The Regulating Function Of MBF1s On The Stress Response Of Primary Eukocytes Affected By 14-3-3 Proteins

Multiprotein bridge factor 1(MBF1)is a transcriptional coactivator evolutionarily conserved in animals,plants and fungi.Through bridging transcriptional factor and TATA-binding protein,MBF1 has been reported to play an important role in the transcriptional regulation of multiple stress-responsive genes,and in the responsiveness to stress.Studies of MBF1 in primary eukaryotes at present are mainly focused on its interaction with transcriptional factors.There is not report yet regarding the role of MBF1 in the copper(Cu)stress response,especially how MBF1 is regulated by 14-3-3 protein.Physarum MBF1(PMBF1)is a protein capable of interacting with 14-3-3 protein in primitive eukaryote Physarum polycephalum(P14-3-3),the role of which,however,remains to be elucidated.1.To study the function of PMBF1 and MBF1 from yeast(Mbf1p),we first tested the ability of PMBF1 in response to stress by recombinant DNA technology.Next we developed Mbf1p-deficient yeast strain mbf1p? and PMBF1-rescued strain mbf1p?+pPMBF1 through gene knockout technique,and performed proteomic and transcriptomic analysis of these strains under Cu stress.A number of genes and pathways involved in the regulation of PMBF1 and Mbf1 p under Cu stress were identified.It has been reported that there is interaction between Mbf1 p and yeast 14-3-3 protein Bmh1 and Bmh2.We had described previously that there was interaction between PMBF1 and P14-3-3.To elucidate the effect of P14-3-3 on MBF1 in stress response,we investigated the peptides and key amino acids involved in the interaction between PMBF1 and P14-3-3,Mbf1 p and Bmh1,and Mbf1 p and Bmh2.Finally,we developed P14-3-3-deficient yeast strain and P14-3-3-rescued strain,and assessed the role of P14-3-3 in response to stress.The results are described below:1.Recombination expression of PMBF1 enhanced the response of E.coli under NaCl stress,and the response of yeast under Cu,heat,and oxidative stress,indicating that PMBF1,with similar function as Mbf1 p in stress response,is a member of MBF1 family.The results of Two-Dimensional Mass Spectrometry(2D-MS)showed that the up-regulated proteins were involved in metabolism,while the down-regulated ones were involved in stress response,indicating that yeast responses to Cu stress by enhancing its metabolism.Additionally,the proteins involved in Cu stress response in yeast were also involved in several aspects including gene replication,transcription,translation and glucose metabolism,and signaling transduction,among which the peroxisomal pathway is the main one under Cu stress.2.To investigate the mechanism by which Mbf1 p responses to Cu stress,we performed transcriptomic analysis of Mbf1p-deficient yeast strain under Cu stress.The results showed that there were 567 genes deregulated under Cu stress.Among them,120 genes were up-regulated,while 447 genes were down-regulated,which was generally consistent with that available in 2D-MS studies.These deregulated proteins under Cu stress were involved in the DNA replication,transcriptional regulation,ribosomal large and small subunits,and oxidative phosphorylation.Oxidative phosphorylation pathway was one of the main pathways involved in Mbf1 p regulation.Additionally,cytochrome c oxidase subunit I(COXI)may be the same member between Cu stress response pathway and Mbf1 p regulatory pathway.These results indicated that oxidation was the main damage to yeast under Cu stress,and oxidative phosphorylation was the key pathway through which Mbf1 p mediated stress response.3.The results of protein-protein interaction indicated that Mbf1 p interacted with Bmh1,but not with Bmh2.ILe78 in PMBF1 and Pro107 in Bmh1 are the key residues responsible for the interaction.Moreover,Ser190 in P14-3-3 is the key phosphorylation site in the interaction with PMBF1.It shares a common feature that the key residues for interaction between PMBF1 and P14-3-3,and Mbf1 p and P14-3-3 all belong to hydrophobic amino acids.4.Overexpression of P14-3-3 protein enhanced the sensitivity of yeast under heat stress.Deprivation of yeast 14-3-3 proteins Bmh1 or Bmh2 increased the yeast tolerance upon high temperature,whereas introducing P14-3-3 gene into the deficient strain rescued the sensitivity of yeast to heat stress,indicating that P14-3-3 has similar function as yeast 14-3-3 protein.5.Introducing PMBF1 gene rescued the tolerance of Mbf1p-deficient strain under heat stress.Additionally,introducing PMBF1 gene not only rescued,but also enhanced the tolerance of mbf1p?+rad24? strain under heat stress,indicating that PMBF1 plays a similar role as Mbf1 p in stress response.6.Simultaneously introducing PMBF1 and P14-3-3 genes not only rescued,but also enhanced the tolerance of mbf1p?+rad24? strain under heat stress,when compared with the control yeast,indicating that the responses of PMBF1 to stress is dependent on its interaction with P14-3-3 protein,and that PMBF1 was regulated by P14-3-3.This study lays a solid foundation for further understanding the mechanism by which MBF1 responses to stress in primary eukaryotes,and the regulatory effects of 14-3-3 on MPF1 in stress response.It is also of great significance for enhancing the capability of eukaryotic microbes to respond to and survive stress,and thus promoting its application.