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Title: Investigating interactions between the endosomal ZFYVE proteins Endofin and SARA and the ESCRT accessory protein HDPTP
Author: Walker, Louise
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2014
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The Endosomal Sorting Complex Required for Transport (ESCRT) proteins are a series of four protein complexes (numbered ESCRT-0 to III) which aid the endosome in sorting internalised receptors. The ESCRT proteins recognise receptors which have been tagged with ubiquitin to help sort between receptors which need to be recycled from those which are to be degraded. The ESCRT proteins are aided in their function by HDPTP, a Bro1-domain protein which can act as a scaffold by replacing one ESCRT complex with another on the Epidermal Growth Factor Receptor (EGFR).The proteins Endofin and SARA both bind to the endosome membrane using a FYVE domain, and were identified in a commercial yeast-2-hybrid screen as potential interacting partners for HDPTP. Functionally, SARA has been suggested as a signalling anchor in the Transforming Growth Factor β (TGFβ) pathway; Endofin performs the same role in the related Bone Morphogenic Protein (BMP) pathway. Additionally, both Endofin and SARA have been suggested to have roles in EGFR signalling and have independently been implicated as members of alternative complexes to the first ESCRT complex, ESCRT-0.The results presented in this thesis confirm that Endofin and SARA can both interact with the Bro1-V domain of HDPTP. Both Endofin and SARA can recruit exogenously expressed, full length HDPTP to structures which contain endocytic markers. Further characterisation shows that Endofin requires an α-helix at its N-terminus to interact with several regions in the HDPTP Bro1-V domain. Knockdown studies show that Endofin co-operates with SARA and possibly ESCRT-0 in regulating HDPTP localisation to the endosome. It is also shown that Endofin and SARA can influence positioning of certain organelles, especially lysosomes. However, no influence of either Endofin or SARA on endosome morphology or distribution has been found. The results also indicate that Endofin can bind to the EGFR and is phosphorylated downstream of EGFR signalling but does not directly influence EGFR trafficking or signalling. Overall, this project has discovered two novel interacting partners for HDPTP: Endofin and SARA. The results also contribute to the understanding of the functions that Endofin and SARA perform at the endosome and during cell signalling.
Supervisor: Woodman, Philip ; Delneri, Daniela Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available