Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation

Abstract

Macroautophagy is an intracellular degradation process that requires multiple autophagy-related () genes. In this study, we performed a genome-wide screen using the autophagic flux reporter GFP-LC3-RFP and identified as a novel gene. TMEM41B is a multispanning membrane protein localized in the endoplasmic reticulum (ER). It has a conserved domain also found in vacuole membrane protein 1 (VMP1), another ER multispanning membrane protein essential for autophagy, yeast Tvp38, and the bacterial DedA family of putative half-transporters. Deletion of blocked the formation of autophagosomes at an early step, causing accumulation of ATG proteins and small vesicles but not elongating autophagosome-like structures. Furthermore, lipid droplets accumulated in-knockout (KO) cells. The phenotype of -KO cells resembled those of-KO cells. Indeed, TMEM41B and VMP1 formed a complex in vivo and in vitro, and overexpression of VMP1 restored autophagic flux in -KO cells. These results suggest that TMEM41B and VMP1 function together at an early step of autophagosome formation.

Stasimon contributes to the loss of sensory synapses and motor neuron death in a mouse model of spinal muscular atrophy

“…Recent studies have shown that Stasimon is an ER-resident six-transmembrane pass protein that is essential for mouse embryonic development (Van Alstyne et al, 2018b) and functions in autophagy (Moretti et al, 2018;Morita et al, 2018;Shoemaker et al, 2019). However, we found no evidence for overt autophagy dysregulation in motor circuit neurons of SMA mice.…”

“…We then analyzed by Western blot the levels of the autophagy proteins p62 and LC3 in WT and StasRNAi NIH3T3 cells under basal conditions and following autophagy induction with serum starvation or inhibition by Bafilomycin A1 treatment ( Figure S2B). We found that the levels of p62 and lipidated LC3 (LC3-II) were moderately increased in Stasimon-deficient relative to WT cells, consistent with dysregulation of autophagy (Moretti et al, 2018;Morita et al, 2018;Shoemaker et al, 2019). However, Western blot analysis showed no changes in the levels of both p62 and LC3 in spinal cords from WT and SMA mice at P11 ( Figure S2C).…”

“…Recent studies have shown that Stasimon is required for autophagy in cultured mammalian cells (Moretti et al, 2018;Morita et al, 2018;Shoemaker et al, 2019). Therefore, we sought to investigate the possible involvement of Stasimon-dependent autophagy dysregulation in SMA pathogenesis.…”

“…Furthermore, we and others have found that SMN deficiency disrupts U12 splicing (Boulisfane et al, 2011;Doktor et al, 2017;Jangi et al, 2017;Lotti et al, 2012), and both misprocessing and reduced expression of Stasimon mRNA was found in disease-relevant motor circuit neurons of SMA mice . Recent studies have also shown that Stasimon is a transmembrane protein localized to the ER that is essential for mouse embryonic development (Van Alstyne et al, 2018b) and involved in autophagy (Moretti et al, 2018;Morita et al, 2018;Shoemaker et al, 2019). However, Stasimon's normal requirement in the mammalian motor circuit and a direct involvement in pathogenesis of SMA mouse models have not been established.…”

Autophagy dark genes: Can we find them with machine learning?

“…However, recent studies have increasingly emphasized the existence of systems controlling and executing autophagy in mammalian cells that are quite different from those in yeast [3]. Among wellaccepted examples, are several autophagy factors absent in yeast that have been identified in organisms from C. elegans to H. sapiens, including ATG101 [4], FIP200 [5], VMP1 [6], EPG5 [7], Stx17 [8,9], and TMEM41B [10][11][12].…”