Functional and structural analysis of MOZART1, an essential MTOC regulator
thesisposted on 05.09.2014, 15:25 by Deepsharan Kaur Dhani
γ-Tubulin plays a universal role in microtubule nucleation from microtubule organizing centers (MTOCs), such as the centrosome in higher eukaryotes and the spindle pole body (SPB) in fungi. The γ-Tubulin functions as part of a multiprotein complex called the γ-tubulin Ring complex (γ-TuRC), consisting of γ-tubulin complex protein (GCP) 1–6, where GCP1 is γ-tubulin. It has been shown that GCP1–3 are core components as they are essential and are absolutely required for the γ-TuRC assembly, whereas the other three GCPs are not. Recently, a novel component of the γ-TuRC, MOZART1, has been identified in humans and plants. In humans, MOZART1 plays an essential role in mitotic spindle formation as a component of the γ-TuRC. The precise functional role of MOZART1 is still yet unknown. In this thesis, I therefore aimed to reveal its mode of action at a molecular level. For this purpose, I chose to exploit a powerful model system, fission yeast, which allows us to take multidisciplinary approaches. The mzt1 (also known as tam4) gene, which encodes fission yeast MOZART1 homologue, is essential. I localised Mzt1 during mitosis and meiosis and found it at the MTOCs. It also coimmunoprecipitates with γ-tubulin from cell extracts. I also determined the starting methionine of the mzt1 gene which encodes a 64–amino acid peptide. Depletion of Mzt1 leads to aberrant microtubule structure, including malformed mitotic spindles and impaired interphase microtubule array. It also causes cytokinesis defects, suggesting a role of the γ-tubulin complex (γ-TuC) in the regulation of cytokinesis. Yeast two-hybrid analysis shows that Mzt1 interacts with GCP3Alp6, a fission yeast homologue of GCP3. Biophysical methods demonstrate that there is a direct interaction between recombinant Mzt1 and the N-terminal region of GCP3Alp6. In addition Mzt1 protein tends to oligomerize into trimer or hexamer. Collectively I propose that Mzt1 oligomer directly interacts with GCP3Alp6 and stabilizes the γ-TuC to facilitate microtubule regulation.