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Characterization of Ubiquitin-Activating Enzyme Uba1 in the Nucleus by Its Mammalian Temperature-Sensitive Mutant

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  • Kimihiko Sugaya
  • Yoshie Ishihara
  • Sonoe Inoue
  • Hideo Tsuji

Abstract

Temperature-sensitive (ts) CHO-K1 mutant tsTM3 exhibits chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. Previously, complementation tests with other mutants showed that tsTM3 harbors a genetic defect in the ubiquitin-activating enzyme Uba1. Sequence comparison of the Uba1 gene between wild-type and mutant cells in this study revealed that the mutant phenotype is caused by a G-to-A transition that yields a Met-to-Ile substitution at position 256 in hamster Uba1. The ts defects in tsTM3 were complemented by expression of the wild-type Uba1 tagged with green fluorescent protein. Expression of the Uba1 primarily in the nucleus appeared to rescue tsTM3 cells. Incubation at 39°C resulted in a decrease of nuclear Uba1 in tsTM3 cells, suggesting that loss of Uba1 in the nucleus may lead to the ts defects. Analyses with the fluorescent ubiquitination-based cell cycle indicator revealed that loss of function of Uba1 leads to failure of the ubiquitin system in the nucleus. Incubation at 39°C caused an increase in endogenous geminin in tsTM3 cells. A ts mutation of Uba1 found in tsTM3 cells appears to be a novel mutation reflecting the important roles of Uba1 in nucleus.

Suggested Citation

  • Kimihiko Sugaya & Yoshie Ishihara & Sonoe Inoue & Hideo Tsuji, 2014. "Characterization of Ubiquitin-Activating Enzyme Uba1 in the Nucleus by Its Mammalian Temperature-Sensitive Mutant," PLOS ONE, Public Library of Science, vol. 9(5), pages 1-12, May.
  • Handle: RePEc:plo:pone00:0096666
    DOI: 10.1371/journal.pone.0096666
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    References listed on IDEAS

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    1. Jianping Jin & Xue Li & Steven P. Gygi & J. Wade Harper, 2007. "Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging," Nature, Nature, vol. 447(7148), pages 1135-1138, June.
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