The Roles Of MTM1 Gene On Nickel Metabolism In Saccharomyces Cerevisiae Cell

Nickel element is very rich in the earth,which is second only to silicon,oxygen,iron and magnesium.With the wide application of nickel in industrial production and human life,nickel pollution to the environment has been paid more and more attention to studying around the world.Although nickel is widely distributed in nature,it is extremely low in the human body,and excessive nickel can cause serious toxic effects on organisms.Nickel not only plays an important role in energy metabolism and nitrogen cycle,but also is an essential element for the growth and development of organisms.Nickel ion has been more comprehensively studied as a cofactor for various enzymes in prokaryote,but the metabolic mechanism of nickel in eukaryote is not clear and needs further analysis.Therefore,the research of nickel metabolism is significant from both the life mechanism studying and solving the realistic environmental nickel pollution.This project used eukaryotic Saccharomyces cerevisiae cells as a research model and utilized molecular biology and genetics methods to fully clarify the metabolic mechanism of nickel ions.Spontaneous mutant strains were generated with excess nickel culture and whole genome DNA was sequenced to discover nickel metabolism related genes by bioinformatics.The interaction mechanisms between nickel related genes and nickel ion were elucidated.The growth assay shows that the MTM1 gene knockout strain(mtm1?)grow significantly better than that of wild type strain under high nickel conditions,accordingly,the overexpression of MTM1 gene makes it sensitive to nickel.The ICP-MS data indicates that the content of nickel in the mtm1? strain is not changed with that of wild type strain,while the nickel content in the mitochondria of knockout strain is dramatically lower than that of wild type strain when treated with nickel.The in vivo experiments demonstrate that nickel ions can restore the activity of manganese-containing superoxide dismutase(SOD2),but there is the same change when nickel treatment in vitro.The experiments of q RT-PCR and ?-galactosidasereporter gene suggest that the expression level of SOD2 m RNA in mtm1? strain is higher than that of wild type strain,however,nickel treatments down-regulate transcriptional level of SOD2 m RNA in mtm1? strain.MTM1 gene knockout increases oxidative stress in cells,but increased oxidative stress does not affect the resistance of mtm1 ? strain to nickel.Western blotting suggests that excess nickel ion treatment distinctly reduces the stability of Mtm1 protein,while green fluorescent protein GFP labeling reveals that nickel ion does not change the intracellular localization of Mtm1 protein.This study confirms that the MTM1 gene in Saccharomyces cerevisiae regulates the distribution of nickel ions in mitochondria,and nickel ions restore the activity of SOD2 leading to mtm1? strain high resistance to nickel ion.Meanwhile,the localization of Mtm1 protein in cells is not changed by excess nickel ion,but significantly decrease the stability of Mtm1 protein.Our research proves that MTM1 gene is associated with the nickel metabolism in eukaryote for the first time and provides a theoretical basis for the treatment of diseases related to nickel metabolism.