title: 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 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 activation of fungal sterol regulatory element-binding protein transcription factors. The Dsc E3 ligase contains five integral membrane subunits and structurally resembles ER-associated degradation E3 ligases. Saccharomyces 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 sterol regulatory element-binding protein 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 was required in cells lacking the multivesicular body pathway and under conditions of ubiquitin depletion. To identify candidate substrates, we performed quantitative diGly proteomics using stable isotope labeling by amino acids in cell culture to survey ubiquitylation in wild-type and tul1Δ cells. We identified 3116 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 tul1Δ cells. Correcting the diGly dataset for these differences increased the number of Tul1-dependent ubiquitylation sites. Together, our data demonstrate that the Tul1 E3 ligase functions in protein homeostasis under non-stress conditions and support a role in protein quality control. This quantitative diGly proteomics methodology will serve as a robust platform for screening for stress conditions that require Tul1 E3 ligase activity.
authors: From the ‡Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205;
authors: Zongtian
authors: Z
authors: Tong
authors: §McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205;
authors: Min-Sik
authors: MS
authors: Kim
authors: §McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; ¶Departments of Biological Chemistry, Oncology and Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; ‖Adrienne Helis Malvin Medical Research Foundation, New Orleans, Louisiana 70130; **Diana Helis Henry Medical Research Foundation, New Orleans, Louisiana 70130.
authors: Akhilesh
authors: A
authors: Pandey
authors: From the ‡Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; peter.espenshade@jhmi.edu.
authors: Peter J
authors: PJ
authors: Espenshade
mesh_terms: D051187:Desmocollins / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D060746:Endoplasmic Reticulum-Associated Degradation; D006056:Golgi Apparatus / Q000378:metabolism; D007553:Isotope Labeling; D017434:Protein Structure, Tertiary; D040901:Proteomics; D050765:Qa-SNARE Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D051779:Sterol Regulatory Element Binding Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D051187:Desmocollins; C081224:PEP12 protein, S cerevisiae; D050765:Qa-SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D051779:Sterol Regulatory Element Binding Proteins; D025801:Ubiquitin; C000595516:Ubx3 protein, S cerevisiae; C461959:TUL1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases