Autophagy-related Protein 8 (Atg8) Family Interacting Motif in Atg3 Mediates the Atg3-Atg8 Interaction and Is Crucial for the Cytoplasm-to-Vacuole Targeting Pathway

Masaya Yamaguchi,Nobuo N Noda,Hitoshi Nakatogawa,Hiroyuki Kumeta,Yoshinori Ohsumi,Fuyuhiko Inagaki

doi:10.1074/jbc.m110.113670

Abstract

The autophagy-related protein 8 (Atg8) conjugation system is essential for the formation of double-membrane vesicles called autophagosomes during autophagy, a bulk degradation process conserved among most eukaryotes. It is also important in yeast for recognizing target vacuolar enzymes through the receptor protein Atg19 during the cytoplasm-to-vacuole targeting (Cvt) pathway, a selective type of autophagy. Atg3 is an E2-like enzyme that conjugates Atg8 with phosphatidylethanolamine. Here, we show that Atg3 directly interacts with Atg8 through the WEDL sequence, which is distinct from canonical interaction between E2 and ubiquitin-like modifiers. Moreover, NMR experiments suggest that the mode of interaction between Atg8 and Atg3 is quite similar to that between Atg8/LC3 and the Atg8 family interacting motif (AIM) conserved in autophagic receptors, such as Atg19 and p62. Thus, the WEDL sequence in Atg3 is a canonical AIM. In vitro analyses showed that Atg3 AIM is crucial for the transfer of Atg8 from the Atg8∼Atg3 thioester intermediate to phosphatidylethanolamine but not for the formation of the intermediate. Intriguingly, in vivo experiments showed that it is necessary for the Cvt pathway but not for starvation-induced autophagy. Atg3 AIM attenuated the inhibitory effect of Atg19 on Atg8 lipidation in vitro, suggesting that Atg3 AIM may be important for the lipidation of Atg19-bound Atg8 during the Cvt pathway.

Highlights

Targets that include aggregated proteins, surplus or damaged organelles, and even invasive bacterial cells [1]
We have shown that autophagy-related protein 8 (Atg8) and its mammalian ortholog LC3 recognize the WXXL sequence conserved in Atg19 and p62, autophagic receptors for aminopeptidase 1 (Ape1) and ubiquitinated protein aggregates, respectively, in a similar manner [15, 16]
Atg8 R28A, L50A, P52A, and R67A mutants, which were defective in the interaction with Atg19AIM [16],3 were severely impaired in binding to Atg3 (Fig. 2C; supplement Fig. S4B). These results clearly indicate that the type Atg3 (Fig. 3A; supplement Fig. S4C). These results show that the interaction between Atg3 and Atg8 through Atg3AIM is not required for Atg3 to receive Atg8 from Atg7

Summary

Introduction

Targets that include aggregated proteins, surplus or damaged organelles, and even invasive bacterial cells [1]. Atg8-Atg19AIM and LC3-p62AIM interactions are crucial for the selective transport of Ape1 and protein aggregates to the vacuole/lysosome through the Cvt pathway and autophagy, respectively [16, 18]. To elucidate the significance of Atg3AIM in the Atg8 system, in vitro conjugation assays were performed using wild-type and W270A mutant Atg3s.

Results

Conclusion