Structure of the Novel C-terminal Domain of Vacuolar Protein Sorting 30/Autophagy-related Protein 6 and Its Specific Role in Autophagy
ABSTRACT Vacuolar protein sorting 30 (Vps30)/autophagy-related protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.
- SourceAvailable from: Yoshinori Ohsumi
[Show abstract] [Hide abstract]
- "Among them, 18 ATG genes encode core components required for autophagosome formation (Atg1–10, 12–14, 16–18, 29, and 31), the majority of which localize at least partly to the preautophagosomal structure (PAS), the nucleation site from which the autophagosome originates (Suzuki et al., 2001, 2007). These 18 proteins are classified into six functional groups: Atg1 kinase and its regulators, the PI3-kinase complex, the Atg9 vesicle, the Atg2–Atg18 complex, and two ubiquitin-like conjugation systems (Nakatogawa et al., 2009). "
ABSTRACT: Autophagy is a conserved eukaryotic process of protein and organelle self-degradation within the vacuole/lysosome. Autophagy is characterized by the formation of an autophagosome, for which Vps34-dervied phosphatidylinositol 3-phosphate (PI3P) is essential. In yeast, Vps34 forms two distinct protein complexes: complex I, which functions in autophagy, and complex II, which is involved in protein sorting to the vacuole. Here we identify and characterize Atg38 as a stably associated subunit of complex I. In atg38Δ cells, autophagic activity was significantly reduced and PI3-kinase complex I dissociated into the Vps15-Vps34 and Atg14-Vps30 subcomplexes. We find that Atg38 physically interacted with Atg14 and Vps34 via its N terminus. Further biochemical analyses revealed that Atg38 homodimerizes through its C terminus and that this homodimer formation is indispensable for the integrity of complex I. These data suggest that the homodimer of Atg38 functions as a physical linkage between the Vps15-Vps34 and Atg14-Vps30 subcomplexes to facilitate complex I formation.The Journal of Cell Biology 10/2013; 203(2):299-313. DOI:10.1083/jcb.201304123 · 9.69 Impact Factor
[Show abstract] [Hide abstract]
- "The preparation of yeast protein extracts was carried out as previously reported  . Immunoblotting was performed using anti-Ape1 antiserum (API-2, Hamasaki and Ohsumi, unpublished), anti-Atg2 antiserum , anti-HA antiserum (Covance, 16B12), anti-Atg8 (IN-13) or anti-Pgk1 antiserum (Invitrogen/Molecular Probes). "
ABSTRACT: The Atg2-Atg18 complex is essential for autophagosome formation in Saccharomyces cerevisiae. In this paper, we show that partial induction of autophagy can proceed in cells expressing engineered variants of Atg2 capable of localizing to the pre-autophagosomal structure (PAS) in the absence of Atg18. Specifically, through the construction of fusion proteins, we show that the fusion to Atg2 of either the phosphatidylinositol 3-phosphate-binding FYVE domain or the core autophagy protein Atg8 allowed limited Atg18-independent recovery of autophagosome formation. These results indicate that effective targeting of Atg2 to the PAS can compensate for loss of Atg18 function in autophagy.FEBS letters 06/2012; 586(16):2473-8. DOI:10.1016/j.febslet.2012.06.008 · 3.34 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Autophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by an autophagosome and delivered to a lysosome/vacuole. Atg18 plays a critical role in autophagosome formation as a complex with Atg2 and phosphatidylinositol 3-phosphate (PtdIns(3)P). However, little is known about the structure of Atg18 and its recognition mode of Atg2 or PtdIns(3)P. Here, we report the crystal structure of Kluyveromyces marxianus Hsv2, an Atg18 paralog, at 2.6 Å resolution. The structure reveals a seven-bladed β-propeller without circular permutation. Mutational analyses of Atg18 based on the K. marxianus Hsv2 structure suggested that Atg18 has two phosphoinositide-binding sites at blades 5 and 6, whereas the Atg2-binding region is located at blade 2. Point mutations in the loops of blade 2 specifically abrogated autophagy without affecting another Atg18 function, the regulation of vacuolar morphology at the vacuolar membrane. This architecture enables Atg18 to form a complex with Atg2 and PtdIns(3)P in parallel, thereby functioning in the formation of autophagosomes at autophagic membranes.Journal of Biological Chemistry 07/2012; 287(38):31681-90. DOI:10.1074/jbc.M112.397570 · 4.57 Impact Factor