SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.DOI: http://dx.doi.org/10.7554/eLife.17290.001

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well-characterized autophagy-related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2- and DFCP1-positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and β-oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER-localized regulator of autophagosome biogenesis and lipid mobilization.

SUMMARY Iron is vital for many homeostatic processes and its liberation from ferritin nanocages occurs in the lysosome. Studies indicate that ferritin and its binding partner nuclear receptor coactivator-4 (NCOA4) are targeted to lysosomes by a form of selective autophagy. By using genome-scale functional screening we identify an alternative lysosomal transport pathway for ferritin that requires FIP200, ATG9A, VPS34 and TAX1BP1 but lacks involvement of the ATG8 lipidation machinery that constitutes classical macroautophagy. TAX1BP1 binds directly to NCOA4 and is required for lysosomal trafficking of ferritin under basal and iron depleted conditions. Under basal conditions ULK1/2-FIP200 controls ferritin turnover but its deletion leads to TAX1BP1-dependent activation of TBK1 which regulates redistribution of ATG9A to the Golgi enabling continued trafficking of ferritin. Cells expressing an Amyotrophic Lateral Sclerosis (ALS)-associated TBK1 allele are incapable of degrading ferritin suggesting a novel molecular mechanism that explains the presence of iron deposits in patient brain biopsies.

SignificanceIn mitophagy, damaged mitochondria are targeted for disposal by the autophagy machinery. PARKIN promotes signaling of mitochondrial damage to the autophagy machinery for engagement, and PARKIN mutations cause Parkinson’s disease, possibly because damaged mitochondria accumulate in neurons. Because regulation of PARKIN abundance and the impact on signaling are poorly understood, we performed a genetic screen to identify PARKIN abundance regulators. Both positive and negative regulators were identified and will help us to further understand mitophagy and Parkinson’s disease. We show that some of the identified genes negatively regulate PARKIN gene expression, which impacts signaling of mitochondrial damage in mitophagy. This link between transcriptional repression and mitophagy is also apparent in neurons in culture, bearing implications for disease.