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

Abstract

Autophagy-related protein 8 (ATG8) is a highly conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and a number of 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 protein–protein interaction resource, obtained using in planta immunoprecipitation (IP) followed by mass spectrometry (MS), 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 (AIM). Additional proteomics experiments indicated that the N-terminal β-strand shapes the broader 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.

Host-interactor screens ofPhytophthora infestansRXLR proteins reveal vesicle trafficking as a major effector-targeted process

“…For the first replicate, we followed the in-gel digestion protocol as previously reported (Petre et al, 2015). For the second replicate, we performed on-beads digestion as described previously (Zess et al, 2019). Both methods yielded similar results ( Figure S9, Dataset 3).…”

“…To achieve our aim, we performed an in planta coIP/MS screen in the model plant Nicotiana benthamiana, which has emerged as an optimal experimental system for cell biology and biochemistry studies of P. infestans-host interactions (Bozkurt and Kamoun 2020). In addition, N. benthamiana is particularly appropriate for large-scale coIP/MS screens thanks to rapid transient protein expression using Agrobacterium tumefaciens (agroinfiltration) and the availability of a genome sequence that improves MS proteome predictions (Goodin et al, 2008;Derevnina et al, 2019;Zess et al, 2019). Using this approach, we generated a host proteinpathogen effector interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins.…”

The Irish potato famine pathogen subverts host vesicle trafficking to channel starvation-induced autophagy to the pathogen interface

“…JOKA2:BFP, BFP:EV, BFP:PexD54, BFP:ATG8CL, ATG9:GFP, 3xHA:EV, JOKA2:3xHA constructs were described in (Dagdas et al, 2016). GFP:ATG8 1.1, GFP:ATG81.2, GFP:ATG8 2.2, GFP:ATG8 3.1, GFP:ATG8 3.2, GFP:ATG8 4 were described in (Zess et al, 2019).…”

“…PexRD54 carries a canonical C-terminal ATG8 interacting motif (AIM) that is typically found on autophagy cargo receptors to bind ATG8 (Maqbool et al, 2016). Among the diverse set of potato ATG8 members (Kellner et al, 2017; Zess et al, 2019), PexRD54 preferentially binds the ATG8CL isoform and outcompetes Joka2/NBR1 from ATG8CL complexes, thereby disarming defense-related autophagy at the pathogen interface. Intriguingly, PexRD54 does not fully shutdown autophagy as has been shown for animal pathogens that suppress autophagy (Choy et al, 2012; Kimmey and Stallings, 2016; Real et al, 2017; Xu et al, 2019).…”

Peer Review #1 of "A novel approach to immunoapheresis of C3a/C3 and proteomic identification of associates (v0.1)"

“…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).…”