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.

Fig. 1.

S. cerevisiae Dsc homologs. A, schematics of Dsc homologs in which black boxes represent predicted transmembrane domains. Predicted signal sequence (SS) and conserved domains are indicated. Underlining denotes amino acids used as antigens for antibody production. B, Pearson correlation coefficients between genetic signatures of S. cerevisiae DSC homologs from DRYGIN database (45). Genetic interactions were tested in a pairwise fashion such that each gene yielded two datasets. The database default setting for significance is CC > 0.1, and dashes denote the absence of a correlation coefficient in database.

Fig. 2.

Tul1 and Dsc3 expression requires E3 ligase subunits. A, microsomal extracts from wild-type, tul1Δ, dsc2Δ, dsc3Δ, and ubx3Δ cells immunoblotted with anti-Tul1, anti-Dsc2, anti-Dsc3, and anti-Ubx3 IgG. B, microsomal extracts from wild-type, tul1Δ, dsc2Δ, dsc3Δ, and ubx3Δ cells were treated in the presence or absence of PNGase F and immunoblotted with anti-Tul1 IgG. C, indicated strains were treated with proteasome inhibitor MG-132 (100 μm) or dimethyl sulfoxide for 2 h. Microsomal extracts were prepared and immunoblotted with anti-Tul1 and anti-Dsc3 IgG. D, microsomal extracts from the indicated strains were prepared and immunoblotted with anti-Tul1 and anti-Dsc3 IgG.

Fig. 3.

Tul1 E3 ligase subunits form distinct subcomplexes. A, digitonin-solubilized extracts from wild-type, tul1Δ, dsc2Δ, dsc3Δ, and ubx3Δ cells were prepared, and proteins associated with Dsc2 were immunopurified by anti-Dsc2 affinity-purified antibodies. Equal amounts of input and 5× bound fractions were immunoblotted using HRP-conjugated anti-Tul1, anti-Dsc2, anti-Dsc3, and anti-Ubx3 antibodies. B, C, proteins associated with Dsc3 or Ubx3, respectively, were immunopurified as described in A using anti-Dsc3 or anti-Ubx3 affinity-purified antibodies. Equal amounts of input and 5× bound fractions were immunoblotted using HRP-conjugated anti-Tul1, anti-Dsc2, anti-Dsc3, and anti-Ubx3 antibodies. D, model illustrating the results from co-immunoprecipitation experiments in A–C. Complexes and subcomplexes observed in wild-type and mutant cells are indicated. Contacts do not denote direct protein–protein interactions.

Fig. 4.

Tul1 E3 ligase complex displays genetic interactions with the multivesicular body pathway. A, indicated strains expressing GFP-tagged mutant form of Pep12 (Pep12D) were imaged using fluorescence microscopy. Staining of vacuolar lumen or limiting membrane is visible. B, indicated single and double mutant yeast strains were spotted on YPD rich medium at 37 °C and grown for 2 days. C, 5-fold serial dilutions of indicated single and double mutants carrying empty vector (pRS426) or expressing 6HIS-myc-ubiquitin (pUB221) were spotted on YPD rich medium with no added copper at 30 °C or 37 °C and grown for 2 days.

Fig. 5.

Schematic of workflow for quantification of ubiquitylated peptides.

Fig. 6.

Identification of candidate Tul1 substrates using quantitative mass spectrometry. A, Venn diagram comparing ubiquitylation sites found in this study and by Beltrao et al. (22). B, scatter plot of ubiquitylated peptide ratios (wild-type/tul1Δ) from two replicate experiments (n = 2289). C, expression of YKL033W-A mRNA in wild-type and tul1Δ cells. cDNA synthesized from total RNA (1 μg) was quantified via real-time PCR. YKL033W-A mRNA expression was normalized to ACT1 and shown as fold change relative to wild-type cells. Data are the average of three biological replicates, and error bars show standard error among biological replicates.