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Identification of E3 ligase substrates and PROTAC-induced ubiquitylation sites using Proximity-based Identification of Ubiquitin Sites (PrIUS)

ABSTRACT: The ubiquitin system regulates virtually all cellular processes, yet the vast majority of ubiquitylation sites identified in the human proteome cannot be attributed to specific E3 ligases. This knowledge gap hampers our understanding of ubiquitin signaling and the development of therapeutics leveraging the ubiquitylation system. Here we present Proximity-based Identification of Ubiquitin Sites (PrIUS), a versatile mass spectrometry-based approach combining proximity-dependent biotinylation (BioID) with ubiquitin remnant (diGly) enrichment. PrIUS enables simultaneous identification of E3 ligase interactors and direct mapping of substrate ubiquitylation sites within a single workflow. Using the E3 ligase NEDD4L, we demonstrated that E3 interactomes are inherently rich for diGly-modified peptides that remain largely undetectable without PTM-specific enrichment. PrIUS identified hundreds of high-confidence ubiquitylation sites, successfully distinguished substrates from non-substrate interactors (including adapters and E2 enzymes), and identified non-degradative ubiquitylation events. We also demonstrate utility for characterizing targeted protein degrader mechanisms by precisely mapping PROTAC-induced ubiquitylation sites. PrIUS thus provides a useful approach for elucidating E3-substrate relationships and characterizing mechanisms for emerging degrader therapeutics at amino acid resolution.

INSTRUMENT(S): timsTOF Pro 2, timsTOF HT, timsTOF SCP

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Matthew Weitzman

PROVIDER: MSV000101010 | MassIVE | Sun Mar 01 19:14:00 GMT 2026

REPOSITORIES: MassIVE

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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.

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Identification of E3 ligase substrates and PROTAC-induced ubiquitylation sites using Proximity-based Identification of Ubiquitin Sites (PrIUS)

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