The insufficiency of ATG4A in macroautophagy

Nathan Nguyen,Taryn J Olivas,Antonio Mires,Jiaxin Jin,Shenliang Yu,Lin Luan,Shanta Nag,Karlina J Kauffman,Thomas J Melia

doi:10.1074/jbc.ra120.013897

Abstract

During autophagy, LC3 and GABARAP proteins become covalently attached to phosphatidylethanolamine on the growing autophagosome. This attachment is also reversible. Deconjugation (or delipidation) involves the proteolytic cleavage of an isopeptide bond between LC3 or GABARAP and the phosphatidylethanolamine headgroup. This cleavage is carried about by the ATG4 family of proteases (ATG4A, B, C, and D). Many studies have established that ATG4B is the most active of these proteases and is sufficient for autophagy progression in simple cells. Here we examined the second most active protease, ATG4A, to map out key regulatory motifs on the protein and to establish its activity in cells. We utilized fully in vitro reconstitution systems in which we controlled the attachment of LC3/GABARAP members and discovered a role for a C-terminal LC3-interacting region on ATG4A in regulating its access to LC3/GABARAP. We then used a gene-edited cell line in which all four ATG4 proteases have been knocked out to establish that ATG4A is insufficient to support autophagy and is unable to support GABARAP proteins removal from the membrane. As a result, GABARAP proteins accumulate on membranes other than mature autophagosomes. These results suggest that to support efficient production and consumption of autophagosomes, additional factors are essential including possibly ATG4B itself or one of its proteolytic products in the LC3 family.

Highlights

Macroautophagy is a conserved pathway for intracellular degradation [1,2,3,4] and is highly induced under amino acid starvation [1, 5,6,7,8]
The redundancy of function across the ATG4 family limits the useful interpretation of single knockouts, and we have created a gene-edited HEK293 cell line that we call our ATG4 quadruple knockout (ATG4QKO)
We previously showed that re-expression of ATG4B in this background is sufficient to rescue LC3 and GABARAP protein priming and lipidation and even to support flux of these proteins in a bafilomycin A1 (Baf A1)–dependent way, suggesting restoration of autophagy [29]

Summary

Introduction

Macroautophagy is a conserved pathway for intracellular degradation [1,2,3,4] and is highly induced under amino acid starvation [1, 5,6,7,8]. In principle, the ATG4QKO-ATG4A cell line that primes GABARAP, GL1, and GL2 and accumulates lipidated forms of all three proteins might support macroautophagy.

Results

Conclusion