Ubc9 is essential for viability of higher eukaryotic cells.
Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins. The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IkappaBalpha, MDM2, p53, and PML, are not present in yeasts. To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, we constructed a conditional UBC9 mutant of chicken DT40 cells containing the UBC9 transgene under control of a tetracycline-repressible promoter and characterized their loss of function phenotypes. Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug. In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, we did not observe accumulation of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9 but we did observe an increase in the number of cells containing multiple nuclei, indicating defects in cytokinesis. A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment.
Hayashi
Tomoko
T
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan.
Seki
Masayuki
M
Maeda
Daisuke
D
Wang
Wensheng
W
Kawabe
Yoh-ichi
Y
Seki
Takahiko
T
Saitoh
Hisato
H
Fukagawa
Tatsuo
T
Yagi
Hideki
H
Enomoto
Takemi
T
D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology; D002453:Cell Cycle / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival / Q000502:physiology*; D002645:Chickens; D020090:Chromosome Segregation; D005434:Flow Cytometry; D017404:In Situ Hybridization, Fluorescence; D008025:Ligases / Q000235:genetics* / Q000378:metabolism*; D010641:Phenotype; D011401:Promoter Regions, Genetic; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D013752:Tetracycline / Q000378:metabolism; D019076:Transgenes; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D000900:Anti-Bacterial Agents; D025842:SUMO-1 Protein; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D013752:Tetracycline