A Universal Strategy for Proteomic Studies of SUMO and Other Ubiquitin-like Modifiers

David H. Russell,William K. Russell,Germán Rosas-Acosta,Van G. Wilson,Adeline Deyrieux

doi:10.1074/mcp.m400149-mcp200

David H. Russell, William K. Russell

Open Access

https://doi.org/10.1074/mcp.m400149-mcp200

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Abstract

Post-translational modification by the conjugation of small ubiquitin-like modifiers is an essential mechanism to affect protein function. Currently, only a limited number of substrates are known for most of these modifiers, thus limiting our knowledge of their role and relevance for cellular physiology. Here, we report the development of a universal strategy for proteomic studies of ubiquitin-like modifiers. This strategy involves the development of stable transfected cell lines expressing a double-tagged modifier under the control of a tightly negatively regulated promoter, the induction of the expression and conjugation of the tagged modifier to cellular proteins, the tandem affinity purification of the pool of proteins covalently modified by the tagged modifier, and the identification of the modified proteins by LC and MS. By applying this methodology to the proteomic analysis of SUMO-1 and SUMO-3, we determined that SUMO-1 and SUMO-3 are stable proteins exhibiting half-lives of over 20 h, demonstrated that sumoylation with both SUMO-1 and SUMO-3 is greatly stimulated by MG-132 and heat shock treatment, demonstrated the preferential usage of either SUMO-1 or SUMO-3 for some known SUMO substrates, and identified 122 putative SUMO substrates of which only 27 appeared to be modified by both SUMO-1 and SUMO-3. This limited overlapping in the subset of proteins modified by SUMO-1 and SUMO-3 supports that the SUMO paralogues are likely to be functionally distinct. Three of the novel putative SUMO substrates identified, namely the polypyrimidine tract-binding protein-associated splicing factor PSF, the structural microtubular component alpha-tubulin, and the GTP-binding nuclear protein Ran, were confirmed as authentic SUMO substrates. The application of this universal strategy to the identification of the pool of cellular substrates modified by other ubiquitin-like modifiers will dramatically increase our knowledge of the biological role of the different ubiquitin-like conjugations systems in the cell.

Highlights

Post-translational modification by the conjugation of small ubiquitin-like modifiers is an essential mechanism to affect protein function
To make the strategy applicable to the identification of the pool of cellular proteins targeted by any ubiquitin-like modifier, a commercially available tandem affinity purification (TAP) tag was added to the N-terminal region of SUMO by placing the genes for SUMO-1 and SUMO-3 in the pBAC-2cp vector (Novagen, Inc)
The overall strategy is clearly applicable to the proteomic analysis of the pool of proteins modified by any other ubiquitin-like modifier, and represents a universal strategy for proteomic studies of ubiquitin-like modifiers

Summary

EXPERIMENTAL PROCEDURES

Development of Stable Transfected Cell Lines and Flow Cytometry—All cells lines used in these studies were grown in complete medium containing 1ϫ Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Gemini Bio-Products, Woodland, CA), in a humidified incubator at 37 °C, 5% CO2, unless otherwise indicated. Protein and Transcript Stability Analyses—For pulse-chase experiments, the F293-SUMO cell lines were plated at 3 ϫ 106 cells per flask in 25-cm flasks, Tet induced for 24 h, starved for 1 h, pulselabeled with 200 ␮Ci of trans-35S label (MP Biomedicals, Irvine, CA) for 1 h, washed and chased in unlabeled complete medium, and collected at different times post-chase. The neutralized eluate was incubated with S-protein agarose (Novagen, Inc.) in a circular rocker for 16 h at 4 °C, washed with 90 bead volumes of 1ϫ PBS, and the bound TAP-purified proteins were either eluted with 2 volumes of 4ϫ SDS-PAGE sample buffer (100 mM Tris, 20% glycerol, 8% SDS, 0.02% bromphenol blue, 4% ␤-mercaptoethanol) or by digestion with EKMaxTM Enterokinase (Invitrogen Corp.) in 1ϫ enterokinase reaction buffer (50 mM Tris pH 8.0, 1 mM CaCl2) for 16 h at 37 °C. Any protein found in the control sample and the F293-SUMO cell lines was removed from the list of identified proteins

RESULTS

DNA topoisomerase I

Molecular function unclassified

Rotamer strain as a determinant of protein structural specificity

Similar to stratifin

DISCUSSION