A HORMA domain in Atg13 mediates PI 3-kinase recruitment in autophagy - PubMed (original) (raw)

A HORMA domain in Atg13 mediates PI 3-kinase recruitment in autophagy

Christine C Jao et al. Proc Natl Acad Sci U S A. 2013.

Abstract

Autophagy-related 13 (Atg13) is a key early-acting factor in autophagy and the major locus for nutrient-dependent regulation of autophagy by Tor. The 2.3-Å resolution crystal structure of the N-terminal domain of Atg13 reveals a previously unidentified HORMA (Hop1p, Rev1p and Mad2) domain similar to that of the spindle checkpoint protein Mad2. Mad2 has two different stable conformations, O-Mad2 and C-Mad2, and the Atg13 HORMA structure corresponds to the C-Mad2 state. The Atg13 HORMA domain is required for autophagy and for recruitment of the phosphatidylinositol (PI) 3-kinase subunit Atg14 but is not required for Atg1 interaction or Atg13 recruitment to the preautophagosomal structure. The Atg13 HORMA structure reveals a pair of conserved Arg residues that constitute a putative phosphate sensor. One of the Arg residues is in the region corresponding to the "safety belt" conformational switch of Mad2, suggesting conformational regulation of phosphate binding. These two Arg residues are essential for autophagy, suggesting that the Atg13 HORMA domain could function as a phosphoregulated conformational switch.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

The HORMA Domain of Atg13 is required for autophagy but not for Atg13 localization. (A) Domain representation of S. cerevisiae Atg13. The HORMA domain is labeled and represented as a rounded rectangle. The unstructured region is represented as a straight line. Phosphorylation sites within the unstructured region are indicated. Amino acid numbers corresponding to the boundaries of the HORMA domain and the Atg1/Atg17 binding region are labeled. (B) Schematic of the organization of the Atg1 complex. This complex is a dimer of pentamers, with a single pentamer shown here. The loci of the previously identified PAS scaffolding and protein phosphorylation functions are indicated. The positioning of the HORMA domain identified in this study is highlighted and its PI 3-kinase recruitment activity annotated. (C) GFP-Atg8 processing assay as monitored by Western blot against GFP. The GFP-Atg8 and GFP bands are labeled. (D) Pho8Δ60 assay to monitor autophagy was performed in SMD (white) and SD-N (gray). Samples were normalized to the activity of Atg13 in starved cells. (E) Representative microscopy images of atg13Δ cells transformed with ATG13-GFP, ATG13 ΔHORMA -GFP, and ATG13 HORMA -GFP in the presence of rapamycin treatment. (F) Quantification of the Atg13 puncta from E. A total of three trials with 100 cells counted per trial. Error bars in D and F represent the SD of triplicate experiments.

Fig. 2.

Fig. 2.

The HORMA domain of Atg13 is necessary for the recruitment of downstream factors in autophagy. (A) Representative microscopy images of atg11Δ atg13Δ ATG14-GFP cells transformed with vector, ATG13, and ATG13 ΔHORMA and treated with rapamycin. (B) Quantification of the Atg14 puncta from A using a brightness threshold of fourfold above the average background fluorescence. A total of three trials with 100 cells counted per trial were made. (C) Representative microscopy images of atg11Δ atg13Δ GFP-ATG8 cells transformed with vector, ATG13, and ATG13 ΔHORMA and treated with rapamycin. (D) Quantification of the Atg8 puncta from C. A total of three trials with 100 cells counted per trial. Error bars in B and D represent the SD of triplicate experiments.

Fig. 3.

Fig. 3.

Crystal structure of the Atg13 HORMA domain. (A) Ribbon diagram of the Atg13 HORMA domain. Regions structurally related to MAD2 are colored in cyan except the safety belt region, which is highlighted in purple. β4’, β4’’, and β8’’ of the Atg13 HORMA domain, which are unique to Atg13, are colored in green. (B) Sequence alignment of the Atg13 HORMA domain with the secondary structure elements colored as in A. The two arginine residues located within the sulfate binding site are marked by dots. Species are abbreviated as follows: L. thermotolerans (Lt), Kluyveromyces lactis (Kl), S. cerevisiae (Sc), Schizosaccharomyces pombe (Sp), and Homo sapiens (Hs). (C) Surface representation of the HORMA domain colored according to electrostatic potential, with saturating blue and red at ±7.5 kT/e. The sulfate ion is represented as spheres. (D) Close-up view of the sulfate binding site. The sulfate is coordinated by the side chain of R118 and backbone NH of L208 and L209. Hydrogen bonds are represented as dashed lines. R205 contributes to the overall positive charge of the binding pocket but is not within hydrogen bonding distance.

Fig. 4.

Fig. 4.

Unexpected structural homology between Atg13 and C-Mad2. (A) Ribbon diagram of the HORMA domain of Atg13 colored as is shown in Fig. 3_A_. (B) Ribbon diagram of the closed state of Mad2 oriented identical to Atg13 in A, with the Mad1 binding peptide shown in dark blue. Regions structurally related to the HORMA domain of Atg13 are colored in cyan except the safety belt region, which is highlighted in purple. Regions unique to Mad2 are colored in yellow. Coordinates taken from Protein Data Bank (PDB) ID 1GO4. (C) Ribbon diagram of the open state of Mad2 oriented identical to Atg13 in A and colored exactly as in B. Coordinates taken from PDB ID 2V64. (D–F) Topology diagrams for the Atg13 HORMA domain (D), closed state of Mad2 (E), and open state of Mad2 (F) colored exactly as in A–C.

Fig. 5.

Fig. 5.

The putative phosphate sensor of the Atg13 HORMA domain is required for autophagy and PI 3-kinase recruitment. (A) GFP-Atg8 processing assay of _atg11_Δ _atg13_Δ cells transformed with ATG13 wild type and mutants designed to disrupt the putative phosphate binding site. GFP-Atg8 processing was monitored by Western blot against GFP. The GFP-Atg8 and GFP bands are labeled. (B) Pho8Δ60 assay to monitor autophagy was performed in SMD (white) and SD-N (gray) using atg11_Δ_atg13_Δ cells transformed with the same ATG13 constructs as in A. Samples were normalized to the activity of Atg13 in starved cells. (C) Representative images of atg11Δ atg13Δ ATG14-GFP cells transformed with vector, and ATG13 (as shown also in Fig. 2_A) and mutant ATG13 alleles, and treated with rapamycin. (D) Quantification of the Atg14 puncta from C using the procedure described in Fig. 2. A total of three trials with 100 cells counted per trial. Error bars in B and C represent the SD of triplicate experiments.

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