Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Summary

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.

Abstract

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.

Introduction

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, especi....

Protocol

1. SUMO detection in fixed tissue culture cells using recombinant KmUTAG-FL SUMO-trapping protein

1. Grow tissue culture cells of choice on 22 mm round cover slips in 6-well TC plates until 70%–80% confluent. Perform steps 1.2–1.8 in the 6-well plate.
2. Wash cells briefly with 1 mL of DPBS (Dulbecco's phosphate-buffered saline)
3. For fixation, prepare a fresh solution of 4% Paraformaldehyde (PFA) solution (diluted in DPBS). To fix cells, add 2 mL 4% PFA in DPBS to each well. Incub.......

Discussion

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.......

Materials

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)