector to express BmHSP20.8. The experiment that BmHSP20.8was degraded by trypsin indicated BmHSP20.8could not form large oligomers. Through the synthase (CS) thermal aggregation experiment we found that BmHSP20.8protein had molecular chaperone activity. BmHSP20.8is located in mitochondria, and as a molecular chaperone it may interact with other mitochondrial proteins. We transfected pIEX-1-BmHSP20.8vector to BmN cells and expressed it successfully. Western blot analysis showed that the recombinant HIS-BmHSP20.8protein was successfully detected in the co-immunoprecipitation.The SDS-PAGE analysis showed that there was a specific protein associated with BmHSP20.8among100kDa to130kDa, which laid a foundation for identification of the proteins interacted with BmHSP20.8.