Novel mechanisms regulating the membrane recruitment of the ATG8 conjugation machinery
Item statusRestricted Access
Embargo end date31/07/2022
Dudley, Leo J.
Autophagy is a highly conserved eukaryotic process that maintains cell viability by targeting excess or damaged cytoplasmic material for lysosomal degradation. This process involves the generation of a double-membraned organelle, termed the autophagosome, which grows around cellular cargo destined for lysosomal delivery. Underpinning the efficient biogenesis of autophagosomes is the activity of the ATG12-ATG5/ATG16L1 protein complex (the ATG5 complex), which catalyses the conjugation of ATG8 proteins to lipid headgroups on the growing autophagosome. However, the mechanism by which the ATG5 complex is recruited to autophagic membranes, the site where it enacts its activity, is not fully understood under specific settings. The research described in this thesis aims to identify novel mechanisms that mediate the recruitment of the mammalian ATG5 complex to autophagic structures following the induction of canonical autophagy. Over the course of these studies, several interesting discoveries were made. Firstly, a PI3P-binding site located in the coiled-coil domain of ATG16L1 was identified. Functionally, this interaction mediates the efficient recruitment of the ATG5 complex to autophagic membranes and, consequently, determines whether autophagy occurs. Secondly, further characterisation of WIPI2, a factor implicated in recruiting the ATG5 complex to autophagic structures, provided novel insights into the functions of this protein during autophagosome biogenesis. Thirdly, several proteins of the ATG8 conjugation machinery were tagged with a newly bioengineered proximity-labelling enzyme, termed TurboID, leading to the generation of a promising list of potential interactors for these factors in mammalian cells. As a whole, this thesis highlights additional activities for various autophagy proteins in facilitating autophagosome biogenesis.