Joachim, JF; (2015) Elucidating the function of WAC in macroautophagy through investigating the WAC-GM130 interaction. Doctoral thesis , UCL (University College London).

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Abstract

Macroautophagy, hereafter called autophagy, is an intracellular degradation pathway that functions to degrade bulk material and recycle macromolecules to maintain cell homeostasis. Autophagy has a protective role and is also implicated in disease processes such as cancer, neurodegeneration and pathogen clearance. Autophagy is characterised by the formation of a double-membrane structure in response to stress insults such as amino acid starvation, which encapsulates cargo to form an enlarged vesicle called an autophagosome. The autophagosome fuses with the lysosome for degradation of the contents. Although the core protein machinery of autophagy has been known for some time, the regulation of this process is not fully understood. WAC was previously identified as a novel positive regulator of starvation-induced autophagy. However the mechanism by which WAC regulates autophagy is completely unknown. It is known that WAC performs functions both in the nucleus and on the Golgi. In order to elucidate the function of WAC in autophagy, I performed a study of WAC function by a combination of microarray transcriptomic analysis and immunoprecipitation coupled with mass spectrometry to identify genes regulated by WAC and novel WAC interaction partners, respectively. WAC is not a potent regulator of autophagy gene expression. However, I identified GM130, a negative regulator of autophagy, as a novel WAC interaction partner. I show here that WAC promotes ULK1 activation whereas GM130 inhibits the early autophagy stages. GM130 is the receptor for WAC on the Golgi and binds WAC directly. This interaction is mediated by the C-terminal regions of both proteins which contain predicted coiled-coil domains. WAC and GM130 both interact with the autophagy protein GABARAP, indirectly and directly respectively. WAC regulates a novel centrosomally-localised pool of GABARAP, GABARAP binding to GM130 and GABARAP localisation to the Golgi. This centrosomal pool of GABARAP, is able to translocate to autophagic structures during starvation. In addition I describe a GABARAP mediated activation of the ULK1 complex. Through the WAC-GM130-GABARAP interplay, I propose that WAC promotes a centrosome to autophagosome GABARAP trafficking step that may in addition maintain ULK1 activation during starvation-induced autophagy.

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