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Title: Investigating the interactions of Pex3p in Saccharomyces cerevisiae
Author: Hutchinson, John
ISNI:       0000 0004 6060 6231
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2016
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Active partitioning of organelles during cell division is conserved throughout the eukaryotic kingdom and has been suggested to play roles in both ageing and cellular fate determination. However, the mechanisms by which each organelle is segregated vary depending on the organelle in question. Like most organelles, a myosin motor, Myo2p, is required for peroxisomal transport in S. cerevisiae and a cellular anchor, Inp1p, is required for retention. Furthermore, the method for segregation of peroxisomes during cell division relies on proteins found to be within the interactome subnetwork of Pex3p (i.e. proteins found to interact with, or be reliant on another protein's interaction with, Pex3p). Pex3p is an integral peroxisomal membrane protein that acts as a hub to which a number of proteins bind. The processes it is involved in encompass the whole lifespan of a peroxisome, ranging from the de novo formation at the endoplasmic reticulum, continuing through growth and peroxisomal membrane protein import through its interaction with Pex19p; during the maintenance of peroxisomes during cell division to provide equal segregation through its interactions with Inp1p for retention and its involvement in Myo2p recruitment via Pex19p; and ending in selective peroxisomal breakdown, also known as pexophagy via its interaction with Atg36p. There are already precedents for individual proteins acting as such a hub for other proteins which are regulators of different aspects of an organelle's function. Through a truncation screen, a region within Inp1p required for co-localisation to Pex3p was identified (aa310-317). This region was found to be conserved throughout other Inp1ps found in different budding yeast species. Furthermore, in vivo and in vitro techniques determined that this region was required for interacting with Pex3p. Further research found that this binding motif bares close similarity to the known Pex3p binding motif contained within Pex19p in H. sapiens. This motif too appears conserved throughout eukaryotic Pex19ps. In vivo and in vitro techniques were again used to determine that this motif within Pex19p was required for Pex3p interaction in S. cerevisie, and therefore required for Pex19p function. It was demonstrated in vivo that overexpression of Inp1p traps Pex3p at the periphery of the cell, leading to an absence of peroxisomes. In vitro and in vivo data suggests that this may be due to Pex19p being unable to access Pex3p when there is an overwhelming presence of Inp1p, due to saturation of a possible shared binding site. However, it was also shown that other Pex3p binding proteins, such as Atg36p, are still able to access Pex3p under these conditions. However, attempts to unravel the coordination of these processes have thus far yielded no concrete solution for the underlying mechanism of how the variety of processes in which Pex3p is involved are orchestrated.
Supervisor: Hettema, Ewald Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available