Identification and Characterization of autophagic and non-autophagic Interaction Partners of Atg18
von Lisa Marquardt
Datum der mündl. Prüfung:2021-05-21
Betreuer:Prof. Dr. Michael Thumm
Gutachter:Prof. Dr. Michael Thumm
Gutachter:Prof. Dr. Blanche Schwappach
EnglischThe PROPPIN Atg18 belongs to the WD40 repeat containing family and folds into a typical seven-bladed beta-propeller. Conserved regions at blade 5 and 6 allow for binding to phosphatidylinositolphosphates and mediate the association with the membrane. WD40 repeat proteins often act as platform for protein-protein interactions, which turns Atg18 into an efficient PtdInsP effector. In yeast the protein fulfills two distinct functions: it is essential for the process of autophagy but also regulates membrane fission events at the vacuole. Autophagy is a physiological process to degrade cellular components, either cytosolic proteins or whole organelles. This is necessary in normal cellular homeostasis to regulate protein turnover but also helps the cell in adapting to extracellular stress such as nutrient scarcity by mobilizing metabolic precursors. During the process cargo is engulfed in a double-membraned sphere that fuses at the edges to form an autophagosome. Fusion with the vacuole releases the cargo enveloped by a single-membrane into the vacuolar lumen to be degraded. Atg18 is essential for membrane expansion during the early stages of autophagosome formation. The second function of Atg18 is at the vacuolar membrane: Atg18 forms a complex with the vacuolar PtdIns 5-kinase and its regulators Vac7 and Vac14 as well as the phosphatase Fig4. Here, it regulates the synthesis of PtdIns(3,5)P2 by providing negative feedback to the kinase. It is also thought to promote fission events by inducing membrane curvature with its hydrophobic loop inserted into the membrane. Not much is known about the mechanisms behind the regulation of vacuolar morphology mediated by Atg18. Therefore, a proximity-dependent labeling assay combined with SILAC was performed to identify potential interaction partners of Atg18. Several new proteins potentially in a complex with Atg18 were identified and three of them could be validated by independent experiments. One of them was chosen for further analyses. Vps35 is a component of the retromer complex, which mediates retrograde transport from the endosome and the vacuole of Golgi-resident membrane proteins. Another protein identified in the BioID approach was the sorting nexin Snx3, which forms a complex with retromer and mediates contact with the membrane. Atg18 was shown to be a cargo of this trafficking pathway, as a defect in retromer – deletion of VPS35 -caused mislocalization of the PROPPIN to the vacuolar and autophagosomal membrane. This indicates involvement of the retromer complex in the recycling of autophagosomal proteins, or at least Atg18, after autophagy. This could occur either at the autophagosomal membrane or at the vacuolar membrane after autophagosome-vacuole fusion. Interestingly, the only known integral membrane protein located to the PAS, Atg9, was also observed to interact with Vps35. Most likely, recycling of Atg9 is not mediated directly by the retromer complex, as deletion of Vps35 does not cause Atg9 mislocalization to the vacuolar membrane. However, it was shown that retrograde trafficking mediated by the retromer complex is involved in recycling proteins of the autophagic machinery.
Keywords: Atg18; Vps35; Retromer; BioID; Atg9; autophagy; SILAC