Abstract
In autophagy, Atg proteins organize the pre-autophagosomal structure (PAS) to initiate autophagosome formation. Previous studies in yeast revealed that the autophagy-related E3 complex Atg12-Atg5-Atg16 is recruited to the PAS via Atg16 interaction with Atg21, which binds phosphatidylinositol 3-phosphate (PI3P) produced at the PAS, to stimulate conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine. Here, we discover a novel mechanism for the PAS targeting of Atg12-Atg5-Atg16, which is mediated by the interaction of Atg12 with the Atg1 kinase complex that serves as a scaffold for PAS organization. While autophagy is partially defective without one of these mechanisms, cells lacking both completely lose the PAS localization of Atg12-Atg5-Atg16 and show no autophagic activity. As with the PI3P-dependent mechanism, Atg12-Atg5-Atg16 recruited via the Atg12-dependent mechanism stimulates Atg8 lipidation, but also has the specific function of facilitating PAS scaffold assembly. Thus, this study significantly advances our understanding of the nucleation step in autophagosome formation.
Highlights
Macroautophagy is a major route for transport of intracellular material into lysosomes or vacuoles in almost all eukaryotes (Ohsumi, 2014; Yang and Klionsky, 2010)
We identified the Atg1 kinase complex, which forms a scaffold for pre-autophagosomal structure (PAS) organization, as a novel interaction partner of the Atg16 complex, and found that this intercomplex interaction collaborates with the phosphatidylinositol 3-phosphate (PI3P)-dependent mechanism to recruit the Atg16 complex to the PAS
The Atg16 complex was visualized by Atg5-GFP, and the PAS was labeled with the scaffold protein Atg17 fused with mCherry (Suzuki et al, 2007)
Summary
Macroautophagy (hereafter autophagy) is a major route for transport of intracellular material into lysosomes or vacuoles in almost all eukaryotes (Ohsumi, 2014; Yang and Klionsky, 2010). Among the over 40 Atg proteins that have been identified to date, 19 are directly involved in the biogenesis of the autophagosome induced under starvation (Nakatogawa et al, 2009; Ohsumi, 2014; Yang and Klionsky, 2010). These ‘core’ Atg proteins constitute six functional units: (i) the Atg kinase complex; (ii) Atg vesicles; (iii) phosphatidylinositol (PI) 3-kinase (PI3K) complex I ; (iv) the Atg2-Atg complex; (v) the Atg conjugation system; and (vi) the Atg
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