A Novel Method to Analyze Nucleocytoplasmic Transport In Vivo by Using Short Peptide Tags

Regulated nucleocytoplasmic transport is of vital importance for maintaining the physiology of the cell, and disturbed nucleocytoplasmic shuttling of certain proteins has been found in a variety of diseases including cancer. The most frequently used procedure to analyze those processes is to fuse the protein of interest to a fluorescent protein such as GFP (green fluorescent protein)—a technique that is prone to impair normal protein function and subcellular localization. We report a novel approach to monitor nucleocytoplasmic transport processes in vivo by combining short TetR inducing peptide tags (TIP) with a TetR-controlled reporter gene in a human cell line. The technology is exemplified by demonstrating nucleocytoplasmic shuttling of the glucocorticoid receptor and activity of two further TIP fusions to cancer-related proteins. The technology presented provides the basis for efficient screening systems to isolate compounds altering the nucleocytoplasmic distribution of a protein of interest. [Display omitted] ► Nucleocytoplasmic transport is essential for maintaining the physiology of the cell. ► TIP peptides and a Tet-responsive reporter gene are used to monitor nuclear transport. ► Nuclear transport of the glucocorticoid receptor is analyzed quantitatively. ► Reporter gene activity correlates with protein localization of a GFP fusion protein. ► The method can be used to characterize nuclear proteins without the need of GFP fusions.