Effects of inhibitors of RNA and protein synthesis on the subcellular distribution of the eukaryotic translation initiation factor, eIF-5A, and the HIV-1 Rev protein.
The subcellular distributions of the endogenous eukaryotic translation initiation factor, eIF-5A, and Rev, a protein of the human immunodeficiency virus proposed to interact with eIF-5A, were studied in COS-7 cells treated with inhibitors of RNA or protein synthesis. We have previously shown that transiently expressed Rev is localized in the nucleolus, whereas eIF-5A is primarily in the cytoplasm. The subcellular localization of Rev was not affected by treatment with protein synthesis inhibitors (cycloheximide, CHX, 10 micrograms/ml; puromycin, 10 micrograms/ml), although its location changed from predominantly the nucleolus to the cytoplasm after treatment with RNA synthesis inhibitors (actinomycin D, 4 micrograms/ml, and 5,6-dichloro-1 beta-D-ribofuranosylbenzimidazole, DRB; 0.1 mM), as previously reported. In contrast, none of the RNA synthesis inhibitors (alpha-amanitin, 10 micrograms/ml; actinomycin D, 4 micrograms/ml, and DRB, 0.1 mM) caused any significant changes in the subcellular distribution pattern of eIF-5A. However, treatment with puromycin, a protein synthesis inhibitor known to dissociate ribosomes, dramatically altered the subcellular distribution pattern of eIF-5A in 30% of the cell population. In these cells, the staining of eIF-5A was changed from an endoplasmic reticulum (ER) net work-like perinuclear structure to a patched dotted pattern dispersed throughout the cytoplasm. This change was not observed in the same cells stained for calnexin, an ER resident protein, nor in cells treated with CHX, which freezes the ribosomes to block protein synthesis. Our data suggest that eIF-5A does not shuttle between the nucleus and cytoplasm in the same way as Rev. Our findings are consistent with our previous conclusion that eIF-5A is associated with the ER through ribosomes and support a role for eIF-5A in protein synthesis.
Shi
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Department of Biological Chemistry, Merck Research Laboratories, West Point, Pa 19486, USA. xiao-ping-shi@merck.com
Yin
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Waxman
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D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D015694:Gene Products, rev / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012313:RNA / Q000096:biosynthesis*; D016601:RNA-Binding Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D000097574:Eukaryotic Translation Initiation Factor 5A
D015694:Gene Products, rev; D019384:Nucleic Acid Synthesis Inhibitors; D010448:Peptide Initiation Factors; D011500:Protein Synthesis Inhibitors; D012313:RNA; D016601:RNA-Binding Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D000097574:Eukaryotic Translation Initiation Factor 5A