The Study Of Relationship Between Yeast MBF1 And BMH2 In Metabolic Regulat Ion

Multiprotein bridging factor 1?MBF1?is a evolutionarily conserved non-DNA binding transcription coactivator in archaea and eukaryotes and could enhance transcription by bridging different transcription factors and TATA-box binding protein TBP. However the reported researches mainly focused on the functions of MBF1 in plants and animals, and little was known about the functions about microbial MBF1. It was found that BMH2 was one protein associative with yMBF1,and both are involved in the regulation of stress responses. While so far no further study about their interactions and the relationship between the life of yeast have be reported.In order to know the functions of yMBF1 and BMH2 in metabolic regulation and the relationship of them.By GST-pull down technique, this study identified the potential proteins interacting with yMBF1 and BMH2 in Saccharomyces cerevisiae S288 c, respectively. yMBF1 with its target proteins involved in the transcriptional regulation of meiosis?cell cycle and spotulation; and also related to the oxidative phosphorlation and oxidative stress and so on.BMH2 extensively involved in DNA replication,transcription,translation,glucosemetabolism,cytoskeleton,endocytosis,meio sis,cell cycle, growth and other regulation.The pull down results showed yMBF1 interacts with BMH2 in vitro.In addition, in this study, we have identified several potential transcription factors interacting with yMBF1,which provided a basis on the study of transcriptional regulation mechanism that yMBF1 and BMH2 involved.In order to understand the relationship of yMBF1 and BMH2 in metabolic regulation network, it was cross-analysised that the proteins interacting with BMH2 and yMBF1, in which 43 proteins were found that could both interact with yMBF1 and BMH2.The function of these proteins involved in chromosome structure, transcription, oxidative phosphorylation, stress response, cell cycle and so on.To understand functions of BMH2 and yMBF1 in heat stress, this study observed the growth of bmh?-? /?+?, mbf?-? /?+? strains under 55?.And the result showed that yMBF1 and BMH2 are both involved in the regulation of the heat stress response,but have a relationship of mutual restraint:MBF1 is necessary for yeast against heat stress and BMH2 restricted thermal stress tolerance for yeast.In the early study, we found that yMBF1 can improve C u²⁺ tolerance for yeast. In this study we found a C u2 + associated protein ICS2 is one of BMH2 the target protein; and further study showed that deletions of bmh2 could reduce C u²⁺ tolerance of yeast.Transcriptome sequencing analysis revealed that the lack of mbf1 caused a down-regulation expression of ics2, which may be regulated by mbf1. In the stress study, another yeast 14-3-3protein BMH1 which has 93% homology to BMH2, was as control and found that they showed obvious differences in heat stress and C u²⁺ stress. In the heat stress, they showed a negative correlation regulation,while BMH2 played a major role; and in the Cu²⁺ Stress, BMH2 showed a positive correlation, whereas BMH1 was kind of negative correlation.In summary, this study have a preliminary study about the functions of yMBF1 and BMH2 in metabolic regulation by proteomic analysis and discussed the potential relation in metabolic regulation; Futher the roles of yMBF1 and BMH2 were verified in stress response.At last we screened two monoclonal yMBF1 antibodies by antibody microarray technology, which were named P12 and P14, corresponding to epitope RGNNIGSPLGAPKK; and we also purified BMH2 monoclonal antibodies from mouse ascites.These contribute to a further study in vivo about functions of yMBF1 and BMH2 in metabolic regulation and their interactions.