Subcellular localization of the hypusine-containing eukaryotic initiation factor 5A by immunofluorescent staining and green fluorescent protein tagging

Eukaryotic initiation factor 5A (eIF-5A) is the only protein in nature that contains hypusine, an unusual amino acid residue formed posttranslationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Although the eIF-5A gene is essential for cell survival and proliferation, the precise function and localization of eIF-5A remain unclear. In this study, we have determined the subcellular distribution of eIF-5A by indirect immunofluorescent staining and by direct visualization of green Fluorescent protein tagged eIF-5A (GFP-eIF5A). Immunofluorescent staining of the formaldehyde-fixed cells showed that eIF-5A was present in both the nucleus and cytoplasm. Only the nuclear eIF-5A was resistant to Triton extraction. Direct visualization of GFP tagged eIF-5A in living cells revealed the same whole-cell distribution pattern. However, a fusion of an additional Pyruvate Kinase (PK) moiety into GFP-eIF-5A precluded the nuclear localization of GFP-PK-eIF-5A fusion protein. Fusion of the GFP-PK tag with three different domains of eIF-5A also failed to reveal any nuclear localization of the fusion proteins, suggesting the absence of receptor-mediated nuclear import. Using interspecies heterokaryon fusion assay, we could detect the nuclear export of GFP-Rev, but not of GFP-eIF-5A. The whole-cell distribution pattern of eIF-5A was recalcitrant to the treatments that included energy depletion, heat shock, and inhibition of transcription, translation, polyamine synthesis, or CRM1-dependent nuclear export. Collectively, our data indicate that eIF-5A gains nuclear entry via passive diffusion, but it does not undergo active nucleocytoplasmic shuttling.