The molecular organization of the phagophore assembly site

Abstract

Autophagy is a degradative pathway crucial for multiple cellular processes and it is implicated in numerous diseases. Despite great efforts being made to understand the fundamental aspects of this pathway, many questions still remain unanswered. For instance, the origin of the phagophore assembly site (PAS) is unknown as well as the source of the membranes composing autophagosomes. Moreover, the precise molecular function of many Atg proteins is not well understood. Understanding the mechanism underlying autophagy is of crucial importance not only because it is involved in a multitude of physiological processes, but also because it is implicated in numerous diseases such as neurodegenerative disorders and cancer. In this thesis some fundamental questions are addressed regarding the origin of the phagophore assembly site (PAS) and the regulated recruitment of Atg18 and Atg2 to this structure using yeast Saccharomyces cerevisiaeas a model organism. The first part of this thesis is a review that provides an overview of the roles of the cytoskeleton network in autophagy in both yeast and mammalian cells. In mammalian cells, in particular, this cellular scaffold is critical for the movement of complete autophagosomes towards cellular locations where endosomes and lysosomes concentrate. The mechanism of this transport still needs elucidation and some possible models are described in this part of the thesis. Next, we have focused on Atg18, a protein essential for autophagy but also for vacuole homeostasis and probably endosomal functions. We investigated how Atg18 recruitment to the PAS is regulated and found that Atg18 via its β-propeller is able to bind both Atg2 and phosphoinositides. In this chapter we propose a model where the Atg18 β-propeller provides organelle specificity by binding two determinants on the target membrane, thus underlining the potential capacity of specific β-propellers to form protein-lipid complexes. We also characterized the interaction between Atg2 and Atg9. Atg2 is implicated in Atg9 retrieval from the PAS. We have identified the binding motifs in Atg2 and Atg9 and generated a number of Atg9-binding mutants that will allow us to study the function of the Atg2-Atg9 interaction in autophagy and provide a useful tool for studying Atg9 retrieval. Furthermore, we highlighted the contribution of Atg9 to the early stages of autophagosome biogenesis by investigating the role of this protein in the formation of the PAS. We elucidate that in yeast the PAS originates from Atg9-positive clusters of vesicles and tubules that we called Atg9 reservoirs. Translocation of one or more reservoirs in proximity to the vacuole, together with the successive recruitment of other Atg proteins, leads to the generation of the PAS.