Functional and structural analysis of gamma-tubulin complexes

• Applicants: Dr. Stefan Pfeffer; Professor Dr. Elmar Schiebel
• Subject Area: Cell Biology
• Term: since 2009
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 133570075

In all eukaryotes, gamma-tubulin complexes (gamma-TuCs) promote the assembly of microtubules (MTs) from alphabeta-tubulin subunits. As such they impact on the structure, dynamics, number and orientation of MTs within cells and are therefore essential for central cellular processes, such as mitosis and meiosis. Despite these absolutely essential functions, structural information on gamma-TuCs is very limited and mostly restricted to a medium resolution cryo-electron microscopy (EM) reconstruction of the small gamma-tubulin complex (gamma-TuSC) from budding yeast. However, MT nucleation in budding yeast is untypical, because the otherwise essential MOZART component of the MT nucleation machinery is not present. For the more complex gamma-tubulin ring complex (gamma-TuRC) of higher eukaryotes, structural information is restricted to a low-resolution cryo-electron tomography study and X-ray structures for the isolated gamma-tubulin and GCP4 subunits. Aiming to understand MT formation on a structural level, the laboratories of Dr. S. Pfeffer and Prof. E. Schiebel have teamed up to elucidate the structures of various gamma-TuCs using cryo-EM. In this proposal, we present extensive structural data, including cryo-EM reconstructions of the Candida albicans gamma-TuSC at near-atomic resolution and the gamma-TuRC from Xenopus laevis at ~ 5 Å resolution. These preliminary data will serve as a blueprint for determining cryo-EM structures of i) the C. albicans gamma-TuSC in complex with the additional MOZART and Spc110 components of the MT nucleation machinery, ii) the budding yeast gamma-TuSC in complex with the MT polymerase Stu2, iii) the X. laevis gamma-TuRC at near-atomic resolution and finally iv) the human gamma-TuRC. With these structures, we will be able to clarify long-standing questions in the field. We will address how MOZART and Spc110 impact on the structure of the gamma-TuSC allowing its oligomerization into a MT nucleation competent ring. We will elucidate how Stu2 contributes to ring complex and MT formation in budding yeast. We will confirm the preliminary subunit architecture for the 14 GCP proteins (GCP2-6) in the X. laevis gamma-TuRC obtained based on our current cryo-EM density and address in detail the role of the large 900 amino acid insertion in GCP6 as well as additional, novel subunits of the gamma-TuRC identified using quantitative mass spectrometry and confirmed in our cryo-EM structure. We will elucidate on a structural level how the CM1 motif of the microcephaly protein CDK5RAP2 activates the vertebrate gamma-TuRC and finally, based on these structural insights, we aim to establish heterologous expression of the complete human gamma-TuRC in insect cells to gain access to a manipulatable system for follow-up studies. In conclusion, this project will decisively contribute to our understanding of the structure-function relationship of gamma-TuCs from yeast to human cells.

• DFG Programme: Research Grants