Dataset Information

Sequential Peptide Immunopurification Methodology to Monitor the Crosstalk between SUMOylation and Ubiquitylation

ABSTRACT: We describe a method that permits the identification of proteins that are modified by both SUMOylation and ubiquitylation to better understand the role of this crosstalk. The procedure requires 3 days when starting from cell pellets and can yield more than 8000 SUMO sites and 3500 ubiquitin sites from 16 mg of cell extract.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Francis McManus

LAB HEAD: Pierre Thibault

PROVIDER: PXD006545 | Pride | 2018-03-27

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	FM_SUMO_SCX1_CONTROL_I1.raw	Raw
	FM_SUMO_SCX1_CONTROL_I2.raw	Raw
	FM_SUMO_SCX1_HEATSHOCK_I1.raw	Raw
	FM_SUMO_SCX1_HEATSHOCK_I2.raw	Raw
	FM_SUMO_SCX1_MG132_I1.raw	Raw

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Publications

Identification of cross talk between SUMOylation and ubiquitylation using a sequential peptide immunopurification approach.

McManus Francis P FP Lamoliatte Frédéric F Thibault Pierre P

Nature protocols 20171019 11

Ubiquitin and ubiquitin-like modifiers (UBLs) such as small ubiquitin-like modifier (SUMO) can act as antagonists to one another by competing to occupy similar residues in the proteome. In addition, SUMO and ubiquitin can be coupled to each other at key lysine residues to form highly branched protein networks. The interplay between these modifications governs important biological processes such as double-strand break repair and meiotic recombination. We recently developed an approach that permit ...[more]

PMID: 29048423

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Project description:Maintenance of protein homeostasis is essential for cellular survival. Central to this regulation are mechanisms of protein quality control in which misfolded proteins are recognized and degraded by the ubiquitin-proteasome system. One well-studied protein quality control pathway requires ER resident, multi-subunit E3 ubiquitin ligases that function in ER-associated degradation (ERAD). Using fission yeast, our lab identified the Golgi Dsc E3 ligase as required for proteolytic activation of fungal sterol regulatory element-binding protein (SREBP) transcription factors. The Dsc E3 ligase contains 5 integral membrane subunits and structurally resembles ERAD E3 ligases. S. cerevisiae codes for homologs of Dsc E3 ligase subunits, including the Dsc1 E3 ligase homolog Tul1 that functions in Golgi protein quality control. Interestingly, S. cerevisiae lacks SREBP homologs, indicating that novel Tul1 E3 ligase substrates exist. Here, we show that the S. cerevisiae Tul1 E3 ligase consists of Tul1, Dsc2, Dsc3, and Ubx3 and define Tul1 complex architecture. Tul1 E3 ligase function required each subunit as judged by vacuolar sorting of the artificial substrate Pep12D. Genetic studies demonstrated that Tul1 E3 ligase is required in cells lacking the multivesicular body pathway and under conditions of ubiquitin depletion. To identify candidate substrates, we used quantitative diGly proteomics to survey ubiquitylation in wild-type and tul1∆ cells. We identified 3,116 non-redundant ubiquitylation sites, including 10 sites in candidate substrates. Quantitative proteomics found 4.5% of quantified proteins (53/1172) to be differentially expressed in tul1∆ cells. Correcting the diGly dataset for these differences increased the number of Tul1-dependent ubiquitylation sites. Together, these data demonstrate that the Tul1 E3 ligase plays a minor role in protein homeostasis under non-stress conditions, consistent with functions in protein quality control. Furthermore, this quantitative diGly proteomics methodology serves as a robust platform to screen for stress conditions that require Tul1 activity.