Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

Joanne Durgan,Alf H Lystad,Katherine Sloan,Sven R Carlsson,Michael I Wilson,Elena Marcassa,Rachel Ulferts,Judith Webster,Andrea F Lopez-Clavijo,Michael J Wakelam,Rupert Beale,Anne Simonsen,David Oxley,Oliver Florey

doi:10.1016/j.molcel.2021.03.020

Joanne Durgan, Alf H Lystad + Show 12 more

Open Access

https://doi.org/10.1016/j.molcel.2021.03.020

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Abstract

SummaryAutophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific “molecular signature” for the non-canonical autophagy pathway.

Highlights

A defining feature of autophagy is the lipidation of ATG8, a family of ubiquitin-like proteins including mammalian LC3A/B/B2/C and GABARAP/L1/L2 (Johansen and Lamark, 2020; Mizushima, 2020)
GFP-tagged ATG8s were expressed in different cell lines, treated with different stimuli, to drive ATG8 lipidation associated with either canonical autophagy or conjugation of ATG8 to single membranes (CASM) (Figure 1)
Canonical autophagy was induced in wild-type (WT) cells expressing GFP-hLC3A, by co-treatment with mTOR (PP242) and V-ATPase (BafA1) inhibitors, which induce and accumulate autophagosomes, respectively

Summary

Introduction

A defining feature of autophagy is the lipidation of ATG8, a family of ubiquitin-like proteins including mammalian LC3A/B/B2/C and GABARAP/L1/L2 (Johansen and Lamark, 2020; Mizushima, 2020). A ubiquitin-like conjugation system, composed of ATG7, ATG3, and ATG16L1/12/5, drives the covalent ligation of this glycine to a lipid, phosphatidylethanolamine (PE), through an amide bond to its headgroup (Figure S1A) (Ichimura et al, 2000; Kirisako et al, 2000). This unique posttranslational modification recruits ATG8 to autophagosomal membranes, where it modulates cargo loading and maturation (Johansen and Lamark, 2020; Nguyen et al, 2016). The mechanism underlying cellular specificity is not fully understood, but physiological pH and phospholipid composition may prohibit alternative lipidation to PS (Nakatogawa et al, 2008; Oh-oka et al, 2008)

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