THESIS
2018
ix, 51 pages : illustrations (some color) ; 30 cm
Abstract
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...[
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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
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