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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.

SUBMITTER: Zess EK

PROVIDER: S-EPMC6675122 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description:GFP-IP based interactome of potato ATG8 isoforms following transient expression in Nicotiana benthamiana.

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Biochemical specialization of an ATG8 isoform in Nicotiana benthamiana

Project description: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 protein-protein 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, particularly a single amino acid polymorphism, underpins binding specificity to the protein PexRD54. This isoleucine to valine substitution perturbed the hydrophobic pocket that accommodates the conserved ATG8 interacting motif of PexRD54. Additional proteomics experiments indicated that the N-terminal β-strand impacts ATG8 interactor profile by defining interaction specificity with about ~100 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise one layer of specificity in the regulation of selective autophagy pathways in plants.

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Nonuple atg8 mutant provides genetic evidence for functional specialization of ATG8 isoforms in Arabidopsis thaliana

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Functional specialization of sensory cilia by an RFX transcription factor isoform.

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