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Nuclear localization of ubiquitin‐activating enzyme Uba1 is characterized in its mammalian temperature‐sensitive mutant
Author(s) -
Sugaya Kimihiko,
Ishihara Yoshie,
Inoue Sonoe
Publication year - 2015
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/gtc.12257
Subject(s) - biology , microbiology and biotechnology , mutant , nuclear protein , mutation , cell nucleus , nuclear localization sequence , nucleus , gene , genetics , transcription factor
In our previous study, a point mutation in Uba1 , the gene encoding ubiquitin‐activating enzyme, was identified in temperature‐sensitive (ts) CHO ‐K1 mutant ts TM 3 cells, which led to a Met‐to‐Ile substitution at amino acid 256 in Uba1 protein. Characterization of this mutant revealed a deficiency of nuclear Uba1 and impaired ubiquitination in the nucleus. The ts defects in ts TM 3 were complemented by the expression of the wild‐type Uba1 tagged with green fluorescent protein ( GFP ). In this study, the expression and localization of Uba1 were investigated using the various forms of Uba1 tagged with GFP . Western blot analysis and confocal microscopy revealed that nuclear localization of Uba1, as well as even the modified and truncated forms of Uba1, appears to be essential to rescue ts TM 3 cells. The localization of Uba1 in the nucleus, even if it was a small amount, was proportional to the efficiency of complementation of ts TM 3 cells. Uba1 plays an important role in the nucleus, and a ts mutation found in ts TM 3 cells appears to result in the loss of localization of Uba1 in the nucleus.