An atypical interaction mode for Arabidopsis ATG8f with the adaptor protein SH3P2 has been identified.
ATG8f interacts specifically with SH3P2 but not with other SH3 proteins.
Binding of ATG8f to the autophagic receptor NBR1 induces unique conformational changes in ATG8f.
The ubiquitin-associated domain of NBR1 competes with the SH3 domain of SH3P2 for binding to ATG8f.
Distinct interaction interfaces are utilized by ATG8f when binding to NBR1 compared to SH3P2.
The -like motif of SH3P2 is crucial for its recruitment to the phagophore membrane but not necessary for its endocytosis trafficking.
Simplified
In selective macroautophagy/, cargo receptors are recruited to the forming autophagosome by interacting with Atg8 (autophagy-related 8)-family proteins and facilitate the selective sequestration of specific cargoes for autophagic degradation. In addition, Atg8 interacts with a number of adaptors essential for autophagosome biogenesis, including ATG and non-ATG proteins. The majority of these adaptors and receptors are characterized by an Atg8-family interacting motif () for binding to Atg8. However, the molecular basis for the interaction mode between ATG8 and regulators or cargo receptors in plants remains largely unclear. In this study, we unveiled an atypical interaction mode for Arabidopsis ATG8f with a plant unique adaptor protein, SH3P2 (SH3 domain-containing protein 2), but not with the other two SH3 proteins. By structure analysis of the unbound form of ATG8f, we identified the unique conformational changes in ATG8f upon binding to the AIM sequence of a plant known autophagic receptor, NBR1. To compare the binding affinity of SH3P2-ATG8f with that of ATG8f-NBR1, we performed a gel filtration assay to show that ubiquitin-associated domain of NBR1 outcompetes the SH3 domain of SH3P2 for ATG8f interaction. Biochemical and cellular analysis revealed that distinct interfaces were employed by ATG8f to interact with NBR1 and SH3P2. Further subcellular analysis showed that the AIM-like motif of SH3P2 is essential for its recruitment to the phagophore membrane but is dispensable for its trafficking in endocytosis. Taken together, our study provides an insightful structural basis for the ATG8 binding specificity toward a plant-specific autophagic adaptor and a conserved autophagic receptor.ATG, autophagy-related; AIM, Atg8-family interacting motif; BAR, Bin-Amphiphysin-Rvs; BFA, brefeldin A; BTH, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; CCV, clathrin-coated-vesicle; CLC2, clathrin light chain 2; Conc A, concanamycin A; ER, endoplasmic reticulum; LDS, LIR docking site; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; LIR, LC3-interacting region; PE, phosphatidylethanolamine; SH3P2, SH3 domain containing protein 2; SH3, Src-Homology-3; UBA, ubiquitin-associated; UIM, ubiquitin-interacting motif. Abbreviations:
Key numbers
11.3 ml
Binding Affinity Comparison
Elution volume of ATG8f-NBR1-UBA complex in gel filtration assay.
12.3 ml
Distinct Interaction Surfaces
Elution volume of ATG8f-SH3P2-SH3 complex in gel filtration assay.
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