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Updated: Jan 19, 2026

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A
Published on: June 10, 2020
CENP-A Ubiquitylation Is Indispensable to Cell Viability
Yohei Niikura1, Risa Kitagawa2, Lei Fang3
1Greehey Children's Cancer Research Institute, Department of Molecular Medicine, UT Health Science Center San Antonio, 8403 Floyd Curl Drive, San Antonio, TX 78229-3000, USA; MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, Nanjing, Jiangsu Province 210061, China.
Abstract:
CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity, but how CENP-A is deposited at the centromere remains obscure. We previously reported that CENP-A K124 ubiquitylation, mediated by the CUL4A-RBX1-COPS8 complex, is essential for CENP-A deposition at the centromere. However, a recent report stated that CENP-A K124R mutants show no defects in centromere localization and cell viability. In the present study, we found that EYFP tagging induces additional ubiquitylation of EYFP-CENP-A K124R, which allows the mutant protein to bind to HJURP. Using a previously developed conditional CENP-A knockout system and our CENP-A K124R knockin mutant created by the CRISPR-Cas9 system, we show that the Flag-tagged or untagged CENP-A K124R mutant is lethal. This lethality is rescued by monoubiquitin fusion, indicating that CENP-A ubiquitylation is essential for viability.
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