Physiological pH and Acidic Phospholipids Contribute to Substrate Specificity in Lipidation of Atg8

Hitoshi Nakatogawa,Kyoko Oh-Oka,Yoshinori Ohsumi

doi:10.1074/jbc.m801836200

Abstract

Yeast Atg8 and its mammalian homolog LC3 are ubiquitin-like proteins involved in autophagy, a primary pathway for degradation of cytosolic constituents in vacuoles/lysosomes. Whereas the lipid phosphatidylethanolamine (PE) was identified as the sole in vivo target of their conjugation reactions, in vitro studies showed that the same system can mediate the conjugation of these proteins with phosphatidylserine as efficiently as with PE. Here, we show that, in contrast to PE conjugation, the in vitro phosphatidylserine conjugation of Atg8 is markedly suppressed at physiological pH. Furthermore, the addition of acidic phospholipids to liposomes also results in the preferential formation of the Atg8-PE conjugate. We have successfully captured authentic thioester intermediates, allowing us to elucidate which step in the conjugation reaction is affected by these changes in pH and membrane lipid composition. We propose that these factors contribute to the selective formation of Atg8-PE in the cell.

Highlights

Various cellular activities involve post-translational modifications of proteins, among which ubiquitin and ubiquitin-like protein (Ubl)2 systems are remarkably versatile both in regard to the processes they regulate and their mechanisms [1]
Further analyses suggest that physiological pH conditions evoke the intrinsic substrate specificity of Atg3 and that acidic phospholipids facilitate the binding of the Atg8-Atg3 thioester intermediate to the membrane
Acidic Phospholipids Facilitate the Membrane Binding of Thioester Intermediates—We investigated how acidic phospholipids facilitated the production of Atg8-PE

Summary

Introduction

Various cellular activities involve post-translational modifications of proteins, among which ubiquitin and ubiquitin-like protein (Ubl) systems are remarkably versatile both in regard to the processes they regulate and their mechanisms [1]. Atg catalyzes the deconjugation of Atg8-PE and recycles and/or regulates Atg8 [5] Both of these Ubl conjugates are localized on intermediate structures of autophagosomes and are thought to play essential roles in their formation. We reconstituted the Atg conjugation system using purified proteins and PE-containing liposomes [15] and found that Atg forms an oligomer in response to PE conjugation, leading to the tethering together and hemifusion of the liposomes [16] This function of Atg was suggested to be involved in the expansion of autophagosomal membranes. Mammalian homologs of Atg, including LC3, are conjugated to PS as efficiently as PE in a similar in vitro system, even though the lipidated form of LC3 purified from cultured cells contained only PE [18] These results suggested that there exists a mechanism that directs Atg conjugation preferentially to PE in the cell. Further analyses suggest that physiological pH conditions evoke the intrinsic substrate specificity of Atg and that acidic phospholipids facilitate the binding of the Atg8-Atg thioester intermediate to the membrane

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Results

Conclusion