Tuesday, April 16, 2024
4/16/2024
Project Summary Selective autophagy pathways use cargo receptors to degrade organelles, organelle subdomains, and misfolded proteins that fail to be degraded by the proteosome. Endoplasmic reticulum (ER) autophagy (also called ER-phagy) is a selective autophagy pathway that acts in ER quality control. ER-phagy cargo receptors connect an ER domain to the autophagosome biogenesis machinery via their ability to bind Atg8 family members (LC3 or GABARAP in mammals). The studies in this proposal are aimed at addressing two important unanswered questions in the field. First, we will ask how the conserved yeast ER-phagy cargo receptor, Atg40, fragments domains of the ER that it targets for degradation. Second, we will ask how Atg40 loads ER domains into autophagosomes, sealed double-membrane structures that are delivered to vacuoles (yeast) or lysosomes (mammals) for degradation. We have found that a non-canonical form of the yeast COPII coat subcomplex, Sec23-Lst1, works with Atg40 to package ER domains into autophagosomes during ER-phagy. The COPII coat is a multi-subunit coat complex that is known for its role in sorting ER proteins into transport carriers that traffic on the secretory pathway. We identified a novel role for Sec23-Lst1 in ER-phagy that is independent of its role in secretion. While the reticulon homology domain of Atg40 has been implicated in ER fission, it is unclear if Atg40 requires Sec23-Lst1 to fragment ER domains. An in vitro approach is needed to unambiguously answer this question. We have found that a variation of the COPII coat in vitro vesicle budding assay can be used to assess the requirements for Atg40-mediated ER fission. The COPII coat is formed by the sequential interactions of the Sar1 GTPase and cytoplasmic coat subcomplexes. We will use this in vitro assay to address if Atg40, Sec23- Lst1 and Atg8 are all needed for fission. Additionally, we will determine if purified Sar1, and other purified cytoplasmic COPII subcomplexes are also required. A long-term goal of these studies is to develop a similar in vitro ER fragmentation assay with mammalian COPII coat subcomplexes. To address how Atg40 sequesters membrane domains into autophagosomes, we will take advantage of an unusual phenotype we observed in lipid droplet (LD) deficient cells. Lipid droplets are ER-derived organelles that contain a neutral lipid core, triacylglycerides (TAG) and sterol esters (SE), surrounded by a phospholipid monolayer. When yeast cells are devoid of LD, resident ER membrane proteins fail to be delivered to the vacuole via ER-phagy. This defect appears to be due to the inability of the cargo receptor, Atg40, to sequester ER domains into autophagosomes. We will perform biochemical, genetic and localization studies to ask how LD are needed to couple Atg40 to its cargo. A long-term goal of these studies will be to address the role of LD in mammalian ER-phagy. These studies will be relevant to variety of metabolic disorders in humans, including diabetes and obesity.
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