title: Characterization of ubiquitin-activating enzyme Uba1 in the nucleus by its mammalian temperature-sensitive mutant.
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.
authors.affiliation: Research Center for Radiation Protection, National Institute of Radiological Sciences, Chiba, Japan; Fukushima Project Headquarters, National Institute of Radiological Sciences, Chiba, Japan.
authors.forename: Kimihiko
authors.initials: K
authors.lastname: Sugaya
authors.affiliation: Research Center for Radiation Protection, National Institute of Radiological Sciences, Chiba, Japan; Fukushima Project Headquarters, National Institute of Radiological Sciences, Chiba, Japan.
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Ishihara
authors.affiliation: Research Center for Radiation Protection, National Institute of Radiological Sciences, Chiba, Japan; Fukushima Project Headquarters, National Institute of Radiological Sciences, Chiba, Japan.
authors.forename: Sonoe
authors.initials: S
authors.lastname: Inoue
authors.affiliation: Fukushima Project Headquarters, National Institute of Radiological Sciences, Chiba, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Tsuji
mesh_terms: D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D009154:Mutation; D013696:Temperature; D044764:Ubiquitin-Activating Enzymes / Q000235:genetics / Q000378:metabolism*
chemical_list: D044764:Ubiquitin-Activating Enzymes