Dataset Information

Identification of Candidate Substrates for the Golgi Tul1 E3 Ligase Using Quantitative diGly Proteomics in Yeast

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD000918 | Pride | 2015-11-12

REPOSITORIES: Pride

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Identification of candidate substrates for the Golgi Tul1 E3 ligase using quantitative diGly proteomics in yeast.

Tong Zongtian Z Kim Min-Sik MS Pandey Akhilesh A Espenshade Peter J PJ

Molecular & cellular proteomics : MCP 20140730 11

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 endoplasmic reticulum (ER)-resident, multi-subunit E3 ubiquitin ligases that function in ER-associated degradation. Using fission yeast, our lab identified the Golgi Dsc E3 ligase as required for proteolytic ac ...[more]

PMID: 25078903

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Project description:In eukaryotic cells, short-lived, regulatory and misfolded or denatured proteins are degraded by the ubiquitin–proteasome system (UPS), a highly regulated mechanism including the active marking of proteins for proteasomal degradation by ubiquitin. The UPS is critical in regulating proteostasis and dysfunctioning of this system has been implicated in diseases such as cancer and neurodegenerative disorders. We have investigated the effects of proteasome malfunctioning in Drosophila by monitoring the global proteome and ubiquitinome using SILAC proteomics. The proteasome was inactivated by either chemical inhibitors or by dsRNA mediated knockdown of specific proteasome subunit constituents. Upon proteasome perturbation both the global proteome and the ubiquitinome were remodeled to a great extent, albeit that the overall impact on the ubiquitinome was much more dramatic. The abundances of ~10% of all observable proteins were increased as a result of both de novo synthesis and of accumulation of ubiquitinated proteins. In the ubiquitinome analysis ~14,000 diGly peptides were identified and most of these could also be quantified. The far majority of diGly peptides showed increased levels, indicating that the pool of ubiquitinated proteins is highly dynamic. Interestingly, because of the high level of detail the ubiquitination dynamics of individual proteins could be explored. Several examples (e.g., Rps3 and H2B) will be discussed, in which different target lysine residues show either increased or decreased ubiquitination on the same protein, suggesting the occurrence of simultaneous and possibly functionally different ubiquitination events. In conclusion, the combination of quantitative proteomic methods with dsRNA mediated knockdown of individual subunits of the 26S proteasome offers a powerful tool to study the dynamics of the (modified) proteome upon perturbation of the UPS. This strategy opens up new avenues for the dissection of the mode of action of this cellular machinery, both under proteostasis and proteotoxic conditions.

			Action	DRS
	MaxQuant_Tul1D_diGly.zip	Other
	MaxQuant_Tul1D_whole.zip	Other
	Tul1D_diGly-K_r1.raw	Raw
	Tul1D_diGly-K_r2.raw	Raw
	Tul1D_whole_r1_b01.raw	Raw