Genome-wide Screening For Genes Associated With NaCl-resistance And The Mechanism Research In Fission Yeast

Objective:Ions are essential for the growth of cells in both prokaryotes and eukaryotes,but excessive amounts of ions may be toxic to cells.Maintaining intracellular ionic homeostasis is a crucial cellular function.In the human body,the imbalance in sodium ion homeostasis caused by genetic defects increases the risk of developing hypertension.In agriculture,salt stress will inhibit crops growth and kill the crops,whereas the use of genetic engineering to modify proteins involved in sodium homeostasis may lead to increased salt resistance in crops.Therefore,studies of sodium ion homeostasis have great significance in many fields including medicine,microbiology and agriculture.Studies on ion homeostasis in yeast cells have not only helped us to understand how cells properly regulate metal ion homeostasis,but also contributed to the identification of homologous systems in humans,which would provide ideas and clues for the study of human diseases related to disruption of ion homeostasis in human cells.Methods:In this study,the model organism Schizosaccharomyces pombe was used to screen and identify gene deletion mutants exhibiting NaCl resistance in the whole genome.Screening and identification of gene deletion mutants resistant to NaCl were performed by spot assay.Bioinformatics methods were used to analyze gene function and biological processes.Defects in membrane trafficking function of gene deletion mutants were identified by fluorescence microscopy.Calcineurin activity was examined in living cells to study the possible existence of gene deletion mutants relevant mechanisms.The cytoplasmic calcium concentration was determined by using aequorin.And once again validated by spot assay to obtain phenotypes of deletion mutants for other drugs and ions.Results:We identified 34 NaCl-resistant strains.Among them,the largest group that consists of 10 genes related to membrane trafficking and 7 out of 10 genes are ESCRT proteins.The ESCRT mutants exhibiting sodium resistance also exhibit defects in vacuolar fusion,and observed obstacles in the transfer process of the Golgi-to-vacuolar.To further understand the role of the ESCRT system in various ion homeostasis,we examined sensitivity of these ESCRT mutants to various cation ions other than NaCl,including LiCl,CaCl₂,CoCl₂,MgCl₂,NiSO₄ and MnCl₂.While these ESCRT mutants showed resistance to LiCl,CoCl₂ and MgCl₂,they showed sensitivity to CaCl₂,NiSO₄ and MnCl₂.Then we examined sensitivity of these ESCRT mutants to various drugs which are known to inhibit the growth of fission yeast cells.While these ESCRT mutants were more or equally sensitive to most of the drugs tested as compared to the wild type,they showed resistance to some drugs such as fluorouracil,tamoxifen and amiodarone.We further studied calcineurin activity in ESCRT mutants using the calcineurin-dependent response element?CDRE?and found that NaCl concentration-dependently enhances calcineurin activity in cells,and the basis of ESCRT mutants were more active than wild type.And the concentration of cytoplasmic calcium in the ESCRT mutants were higher than that in the wild type.Conclusions:1.We screened and identified 34 NaCl resistance gene deletion mutants.Among them,the largest one gene functional group consists of 10 genes involved in membrane transport,and 7 out of 10 genes are part of the ESCRT systems.2.The ESCRT mutants showed defects of vacuolar fusion and Golgi to vacuolar transport process.3.The ESCRT system plays an important role in drug/ion sensitivity.4.In ESCRT mutants,the calcineurin activity were higher than the wild type.5.The concentration of cytoplasmic calcium in the ESCRT mutants were higher than that in the wild type.