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.
Highlights
Autophagy is an umbrella term for a broad family of trafficking pathways that transport cytoplasmic material to the lysosome for destruction
The inventory of factors involved in autophagy has been further expanded by the discovery of additional autophagy factors not found in yeast, such as ATG101, EPG5, and vacuole membrane protein 1 (VMP1) [5,6,7,8]
The red fluorescent protein (RFP) to green fluorescent protein (GFP) fluorescence ratio (Red:Green ratio) of tfLC3 is a widely used metric for autophagic flux predicated on the selective quenching of GFP in low pH environments, such as the lysosomal lumen (Fig 1A)
Summary
Autophagy is an umbrella term for a broad family of trafficking pathways that transport cytoplasmic material to the lysosome for destruction. The first factors identified in autophagosome biogenesis, the so-called autophagy-related (ATG) factors, were originally identified by genetic screens in yeast [1,2,3]. This list eventually matured to its current state that includes approximately 40 yeast ATGs and distinguishes “core” factors required for all forms of autophagy from those required only for specific substrates (e.g., a damaged mitochondrion) [4]. The inventory of factors involved in autophagy has been further expanded by the discovery of additional autophagy factors not found in yeast, such as ATG101, EPG5, and vacuole membrane protein 1 (VMP1) [5,6,7,8]. Whether additional autophagy factors have yet to be discovered remains an open question
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