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 func-tions of PAZ, MID and Piwi domains of eukaryotic AGO proteins have been described, but the functions of the N domain remain less clear. Here, we use yeast two-hybrid screening with the N domain of the founding member of the AGO protein family, Arabidopsis AGO1, to reveal that it interacts with many factors involved in regulated pro-teolysis. Interaction with a large group of proteins, including the autophagy cargo recep-tors 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 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.