Characterization of ubiquitin-activating enzyme Uba1 in the nucleus by its mammalian temperature-sensitive mutant

PLoS One. 2014 May 7;9(5):e96666. doi: 10.1371/journal.pone.0096666. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Cell Nucleus / metabolism*
  • Cricetinae
  • Cricetulus
  • Mutation*
  • Temperature
  • Ubiquitin-Activating Enzymes / genetics
  • Ubiquitin-Activating Enzymes / metabolism*

Substances

  • Ubiquitin-Activating Enzymes

Grant support

This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 23570012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.