The subcellular distribution of eukaryotic translation initiation factor, eIF-5A, in cultured cells
The subcellular distribution of eukaryotic translation initiation factor, eIF-5A, in cultured cells
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DOI：
10.1006/excr.1996.0185
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发表时间：
1996-06-15
影响因子：
3.7
通讯作者：
Waxman, L
中科院分区：
文献类型：
--
作者：
Shi, XP;Yin, KC;Waxman, L
为了深入了解真核翻译起始因子eIF-5A的作用，我们使用高效的抗eIF-5A的单特异性多克隆抗体研究了该蛋白在几种培养细胞类型和细胞周期不同阶段的亚细胞分布。使用间接免疫荧光和共聚焦显微镜结合亚细胞分离的研究表明，eIF-5A主要定位于细胞质。在细胞周期的不同阶段，eIF-5A的胞质位置没有明显改变，eIF-5A的亚细胞分布模式也没有因病毒癌基因转化而改变。细胞分离实验证实，胞浆中存在两组eIF-5A，一组为可溶性组分，另一组为与内膜结合的组份。通过与内质网(ER)的驻留蛋白Calnexin抗体的双重免疫荧光染色，我们证明eIF-5A的膜结合部分与内质网而不是与细胞骨架共定位，人类免疫缺陷病毒1型(HIV-1)的调节蛋白Rev的表达并不改变内源性eIF-5A在这些细胞中的亚细胞分布。EIF-5A在所有被检测的组织和细胞中都被检测到，包括从非洲爪哇卵母细胞中提取的提取物。我们的结果表明eIF-5A是一种普遍存在的细胞质蛋白，提示eIF-5A的一个功能位点可能与内质网有关。(C)1996年学术出版社。
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 eIF-5A. Studies using indirect immunofluorescence and confocal microscopy 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 experiments identified two populations of eIF-5A in the cyto plasm, a soluble fraction and a fraction bound to internal membranes. By double immunofluorescence staining with an antibody against calnexin, a resident protein of the endoplasmic reticulum (ER), we demonstrate that the membrane-bound fraction of eIF-5A colocalizes with the ER and not with the cytoskeleton, Expression of Rev, a regulatory protein of human immunodeficiency virus type 1 (HIV-1), does not alter the subcellular distribution of endogenous eIF-5A in these cells. eIF-5A is detected in all tissues and cells examined including extracts prepared from Xenopus oocytes. Our results indicate that eIF-5A is a ubiquitous cytoplasmic protein and suggest that a site of eIF-5A function is likely to be in association with the ER. (C) 1996 Academic Press, Inc.