Arkadia/RNF111 is a SUMO-targeted ubiquitin ligase with preference for substrates marked with SUMO1-capped SUMO2/3 chain

Annie M Sriramachandran,Gerrit J K Praefcke,R Jürgen Dohmen,Katrin Meyer-Teschendorf,Niels H Gehring,Stefan Pabst,Helle D Ulrich,Kay Hofmann

doi:10.1038/s41467-019-11549-3

Annie M Sriramachandran, Gerrit J K Praefcke + Show 6 more

Open Access

https://doi.org/10.1038/s41467-019-11549-3

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Abstract

Modification with SUMO regulates many eukaryotic proteins. Down-regulation of sumoylated forms of proteins involves either their desumoylation, and hence recycling of the unmodified form, or their proteolytic targeting by ubiquitin ligases that recognize their SUMO modification (termed STUbL or ULS). STUbL enzymes such as Uls1 and Slx5-Slx8 in budding yeast or RNF4 and Arkadia/RNF111 in humans bear multiple SUMO interaction motifs to recognize substrates carrying poly-SUMO chains. Using yeast as experimental system and isothermal titration calorimetry, we here show that Arkadia specifically selects substrates carrying SUMO1-capped SUMO2/3 hybrid conjugates and targets them for proteasomal degradation. Our data suggest that a SUMO1-specific binding site in Arkadia with sequence similarity to a SUMO1-binding site in DPP9 is required for targeting endogenous hybrid SUMO conjugates and PML nuclear bodies in human cells. We thus characterize Arkadia as a STUbL with a preference for substrate proteins marked with distinct hybrid SUMO chains.

Highlights

Modification with SUMO regulates many eukaryotic proteins
The first one contains a chain of four SUMO1 moieties (4xSUMO1-GFP-HA), the second one has a SUMO2-capped 3xSUMO1 chain. Both types of substrates were relatively poorly targeted by Arkadia upon co-expression in yeast (Supplementary Fig. 5). These results indicate that, while SUMO1 is important at the distal end of a SUMO2 chain for efficient targeting by Arkadia, SUMO1 chains do not represent good targeting signals for this SUMO-targeted ubiquitin ligases (STUbLs) supporting our notion that SUMO1-capped SUMO2 chains are the preferred recognition signal of this ligase
Our in vivo targeting experiments with Arkadia and substrates wherein the respective residues in the distal SUMO moieties of a chain were mutated (SUMO1-H75D and SUMO2-D71 into histidine (D71H)) showed that these changes led to an inversion of targeting efficiency (Fig. 4)

Summary

Introduction

Modification with SUMO regulates many eukaryotic proteins. Down-regulation of sumoylated forms of proteins involves either their desumoylation, and recycling of the unmodified form, or their proteolytic targeting by ubiquitin ligases that recognize their SUMO modification (termed STUbL or ULS). STUbL enzymes such as Uls[1] and Slx5-Slx[8] in budding yeast or RNF4 and Arkadia/RNF111 in humans bear multiple SUMO interaction motifs to recognize substrates carrying poly-SUMO chains. Using yeast as experimental system and isothermal titration calorimetry, we here show that Arkadia selects substrates carrying SUMO1-capped SUMO2/3 hybrid conjugates and targets them for proteasomal degradation. We and others have identified a class of RING-type ubiquitin ligases, named SUMO-targeted ubiquitin ligases (STUbLs), that recognize and ubiquitylate SUMO modified proteins[5,6,7,8,9,10,11] Two such STUbLs, Uls[1] and the heterodimeric Slx5-Slx[8] (Uls2), which bear multiple short SUMO interaction motifs (SIMs), were identified in Saccharomyces cerevisiae[5,6]. Using engineered test substrates bearing different types of poly-SUMO chains, we discover that Arkadia represents a distinct type of STUbL with a preference for substrates carrying hybrid SUMO1-capped SUMO2/3 chains

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