THESIS
2020
xiii, 108 pages : illustrations (some color) ; 30 cm
Abstract
The γ-tubulin ring complex (γ-TuRC) plays a principal role in microtubule nucleation and
anchoring microtubules to microtubule-organizing centers. Proper regulation of the activity
and localization of γ-TuRCs is crucial for the spatial and temporal organization of
microtubules. I developed a γ-TuRC purification protocol using the tandem affinity
purification method. Using the γ-TuRC-binding region of the γ-TuRC-associated protein
CDK5RAP2 and NEDD1, I purified γ-TuRCs and identified a number of γ-TuRC-associated
protein candidates by mass spectrometry and analyzed the proteins by sorting them according
to their identified abundance. Specifically, I found the co-existence of CDK5RAP2 and
NEDD1 in γ-TuRCs and their cooperative effect on microtubule nucleation. New
post-translatio...[
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The γ-tubulin ring complex (γ-TuRC) plays a principal role in microtubule nucleation and
anchoring microtubules to microtubule-organizing centers. Proper regulation of the activity
and localization of γ-TuRCs is crucial for the spatial and temporal organization of
microtubules. I developed a γ-TuRC purification protocol using the tandem affinity
purification method. Using the γ-TuRC-binding region of the γ-TuRC-associated protein
CDK5RAP2 and NEDD1, I purified γ-TuRCs and identified a number of γ-TuRC-associated
protein candidates by mass spectrometry and analyzed the proteins by sorting them according
to their identified abundance. Specifically, I found the co-existence of CDK5RAP2 and
NEDD1 in γ-TuRCs and their cooperative effect on microtubule nucleation. New
post-translational modifications (PTM) on γ-TuRC proteins were also identified through mass
spectrometry. Unexpectedly, 3 cysteine sites of γ-tubulin, C26, C138 and C201 were found
phosphorylated. I investigated the function of these phosphorylation events and found that the
phosphor-mimetic mutant C26D cannot be integrated into γ-TuRC whereas the
non-phosphorylatable mutant C26S and wide type γ-tubulin can. Further experiments showed
that γ-tubulin is important for the interaction between GCPs. I also found that microtubule
nucleation is inhibited when γ-TuRC contains C138D mutant. Taken together, cysteine
phosphorylation on γ-tubulin participates in the microtubule organization through controlling
the assembly and microtubule nucleation ability of γ-TuRC.
In summary, I have purified γ-TuRC and identified several potential regulatory proteins and
PTM of γ-TuRC through mass spectrometry. In addition, I found the cooperative effect of
CDK5RAP2 and NEDD1 and characterized the identified cysteine phosphorylation on
γ-tubulin and found their inhibitory functions for γ-TuRC activity. These findings provide
novel candidates working in microtubule organization, unveil the existence and importance of
the newly identified modification and demonstrate new regulation mechanism for γ-TuRC
assembly and function.
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