Study On The Regulation Of Leucine Metabolism By Deacetylase Hda1 In Saccharomyces Cerevisiae

Leucine is one of the most abundant essential amino acids in human that plays roles in protein synthesis and degradation as a regulator,regulation of energy metabolism and substrate supplying for protein synthesis.It is very important to maintain leucine homeostasis for the organisms' normal metabolism due to leucine deficiency leads to serious growth defect.There are a variety of protein modification types in the organism,which regulate various physiological and pathological processes of the cells.The acetylation is an important post-translational modification of proteins attracting extensive attention,involving a number of important physiological functions such as transcriptional regulation,signaling pathway,metabolism,protein activity and pathogenic microbial infection.In the investigation process of nutrituion metabolism,for the first time,we have discovered deacetylase Hda1 is related in regulation of leucine metabolism.In this thesis,Saccharomyces cerevisiae was used as a model organism to study the regulation of deacetylase Hda1 in leucine metabolism by molecular biology and metabolomics.The experimental results show that the HDA1 knockout strain shows growth defect in SC medium and its leucine content is significantly reduced compared with that of wild type strain.The hda1? cells resume growth in SC media,supplied with excess leucine,but other nitrogen sources such as amino acids display no recovery effect.The hda1? mutant strain was screened by spontaneous mutation on SC medium plate,and the mutant leucine content restores to the wild type level.Whole-genome sequencing of spontaneous mutant strains revealed point mutations in the permease SEO1(Seo1-A242G),and high-affinity leucine transporter BAP2(Bap2-D573N),while turnover protein COS4 indicates expression level change.Overexpressions of SEO1,BAP2 and COS4 in hda1? cells restore growth on SC medium,respectively.The m RNA expression level and Lac-Z reporter gene assay showed that the m RNA expression levels of BAP2 and COS4 in hda1? cells are significantly higher than those of wild type;while leucine induces the expression of SEO1,BAP2 and COS4 m RNA levels.The protein levels of SEO1 and mutant SEO1 suggest no dramatical difference between wild-type and hda1? cells by western blotting assay.The results of this study demonstrate that the deacetylase Hda1 regulates leucine uptake by regulating SEO1,BAP2 and COS4 resulting in affecting cell growth.The clarification of this mechanism will provide a new interpretation and insight for the auxotrophic and leucine related diseases.