The Degradation Pathway of the Mitophagy Receptor Atg32 Is Re-Routed by a Posttranslational Modification

Abstract

The outer mitochondrial membrane protein Atg32 is the central receptor for mitophagy, the mitochondria-specific form of autophagy. Atg32 is an unstable protein, and is rapidly degraded under conditions in which mitophagy is not induced. Here we show that Atg32 undergoes a posttranslational modification upon induction of mitophagy. The modification is dependent on the core autophagic machinery, including Atg8, and on the mitophagy-specific adaptor protein Atg11. The modified Atg32 is targeted to the vacuole where it becomes stabilized when vacuolar proteases are deficient. Interestingly, we find that this degradation pathway differs from the degradation pathway of non-modified Atg32, which neither involves vacuolar proteases, nor the proteasome. These analyses reveal that a posttranslational modification discriminates a form of Atg32 targeting mitochondria for mitophagy from that, which escapes mitophagy by rapid degradation.

Fig 1. Yeast expressing Atg32 constructs for IgG affinity chromatography.

(A) Topologies of Atg32^ZZ, ^ZZAtg32 and ^ZZAtg32_IMS constructs. ZZ, two repeats of the IgG-binding domain of protein A (Z domain), TMD, transmembrane domain, OM, outer membrane, IMS, intermembrane space. (B) Expression of Atg32^ZZ, ^ZZAtg32 and ^ZZAtg32_IMS constructs. Lysates of yeast cells grown in indicated media were analyzed by Western blotting with antibodies against the IgG-binding domain (α-PAP) and mitochondrial proteins Tim21 and Cox13, as a control. (C) Analysis of mitochondrial turnover (mitophagy assay). Mito-GFP (DHFR-GFP fused to the presequence of N. crassa F₁F_O ATPase subunit 9) was expressed in the respective yeast strains and mitophagy was induced by starvation in nitrogen-free media for 6h. Cell lysates were analyzed by Western blotting with antibodies directed against GFP. (D) Mitophagy assay as in (C) after induction of mitophagy by rapamycin treatment (1 μg/ml) for the indicated time points. (E) Total, unbound, and eluate fractions of IgG affinity chromatography using the indicated yeast strains were analyzed by Western blot using the α-PAP antibody.

Fig 2. Blocking vacuolar degradation stabilizes modified Atg32 upon mitophagy induction.

(A) Yeast cells expressing ^ZZAtg32 were grown in medium lacking any nitrogen source for the indicated time points. Cells were harvested and proteins were analyzed by Western blotting after SDS gel electrophoresis using the α-PAP antibody to detect Atg32, with Tim50 as a control. Arrow indicates the modified form of Atg32. (B) Analysis of Atg32 modification as in (A) after induction of mitophagy using 1 μg/ml rapamycin for indicated time points. (C) Mitophagy assay as shown in (Fig 1C) after indicated time points of rapamycin treatment. (D) Atg32 modification in post log cell lysates, analyzed as in (Fig 2A). (E) Mitophagy assay in cells grown until post log phase for the indicated time points.

Fig 3. Autophagy-specific genes are required for Atg32 modification.

(A) Schematic representation of the sequence of events involved in mitophagy. (B) pep4Δ strains expressing ^ZZAtg32 and carrying an additional deletion in the indicated gene were induced for mitophagy by 1 μg/ml rapamycin treatment. Cell lysates were analyzed by SDS PAGE and immunoblotting with the α-PAP antibody. Arrows indicate modification products. Asterisk indicates a second modification of Atg32. (C) Indicated yeast strains expressing the ^ZZAtg32 construct were treated with 1 μg/ml rapamycin for 0 or 60 min and analyzed by Western blotting using the α-PAP antibody. WT–wild-type, ΔΔ –double deletion mutant atg11Δ pep4Δ. Arrow indicates a modification product. (D) Experiment as in (C) using cells expressing the ^ZZAtg32_IMS construct.

Fig 4. Atg32 modification and degradation is not affected by the defects in the proteasome.

(A) Yeast expressing the ^ZZAtg32 construct and the indicated mutation in a degradation pathway were analyzed by Western blot after treatment with 1 μg/ml rapamycin for the indicated time periods. Immunoblotting for Atg32 with the α-PAP antibody. WT–wild-type, pre*–pre1-1 pre2-2 mutant. Arrows indicate modification products. (B) The ^ZZAtg32 degradation upon mitophagy induction by rapamycin was analyzed in the presence and absence of MG132. Accumulation of ubiquitinated proteins upon administration of MG132 was documented using an ubiquitin specific antibody. (C) Yeast cells expressing HA-Ubiquitin were treated with MG132 or with DMSO as a control. Cell extracts were submitted to HA-affinity isolation and eluates were analyzed by western blot using an HA specific antibody. Unconjugated HA-Ub in the low molecular range was detected as a control. (D) Mitophagy was induced by Rapamycin treatment and cell extracts were treated with alkaline Phosphatase and phosphatase inhibitor as a control (left panel). Cell extracts were treated with the deubiquitinase USP2 or treated with the inhibitor NEM as a control (right panel). (E) Model of Atg32 modification.