Abstract |
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.
|
Authors | David Li-En Jao, Kuang Yu Chen |
Journal | Journal of cellular biochemistry
(J Cell Biochem)
Vol. 86
Issue 3
Pg. 590-600
( 2002)
ISSN: 0730-2312 [Print] United States |
PMID | 12210765
(Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
|
Copyright | Copyright 2002 Wiley-Liss, Inc. |
Chemical References |
- Eukaryotic Initiation Factors
- Karyopherins
- Luminescent Proteins
- Nuclear Localization Signals
- Peptide Initiation Factors
- RNA-Binding Proteins
- Receptors, Cytoplasmic and Nuclear
- Calnexin
- Green Fluorescent Proteins
- hypusine
- Lysine
|
Topics |
- 3T3 Cells
- Active Transport, Cell Nucleus
- Animals
- COS Cells
- Calnexin
(analysis)
- Cell Nucleus
(chemistry, metabolism)
- Cell Survival
- Eukaryotic Initiation Factors
(analysis, immunology, metabolism)
- Fluorescent Antibody Technique, Indirect
- Green Fluorescent Proteins
- HeLa Cells
- Humans
- Karyopherins
(analysis)
- Luminescent Proteins
(analysis)
- Lysine
(analogs & derivatives, analysis)
- Mice
- Nuclear Localization Signals
- Peptide Initiation Factors
(analysis, immunology, metabolism)
- Protein Transport
- RNA-Binding Proteins
- Receptors, Cytoplasmic and Nuclear
- Eukaryotic Translation Initiation Factor 5A
- Exportin 1 Protein
|