A nuclear export sequence in GPN-loop GTPase 1, an essential protein for nuclear targeting of RNA polymerase II, is necessary and sufficient for nuclear export

Biochim Biophys Acta. 2012 Oct;1823(10):1756-66. doi: 10.1016/j.bbamcr.2012.07.001. Epub 2012 Jul 14.


XAB1/Gpn1 is a GTPase that associates with RNA polymerase II (RNAPII) in a GTP-dependent manner. Although XAB1/Gpn1 is essential for nuclear accumulation of RNAPII, the underlying mechanism is not known. A XAB1/Gpn1-EYFP fluorescent protein, like endogenous XAB1/Gpn1, localized to the cytoplasm but it rapidly accumulated in the cell nucleus in the presence of leptomycin B, a chemical inhibitor of the nuclear transport receptor Crm1. Crm1 recognizes short peptides in substrate proteins called nuclear export sequences (NES). Here, we employed site-directed mutagenesis and fluorescence microscopy to assess the functionality of all six putative NESs in XAB1/Gpn1. Mutating five of the six putative NESs did not alter the cytoplasmic localization of XAB1/Gpn1-EYFP. However, a V302A/L304A double mutant XAB1/Gpn1-EYFP protein was clearly accumulated in the cell nucleus, indicating the disruption of a functional NES. This functional XAB1/Gpn1 NES displays all features present in most common and potent NESs, including, in addition to Φ1-Φ4, a critical fifth hydrophobic amino acid Φ0. Therefore, in human Gpn1 this NES spans amino acids 292-LERLRKDMGSVAL-304. XAB1/Gpn1 NES is remarkably conserved during evolution. XAB1/Gpn1 NES was sufficient for nuclear export activity, as it caused a complete exclusion of EYFP from the cell nucleus. Molecular modeling of XAB1/Gpn1 provided a mechanistic reason for NES selection, as functionality correlated with accessibility, and it also suggested a mechanism for NES inhibition by intramolecular masking. In conclusion, we have identified a highly active, evolutionarily conserved NES in XAB1/Gpn1 that is critical for nucleo-cytoplasmic shuttling and steady-state cytoplasmic localization of XAB1/Gpn1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus
  • Amino Acid Sequence
  • Cell Nucleus / enzymology*
  • Conserved Sequence / genetics
  • Evolution, Molecular
  • GTP-Binding Proteins / chemistry
  • GTP-Binding Proteins / metabolism
  • Genes, Reporter
  • HEK293 Cells
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Export Signals*
  • Protein Structure, Secondary
  • RNA Polymerase II / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Structure-Activity Relationship


  • Nuclear Export Signals
  • Recombinant Fusion Proteins
  • RNA Polymerase II
  • GPN1 protein, human
  • GTP-Binding Proteins