TY - JOUR

T1 - The N-coil and the globular N-terminal domain of plant ARGONAUTE1 are interaction hubs for regulatory factors

AU - Bressendorff, Simon

AU - Kausika, Swathi

AU - Sjøgaard, Ida Marie Zobbe

AU - Oksbjerg, Emilie Duus

AU - Michels, Alec

AU - Poulsen, Christian

AU - Brodersen, Peter

N1 - Publisher Copyright: © 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

PY - 2023

Y1 - 2023

N2 - 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.

AB - 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.

KW - argonaute

KW - autophagy

KW - N domain

KW - regulated proteolysis

KW - ubiquitin proteasome system

U2 - 10.1042/BCJ20230025

DO - 10.1042/BCJ20230025

M3 - Journal article

C2 - 37278687

AN - SCOPUS:85164259303

VL - 480

SP - 957

EP - 974

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 13

ER -