Microtubule skeleton plays essential roles in eukaryotic cells. Microtubule is nucleated by γ-tubulin which functions in γ-tubulin ring complexes (γ-TuRCs). Recently the structural analysis of the subunits of γ-TuRC and some regulatory proteins of γ-TuRCs have been reported. But the mechanism of γ-TuRC-mediated microtubule nucleation and its regulation are mostly unclear. Isolation of γ-TuRC has boosted the understanding of γ-TuRC in various aspects and large-scale isolation is required for potential structure determination of γ-TuRC. In my research, I addressed the stability of isolated γ-TuRC, evaluated γ-TuRC isolation from porcine brain, and optimized γ-TuRC enrichment via PEG precipitation which is necessary to achieve high-yield isolation. These experiments demonstrated tha...[ Read more ]

Microtubule skeleton plays essential roles in eukaryotic cells. Microtubule is nucleated by γ-tubulin which functions in γ-tubulin ring complexes (γ-TuRCs). Recently the structural analysis of the subunits of γ-TuRC and some regulatory proteins of γ-TuRCs have been reported. But the mechanism of γ-TuRC-mediated microtubule nucleation and its regulation are mostly unclear. Isolation of γ-TuRC has boosted the understanding of γ-TuRC in various aspects and large-scale isolation is required for potential structure determination of γ-TuRC. In my research, I addressed the stability of isolated γ-TuRC, evaluated γ-TuRC isolation from porcine brain, and optimized γ-TuRC enrichment via PEG precipitation which is necessary to achieve high-yield isolation. These experiments demonstrated that γ-TuRC is more abundant in human cell line than in porcine brain tissue, and γ-TuRCs isolated from human cell line is more stable and purer. I also studied the control of γ-tubulin by the ubiquitin-proteasome pathway. Mutation of some potential sites could reduce the ubiquitination level of γ-tubulin. The degradation of γ-tubulin can be blocked either by the proteasome inhibitor MG132, or the inhibitor of cullin-RING E3 ligases MLN4924. These results indicate that γ-tubulin undergoes ubiquitination and proteasomal degradation that is dependent of the activity of certain cullin-RING E3 ligase. Moreover, the depletion of the binding partner GCP2 or GCP3 caused co-depletion of γ-tubulin, and vice versa. This suggests that degradation of γ-tubulin is coordinated with disassembly of γ-tubulin complexes.

Key words: γ-tubulin, γ-tubulin ring complex, ubiquitination, degradation