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 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 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 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.