THE SUBCELLULAR-DISTRIBUTION OF EUKARYOTIC TRANSLATION INITIATION-FACTOR, EIF-5A, IN CULTURED-CELLS

To gain insight into the role of the eukaryotic translation initiation factor, eIF-5A, we investigated the subcellular distribution of this protein in several cultured cell types and at different stages of the cell cycle using a highly potent monospecific polyclonal antibody to e IF-5A. Studies using indirect immunofluorescence and confocal microsco py in conjunction with subcellular fractionation demonstrate that eIF- 5A is primarily localized in the cytoplasm of cells. This cytoplasmic location of eIF-5A is not significantly altered in different stages of the cell cycle and the subcellular distribution pattern of eIF-5A is not changed by viral oncogene transformation. Cell fractionation exper iments identified two populations of eIF-5A in the cyto plasm, a solub le fraction and a fraction bound to internal membranes. By double immu nofluorescence staining with an antibody against calnexin, a resident protein of the endoplasmic reticulum (ER), we demonstrate that the mem brane-bound fraction of eIF-5A colocalizes with the ER and not with th e cytoskeleton, Expression of Rev, a regulatory protein of human immun odeficiency virus type 1 (HIV-1), does not alter the subcellular distr ibution of endogenous eIF-5A in these cells. eIF-5A is detected in all tissues and cells examined including extracts prepared from Xenopus o ocytes. Our results indicate that eIF-5A is a ubiquitous cytoplasmic p rotein and suggest that a site of eIF-5A function is likely to be in a ssociation with the ER. (C) 1996 Academic Press, Inc.