Structure and function of yeast Atg20, a sorting nexin that facilitates autophagy induction

Abstract

The Atg20 and Snx4/Atg24 proteins have been identified in a screen for mutants defective in a type of selective macroautophagy/autophagy. Both proteins are connected to the Atg1 kinase complex, which is involved in autophagy initiation, and bind phosphatidylinositol-3-phosphate. Atg20 and Snx4 contain putative BAR domains, suggesting a possible role in membrane deformation, but they have been relatively uncharacterized. Here we demonstrate that, in addition to its function in selective autophagy, Atg20 plays a critical role in the efficient induction of nonselective autophagy. Atg20 is a dynamic posttranslationally modified protein that engages both structurally stable (PX and BAR) and intrinsically disordered domains for its function. In addition to its PX and BAR domains, Atg20 uses a third membrane-binding module, a membrane-inducible amphipathic helix present in a previously undescribed location in Atg20 within the putative BAR domain. Taken together, these findings yield insights into the molecular mechanism of the autophagy machinery.

Keywords: autophagy; vacuole; yeast.

Fig. 1.

Atg20 is essential for the efficient initiation of bulk autophagy. (A) WT (WLY176) and atg20Δ (HPY063) cells expressing Pho8Δ60 were shifted from nutrient-rich conditions to SD-N medium for 4 h. (B) Autophagy as measured by the GFP-Atg8 processing assay in WT (SEY6210) and atg20Δ (D3Y009) cells. Cells transformed with the plasmid (pRS426) carrying a GFP-Atg8 construct were grown in rich selective medium and then starved for 1, 2, and 3 h. The free GFP:total GFP ratio was measured and normalized to that of WT after 3 h of starvation (100%). (C) Kinetics of prApe1 maturation in nutrient-rich medium and after the shift to SD-N medium for the indicated times. The vac8∆ atg20∆ (HPY079) strain was compared with the vac8Δ (CWY230) strain. The atg11Δ (SEY6210) strain served as a negative control, and Pgk1 served as a loading control. Quantitative evaluation of western blot data was done with three to four independent experiments carried out in nutrient-rich and nitrogen starvation conditions. Error bars represent the SD from three to four independent experiments. Statistical significance was tested using the unpaired two-tailed Student’s t test: **P < 0.01; ***P < 0.005.

Fig. 2.

Bioinformatics and biochemical analysis of structured and intrinsically disordered domains in Atg20. (A) Domain representation of Atg20 that incorporates the results of the protein amino acid sequence analyses by the PONDR-FIT, MetaDisorderMD2, protein-protein BLAST, MoRFPred, and ANCHOR algorithms. (B, Left) Far UV circular dichroism spectrum of the recombinant purified Atg20[FR]. (B, Right) One-dimensional ¹H NMR spectrum of Atg20[FR]. (C) Empty vector, the plasmids pCuGFP-Atg20(426), pCuGFP-Atg20[ΔFR](426), pCuGFP-Atg20[Δ380–480](426), pCuGFP-Atg20[Δ533–632](426), and pCuGFP-Atg20[Aroma](426) were transformed into atg20Δ (D3Y009) cells and examined for prApe1 processing in rich selective medium. The atg11Δ (SEY6210) strain served as a negative control. Quantification of the Ape:prApe1 ratio was determined from three independent experiments. Error bars represent SDs. Statistical significance was tested using the unpaired two-tailed Student’s t test: **P < 0.05; ***P < 0.005. (D) Comparison of PONDR-FIT scores for WT (black) and Atg20[Aroma] (magenta).

Fig. 3.

Mapping of the Atg11 and Snx4 binding sites on Atg20. (A and B) Coprecipitation of HA-Atg11 by PA-Atg20. The plasmids pCuPA(424), pCuPA-Atg20(424), pCuPA-Atg20[ΔFR](424), pCuPA-Atg20[Δ380–480](424), and pCuPA-Atg20[Δ533–632](424) were transformed into MKO (YCY123) cells and coexpressed with a plasmid encoding HA-Atg11 (pCuHA-Atg11; 416) under the CUP1 promotor. (C–E) Coprecipitation of GFP-Atg20 by PA-Snx4. The plasmids pCuGFP(426), pCuGFP-Atg20(426), pCuGFP-Atg20[ΔFR](426), pCuGFP-Atg20[Δ380–480](426), pCuGFP-Atg20[Δ533–632](426), and pCuGFP-Atg20[533-640](426) were transformed into MKO (YCY123) cells and coexpressed under the CUP1 promotor with a plasmid encoding PA-Snx4 (pCuPA-Snx4; 424). For A–E, cells were cultured in SMD, and cell lysates were prepared and incubated with IgG-Sepharose for affinity purification. The proteins were separated by SDS/PAGE and detected with the indicated antibody. (F) Schematic representation depicting the Atg11-Atg20-Snx4 trimer, based on the results presented in the figure.

Fig. 4.

Atg20 is a posttranslationally modified protein. (A) Schematic domain representation of Atg20 with all PTM sites that were experimentally detected by LC-MS/MS analysis. (B) Functionality of Atg20 variants in the Cvt pathway analyzed by the prApe1 maturation assay. The SEY6210 atg20Δ strain was transformed with the plasmids pCuGFP(426), pCuGFP-Atg20(426), pCuGFP-Atg20[10STA](426), and pCuGFP-Atg20[4KR](426). Cells were cultured in rich selective medium. (C) Autophagy induction examined for WT and PTM mutants of Atg20 using the prApe1 maturation assay. The SEY6210 atg20∆ vac8∆ strain was transformed with the same plasmids as indicated in B. Cells were cultured in rich selective medium and then shifted to SD-N medium for 0.5 h. The atg11Δ (SEY6210) strain served as a negative control, and Pgk1 served as a loading control. Quantification of the Ape:prApe1 ratio was determined from three independent experiments. Error bars represent SDs. Statistical significance was tested using the unpaired two-tailed Student’s t test: ***P < 0.005.

Fig. 5.

Structural characterization of Atg20. (A) Sedimentation velocity AUC recorded on the full-length Atg20-Snx4 heterodimer. (B) In-line SEC with SAXS data recorded on the Atg20_156–640-Snx4_21–423 heterodimer. (Inset) The Guinier region for these data. SAXS data calculated from the Atg20-Snx4 ensemble homology model in F are shown in red. (C) Pair distance distribution function calculated from the SEC-SAXS data in B. (D and E) Homology modeling of structurally stable domains in Atg20. (D) Structural alignment of the crystal structure of the PX domain of SNX1 (gray) with the homology model of the PX domain of Atg20 (blue). (E) Structural alignment of the crystal structure of the BAR domain of SNX1 (gray) with the homology model of the BAR domain of Atg20 (blue). The amino acid residues of Atg20 mutated to Glu (Tyr177, Tyr180, Ile382, Tyr385, Tyr469, Leu472, Phe539, and Phe542) are shown (black). Phe539 and Phe542 are within MoRF5/BR10 that maps on the putative α5 helix of the BAR-GAP. (F) Atg20-Snx4 homology model of the BAR domain dimer. Atg20 is shown in blue; Snx4, in red. (G) Atg20-Snx4 ensemble model generated using BilboMD. The BAR domains of the different ensemble models are superimposed. The PX domain and linker from each model are shown in a different shade of blue (Atg20) or red (Snx4).

Fig. 6.

The membrane-induced AH in Atg20. (A) Helical wheel representation of the amino acid sequence in Atg20 that corresponds to MoRF5/BR10. Black arrows indicate the double mutation F539E, F542E. Yellow indicates hydrophobic amino acid residues; green, polar; red, negatively charged; blue, positively charged. (B) The vac8∆ atg20∆ (HPY079) cells transformed with the plasmids {pCuGFP(426), pCuGFP-Atg20(426), pCuGFP-Atg20[Y177E Y180/E](426), pCuGFP-Atg20[I382E Y385E](426), pCuGFP-Atg20[Y469E L472E](426), and pCuGFP-Atg20[F539E F542E](426)} were cultured in rich selective medium and then nitrogen-starved for 0.5 h. Error bars represent SD from three independent experiments. (C) Liposome sedimentation assay for the recombinant Atg20-Snx4 heterodimer, in which Atg20 was either the WT or F539E F542E mutant, with Folch liposomes of varying diameter. (D) In vitro reconstitution of yeast microsomes, isolated from SEY6210 atg20Δ snx4Δ cells, with the recombinant purified heterodimer Atg20-Snx4, in which Atg20 was either WT or mutant including the F539E F542E mutation. Supernatant (S) and pellet (P) were obtained by ultracentrifugation and analyzed by western blot analysis. (E, Upper) Representative negative stain EM images of 1.0-μm vesicles incubated with WT Atg20-Snx4 or mutant Atg20-Snx4 [F539E F542E] heterodimer. (Scale bars: 0.2 µm.) (E, Lower) Quantification of lipid tube length from grid squares, which are 484 µm². Data from each trial were normalized to the tube length from WT. Error bars represent the SD from three independent experiments. Statistical significance was tested using the unpaired two-tailed Student’s t test: **P < 0.01; ***P < 0.005.

Fig. 7.

Proposed molecular mechanism of Atg20 function depicted by a schematic model of the Atg11-Atg20-Snx4 heterotrimer interacting with the PtdIns3P-enriched membrane in yeast S. cerevisiae. The PX domain of Atg20 acts as a lipid-selective module that interacts with PtdIns3P. The BAR domain and AH of Atg20 are membrane-sensing and -remodeling modules that detect and stabilize a membrane curvature. The lipid-binding modules are the main targets of acetylation, whereas phosphorylation is located predominantly in IDPRs of Atg20. Together, these two PTMs modulate the optimal Atg20 conformation that uses the disordered N terminus (FR domain) and the first segment of the BAR domain (region 380–480) to interact with Atg11. Fine-tuning of Atg20 architecture by PTMs is important for the Cvt pathway, in which Atg11 is a critical component. Snx4 forms the heterodimer with Atg20 via binding to its second segment of the BAR domain near the very C terminus. The three lipid-binding modules of Atg20 along with the PX and BAR domain of Snx4 recruit the PtdIns3P-enriched membranes. The rate of this process is critical for the efficient initiation of bulk autophagy.