Visualizing and Quantifying Protein PolySUMOylation
at the Single-Molecule Level
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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