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    Structural and Biophysical Characterisation of Sgt1, An Essential Component in the Assembly of the Yeast Kinetochore

    Willhöft, Oliver (2013) Structural and Biophysical Characterisation of Sgt1, An Essential Component in the Assembly of the Yeast Kinetochore. PhD thesis, Birkbeck, University of London.

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    Abstract

    Sgt1, a co-chaperone of Hsp90, plays a fundamental role in plant immunity, protein degradation and kinetochore assembly in yeast. Sgt1 has a multi-domain architecture, including an N-terminal tetratricopeptide repeat (TPR) domain. Through its modular architecture, Sgt1 is believed to bridge the interaction between two key structural components of the yeast inner kinetochore, Skp1 and Ctf13, and Hsp90. This interaction recruits Hsp90 to Ctf13 for subsequent activation into a complex that is competent to bind centromere DNA. This is an essential first step in the assembly of the yeast kinetochore, since activation of Ctf13 is a prerequisite for the subsequent binding of all other components of the kinetochore. The aim of this project was to gain biophysical and structural insight into Sgt1 and the basis of its interaction with Skp1, utilising X-ray crystallography and biophysical methods. Sgt1 exhibits a novel mode of TPR dimerisation in the structure of an Sgt1:Skp1 complex and a second potential mode of dimerisation in the structure of the Sgt1 TPR domain alone. One of these interfaces utilises yeast specific insertions and was confirmed to exist in solution by mutagenesis s tudies targeting the interface. Biophysical studies show that Sgt1 exists predomina ntly as a dimer in solution, but truncating its C-terminal domains exposes regions that promote trimer formation. The structure of the Sgt1:Skp1 complex shows that the interface with Skp1 comprises the concave groove of the TPR domain. Studies of the stoichiometry of Sgt1:Skp1 suggest that the functionally relevant species is 2:1, though the structure shows a fundamental 1:1 interaction. Targeting this interface by mutagenesis disrupts complex formation and show also that a monomer of Sgt1 is sufficient to bind Skp1. Together, this data is used to present an updated m odel for the role of Hsp90 in kinetochore assembly.

    Metadata

    Item Type: Thesis
    Copyright Holders: The copyright of this thesis rests with the author, who asserts his/her right to be known as such according to the Copyright Designs and Patents Act 1988. No dealing with the thesis contrary to the copyright or moral rights of the author is permitted.
    Depositing User: Acquisitions And Metadata
    Date Deposited: 23 May 2013 14:15
    Last Modified: 01 Mar 2021 11:54
    URI: https://eprints.bbk.ac.uk/id/eprint/40026

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