Mechanistic insights into an atypical interaction between ATG8 and SH3P2 inArabidopsis thaliana

Abstract

In selective macroautophagy/autophagy, 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 (AIM) 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 domaincontaining 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.

Clathrin Light Chains are essential in negative regulation of cell death and immunity in Arabidopsis through interacting with autophagy pathway

“…The LIR/AIM docking site (LDS) presented in the ATG8 protein family is critical for the interactions between ATG8 proteins and autophagic receptors/adaptors, which leads to the recruitment of cargos to autophagosomes. A hydrophobic pocket is highly conserved in the Arabidopsis ATG8 isoforms in the LDS (Figure 8A and Figure 5S; Marshall et al, 2019;and Sun et al, 2022,). Mutations of Y50 and L51 simultaneously at the hydrophobic pocket to Alanine (A) (Y50A, L51A) abolished the LDS binding affinity of ATG8a or ATG8f (Marshall et al, 2019;and Sun et al, 2022).…”

“…A hydrophobic pocket is highly conserved in the Arabidopsis ATG8 isoforms in the LDS (Figure 8A and Figure 5S; Marshall et al, 2019;and Sun et al, 2022,). Mutations of Y50 and L51 simultaneously at the hydrophobic pocket to Alanine (A) (Y50A, L51A) abolished the LDS binding affinity of ATG8a or ATG8f (Marshall et al, 2019;and Sun et al, 2022). Therefore, we generated Y52A/L53A mutant of ATG8h by site-directed mutagenesis.…”

“…Furthermore, SH3P2 also interacts with all ATG8 isoforms in Arabidopsis and an AIMlike motif within the SH3 domain, which is essential for the SH3P2-ATG8f interaction (Zhuang et al, 2013;Sun et al, 2022). Therefore, clathrin can be associated with autophagosomes through interacting with ATG8h/ATG8i directly (Fig.…”

Crossing paths: recent insights in the interplay between autophagy and intracellular trafficking in plants

“…The BAR-domain containing protein SH3P2 has been proposed to behave as ubiquitinbinding protein enabling the endocytosis of ubiquitinated PM-associated proteins, in effect working as a substitute for the missing ESCRT-0 in plants, in addition to interacting with VPS23, a component of ESCRT-I [42]. SH3P2 has also been shown to interact with ATG8 along with PI3P and was proposed to participate in phagophore maturation [43,44]. In good agreement with Zhao et al [34], who identified SH3P2 on the outer-surface of autophagosomes and showed an interaction between SH3P2 and CFS1, SH3P2 was also found to associate to amphisomelike structures [43].…”

The Autophagy Receptor NBR1 Directs the Clearance of Photodamaged Chloroplasts

“…The PB1 (Phox and Bem1 domain) domain present in both SQTM1 and NBR1 mediate their mutual interaction and oligomerization into helical filaments (Ciuffa et al, 2015) which then promote the aggregation of ubiquitylated species (Jakobi et al, 2020; Turco et al, 2021). In addition, mammalian SQSTM1 and NBR1 share a zinc-finger domain (ZZ) that can bind N-terminally arginylated proteins, lysine-48 (K48)- and K63-linked polyubiquitylated proteins, and other cargo (Cha-Molstad et al, 2015; Kwon et al, 2018; Wang et al, 2021), a ubiquitin-associated (UBA) domain with affinity for ubiquitin, and an ATG8-interacting motif (AIM) sequence that binds ATG8 (Seibenhener et al, 2004; Ichimura et al, 2008; Zientara-Rytter et al, 2011; Sun et al, 2022a). NBR1, but not SQSTM1, also contains a Four-Tryptophan (FW) domain, which at least in some fungal species recognizes cargo for selective autophagy.…”