title: Development of a high-throughput method for the systematic identification of human proteins nuclear translocation potential.
abstract: BACKGROUND
  Important clues to the function of novel and uncharacterized proteins can be obtained by identifying their ability to translocate in the nucleus. In addition, a comprehensive definition of the nuclear proteome undoubtedly represents a key step toward a better understanding of the biology of this organelle. Although several high-throughput experimental methods have been developed to explore the sub-cellular localization of proteins, these methods tend to focus on the predominant localizations of gene products and may fail to provide a complete catalog of proteins that are able to transiently locate into the nucleus.
  
  
  RESULTS
  We have developed a method for examining the nuclear localization potential of human gene products at the proteome scale by adapting a mammalian two-hybrid system we have previously developed. Our system is composed of three constructs co-transfected into a mammalian cell line. First, it contains a PCR construct encoding a fusion protein composed of a tested protein, the PDZ-protein TIP-1, and the transactivation domain of TNNC2 (referred to as ACT construct). Second, our system contains a PCR construct encoding a fusion protein composed of the DNA binding domain of GAL4 and the PDZ binding domain of rhotekin (referred to as the BIND construct). Third, a GAL4-responsive luciferase reporter is used to detect the reconstitution of a transcriptionally active BIND-ACT complex through the interaction of TIP-1 and rhotekin, which indicates the ability of the tested protein to translocate into the nucleus. We validated our method in a small-scale feasibility study by comparing it to green fluorescent protein (GFP) fusion-based sub-cellular localization assays, sequence-based computational prediction of protein sub-cellular localization, and current sub-cellular localization data available from the literature for 22 gene products.
  
  
  CONCLUSIONS
  Our reporter-based system can rapidly screen gene products for their ability to be translocated to the nucleus. Large-scale applications of the system presented herein should provide invaluable information for a more complete biological atlas.
authors.affiliation: RIKEN Omics Science Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan. trinhxuanhoat@yahoo.com
authors.forename: Trinh Xuan
authors.initials: TX
authors.lastname: Hoat
authors.forename: Nicolas
authors.initials: N
authors.lastname: Bertin
authors.forename: Noriko
authors.initials: N
authors.lastname: Ninomiya
authors.forename: Shiro
authors.initials: S
authors.lastname: Fukuda
authors.forename: Kengo
authors.initials: K
authors.lastname: Usui
authors.forename: Jun
authors.initials: J
authors.lastname: Kawai
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Hayashizaki
authors.forename: Harukazu
authors.initials: H
authors.lastname: Suzuki
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017930:Genes, Reporter; D006801:Humans; D009687:Nuclear Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
chemical_list: D009687:Nuclear Proteins