Immunofluorescent localization of the ubiquitin-activating enzyme, E1, to the nucleus and cytoskeleton

Abstract

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.

IκBα Physically Interacts with a Cytoskeleton-Associated Protein through Its Signal Response Domain

“…This activation is accompanied by concomitant IB␣ proteolysis. Of note, ubiquitin and ubiquitin-activating enzymes are also localized to microtubules (49,67). During the preparation of this article, Régnier et al identified conserved helix-loop-helix ubiquitous kinase (CHUK) as an IB kinase capable of phosphorylating Ser-32 and Ser-36, modifications that are required for targeted degradation of IB␣ via the ubiquitin-proteasome pathway (54).…”

Identification of a Region within the Ubiquitin-activating Enzyme Required for Nuclear Targeting and Phosphorylation

“…Cell Culture and Transfection-HeLa cells were cultured in Dulbecco's modified Eagle's medium and 10% fetal calf serum and maintained at 37°C and 5% CO 2 in a humidified chamber as described previously (9). Transient transfections were performed using a calcium phosphate coprecipitation method as described by Chen and Okayama (14); cells were processed 40 -48 h following transfection.…”

“…Because Ub requires activation prior to participation in any subsequent reactions, E1 plays a key role in the Ub-conjugating pathway. E1 is localized in both the nucleus and the cytoplasm (8,9) and exists as two isoforms E1a (117 kDa) and E1b (110 kDa) (10,11). To investigate the nature of these isoforms, epitope-tagged cDNA constructs of E1 were generated where the hemagglutinin (HA) epitope tag was placed after the first methionine (amino acid 1; HA1-E1) or after the second methionine (amino acid 40; HA2-E1) (11).…”

UBE1DC1, an ubiquitin‐activating enzyme, activates two different ubiquitin‐like proteins

“…El proteins and their encoding genes have been isolated from rabbit, yeast, wheat, human, and mice. Initially, UBE1 localizes in both the nucleus and the cytoplasm and exists as two isoforms E1a (117 kDa) and E1b (110 kDa) in mammals [Handley et al, 1991;Schwartz et al, 1992;Trausch et al, 1993;Stephen et al, 1997]. In recent studies, at least four kinds of E1 (UBE1, AOS1/UBA2, APP-BP1/UBA3, and APG12) exist, each of which has different function in organism.…”