Arginine68 is an essential residue for the C-terminal cleavage of human Atg8 family proteins

Chao Liu,Jun O Liu,Qiang Huang,Haijie Ma,Jiaxue Wu,Long Yu

doi:10.1186/1471-2121-14-27

Chao Liu, Jun O Liu + Show 4 more

Open Access

https://doi.org/10.1186/1471-2121-14-27

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Abstract

BackgroundAutophagy is a conserved cellular process that degrades and recycles cytoplasmic components via a lysosomal pathway. The phosphatidylethanolamine (PE)-conjugation of the Atg8 protein plays an important role in the yeast autophagy process. In humans, six Atg8 homologs, including MAP1LC3A, MAP1LC3B, MAP1LC3C (refer to LC3A, LC3B, and LC3C hereafter), GABARAP, GABARAPL1, and GABARAPL2 have been reported. All of them can be conjugated to PE through a ubiquitin-like conjugation system, and be located to autophagosomes.ResultsIn this study, we found 3 new alternative splicing isoforms in LC3B, GABARAP, and GABARAPL1, (designated as LC3B-a, GABARAP-a and GABARAPL1-a, respectively). None of them can go through the PE-conjugation process and be located to autophagosomes. Interestingly, compared with LC3B, LC3B-a has a single amino acid (Arg68) deletion due to the NAGNAG alternative splicing in intron 3. Through structural simulations, we found that the C-terminal tail of LC3B-a is less mobile than that of LC3B, thus affecting its C-terminal cleavage by human ATG4 family proteins. Furthermore, we found that Arg68 is an essential residue facilitating the interaction between human Atg8 family proteins and ATG4B by forming a salt bridge with Asp171 of ATG4B. Depletion of this salt bridge reduces autophagosomes formation and autophagic flux under both normal and nutrition starvation conditions.ConclusionsThese results suggest Arg68 is an essential residue for the C-terminal cleavage of Atg8 family proteins during the autophagy process.

Highlights

Autophagy is a conserved cellular process that degrades and recycles cytoplasmic components via a lysosomal pathway
We report the identification and characterization of an essential residue for the autophagic activity of human Atg8 family proteins, which provides potential targets to regulate the autophagy activity in human diseases
To fully explore the members in human Atg8 family proteins, we searched their alternative splicing patterns from the Alternative Splicing and Transcript Diversity (ASTD) database which has been integrated in Ensembl [34]

Summary

Introduction

Autophagy is a conserved cellular process that degrades and recycles cytoplasmic components via a lysosomal pathway. The phosphatidylethanolamine (PE)-conjugation of the Atg protein plays an important role in the yeast autophagy process. All of them can be conjugated to PE through a ubiquitin-like conjugation system, and be located to autophagosomes. Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved cellular process which degrades and recycles cytoplasmic components through the de novo synthesized membrane system and lysosome [1,2]. The ubiquitin-like protein Atg is the only protein that could bind to all autophagic membrane structures, it is commonly used as a marker to trace autophagosomes [12]. Before binding with autophagic membrane structures, newly synthesized Atg protein (pro-Atg8) is processed by a cysteine protease, Atg.

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