Structure-based analyses reveal distinct binding sites for Atg2 and phosphoinositides in Atg18

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.

FIGURE 1.

Crystal structure of Hsv2. A, ribbon diagram of the Hsv2 structure. The seven blades and β-strands are labeled. B, phosphoinositide-binding sites of Hsv2A (left) and Hsv2B (right). The side chains of the site 1 and 2 residues and the bound sulfate are shown as a stick model. The crystallographically adjacent Hsv2 molecule bound to site 2 is shown in yellow. Sites 1 and 2 are shown in dashed circles.

FIGURE 2.

A, structurally annotated sequence alignment of Atg18 homologs. Gaps have been introduced to maximize the similarity. The conserved residues are shaded in black. The secondary structural elements of Hsv2 are shown above the sequence. Sc, S. cerevisiae; Pp, P. pastoris; Hs, H. sapiens. B, mapping of the residues conserved among the Atg18 orthologs on the KmHsv2 structure. The conserved residues are shaded in black. The residues in parentheses are the Atg18 residues that are the structural equivalent of KmHsv2 residues. Blade 2 AB and BC loops as well as sites 1 and 2 are shown in dashed circles.

FIGURE 3.

Localization of mutant Atg18-HG constructs and vacuolar morphology. Exponentially growing atg18Δ cells (TKY1001) expressing the indicated mutant Atg18-HG constructs were labeled with FM4-64 and subjected to fluorescence microscopy. Scale bar = 2 μm.

FIGURE 4.

Mutational effect of the conserved residues of Atg18 on autophagy. A, autophagic activity was estimated using an alkaline phosphatase (ALP) assay (see “Experimental Procedures”). The white and black bars indicate alkaline phosphatase activity at 0 and 4 h after starvation, respectively. Values are the means ± S.D. of three independent experiments. B, total lysates from atg18Δ cells carrying the indicated plasmids were subjected to Western blotting using anti-Ape1, anti-HA, or anti-Pgk1 (loading control) antiserum. To induce autophagy, the cells were treated with rapamycin for 3 h.

FIGURE 5.

The BC loop in blade 2 is essential for the PAS targeting of Atg18. atg18Δ cells carrying integrated mRFP-Ape1 and mutant Atg18-HG constructs were observed by microscopy after rapamycin treatment for 1 h. The arrows indicate the PAS. Scale bar = 2 μm.

FIGURE 6.

Analysis of the interaction between Atg18 and Atg2. Co-immunoprecipitation (IP) experiments were performed as described under “Experimental Procedures.” A, Atg18-HG constructs were pulled down using GFP-Trap magnetic beads. The protein bands for Atg2 and Pgk1 were detected using rabbit IgG and anti-Pgk1 antibody, respectively. The protein bands for Atg18 were detected using anti-GFP or anti-HA antibody. B, Atg2 fused to a TAP tag was pulled down using IgG-conjugated magnetic beads. The protein bands for Atg2, Atg18, and Pgk1 were detected using anti-Atg2, anti-HA, and anti-Pgk1 antibody, respectively.

FIGURE 7.

Schematic representation of the Atg2-Atg18 complex on the membrane. Atg2 (yellow) grips the ring-like structure of Atg18 (green) at blade 2, whereas Atg18 interacts with membrane (dark red) at blades 5 and 6.