Skip to main content
. 2015 Mar 3;112(11):3350–3355. doi: 10.1073/pnas.1421092112

Fig. 1.

Fig. 1.

The HORMA domain of Atg13 binds Atg9. (A) Schematic diagram of Atg13. (B) Atg13 interacts with Atg9. atg13Δ cells expressing GFP or GFP-Atg13 were treated with rapamycin for 1 h. The cells were disrupted using a Multi-beads shocker and were solubilized with 0.1% Nonidet P-40. After a centrifugation at 17,400 × g for 10 min, the supernatants were subjected to immunoprecipitation using anti-GFP magnetic beads. Bound materials were eluted with SDS/PAGE sample buffer and subjected to immunoblotting with antibodies against GFP, Atg1, Atg17, Atg9, Atg14, Atg2, and phosphoglycerate kinase 1 (Pgk1). (C) The Atg13–Atg9 interaction in vivo. atg1Δ atg11Δ atg13Δ atg17Δ atg29Δ atg31Δ cells expressing GFP, GFP-Atg13, GFP-Atg13HORMA (residues 2–268), or GFP-Atg13ΔHORMA (residues 281–738) were treated with rapamycin for 1 h and then were subjected to immunoprecipitation as in B. Bound materials were eluted with SDS/PAGE sample buffer and subjected to immunoblotting with antibodies against GFP, Atg9, and Pgk1. (D) The Atg13 HORMA domain interacts directly with Atg9 in vivo. Cells overexpressing both GFP-Atg13HORMA and Atg9 were treated with rapamycin for 1 h and then were subjected to immunoprecipitation as in B. Bound materials were eluted with SDS/PAGE sample buffer and subjected to SDS/PAGE followed by Coomassie Brilliant Blue (CBB) staining. Asterisks indicate a nonspecific binding protein on anti-GFP magnetic beads. (E) Schematic diagram of Atg9. (F) Yeast two-hybrid analysis of the Atg13–Atg9 interaction. The yeast indicator strain AH109 was transformed with plasmids expressing a transcription activation domain (AD) fused with the N region of Atg9 (residues 2–318), the M region of Atg9 (residues 395–534), or the C region of Atg9 (residues 747–997) and plasmids expressing a DNA-binding domain (BD) fused with full-length Atg13 or the HORMA domain of Atg13. These strains were grown on synthetic dextrose-Leu-Trp (SC-LW) (+Ade) and synthetic dextrose-Leu-Trp-Ade (SC-LWA) (−Ade) agar plates. (G) The Atg9N–Atg13HORMA interaction in vivo. Cells expressing tandem affinity purification tag fused Atg9N (Atg9N-TAP) and Atg13HORMA-GFP under control of their own promoters were treated with rapamycin for 1 h and then were subjected to immunoprecipitation using IgG-coated epoxy Dynabeads. Bound materials were eluted with SDS/PAGE sample buffer and subjected to immunoblotting with antibodies against TAP and GFP.