Phosphorylation of mitophagy and pexophagy receptors coordinates their interaction with Atg8 and Atg11

Abstract

The selective autophagy receptors Atg19 and Atg32 interact with two proteins of the core autophagic machinery: the scaffold protein Atg11 and the ubiquitin-like protein Atg8. We found that the Pichia pastoris pexophagy receptor, Atg30, also interacts with Atg8. Both Atg30 and Atg32 interactions are regulated by phosphorylation close to Atg8-interaction motifs. Extending this finding to Saccharomyces cerevisiae, we confirmed phosphoregulation for the mitophagy and pexophagy receptors, Atg32 and Atg36. Each Atg30 molecule must interact with both Atg8 and Atg11 for full functionality, and these interactions occur independently and not simultaneously, but rather in random order. We present a common model for the phosphoregulation of selective autophagy receptors.

Figure 1

Atg30 interacts with Atg8 through a cryptic AIM, and phosphorylation upstream of the AIM regulates their interaction. (A) IP of GFP–Atg8 (α-GFP), Atg30–Flag (α-Flag) and Pex3 (α-Pex3) under pexophagy conditions. The abundant peroxisome matrix protein, AOX, was used as a negative control. Input: total lysate; ϕ: IP without antibody. (B) Two multiple sequence alignments, including 11 Atg30 homologues and 11 Atg32 homologues (identical residues are indicated with black boxes, and similar residues with grey boxes), and a sequence logo of the combined multiple sequence alignments from Atg30 and Atg32 homologues listed currently in GenBank. (C,D) Pexophagy experiments of Δatg30 (ϕ), wild-type (Atg30) and Atg30 AIM mutant (Atg30^{W73A F76A}) cells were done by fluorescence microscopy, following the degradation of peroxisomes labelled with BFP fused at its C-terminus to the Ser–Lys–Leu peroxisomal targeting signal 1 (BFP–SKL) and biochemically by monitoring peroxisomal thiolase degradation. Vacuoles were labelled with FM4-64. Scale bar, 5 μm. (E) AH109 cells were transformed with two yeast two-hybrid assay plasmids, AD and BD, which encode the indicated domains fused with Atg30, Atg32 and Atg8 or an empty vector, as negative controls and grown on +His and −His+40 mM 3-AT plates. (F). Δatg30 (ϕ) and Δatg30 cells complemented with Atg30–HA (Atg30) and several Atg30–HA mutants were immunoprecipitated (α-HA IP) under pexophagy conditions. In addition, α-HA IP of Δatg30 cells (ϕ) and Δatg30 cells complemented with Atg30–HA (Atg30) were incubated with (+) and without (−) λPP. Input: total lysate. (G) Pexophagy in atg30 mutants was monitored by following thiolase levels of oleate-induced peroxisomes after shifting cells to SD-N. aa, amino acid; AD, activation domain; AIM, Atg8-family-interacting motif; AOX, alcohol oxidase; BD, binding domain; GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; λPP, λ protein phosphatase.

Figure 2

Interactions between Atg30, Atg8 and Atg11. (A₁) Atg30 sequences from aa 68–122 of wild-type (Atg30), the S71A mutant (Atg30^S71A) and two different deletions of the sequence between the AIM and phosphosite required for Atg11 binding in Atg30 (Atg30^WDILSSS and Atg30^WSILSSS). (A₂) Pexophagy experiments of Δatg30 cells complemented with appropriate wild-type or mutant Atg30 proteins described in A₁. (A₃) Two-hybrid assays between Atg30 wild-type or mutants (described in A₁) and Atg8. Phosphomimic S71E was included to detect the interaction with Atg8. (B₁) Schematic of the two Atg30 molecules, one with an Atg8-binding site mutated and a second with an Atg11-binding site (A11-BS) mutated, used to complement Δatg30 cells by co-expression. P: indicates phosphorylation in vivo. (B₂) Pexophagy experiments of Δatg30 cells complemented with the two Atg30 molecules described in B₁. (C₁) Schematic of the Atg30 mutations, S71A and S112A that impair Atg8 and Atg11 binding, respectively. (C₂) Pexophagy assays of Δatg30 cells complemented with Atg30 wild-type and mutants. aa, amino acids; AD, activation domain; AIM, Atg8-family-interacting motif; BD, binding domain; HA, haemagglutinin.

Figure 3

Atg8 and Atg11 localization during pexophagy. (A) Large phagophore membrane formation in WT and the Atg30 mutant cells monitored by GFP–Atg8 during pexophagy conditions. (B) Localization of GFP–Atg11 during pexophagy of methanol-induced peroxisomes in cells expressing WT or mutant Atg30 proteins. White arrows indicate correct localization and yellow arrows indicate mislocalization, or in case of Atg8 localization, indicate absence of phagophore membrane elongation. Peroxisomes were labelled with BFP–SKL and vacuoles with FM4-64. Scale bar, 5 μm. GFP, green fluorescent protein; WT, wild type.

Figure 4

Atg32, ScAtg32 and Atg36 use similar interaction mechanisms as Atg30. (A) Mitophagy experiments of WT (PPY12), Δatg5, Δatg32 (ϕ) and Δatg32 complemented with WT Atg32 (Atg32), Atg32 with a deletion of the sequence between the AIM and phosphosite required for Atg11 binding (Atg32^WQVLSSS), Atg32 AIM mutant (Atg32^{W121A V124A}), mutants of the Thr upstream of the Atg32 AIM (Atg32^T119A) and mutants altered in the Ser required for Atg11 binding (Atg32^S159A). The cells were grown in YPL medium and shifted to SD-N. Mitophagy was followed by the transport of Tom20–mCherry to the vacuole by fluorescence microscopy. Mitophagy was classified as −no mitophagy,+few cells show mitophagy, +++ most cells show mitophagy and ++++ almost all the cells show mitophagy (the intensity and numbers of cells containing Tom20–mCherry in the vacuole was considered). Scale bar, 5 μm. (B) P. pastoris Δatg32 cells (ΔPpatg32) expressing Tom20–GFP and expressing the indicated Atg32 mutants were cultured in YPL medium for 12, 18 and 36 h. Mitophagy was monitored by GFP appearance by immunoblotting with α-GFP antibodies. (C) N-terminal sequence of Atg36 manually aligned against the several sequence alignments of Atg30, Atg32 and ScAtg32. Identical residues are indicated with black boxes, and similar residues with grey boxes. (D) Two-hybrid protein–protein interaction analysis of ScAtg36, ScAtg8 and ScAtg11. The receptors were mutated at the AIM (Atg36^{F33A L36A}), at serine(s) upstream of the AIM (Atg36^S31A) and at the Atg11-binding site (Atg36^S97A). (E) S. cerevisiae Δatg36 cells (ΔScatg36) expressing thiolase–GFP and expressing the indicated Atg36 mutants were cultured in oleate medium until mid-log growth and then shifted to SD-N. Pexophagy was monitored by GFP appearance by immunoblotting with α-GFP antibodies. (F) Y3H analysis of ScAtg36 with ScAtg8 and ScAtg11. The Y3H technology is on the basis of the yeast two-hybrid system but with the co-expression of third protein as a competitor and indicated in the figure (NLS–ScAtg11 or NLS–ScAtg8). The positive control was the ScAtg36 mutant affected in Atg11 binding (ScAtg36^S97A) or in Atg8 binding (ScAtg36^S31A), which should be unaffected by the competition of NLS–ScAtg11 or NLS–ScAtg8, respectively. Appropriate auto-activation and interaction controls were also included. AD, activation domain; AIM, Atg8-family-interacting motif; BD, binding domain; GFP, green fluorescent protein; WT, wild type; Y3H, yeast three-hybrid.