Autophagy-Independent Lysosomal Targeting Regulated by ULK1/2-FIP200 and ATG9

Abstract

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.

TMEM 41B is a novel regulator of autophagy and lipid mobilization

“…Core components of mammalian autophagy such as ATG3, ATG5, ATG7, ATG9A, ATG16L1, RB1CC1, or WIPI2 scored as robust regulators of both p62 and NDP52 (Fig 1B and C). TMEM41B was also among the top-scoring regulators of NDP52 ( Fig 1C) and TAX1BP1 [16]. We identified the uncharacterized transmembrane protein TMEM41B as a strong hit for p62 turnover when autophagy was activated with AZD8055 ( Fig 1B).…”

“…We set out to uncover novel regulators of autophagy using the FACSbased pooled CRISPR screening paradigm outlined in Fig 1A. Neuroglioma H4 cells were chosen for their amenability to pooled CRISPR screening and well-profiled autophagy pathway at genomewide scale [15][16][17]. We monitored two endogenous autophagy cargo receptors, namely p62 and NDP52, by immunofluorescence-based staining in Cas9-expressing H4 cells.…”

“…Single clones were selected based on Cas9 expression levels. H4 Cas9 ATG7 clonal KO cells were previously described [16]. H4 Cas9 clone 7 and HeLa Cas9 clone 4 were used for validation studies.…”

“…In addition to defective autophagy, TMEM41B-deficient cells display enlarged LDs and impaired mobilization and mitochondrial b-oxidation of fatty acids (Figs 3 and 4). Interestingly, levels of the autophagy cargo receptors NDP52 and TAX1BP1 [16] are already basally increased upon TMEM41B KO, whereas levels of GFP-tagged p62 were reduced [15]. One possibility is that TMEM41B depletion causes a cellular defect, which independently impairs autophagy and lipid mobilization.…”

Disruptive Technology: CRISPR/Cas-Based Tools and Approaches

“…Because high-throughput screens depend on readily scorable phenotypes, such as viral infection, cell death or cell proliferation, screening for nonmalignant genetic diseases with phenotypes that mostly only appear at the organismal level is more difficult. Nevertheless, here CRISPR/Cas screens have already revealed novel genes with therapeutic relevance in β-hemoglobinopathies [128], Parkinson’s disease [130], thromboembolisms [131] and inflammatory responses [132] and have shortlisted genes required for processes as diverse as neuronal fate [133], energy metabolism [134] and ferritinophagy [135], giving pointers to novel disease-related pathways and candidate genes.…”