Detection of proteins sumoylated in vivo and in vitro

Abstract

Small ubiquitin-related modifier (SUMO) is an ubiquitin-like protein that is covalently attached to a variety of target proteins. Unlike ubiquitination, sumoylation does not target proteins for proteolytic breakdown, but is instead involved in regulating multiple protein functional properties including protein-protein interactions and subcellular targeting, to name a few. Protein sumoylation has been particularly well characterized as a regulator of many nuclear processes as well as nuclear structure, making the characterization of this modification vital for understanding nuclear structure and function. Consequently, there has been intense interest in identifying new proteins that are targets of this modification and determining what role it plays in regulating their functions. This chapter presents methodologies for determining whether a particular protein is a substrate of sumoylation, and for identifying the lysine residue(s) where the modification occurs.

Fig. 1

The SUMO conjugation pathway. After they are translated, SUMO proteins must first be processed by a SUMO protease such as Ulp1, which removes four C-terminal residues so that the mature form ends with a glycine. These SUMO proteases are also responsible for removing SUMO groups from proteins. This mature form is then activated in an ATP-dependent manner by forming a thioester bond with a cysteine residue in the SAE2 subunit of the heterodimeric E1 activating enzyme. Following this activation step, the SUMO moiety is transferred to the E2 conjugating enzyme ubc9. In the final step SUMO is transferred in a ligation reaction from ubc9 to substrate proteins, forming an isopeptide bond between the terminal glycine on SUMO and the ε-amino group of a lysine in the target protein. The efficiency of sumoylation of some proteins is enhanced by SUMO ligase E3 proteins, via their ability to bind both ubc9 and the target protein, thereby increasing the kinetics of the SUMO transfer.

Fig. 2

Detection of sumoylation by immunoprecipitation/SUMO-1 Western blot. HSF2 protein was immunoprecipitated from extracts of HeLa cells followed by Western blot using anti-SUMO-1 antibodies. The positions of molecular weight standards are indicated on the left side of the panel. This figure is from our published work (24), used with permission.

Fig. 3

Analysis of SUMO-1 modification by reconstituted in vitro sumoylation reaction. (A) In vitro translated ³⁵S-labeled HSF2 protein was incubated with HeLa cytosol (as a source of E1), Ubc9, SUMO-1, SUMO-2 or with various combinations of each of these, and then subjected to SDS-PAGE followed by autoradiography. The positions of unmodified and SUMO-modified HSF2 are indicated to the right of the panel. (B) In vitro translated ³⁵S-labeled HSF2 protein was subjected to the in vitro SUMO-1 modification assay using either 6xHis-SUMO-1 or GST-SUMO-1 as the SUMO-1 substrate for the reaction. This figure is from our published work (24), used with permission.

Fig. 4

In vitro translated ³⁵S-labeled wildtype HSF2 protein and the HSF2 SUMO-1 consensus site mutants K82R, K139R, and K151R were used as substrates for in vitro SUMO-1 modification reactions. The positions of unmodified and SUMO-modified HSF2 proteins are indicated to the right of the panel. This figure is from our published work (24), used with permission.