Multiplexed, Proteome-Wide Protein Expression Profiling: Yeast Deubiquitylating Enzyme Knockout Strains

Abstract

Characterizing a protein's function often requires a description of the cellular state in its absence. Multiplexing in mass spectrometry-based proteomics has now achieved the ability to globally measure protein expression levels in yeast from 10 cell states simultaneously. We applied this approach to quantify expression differences in wild type and nine deubiquitylating enzyme (DUB) knockout strains with the goal of creating "information networks" that might provide deeper, mechanistic insights into a protein's biological role. In total, more than 3700 proteins were quantified with high reproducibility across three biological replicates (30 samples in all). DUB mutants demonstrated different proteomics profiles, consistent with distinct roles for each family member. These included differences in total ubiquitin levels and specific chain linkages. Moreover, specific expression changes suggested novel functions for several DUB family members. For instance, the ubp3Δ mutant showed large expression changes for members of the cytochrome C oxidase complex, consistent with a role for Ubp3 in mitochondrial regulation. Several DUBs also showed broad expression changes for phosphate transporters as well as other components of the inorganic phosphate signaling pathway, suggesting a role for these DUBs in regulating phosphate metabolism. These data highlight the potential of multiplexed proteome-wide analyses for biological investigation and provide a framework for further study of the DUB family. Our methods are readily applicable to the entire collection of yeast deletion mutants and may help facilitate systematic analysis of yeast and other organisms.

Keywords: COX complex; Orbitrap Fusion; TMT; UBP3; deubiquitinases; high-throughput proteomics; inorganic phosphate pathway; isobaric labeling; quantitative proteomics; ubiquitin.

Figure 1

Proteomic analysis of nine yeast deletion strains in triplicate. (A) Workflow overview of the MS protein analysis of nine DUB deletions and wild type strains grown in biological triplicates. (B) Growth ability in YPD of the nine DUBs deletions compared to a wild type strain. Cells (3 × 10⁴) were spotted in the first raw and 1/3 serial dilutions were made for successive dilutions. (C) Table summarizing peptide and protein quantification for the three biological replicates. Proteins were collapsed to a final protein-level FDR < 1%. (D) Venn diagram representing quantified proteins for each experiment. A total of 3715 proteins were quantified in triplicate. (E) Examples illustrating the expected loss of deleted proteins. Data shown are means with error bars as one standard deviation (n = 3).

Figure 2

Effect of nine individual DUB deletions on the total ubiquitin pool. (A) Sequence of the 76-amino-acid ubiquitin polypeptide encoded in Saccharomyces cerevisiae. Conjugation of multiple ubiquitin moieties can take place at seven different lysine residues (Lys, Lys, Lys, Lys, Lys, Lys, and Lys⁶³) as well as the methionine at the N-termini (all highlighted in red). (B) Relative abundance of the ubiquitin-ribosomal 60S subunit L40A fusion protein in all ten samples and the standard deviation of their mean. (C) Whole cell lysates of WT and nine DUBs deletion strains were immunobloted against ubiquitin. Equal loading was confirmed by stripping the immunoblot and reprobing for actin. (D) Lys and Lys⁶³-ubiquitin linkages were quantified in triplicate across all samples and normalized against total ubiquitin pools.

Figure 3

Overview of protein expression differences in the yeast strains. (A) Heatmap of the 10 DUB deletion proteomes in triplicate. Protein abundance is shown as the Log2 ratio between each sample with respect to the average of reference sample (WT). (B) Principal component analysis of the relative protein abundances of all 30 values. Total explained variance of each principal component is shown in parentheses. (C) Number of significantly changing proteins in each deletion strain compared to wild type levels (adjusted p-value <0.01 and fold-change >1.5).

Figure 4

Effect of individual DUBs deletions on protein abundances. Proteins with statistically significant differences in abundance (adjusted p-value <0.01) are represented for each DUB deletion strain. Genes are annotated in different colors based on their membership in the Biological Processes category in Gene Ontology. Genes that changed significantly in more than one DUB KO are labeled with an asterisk.

Figure 5

UBP3 affects protein expression of many members of the COX complex. (A) Heatmap of all quantified COX subunits (bold) and their assembly factors. (B) Relative abundance of all COX subunits (mean ± SD). (C) Expression of COX subunits was decreased to wild type levels upon Ubp3 overexpression (Ubp3 OE). (D) Ability of wild type and nine DUB deletions to rescue growth defects conferred by respiratory impairment (YPGlycerol -3%). Cells (3 × 10⁴) were spotted in the first raw, and 1/2 serial dilutions were made for successive columns.

Figure 6

DUB deletion affects the inorganic phosphate pathway. (A) Relative changes in expression for some components of the PHO pathway (mean ± SD). (B) Heatmap showing protein changes in the PHO pathway comparing triplicates of wild type, ubp3Δ, and Ubp3 overexpression (Ubp3 OE). Changes are expressed as Log2 ratios to mean of wild type levels. (C) C-termini tagged Pho5-FLAG (left) and Pho84-FLAG (right) expression in different mutants grown in YPD were monitored by immunoblot analysis. Actin was used as loading control. (D) Amount of Pho5-FLAG (top) and Pho84-FLAG (bottom) in the presence and absence of inorganic phosphate was examined by immunoblotting in the indicated strains. For equal loading of the samples, membranes were stripped and reprobed with actin antibody. (E) Cellular localization of Pho4 fused to GFP was monitored by immunofluorescence in WT, ubp3Δ, and Ubp3 OE cells. Controls as described by Carroll et al. were used as a benchmark (top). Pearson correlation of the colocalization signal of at least 100 cells per condition is shown in a box plot (bottom).