A universal stategy for proteomic studies of SUMO and other ubiquitin-like modifiers

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

Research output: Contribution to journalArticle

175 Citations (Scopus)

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 α-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.

Original languageEnglish (US)
Pages (from-to)56-72
Number of pages17
JournalMolecular and Cellular Proteomics
Volume4
Issue number1
DOIs
StatePublished - Jan 2005
Externally publishedYes

Fingerprint

Ubiquitin
Proteomics
Substrates
Proteins
SUMO-1 Protein
Sumoylation
Polypyrimidine Tract-Binding Protein
Post Translational Protein Processing
Tubulin
Nuclear Proteins
GTP-Binding Proteins
Physiology
Guanosine Triphosphate
Shock
Hot Temperature
Purification
Cell Line
Cells

ASJC Scopus subject areas

  • Biochemistry

Cite this

A universal stategy for proteomic studies of SUMO and other ubiquitin-like modifiers. / Rosas-Acosta, Germán; Russell, William; Deyrieux, Adeline; Russell, David H.; Wilson, Van G.

In: Molecular and Cellular Proteomics, Vol. 4, No. 1, 01.2005, p. 56-72.

Research output: Contribution to journalArticle

Rosas-Acosta, Germán ; Russell, William ; Deyrieux, Adeline ; Russell, David H. ; Wilson, Van G. / A universal stategy for proteomic studies of SUMO and other ubiquitin-like modifiers. In: Molecular and Cellular Proteomics. 2005 ; Vol. 4, No. 1. pp. 56-72.
@article{cd56269ff2be4005be7d2518824118ee,
title = "A universal stategy for proteomic studies of SUMO and other ubiquitin-like modifiers",
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 α-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.",
author = "Germ{\'a}n Rosas-Acosta and William Russell and Adeline Deyrieux and Russell, {David H.} and Wilson, {Van G.}",
year = "2005",
month = "1",
doi = "10.1074/mcp.M400149-MCP200",
language = "English (US)",
volume = "4",
pages = "56--72",
journal = "Molecular and Cellular Proteomics",
issn = "1535-9476",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "1",

}

TY - JOUR

T1 - A universal stategy for proteomic studies of SUMO and other ubiquitin-like modifiers

AU - Rosas-Acosta, Germán

AU - Russell, William

AU - Deyrieux, Adeline

AU - Russell, David H.

AU - Wilson, Van G.

PY - 2005/1

Y1 - 2005/1

N2 - 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 α-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.

AB - 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 α-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.

UR - http://www.scopus.com/inward/record.url?scp=14644402420&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=14644402420&partnerID=8YFLogxK

U2 - 10.1074/mcp.M400149-MCP200

DO - 10.1074/mcp.M400149-MCP200

M3 - Article

VL - 4

SP - 56

EP - 72

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 1

ER -