Immunofluorescent localization of the ubiquitin-activating enzyme, E1, to the nucleus and cytoskeleton.
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.
Trausch
J S
JS
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri.
Grenfell
S J
SJ
Handley-Gearhart
P M
PM
Ciechanover
A
A
Schwartz
A L
AL
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D014018:Tissue Distribution; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases
D000911:Antibodies, Monoclonal; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes