Selective autophagy of organelles is critical for cellular differentiation, homeostasis, and organismal health. Autophagy of the ER (ER-phagy) is implicated in human neuropathy but is poorly understood beyond a few autophagosomal receptors and remodelers. By using an ER-phagy reporter and genome-wide CRISPRi screening, we identified 200 high-confidence human ER-phagy factors. Two pathways were unexpectedly required for ER-phagy. First, reduced mitochondrial metabolism represses ERphagy, which is opposite of general autophagy and is independent of AMPK. Second, ER-localized UFMylation is required for ER-phagy to repress the unfolded protein response via IRE1a. The UFL1 ligase is brought to the ER surface by DDRGK1 to UFMylate RPN1 and RPL26 and preferentially targets ER sheets for degradation, analogous to PINK1-Parkin regulation during mitophagy. Our data provide insight into the cellular logic of ER-phagy, reveal parallels between organelle autophagies, and provide an entry point to the relatively unexplored process of degrading the ER network. UFMylation

2 Summary [150 words]Selective degradation of organelles via autophagy is critical for cellular differentiation, homeostasis, and organismal health. Autophagy of the ER (ER-phagy) is implicated in human neuropathy but is poorly understood beyond a few specialized autophagosomal receptors and remodelers. Using an ER-phagy reporter and genome-wide CRISPRi screening, we identified 200 high-confidence factors involved in human ER-phagy. We mechanistically investigated two pathways unexpectedly required for ER-phagy.First, reduced mitochondrial metabolism represses ER-phagy, which reverses the logic of general autophagy. Mitochondrial crosstalk with ER-phagy bypasses the energy sensor AMPK, instead directly impacting ULK1. Second, ER-localized UFMylation is required for ER-phagy that represses the unfolded protein response. The UFL1 ligase is brought to the ER surface by DDRGK1, analogous to PINK1-Parkin regulation during mitophagy. Our data provide insight into the unique cellular logic of ER-phagy, reveal parallels between organelle autophagies, and provide an entry point to the relatively unexplored process of degrading the ER network.unfolded protein response (UPR) did not cause ER-phagy. Only prolonged starvation (16 hours) using Earl's Buffered Saline Solution (EBSS) robustly induced ER-phagy.Using EBSS as an ER-phagy stimulus, we coupled EATR-based FACS screening with genomewide CRISPR transcriptional inhibition (CRISPRi) to identify novel pathways involved in ER-phagy ( Fig 1A-B) (Gilbert et al., 2014;Horlbeck et al., 2016;Liang et al., 2018). Since autophagy is influenced by the availability of cellular energy, we reasoned that complete knockout of ER-phagy regulators via CRISPR cutting could be detrimental to cells and mask interesting players. The variability in sgRNA efficiencies of CRISPRi leads to different knockdown efficiencies, allowing for allelic series and residual function of essential genes involved in cellular energy regulation .As a proof-of-concept, we first assessed the suitability of EATR for CRISPRi screening by conducting a pilot screen with a custom CRISPRi library targeting known autophagy genes (Table S1).We used EATR-based FACS to isolate the top 25% of cells with the most ER-phagy ('enhanced' sort gate), and the bottom 25% of cells with the least ER-phagy ('inhibited' sort gate) ( Fig S1C). This pilot screen successfully identified gRNAs targeting core autophagy genes as required for ER-phagy, and correctly assigned their role in promoting ER-phagy such that knockdown of autophagy components was enriched in the 'inhibited' gate ( Fig S1D) and depleted in the 'enhanced' sort gate ( Fig S1E).We scaled up to perform an unbiased, genome-wide CRISPRi-v2 screen (Gilbert et al., 2014;Horlbeck et al., 2016) for ER-phagy regulators using EATR-FACS. From the gene-level statistics, we defined a high-confidence list of ER-phagy genes by first performing a cutoff at p < 0.01, and then requiring that true hits have opposite phenotypes in the "enhanced" and "inhibited" sort gates relative to the u...