CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor

Shoemaker, Christopher J. and Huang, Tina Q. and Weir, Nicholas R. and Polyakov, Nicole J. and Schultz, Sebastian W. and Denic, Vladimir and Simonsen, Anne (2019) CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor. PLOS Biology, 17 (4). e2007044. ISSN 1545-7885

[thumbnail of file (1).pdf] Text
file (1).pdf - Published Version

Download (4MB)

Abstract

The power of forward genetics in yeast is the foundation on which the field of autophagy research firmly stands. Complementary work on autophagy in higher eukaryotes has revealed both the deep conservation of this process, as well as novel mechanisms by which autophagy is regulated in the context of development, immunity, and neuronal homeostasis. The recent emergence of new clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based technologies has begun facilitating efforts to define novel autophagy factors and pathways by forward genetic screening in mammalian cells. Here, we set out to develop an expanded toolkit of autophagy reporters amenable to CRISPR/Cas9 screening. Genome-wide screening of our reporters in mammalian cells recovered virtually all known autophagy-related (ATG) factors as well as previously uncharacterized factors, including vacuolar protein sorting 37 homolog A (VPS37A), transmembrane protein 251 (TMEM251), amyotrophic lateral sclerosis 2 (ALS2), and TMEM41B. To validate this data set, we used quantitative microscopy and biochemical analyses to show that 1 novel hit, TMEM41B, is required for phagophore maturation. TMEM41B is an integral endoplasmic reticulum (ER) membrane protein distantly related to the established autophagy factor vacuole membrane protein 1 (VMP1), and our data show that these two factors play related, albeit not fully overlapping, roles in autophagosome biogenesis. In sum, our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms.

Item Type: Article
Subjects: OA Digital Library > Biological Science
Depositing User: Unnamed user with email support@oadigitallib.org
Date Deposited: 19 Jan 2023 11:25
Last Modified: 22 May 2024 09:15
URI: http://library.thepustakas.com/id/eprint/122

Actions (login required)

View Item
View Item