PXD000918

PXD000918 is an original dataset announced via ProteomeXchange.

Title	Identification of Candidate Substrates for the Golgi Tul1 E3 Ligase Using Quantitative diGly Proteomics in Yeast
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.
HostingRepository	PRIDE
AnnounceDate	2015-11-12
AnnouncementXML	Submission_2015-11-12_02:51:13.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Akhilesh Pandey
SpeciesList	scientific name: Saccharomyces cerevisiae (Baker's yeast); NCBI TaxID: 4932;
ModificationList	acetylated residue; iodoacetamide derivatized residue; ubiquitinylated lysine
Instrument	LTQ Orbitrap Elite

Revision	Datetime	Status	ChangeLog Entry
0	2014-04-23 01:03:04	ID requested
⏵ 1	2015-11-12 02:51:15	announced

Tong Z, Kim MS, Pandey A, Espenshade PJ, Identification of candidate substrates for the Golgi Tul1 E3 ligase using quantitative diGly proteomics in yeast. Mol Cell Proteomics, 13(11):2871-82(2014) [pubmed]

submitter keyword: SILAC, ubiquitination, protein homeostasis

Akhilesh Pandey
contact affiliation	McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 USA
contact email	pandey@jhmi.edu
lab head
Akhilesh Pandey
contact affiliation	Johns Hopkins University
contact email	pandey@jhmi.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD000918
     1. Label: PRIDE project
     2. Name: Identification of Candidate Substrates for the Golgi Tul1 E3 Ligase Using Quantitative diGly Proteomics in Yeast