AbstractThe formation of the autophagosome is highly dynamic and complex, and requires the coordinated action of many conserved autophagy related (ATG) proteins. The two ubiquitin-like (UBL) conjugation systems, play essential roles in the final stages of autophagosome biogenesis; specifically, during autophagosome shaping, elongation and closure. However, the exact molecular mechanisms that govern these processes are unclear. To provide further insight into the molecules that regulate autophagosome assembly, I have used quantitative SILAC-based proteomics to obtain an unbiased, proteome of the first UBL conjugation system by using ATG5 to identify novel interactors that regulate autophagosome biogenesis. Many of the novel interactors had roles protein trafficking and specifically clathrin-mediated trafficking. The proteomics investigation also highlighted that the formation of the ATG12-ATG5 conjugate is necessary, as many of these interactions were lost when a conjugation- deficient ATG5 mutant was employed. Two interactors that could be validated by immunoblotting—PI2KC2a and HIP1R—were shown to be required autophagy, with each having subtly different effects on basal and starvation-induced autophagic flux. This highlights the close interplay between regulators of clathrin-mediated endocytosis and the autophagy machinery, via the ATG12-ATG5 complex.
The overwhelming enrichment of many proteins involved in clathrin-mediated membrane trafficking led me to hypothesise that the ATG12-ATG5 conjugate has a role in regulating this process. To explore this further, I carried out biotinylation based surface proteomics using triple SILAC labelling. This approach revealed that autophagy has a putative role in the membrane trafficking of specific cell surface proteins via clathrin-mediated trafficking. Surface levels of the amino acid transporters LAT1/4F2hc, were shown to be regulated in an ATG5-dependent fashion and intracellular arginine levels, were shown to depend in part on the status of ATG5 in these cells. We therefore propose a global role for autophagy and or ATG12-ATG5 or unconjugated ATG5 in the regulation of cell surface protein internalisation and/or trafficking, which may in part rely on the interaction of ATG12-ATG5 to key components of the clathrin-mediated trafficking machinery.
|Date of Award||23 Jan 2019|
|Supervisor||Jon D Lane (Supervisor)|