Trausch J S, Grenfell S J, Handley-Gearhart P M, Ciechanover A, Schwartz A L
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri.
Am J Physiol. 1993 Jan;264(1 Pt 1):C93-102. doi: 10.1152/ajpcell.1993.264.1.C93.
Ubiquitin, a 76-amino acid protein, is covalently attached to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Ubiquitin is found within the cytoplasm, nucleus, microvilli, autophagic vacuoles, and lysosomes. The ubiquitin-activating enzyme, E1, catalyzes the first step in ubiquitin conjugation. To date, very little is known about the subcellular distribution of this enzyme. We have utilized immunofluorescence and immunoblotting to examine the cellular distribution of E1 in several eukaryotic cell lines, including HeLa, smooth muscle A7r5, choriocarcinoma BeWo, Pt K1, and Chinese hamster ovary (CHO) E36. E1 was identified in both cytoplasmic and nuclear compartments in all cell lines examined. However, the relative abundance within these compartments differed markedly between the cell lines. Even within a single cell line, nuclear distribution was not uniform, and certain cells demonstrated an absence of nuclear staining. E1 resides predominantly within the nucleus in BeWo. In contrast, its distribution in CHO and Pt K1 cells is mainly cytoplasmic. Within the cytoplasm, three pools of E1 were identified by double-label immunofluorescence. The first of these colocalized with phalloidin, indicating association of E1 with actin filaments. A second cytoplasmic pool colocalized with tubulin and was predominantly perinuclear in its distribution. The third pool associated with intermediate filaments. This suggests that E1 is associated with all three components of the cytoskeleton. The distribution of E1 was unaltered in a mutant line of CHO E36 designated ts20, in which the E1 can be thermally inactivated. The variable distribution of E1 among cell lines, including its apparent cytoskeletal association, suggests pleiotropic functions of this enzyme and the ubiquitin-conjugating system.
泛素是一种由76个氨基酸组成的蛋白质，它通过共价键与异常的和寿命较短的蛋白质相连，从而在真核细胞中标记这些蛋白质以便进行依赖ATP的蛋白水解。泛素存在于细胞质、细胞核、微绒毛、自噬泡和溶酶体中。泛素激活酶E1催化泛素缀合的第一步。迄今为止，关于这种酶的亚细胞分布了解甚少。我们利用免疫荧光和免疫印迹法检测了E1在几种真核细胞系中的细胞分布，包括HeLa细胞系、平滑肌A7r5细胞系、绒毛膜癌BeWo细胞系、Pt K1细胞系和中国仓鼠卵巢（CHO）E36细胞系。在所检测的所有细胞系中，E1在细胞质和细胞核区室中均有发现。然而，这些区室中的相对丰度在不同细胞系之间存在显著差异。即使在单个细胞系中，核分布也不均匀，某些细胞显示无核染色。E1主要存在于BeWo细胞系的细胞核中。相比之下，它在CHO和Pt K1细胞中的分布主要在细胞质中。在细胞质中，通过双标记免疫荧光鉴定出E1的三个池。其中第一个与鬼笔环肽共定位，表明E1与肌动蛋白丝相关。第二个细胞质池与微管蛋白共定位，其分布主要在核周。第三个池与中间丝相关。这表明E1与细胞骨架的所有三个成分相关。在一个名为ts20的CHO E36突变细胞系中，E1的分布未改变，在该细胞系中E1可被热灭活。E1在不同细胞系中的可变分布，包括其明显的细胞骨架关联，表明这种酶和泛素缀合系统具有多效性功能。
