Abstract 1434 "Mapping the interaction between Atg11 and Atg9 in selective autophagy"

Macroautophagy (hereafter, autophagy) is a process of degradation or removal of unwanted cellular components. It consists of two pathways: selective and nonselective autophagy. Nonselective autophagy is induced by starvation and degrades any cargo. Cellular stresses induce selective autophagy, which degrades or recycles a specific cargo. For example, mitophagy is the selective autophagic pathway that targets damaged mitochondria for recycling. Autophagy helps prevent neurodegenerative diseases like Parkinson's or Huntington's, cancer, and infection by bacterial pathogens. It involves the formation of a double-membrane vesicle, an autophagosome, that wraps around the cargo and takes it to the vacuole or lysosome for degradation. Atg11 is the central organizer of the autophagy initiation complex in selective autophagy. It acts as a scaffold protein that interacts with Atg9, Atg1 and other Atg proteins for the formation of the phagophore, the membrane structure that matures into an autophagosome. Atg9 is a critical binding partner of Atg11. It acts as a scramblase protein that flips phospholipids to form the phagophore membrane bilayer. Atg1 also interacts with Atg11 in autophagosome formation. It acts as a protein kinase that provides structure to the autophagy initiation complex and activates other Atg proteins for autophagosome formation. Our research goal is to find mutations that specifically cause a loss of interaction between Atg11 and Atg9 without interfering with Atg11 dimerization or its interaction with Atg1; we are focusing on residues 455-535 of Atg11 as a putative Atg9 interaction site. We performed mutagenesis on four residues in this region of Atg11 that we hypothesized to be important based on a sequence alignment of Atg11 homologs. Then we used yeast-2-hybrid and coimmunoprecipitation to test for a loss of interaction with Atg9. These results for the quadruple mutant (Y468A, D516A, L517A, L526A) showed a loss of interaction between Atg11 and Atg9, and only a partial loss between Atg11 and Atg1. Next, we tested two double mutants. However, Y468A, L526A showed no loss of interaction with Atg9 or Atg1, while D516A, L517A showed at most a partial loss of interaction. Thus, none of these residues could be individually identified as essential for the specific interaction of Atg11 with Atg9. Our next step will be to identify and test other potential residues in Atg11's 455-535 region that may be critical for its interaction with Atg9. This research was funded by NSF RUI grant #2243163 to Dr. Steven K. Backues and by the Eastern Michigan University Graduate College and Chemistry department.