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Title: Structural and functional organisation of ESCRT complexes
Author: Gill, D. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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My structure of the highly conserved heterotrimeric ESCRT-I core provides a blueprint for the assembly of ESCRT-I. The structure shows a fan-like arrangement of three helical hairpins, each corresponding to a different subunit. Vps23/TSG101 is the central hairpin sandwiched between the other subunits, explaining the critical role of its “steadiness box” in the stability of ESCRT-I. Subsequently, Mvb12, a fourth subunit of ESCRT-I was identified in yeast. I find that Mvb12 engages ESCRT-I directly, with nanomolar affinity to form a 1:1:1:1 heterotetramer. Mvb12 plays a critical role in maintaining structural stability of the ESCRT-I complex, through extensive interactions with both the fan-shaped structural core and another region within ESCRT-I built from upstream sequences present in the Vps23 and Vps37 subunits. Yeast ESCRT-I and ESCRT-II interact directly in vitro, however, this association is not detected in yeast cytosol. To gain understanding of the molecular mechanisms of this link, I have characterised the ESCRT-I/-II super-complex and determined the crystal structure of its interface. The link is formed by the Vps28 C-terminus (ESCRT-I) binding with nanomolar affinity to the Vps36-NZF-N zinc-finger domain (ESCRT-II). A hydrophobic patch on the Vps28-CT four-helix bundle contacts the hydrophobic knuckles of the Vps36-NZF-N. Mutation of the ESCRT-I/-II link results in a cargo-sorting defect in yeast, apparently due to the failure to generate ILVs on the maturing endosome. Interestingly, the two Vps36 NZF domains, NZF-N and NZF-C, despite having the same core fold, use distinct surfaces to bind ESCRT-I or ubiquitinated cargo. Mvb12 does not affect the affinity of ESCRT-I for ESCRT-II in vitro. In contrast, deletion of Mvb12 results in formation of a constitutive ESCRT-I/II association in vivo. These data suggest a complex regulatory mechanism for the ESCRT-I/-II link in yeast.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available