Autophagy is known to be required for eukaryotic cells to withstand nutrient starvation by mediating the degradation of cellular components to supply substrates for ATP generation thereby helping cells survive until extracellular nutrient availability returns. Autophagy also mediates forms of quality control within cells by engulfing and degrading protein aggregates and damaged mitochondria that mitigates neurodegeneration in mice. Autophagy substrates become encapsulated by a double membrane structure that then fuses with lysosomes to mediate degradation of the cargo. How autophagosomes form and how their substrates are recognized for engulfment remain poorly understood. We are exploring the regulation and mechanism of gene products including ATG6, ATG13, ATG14 that are known to be required for autophagy. ATG6 is known to form a complex with VPS34 and ATG14 and its recruitment to membranes is dependent on ATG14. We have found that a novel domain of ATG6 that is required for membrane binding and for autophagy induction. We have confirmed that the autophagy activity and membrane targeting function of this domain is conserved in yeast and mammals. Fusion of this domain to the apoptosis inducing protein Bax spontaneously activates Bax indicating that this domain can target mitochondrial membranes. We have also found that Atg6 is phosphorylated specifically upon autophagy induction and we have identified the amino acids phosphorylated allowing us to mutate these sites and determine the role of phosphorylation in autophagy regulation. To more conclusively understand the molecular mechanisms of autophagy we have knocked out ATG5, ATG6, ATG13, and ATG14 in mammalian cells using new Talen nuclease technology. These knock out cells reveal that ATG6 phosphorylation depends on expression of ATG14, a protein that specifically docks ATG6 to membranes and is crucial for autophagosome induction. We are using these knock out cells to understand how autophagosomes form and how they recognize and engulf specific cargo such as damaged mitochondria. In a collaborative project we found that the chaperone, HSP90 is required for the selective autophagy of mitochondria and accumulation of ATG13 on the mitochondria as a step in this process. We also plan to explore if the loss of dopaminergic neurons that can occur in animal models of Parkinson's disease can be rescued by up regulation of autophagy pathways.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2013
Total Cost
$1,214,399
Indirect Cost
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State
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Randow, Felix; Youle, Richard J (2014) Self and nonself: how autophagy targets mitochondria and bacteria. Cell Host Microbe 15:403-11
Shen, Qinfang; Yamano, Koji; Head, Brian P et al. (2014) Mutations in Fis1 disrupt orderly disposal of defective mitochondria. Mol Biol Cell 25:145-59
Yamano, Koji; Fogel, Adam I; Wang, Chunxin et al. (2014) Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy. Elife 3:e01612
Lazarou, Michael; Narendra, Derek P; Jin, Seok Min et al. (2013) PINK1 drives Parkin self-association and HECT-like E3 activity upstream of mitochondrial binding. J Cell Biol 200:163-72
Fogel, Adam I; Dlouhy, Brian J; Wang, Chunxin et al. (2013) Role of membrane association and atg14-dependent phosphorylation in beclin-1-mediated autophagy. Mol Cell Biol 33:3675-88
Huang, Chiu-Hui; Lazarou, Michael; Youle, Richard J (2013) Sequestration and autophagy of mitochondria do not cut proteins across the board. Proc Natl Acad Sci U S A 110:6252-3