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

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.

[1]  J. Weissman,et al.  Differential Scales of Protein Quality Control , 2014, Cell.

[2]  Andrew R. Jones,et al.  ProteomeXchange provides globally co-ordinated proteomics data submission and dissemination , 2014, Nature Biotechnology.

[3]  T. Stearns,et al.  Methods in yeast genetics , 2013 .

[4]  J. Olzmann,et al.  The mammalian endoplasmic reticulum-associated degradation system. , 2013, Cold Spring Harbor perspectives in biology.

[5]  P. Espenshade,et al.  Subunit Architecture of the Golgi Dsc E3 Ligase Required for Sterol Regulatory Element-binding Protein (SREBP) Cleavage in Fission Yeast* , 2013, The Journal of Biological Chemistry.

[6]  S. Fields,et al.  Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation , 2013, Nature Methods.

[7]  Andrea C. Carrano,et al.  Using the Ubiquitin-modified Proteome to Monitor Protein Homeostasis Function , 2013, Molecular & Cellular Proteomics.

[8]  M. K. Lemberg.  Sampling the membrane: function of rhomboid-family proteins. , 2013, Trends in cell biology.

[9]  S. Benkovic,et al.  Replication clamps and clamp loaders. , 2013, Cold Spring Harbor perspectives in biology.

[10]  Steven P. Gygi,et al.  Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization , 2013, Nature.

[11]  J. Olzmann,et al.  Spatial regulation of UBXD8 and p97/VCP controls ATGL-mediated lipid droplet turnover , 2013, Proceedings of the National Academy of Sciences.

[12]  J. Brodsky.  Cleaning Up: ER-Associated Degradation to the Rescue , 2012, Cell.

[13]  Allyson F. O’Donnell.  The Running of the Buls: Control of Permease Trafficking by α-Arrestins Bul1 and Bul2 , 2012, Molecular and Cellular Biology.

[14]  Thomas Sommer,et al.  The Ubiquitin–Proteasome System of Saccharomyces cerevisiae , 2012, Genetics.

[15]  B. André,et al.  Internal Amino Acids Promote Gap1 Permease Ubiquitylation via TORC1/Npr1/14-3-3-Dependent Control of the Bul Arrestin-Like Adaptors , 2012, Molecular and Cellular Biology.

[16]  R. Haguenauer‐Tsapis,et al.  Ubiquitin-Mediated Regulation of Endocytosis by Proteins of the Arrestin Family , 2012, Biochemistry research international.

[17]  T. Sommer,et al.  Finding the will and the way of ERAD substrate retrotranslocation. , 2012, Current opinion in cell biology.

[18]  W. Lim,et al.  Systematic Functional Prioritization of Protein Posttranslational Modifications , 2012, Cell.

[19]  Sean R. Collins,et al.  Hierarchical modularity and the evolution of genetic interactomes across species. , 2012, Molecular cell.

[20]  J. Wade Harper,et al.  Defining human ERAD networks through an integrative mapping strategy , 2011, Nature Cell Biology.

[21]  Junmin Peng,et al.  Analysis of Ubiquitinated Proteome by Quantitative Mass Spectrometry , 2012, Quantitative Methods in Proteomics.

[22]  C. Nusbaum,et al.  Yeast Sterol Regulatory Element-binding Protein (SREBP) Cleavage Requires Cdc48 and Dsc5, a Ubiquitin Regulatory X Domain-containing Subunit of the Golgi Dsc E3 Ligase* , 2011, The Journal of Biological Chemistry.

[23]  Edward L. Huttlin,et al.  Systematic and quantitative assessment of the ubiquitin-modified proteome. , 2011, Molecular cell.

[24]  Michael J. Emanuele,et al.  Global Identification of Modular Cullin-RING Ligase Substrates , 2011, Cell.

[25]  T. Mustelin,et al.  Ubiquitin Ligase Substrate Identification through Quantitative Proteomics at Both the Protein and Peptide Levels , 2011, The Journal of Biological Chemistry.

[26]  J. Olzmann,et al.  Derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant α-1 antitrypsin from the endoplasmic reticulum , 2011, Nature Structural &Molecular Biology.

[27]  Sebastian A. Wagner,et al.  A Proteome-wide, Quantitative Survey of In Vivo Ubiquitylation Sites Reveals Widespread Regulatory Roles* , 2011, Molecular & Cellular Proteomics.

[28]  Scott D Emr,et al.  The ESCRT pathway. , 2011, Developmental cell.

[29]  N. Krogan,et al.  Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex. , 2011, Molecular cell.

[30]  M. Mann,et al.  Andromeda: a peptide search engine integrated into the MaxQuant environment. , 2011, Journal of proteome research.

[31]  Samie R Jaffrey,et al.  Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling , 2010, Nature Biotechnology.

[32]  P. Espenshade,et al.  Sterol Regulatory Element Binding Proteins in Fungi: Hypoxic Transcription Factors Linked to Pathogenesis , 2010, Eukaryotic Cell.

[33]  Gary D Bader,et al.  The Genetic Landscape of a Cell , 2010, Science.

[34]  R. Deshaies,et al.  RING domain E3 ubiquitin ligases. , 2009, Annual review of biochemistry.

[35]  John Rush,et al.  Quantitative Proteomics Reveals the Function of Unconventional Ubiquitin Chains in Proteasomal Degradation , 2009, Cell.

[36]  M. Mann,et al.  MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification , 2008, Nature Biotechnology.

[37]  R. Deshaies,et al.  UBXD7 Binds Multiple Ubiquitin Ligases and Implicates p97 in HIF1α Turnover , 2008, Cell.

[38]  Akhilesh Pandey,et al.  Quantitative proteomics using stable isotope labeling with amino acids in cell culture , 2008, Nature Protocols.

[39]  Richard I. Morimoto,et al.  Adapting Proteostasis for Disease Intervention , 2008, Science.

[40]  P. Espenshade,et al.  SREBP Controls Oxygen-Dependent Mobilization of Retrotransposons in Fission Yeast , 2007, PLoS genetics.

[41]  N. Krogan,et al.  Ubiquitination Screen Using Protein Microarrays for Comprehensive Identification of Rsp5 Substrates in Yeast , 2022 .

[42]  M. Mann,et al.  In-gel digestion for mass spectrometric characterization of proteins and proteomes , 2006, Nature Protocols.

[43]  M. Mann,et al.  Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips , 2007, Nature Protocols.

[44]  J. Huibregtse,et al.  The Deubiquitinating Enzyme Ubp2 Modulates Rsp5-dependent Lys63-linked Polyubiquitin Conjugates in Saccharomyces cerevisiae*> , 2006, Journal of Biological Chemistry.

[45]  Matthias Mann,et al.  Innovations: Functional and quantitative proteomics using SILAC , 2006, Nature Reviews Molecular Cell Biology.

[46]  H. Pelham,et al.  Swf1‐dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation , 2005, The EMBO journal.

[47]  Peter J. Espenshade,et al.  SREBP Pathway Responds to Sterols and Functions as an Oxygen Sensor in Fission Yeast , 2005, Cell.

[48]  M. Mann,et al.  Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics* , 2002, Molecular & Cellular Proteomics.

[49]  H. Pelham,et al.  A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies , 2002, Nature Cell Biology.

[50]  H. Pelham,et al.  Polar transmembrane domains target proteins to the interior of the yeast vacuole. , 2000, Molecular biology of the cell.

[51]  H. Pelham,et al.  A Selective Transport Route from Golgi to Late Endosomes That Requires the Yeast Gga Proteins , 2000, The Journal of cell biology.

[52]  A. Amerik,et al.  The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast. , 1999, Molecular biology of the cell.

[53]  P. Philippsen,et al.  Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae , 1998, Yeast.

[54]  S. Emr,et al.  Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast. , 1996, Molecular biology of the cell.

[55]  D. Ecker,et al.  Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant , 1994, Molecular and cellular biology.

[56]  Janina Maier,et al.  Guide to yeast genetics and molecular biology. , 1991, Methods in enzymology.