title: Visualizing and quantifying protein polySUMOylation at the single-molecule level.
abstract: Protein polySUMOylation, the attachment of small ubiquitin-like modifier (SUMO) chains to the target protein, is associated with a variety of physiological processes. However, the analysis of protein polySUMOylation is often complicated by the heterogeneity of SUMO-target conjugates. Here, we develop a new strategy to visualize and quantify polySUMOylation at the single-molecule level by integrating the tetracysteine (TC) tag labeling technology and total internal reflection fluorescence (TIRF)-based single-molecule imaging. As a proof-of-concept, we employ the human SUMO-2 as the model. The addition of TC tag to SUMO-2 can specifically translate the SUMO-mediated modification into visible fluorescence signal without disturbing the function of SUMO-2. The SUMO monomers display homogeneous fluorescence spots at the single-molecule level, whereas the mixed SUMO chains exhibit nonuniform fluorescence spots with a wide range of intensities. Analysis of the number and the brightness of fluorescence spots enable quantitative measurement of the polySUMOylation degree inside the cells under different physiological conditions. Due to the frequent occurrence of posttranslational modification by polymeric chains in cells, this single-molecule strategy has the potential to be broadly applied for studying protein posttranslational modification in normal cellular physiology and disease etiology.
authors.affiliation: Single-molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences , Shenzhen, Guangdong 518055, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Yang
authors.forename: Chun-yang
authors.initials: CY
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D058207:Sumoylation
chemical_list: D034961:Antigens, Nuclear; D001324:Autoantigens; D011993:Recombinant Fusion Proteins; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; C066642:SP100 protein, human; D003545:Cysteine