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Updated: Jun 20, 2026
A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis
Published on: January 24, 2017
Development of a high-throughput method for the systematic identification of human proteins nuclear translocation
Trinh Xuan Hoat1, Nicolas Bertin, Noriko Ninomiya
1RIKEN Omics Science Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan. trinhxuanhoat@yahoo.com
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.
