SILAC Based Quantitative Proteomics Study Reveals The Regulation Mechanism And Function Of Ubiquitination On Lsb1 In Yeast

Ubiquitin-proteasome system(UPS)is the major proteins degradation machinery with high specificity in eukaryotic cells,which precisely regulates proteins abundance and involves in almost all of the cellular process such as cell cycle,immune response and signal transduction.Protein ubiquitination requires E1-E2-E3 cascade reaction and then transfer ubiquitin(Ub)to protein substrate on varied lysine resides,resulting in macroheterogeneity of protein post-translation modification.The attached ubiquitin can be further modified by the following ubiquitins at different lysine residues to generate ubiquitin chains.Deubiquitinases(DUBs)is an hydrolase that can hydrolyze ubiquitin chains or mono-ub from substrate to reverse the ubiquitination process tomaintain the balance of UPS.The disorder of this reversing process may result severe disease in human being,indicating the importance of this process.However there is rare examples suitable for dissecting the detail regulating process because of technical barrier.In previous research,we systematically evaluated the specificity of DUBs on UbC and ubiquitin linkages in Saceharomyces cerevisiae and found that Ubp2 showed strong preference for protein substrates with K63 ubiquitin linkage.Lsb1 is one of these substrates ubiquitinated on both of the K41 and K79.To completely characterize the ubiquitinated forms of this protein,we tagged 6×his and biotin on the C terminal of LSB1 and tandemly purified(TAP)Lsb1 with Ni-NTA and Streptavidin.SDS-PAGE analysis showed that we got the pure ubiquitination Lsb1 through TAP,indicating our TAP strategies is working.Through LC-MS/MS analysis,we successfully identified Lsb1 and it`s mainly ubiquitin sites,K41 and K79,which was consistent with before research.At the same time,we identified 4 new ubiquitin site,which was K37,K85,K98 and K108.Employing site mutant-SILAC quantificationstrategy,we found that K41 and K79 site happened both K48 and K63-linked chain.And K41 site mainly ubiquitinated by K63-linked chain,while K79 site mainly happened K48-linked chain.In summary,the specific ubiquitin site of Lsb1 happened K48 or K63-linked chain,and Ubp2,Ubp3 and Ubp14 regulated it`s ubiquitin status.In order to expound the molecular mechanism of Ubp2 regulation Lsb1,we do the SILAC-Protein-Protein Interacting research of Lsb1 specific UbC.Through SILAC quantification and strict filter,we got 151 credible interactions of Lsb1.The biology process analysis results of Lsb1 interactions showed that these proteins mainly involved in cellular amino acid metabolic process,protein folding process,cellular ion homeostasis,endocytosis etc.We used same method to research the binding proteins of Lsb1 ubiquitin chain and got the 45 proteins through the same filter conditions.The biology process analysis showed that these proteins mainly involved in branched-chain amino acid biosynthetic process,protein folding,zinc II ion transmembrane transport etc.Finally,the amino acid deficiency plate experiment of Lsb1 and it`s site mutant showed that ubiquitination Lsb1 can participate in the biosythetic process of branched-chain amino acid in yeast.This results consistent with our biology process analysis.And these data was benefit for the further function research of Lsb1 and it`s ubiquitin chains.