Nuclear localization of the ubiquitin-activating enzyme, E1, is cell-cycle-dependent

Abstract

The mechanisms that regulate ubiquitin-mediated degradation of proteins such as the mitotic cyclins at defined stages of the cell cycle are poorly understood. The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by the ubiquitin-activating enzyme, E1. Previously we have described the subcellular localization of this enzyme to both nuclear and cytoplasmic compartments. In the present study, we have used the 1C5 anti-E1 monoclonal antibody in immunofluorescent-microscopy and subcellular-fractionation techniques to examine the distribution of E1 during the HeLa cell cycle. E1 is both cytoskeletal and nuclear during the G1-phase. As the cells progress into S-phase, E1 is exclusively cytoskeletal and has a perinuclear distribution. During G2-phase, E1 reappears in the nucleus before breakdown of the nuclear envelope. In mitotic cells, E1 localizes to both the mitotic spindle and the cytosol, but is absent from the chromosomes. Immunoblot analysis reveals multiple forms of E1 in HeLa whole cell extract. This heterogeneity is not a result of polyubiquitination and may represent inactive pools of E1. Only the characteristic E1 doublet is able to activate ubiquitin. Cell-fractionation studies reveal a differential distribution of specific E1 isoforms throughout the cell cycle. Therefore we propose that the subcellular localization of E1 may play a role in regulating cell-cycle-dependent conjugation of ubiquitin to target proteins.

The ubiquitin-activating enzyme E1-like protein in lung cancer cell lines

“…Furthermore, not all MG37 and GLC-45/UBE1L cells were positive for UBE1L protein expression. Both observations indicate that the BB1 MAb will recognize the UBE1L protein on cytospin slides only when abundantly expressed, as is the case for MG37 and pUBE1Lneo-transfected cells, and that the localization and expression level might be cell cycle dependent as has been described for UBE1 (Grenfell et al, 1994). On Western blot, however, the BB1 MAb is clearly more sensitive, e.g., being able to detect the UBE1L protein also in Ca5I1 cells.…”

Characterization of the interactions of human papillomavirus type 16 E6 with p53 and E6-associated protein in insect and human cells.

“…Alternatively, E6 may have several functions that operate in distinct areas of the cell, like SV40 large T antigen (Butel & Jarvis, 1986), or its localization may even be cell cycle regulated. The localization of some of the other proteins involved in the ubiquitin-dependent pathway is also cell cycle regulated (Amsterdam et al, 1993 ;Grenfell et al, 1994). Whichever alternative is the case, further work is required to clarify these points, which add sophistication to the already elegant mechanisms developed by HPV for the subversion of cellular processes.…”

Ubiquitin System-Dependent Regulation of Growth Hormone Receptor Signal Transduction and Effects of Oxidative Stress

“…E1 is highly concentrated in both yeast (188) and mammalian cell nuclei (189). Nuclear localization signals allow cellcycle-regulated nuclear localization and phosphorylation, with an increase in nuclear distribution in G2 phase (190). The finding that cells expressing a tempe- rature-sensitive E1 undergo cell-cycle arrest provided the first evidence for the physiological significance of ubiquitination (191).…”