Studies On The Ubiquitination Of The Lysosomal Protein TMEPAI And Its Function

TMEPAI(Transmembrane prostate androgen-induced protein)is mainly localized in lysosomes and contributes to the stability of structure and function of lysosomes.Its transportation and localization in cells are related with Nedd4.TMEPAI is highly expressed in many tumor cells and plays an important role in tumorigenesis and development.In order to further study the mechanism of the lysosomal transportation of TMEPAI in tumorigenesis,this paper mainly does the following works:The in vitro ubiquitination assays show that TMEPAI mainly occurs monoubiquitination and the same result is verified by in vivo ubiquitination.Immunofluorescence experiment shows the ubiquitination of TMEPAI affects its transportation to lysosomes.Once it can not be ubiquitined,TMEPAI will be localized in cell membrane.HGS and STAM of TMEPAI interaction protein are screening out by yeast two hybrid system.When HGS was knockdown,TMEPAI has no co-localization with lysosomes and the protein level of TMEPAI decreases significantly.GST pull-down assay shows that WW2,WW3 and WW4 domain of Nedd4 are interacted with TMEPAI.The TMEPAI-Nedd4 complex can be isolated and purified by using superdex-200 molecular sieve.Nedd4L,Smurfl,Smurf2,WWP1,WWP2 and Rsp5 of E3 ubiqutin ligase are interacted with TMEPAI by yeast two hybrid system.Taken together,these results indicate that the intracellular transportation of TMEPAI needs the E3 ubiqutin ligase Nedd4 and ubiquitin binding protein HGS.The interaction site of TMEPAI-Nedd4 can be used as a potential drug target of cancers therapy.