Development of a high-throughput method for the systematic identification of human proteins nuclear translocation potential

BMC Cell Biol. 2009 Sep 22:10:69. doi: 10.1186/1471-2121-10-69.


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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Active Transport, Cell Nucleus
  • Animals
  • Base Sequence
  • Cell Line
  • Genes, Reporter
  • Humans
  • Nuclear Proteins / analysis*
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Transcriptional Activation
  • Two-Hybrid System Techniques*


  • Nuclear Proteins