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.
Jao
David Li-En
DL
Department of Chemistry and Chemical Biology, Joint Graduate Program in Cell and Developmental Biology, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854-8087, USA.
Yu Chen
Kuang
K
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus; D000818:Animals; D019556:COS Cells; D037281:Calnexin / Q000032:analysis; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002470:Cell Survival; D039642:Eukaryotic Initiation Factors / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000032:analysis; D008164:Luminescent Proteins / Q000032:analysis*; D008239:Lysine / Q000031:analogs & derivatives* / Q000032:analysis*; D051379:Mice; D019913:Nuclear Localization Signals; D010448:Peptide Initiation Factors / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D021381:Protein Transport; D016601:RNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D000097574:Eukaryotic Translation Initiation Factor 5A; D000097606:Exportin 1 Protein
D037281:Calnexin; D039642:Eukaryotic Initiation Factors; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D008164:Luminescent Proteins; D008239:Lysine; D019913:Nuclear Localization Signals; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D000097574:Eukaryotic Translation Initiation Factor 5A; D000097606:Exportin 1 Protein; C100028:hypusine