Assay methods for small ubiquitin-like modifier (SUMO)-SUMO-interacting motif (SIM) interactions in vivo and in vitro using a split-luciferase complementation system

Mikako Hirohama; Arnout R D Voet; Takeaki Ozawa; Hisato Saitoh; Yoichi Nakao; Kam Y J Zhang; Akihiro Ito; Minoru Yoshida

doi:10.1016/j.ab.2013.12.009

Assay methods for small ubiquitin-like modifier (SUMO)-SUMO-interacting motif (SIM) interactions in vivo and in vitro using a split-luciferase complementation system

Anal Biochem. 2014 Mar 1:448:92-4. doi: 10.1016/j.ab.2013.12.009. Epub 2013 Dec 11.

Authors

Mikako Hirohama¹, Arnout R D Voet², Takeaki Ozawa³, Hisato Saitoh⁴, Yoichi Nakao⁵, Kam Y J Zhang², Akihiro Ito⁶, Minoru Yoshida⁷

Affiliations

¹ Chemical Genetics Laboratory, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Japan Science and Technology Corporation, CREST Research Project, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan; Department of Chemistry and Biochemistry, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
² Zhang Initiative Research Unit, RIKEN, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
³ Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Bunkyo-ku, Hongo, Tokyo 113-0033, Japan.
⁴ Department of New Frontier Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan.
⁵ Department of Chemistry and Biochemistry, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
⁶ Chemical Genetics Laboratory, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Electronic address: [email protected].
⁷ Chemical Genetics Laboratory, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Japan Science and Technology Corporation, CREST Research Project, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.

PMID: 24333278
DOI: 10.1016/j.ab.2013.12.009

Abstract

SUMOylation is a posttranslational process that attaches a small ubiquitin-like modifier (SUMO) to its target proteins covalently. SUMOylation controls multiple cellular processes through the recognition of SUMO by a SUMO-interacting motif (SIM). In this study, we developed assay systems for detecting noncovalent interactions between SUMO and SIM in cells using split-luciferase complementation. We applied a version of this assay to the detection of in vitro SUMO-SIM interactions using a bacterial expression system. These novel assays enable screening of inhibitors of SUMO-dependent protein-protein interactions, either in vivo or in vitro, in a high-throughput manner.

Keywords: Assay; Protein–protein interactions; SIM; SUMOylation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Carrier Proteins / genetics
Carrier Proteins / metabolism*
Escherichia coli / metabolism
Fluorescent Dyes / chemistry
Genes, Reporter
Humans
Light
Luciferases / chemistry
Luciferases / genetics
Luciferases / metabolism*
Protein Interaction Domains and Motifs
Protein Structure, Tertiary
Recombinant Fusion Proteins / chemistry
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism
SUMO-1 Protein / genetics
SUMO-1 Protein / metabolism*
Spectrometry, Fluorescence*

Substances

Carrier Proteins
Fluorescent Dyes
Recombinant Fusion Proteins
SIMC1 protein, human
SUMO-1 Protein
SUMO1 protein, human
Luciferases