Dataset Information

DDI2 is a ubiquitin-directed endoprotease, responsible for cleavage of transcription factor NRF1

ABSTRACT: The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here we show that DDI2 knock-out cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro, but only when NRF1 protein is poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endo-protease.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Fibroblast

SUBMITTER: Michele Puglia

LAB HEAD: Blagoy Blagoev

PROVIDER: PXD019152 | Pride | 2020-06-24

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20180412Sl_QexXn2_200318_25cmr_Test_FLAG_TR_5pr.raw	Raw
	20180412Sl_QexXn2_200318_25cmr_Test_GST_TR_5pr.raw	Raw
	20180509MP_QexXn2_200418_25cmr_FL_1.raw	Raw
	20180509MP_QexXn2_200418_25cmr_FL_10.raw	Raw
	20180509MP_QexXn2_200418_25cmr_FL_11.raw	Raw

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Publications

DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1.

Dirac-Svejstrup A Barbara AB Walker Jane J Faull Peter P Encheva Vesela V Akimov Vyacheslav V Puglia Michele M Perkins David D Kümper Sandra S Hunjan Suchete S SS Blagoev Blagoy B Snijders Ambrosius P AP Powell David J DJ Svejstrup Jesper Q JQ

Molecular cell 20200609 2

The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These pro ...[more]

PMID: 32521225

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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.