Project Summary The endoplasmic reticulum (ER) undergoes autophagic degradation in response to the accumulation of aggregated proteins within its lumen or in response to starvation. We have proposed that in yeast, actin assembly at sites of contact between the cortical ER (cER) and endocytic pits is required to displace elements of the cER from their association with the plasma membrane so that they can interact with the autophagosome assembly machinery near the vacuole. To test our model, we will determine if the requirement in cER-phagy for actin assembly at endocytic pits can be bypassed by deleting all six genes encoding ER-plasma membrane tethers (DTether). If the role of actin assembly is to push the cER away from the plasma membrane, the DTether mutations should bypass the need for actin assembly in ER-phagy. We will ask if the bypass of actin assembly is specific to cER-phagy or if actin-related defects in other selective autophagy pathways, such as nucleophagy, mitophagy, and pexophagy are also bypassed. As an additional test we will ask if an artificial ER-plasma membrane tether can block cER- phagy in both wild type and the DTether strain. Extensive parallels between ER-phagy in yeast and mammalian cells suggest a conserved mechanism. A long-term goal will be to use siRNA knock downs of actin assembly components in U2OS cells to assess their roles in ER-phagy. We will order all known cER-phagy requirements with respect to the displacement of the cER from the plasma membrane. Our recent data suggests that a vesicle coat adaptor protein associates with a selective ER-phagy receptor prior to the displacement of cER from the plasma membrane. We will develop a bimolecular fluorescence complementation assay to visualize the dynamics of this ER-phagy intermediate in live cells. We will also order the displacement of the cER from the plasma membrane with respect to the super assembly of Atg40 oligomers, a process required for packaging of ER fragments into autophagosomes. We will explore a novel fluorescence complementation assay for super assembly of Atg40 oligomers that will allow visualization in live cells. In total our studies will define the pathway by which the ER is degraded by autophagy. This pathway plays a key role in a variety of human diseases including neurodegenerative disorders and forms of diabetes.