Biology subjects

Oksbjerg, E.

Publications and source records attributed to Oksbjerg, E..

2 recordsLinked to original sources

bioRxiv2023

The N-coil and the globular N-terminal domain of plant ARGONAUTE1 are interaction hubs for regulatory factors

The effector complex of RNA interference (RNAi) contains at its core an ARGONAUTE (AGO) protein bound to a small guide RNA. AGO proteins adopt a two-lobed structure in which the N-terminal (N) and Piwi-Argonaute-Zwille (PAZ) domains make up one lobe, while the middle (MID) and Piwi domains make up the other. Specific biochemical functions of PAZ, MID and Piwi domains of eukaryotic AGO proteins have been described, but the functions of the N-terminal domain remain less clear. Here, we use yeast two-hybrid screening with the N-terminal domain of the founding member of the AGO protein family, arabidopsis AGO1, to reveal that it interacts with many factors involved in regulated proteolysis. Interaction with a large group of proteins, including the autophagy cargo receptors ATI1 and ATI2, requires residues in a short, linear region, the N-coil, that joins the MID-Piwi lobe in the three-dimensional structure of AGO. In contrast, the F-box protein AUF1 interacts with AGO1 independently of the N-coil and requires distinct residues in the globular N domain itself. Mutation of AGO1 residues necessary for interaction with protein degradation factors in yeast stabilizes reporters fused to the AGO1 N-terminal domain in plants, supporting their in vivo relevance. Our results define distinct regions of the N domain implicated in protein-protein interaction, and point to a particular importance of the AGO1 N-coil as a site of interaction with regulatory factors.

molecular biology↗

bioRxiv2018

The Arabidopsis autophagy cargo receptors ATI1 and ATI2 bind to ATG8 via intrinsically disordered regions and are post-translationally modified upon ATG8 interaction

Selective autophagy has emerged as an important mechanism by which eukaryotic cells control the abundance of specific proteins. This mechanism relies on cargo recruitment to autophagosomes by receptors that bind to both the ubiquitin-like AUTOPHAGY8 (ATG8) protein through ATG8 interacting motifs (AIMs) and to the cargo to be degraded. In plants, two autophagy cargo receptors, ATG8 Interacting Protein 1 (ATI1) and 2 (ATI2), were identified early on, but their molecular properties remain poorly understood. Here, we show that ATI1 and ATI2 are transmembrane proteins with long N-terminal intrinsically disordered regions (IDRs). The N-terminal IDRs contain the functional AIMs, and we use nuclear magnetic resonance spectroscopy to directly observe the disorder-order transition of the AIM upon ATG8 binding. Our analyses also show that the IDRs of ATI1 and ATI2 are not equivalent, because ATI2 has properties of a fully disordered polypeptide, while ATI1 has properties consistent with a collapsed pre-molten globule-like conformation Interestingly, wild type ATI1 and ATI2 exist as distinct post-translationally modified forms. Specifically, different forms are detectable upon mutation of the AIM, suggesting that interaction of ATI1 and ATI2 to ATG8 is coupled to a change in their post-translational modification.

molecular biology↗