A subscription to JoVE is required to view this content. Sign in or start your free trial.
SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.
Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.
The purpose of this method is to facilitate the study and analysis of SUMO-conjugated proteins using the recombinant SUMO-trapping UTAG (Ulp domain Tag) protein. As detailed below, UTAG can be used in lieu of other reagents and approaches to purify, detect, and visualize SUMO-modified proteins. Depending on growth conditions, cells may contain hundreds or thousands of proteins that are modified with SUMO or SUMO chains (for review see Kerscher et al. 20061 and Kerscher 20162). This represents a considerable difficulty for the functional analyses of specific SUMO-modified proteins, especially since only a fraction of a particular sumoylation target is actually modified3. In addition to their roles in essential cellular processes such as transcriptional regulation, protein homeostasis, the response to cellular stress, and chromatin remodeling during mitosis and meiosis; it has now become sufficiently clear that SUMO, SUMO-modified proteins, and SUMO pathway components also have potential as biomarkers for pathologies such as cancer and neurodegenerative disorders4,5,6,7. This underscores the need for robust, reliable, and readily available tools and innovative approaches for the detection and functional analysis of SUMO-modified proteins in a variety of cells and samples.
In many systems, SUMO-specific antibodies are the reagents of choice for the detection, isolation and functional analyses of SUMO-modified proteins8,9. However, some commercially available SUMO-specific antibodies are expensive, limited in quantity or availability, prone to exhibit wildly variable affinities and cross-reactivity, and in some instances lack reproducibility10. One alternative approach is the expression of epitope-tagged SUMO in transformed cells and organisms, but linking epitope tags to SUMO may artificially lower its conjugation to protein targets11. Additionally, epitopes are not useful when untransformed cells or tissues are evaluated.
Ulp1 is a conserved SUMO protease from S. cerevisiae that both processes the SUMO precursor and desumoylates SUMO-conjugated proteins12. We developed the UTAG reagent based on the serendipitous observation that a mutation of the catalytic Cysteine (C580S) in Ulp1's SUMO processing Ulp domain (UD) not only prevents SUMO cleavage but also traps SUMO-conjugated proteins with high avidity12. For simplicity, we referred to this carboxy-terminal SUMO-trapping Ulp1(C580S) fragment as UTAG (short for UD TAG). UTAG is a recombinant pan-SUMO trapping protein that represents a useful alternative to anti-SUMO antibodies used for the isolation and detection of SUMO-modified proteins. Importantly, it specifically recognizes natively-folded, conjugated SUMO and not just one or several epitopes on SUMO. To improve both the protein stability and SUMO binding strength of UTAG, we generated a variant of UTAG from the stress-tolerant yeast Kluyveromyces marxianus (Km). KmUTAG tightly binds SUMO-conjugates with nanomolar affinity13. Additionally, kmUTAG is resistant to elevated temperatures (42 °C), reducing agents (5 mM TCEP - Tris(2-carboxyethyl)phosphine hydrochloride), denaturants (up to 2 M Urea), oxidizing agents (0.6% hydrogen peroxide), and non-ionic detergents. This stress tolerance is beneficial during harsh purification condition and prolonged incubation times, ensuring its stability and SUMO-trapping activity. Not surprisingly, however, KmUTAG's SUMO-trapping activity is incompatible with cysteine-modifying reagents, ionic detergents and fully denatured protein extracts. The remarkable affinity and properties of KmUTAG indicate that this reagent may become part of the standard repertoire for the study of sumoylated proteins in multiple species.
Here we provide a simple method to detect SUMO-modified proteins in mammalian cells and nematodes using a recombinant fluorescent mCherry-KmUTAG fusion protein (kmUTAG-fl).
1. SUMO detection in fixed tissue culture cells using recombinant KmUTAG-FL SUMO-trapping protein
2. SUMO detection in fixed nematode gonads using UTAG-fl
KmUTAG-fl is a recombinant, mCherry-tagged SUMO-trapping protein. To produce kmUTAG-fl, we cloned a codon-optimized mCherry-kmUTAG into the pSPOT1 bacterial overexpression plasmid (Figure 1). After induction, the kmUTAG-fl protein was purified on Spot-TRAP, eluted, and frozen until further use. To ensure the SUMO-trapping activity of KmUTAG-fl, we confirmed binding to SUMO1-conjugated beads and precipitation of a SUMO-CAT fusion protein (data not shown, but s...
Here we introduce the use of kmUTAG-fl, a recombinant protein, for functional studies of SUMO in fixed mammalian cells and dissected nematode gonads. KmUTAG-fl is a stress-tolerant pan-SUMO specific reagent that recognizes and traps native SUMO-conjugated proteins and SUMO chains. Since SUMO's tertiary structure is highly conserved it is very likely that SUMO variants from additional model and non-model systems can be analyzed with the kmUTAG-fl reagent. As such, KmUTAG-fl may represent a useful alternative or second...
Recombinant kmUTAG reagents are provided to the research community via Kerafast.com.
We would like to thank all members of the Kerscher lab for their support, Nathalie Nguyen for critical reading of the manuscript, and Lidia Epp for sequencing. This work has been supported by the Commonwealth Research Commercialization fund MF16-034-LS to OK. Research support for W&M students was provided by the Bailey-Huston Research fund, and Charles Center Honors Fellowships to RY and CH.
Name | Company | Catalog Number | Comments |
16% Paraformaldehyde (formaldehyde) aqueous solution | Electron Microscopy Sciences | 30525-89-4 | |
6-Well Cell Culture Plates | Genesee Scientific/Olympus Plastics | 25-105 | |
Alexa Fluor 488 AffiniPure Goat Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 115-545-003 | Used as a secondary antibody for mouse monoclonal antibody |
DPBS, no calcium, no magnesium | Fisher Scientific | Gibco 14190144 | |
Dylight 488 conjugated AffiniPure Goat Anti-Moue IgG (H+L) | Jackson ImmunoResearch | 115-485-146 | Used as a secondary antibody for mouse monoclonal antibody |
Fisherbrand Coverglass for Growth Cover Glasses | Fisherbrand | 12545101 | |
FLUORO-GEL II with DAPI | Electron Microscopy Sciences | 50-246-93) | Mounting media in step 1.11 |
FLUORO-GEL with DABCO | Electron Microscopy Sciences | 17985-02 | With DAPI added to 1 µg/mL; mounting media in step 2.2.5 |
Glycine-HCl | Fisher BioReagents | BP3815 | |
Glycine-HCl | ACROS Organics | 6000-43-7 | |
KmUTAG-fl | Kerafast | KmUTAG reagents are available on Kerafast.com | |
Oneblock Western-CL blocking buffer | Prometheus | 20-313 | |
PBS, Phosphate Buffered Saline, 10X Solution | Fisher BioReagents | BP3994 | |
PNT2 cell line | Sigma-Aldrich | 95012613 | Normal prostate epithelium immortalized with SV40. |
pSPOT1 | (ChromoTek GmbH) | ev-1 | https://www.chromotek.com/fileadmin/user_upload/pdfs/Datasheets/pSpot1_v1.pdf |
SUMO 6F2 | DSHB | SUMO 6F2 | SUMO 6F2 was deposited to the DSHB by Pelisch, F. / Hay, R.T. (DSHB Hybridoma Product SUMO 6F2) |
SUMO protease buffer [10x] | 500 mM Tris-HCl, pH 8.0, 2% NP-40, 1.5 M NaCl | ||
SUMO-2 Antibody 8A2 | DSHB | SUMO-2 8A2 | SUMO-2 8A2 was deposited to the DSHB by Matunis, M. (DSHB Hybridoma Product SUMO-2 8A2) |
TCEP-HCL | GoldBio | 51805-45-9 | Used as a reducing agent at a concentration of 5mM |
Triton X-100 | Fisher BioReagents | 9002-93-1 | Used for permeablization at 0.1% in DPBS/PBS(for worms) |
Request permission to reuse the text or figures of this JoVE article
Request PermissionExplore More Articles
This article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved