N-terminal β-strand underpins biochemical specialization of an ATG8 isoform

Abstract

ATG8 is a highly-conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and numerous proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive proteinprotein interaction resource, obtained using in planta immunoprecipitation followed by mass spectrometry, to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal β-strand-and, in particular, a single amino acid polymorphism-underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this protein's ATG8 interacting motif. Additional proteomics experiments indicated that the N-terminal βstrand shapes the ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins.Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants.

Human LC3 and GABARAP subfamily members achieve functional specificity via specific structural modulations

“…As shown in our phylogenetic tree, nine Arabidopsis proteins were grouped with Atg8 cluster into two groups (Atg8H-I and Atg8A-G), with latter dividing further into two subgroups. In a recent study, potato Atg8 isoforms were also proposed to bind to a distinct set of proteins [44]. The recognition motif for subfamilies, identified in our study, thus also provides a valuable resource for the autophagic community to decode variability within the multi-member Atg8 family.…”

A novel approach to immunoapheresis of C3a/C3 and proteomic identification of associates

“…The methodology used has already been described in earlier studies (Qiao et al, 2016; Wissel et al, 2016; Zess et al, 2018). In brief, the UltiMate 3000 RSLC nano system (Thermo Fisher Scientific, Amsterdam, Netherlands) was combined with Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Bremen, Germany), using a Proxeon nanospray source (Thermo Fisher Scientific, Odense, Denmark).…”

“…A detailed description is available in previous articles (Qiao et al, 2016; Wissel et al, 2016; Zess et al, 2018).…”

“…As search engine MSAmanda v2.0.0.9849 (Dorfer et al, 2014) was used connected with SwissProt human database (20169 sequences; 11315794 residues). Exact parameter settings are indicated in previous work (Zess et al, 2018). The tool ptmRS was used to investigate the locus of the posttranslational peptide modification sites (Taus et al, 2011).…”

Boosting autophagy in sexual reproduction: a plant perspective

“…It is technically challenging and time consuming to generate an Arabidopsis mutant lacking multiple AtATG8 s by conventional genetic mutation, crossing and screening. Recently, Zess et al () employed immunoprecipitation together with mass spectrometry analysis to demonstrate that ATG8 isoforms actually bind to different sets of interactors via their specific N‐terminal β‐strand in potato ( Solanum tuberosum ). Moreover, it was found that diverse populations of transcription factors that are involved in plant development and stress responses bind and regulate the transcription of distinct AtATG8 s in Arabidopsis (Wang et al , ).…”