Research Article | Molecular Biology and Physiology

Gcn5p and Ubp8p Affect Protein Ubiquitylation and Cell Proliferation by Altering the Fermentative/Respiratory Flux Balance in Saccharomyces cerevisiae

Antonella De Palma, Giulia Fanelli, Elisabetta Cretella, Veronica De Luca, Raffaele Antonio Palladino, Valentina Panzeri, Valentina Roffia, Michele Saliola, Pierluigi Mauri, Patrizia Filetici
Sang Yup Lee, Editor
DOI: 10.1128/mBio.01504-20

Article Figures & Data

Figures

  • FIG 1
    FIG 1

    Expression and purification of His6-Ub proteins in S. cerevisiae. (A) Schematic protocol for the expression of His6Ub proteins in strains containing pDJ421 (41). Red oval, origin of replication; blue rectangle, pCUP promoter (LEU cassette). His6-Ub was expressed in CuSO4, purified through an Ni+ column, and analyzed by MS after trypsinization. (B) Western blot analysis showing the eluate of His6-Ub proteins with respect to the controls hybridized with anti-His6 antibody. Total lysates were probed with anti-Ada2p antibody as an internal standard. (C) Venn diagram of Ub protein distributions found in WT (blue), ubp8Δ (yellow), gcn5Δ (green), and ubp8Δ gcn5Δ (pink) strains. The areas of intersection contain proteins common to different strains. The 113 proteins absent in the WT are highlighted by a dashed circle.

  • FIG 2
    FIG 2

    Interactome networks built using STRING database through the mapping of the 103 differentially expressed Ub proteins identified comparing ubp8Δ, gcn5Δ, and ubp8Δ gcn5Δ strains versus the WT condition. The color code of distinct nodes represents the DAve value and the relevant chromatic scale (reported in the figure) and ranges from −2.00 to 0 (dark blue to white) and from 0 to +2.00 (white to dark red). Proteins with DAve (ratio of protein expression) ≥ l0.4l and a DCI (confidence of differential expression) ≥ l5l pass the filters and could be considered differentially expressed in the considered comparison. Positive DAve values indicate proteins down-expressed in mutant strains, while negative DAve values indicate proteins up-expressed in mutant strains. The differentially expressed proteins resulting from the three most interesting pairwise comparisons (WT/ubp8Δ, WT/gcn5Δ, and WT/ubp8Δ and gcn5Δ) have been plotted on a protein-protein interaction network built by means of STRING database (https://string-db.org) (16). Experimentally and computationally predicted interactions were considered for network construction, setting a confidence score of 0.4. Proteins are represented as colored nodes based on their DAve value and highlighted by gray edges; protein-protein interactions are clustered with respect to the functional pathway.

  • FIG 3
    FIG 3

    Major glycolytic enzymes are differentially ubiquitylated in the absence of Gcn5p and Ubp8p. (A) Glycolytic pathway in S. cerevisiae. Enzymes are indicated according to the ubiquitylation color code in Fig. 2B. The color code palette found in ubp8Δ, gcn5Δ, and ubp8Δ gcn5Δ strains is compared to WT for the indicated enzymes. (C) RT-qPCR of PFK1, PFK2, and PYC1 mRNA expression respect to actin in WT, ubp8Δ, and gcn5Δ strains. (D) Ubiquitylation pattern of Pfk1p the essential enzyme required for initiation of glycolytic flux. His6Ub-Pfk1pmyc version (∼120 kDa) was analyzed by Western blotting in the indicated strains hybridized with anti-myc and anti-tubulin (55 kDa) as internal loading control. The lower panel shows the eluted profiles of His6Ub-Pfk1pmyc in different strains.

  • FIG 4
    FIG 4

    Loss of Gcn5p and Ubp8p causes defects in glycolysis with poor growth in low sugar. (A) Serial dilutions (1:10) of the indicated strains grown for several days on medium containing high (2%) and low (0.1%) glucose show defects in the absence of Gcn5p, Ubp8p, and both not rescued by the addition of lactate and ethanol. (B) Growth of liquid cultures in 2% (black) and 0.1% glucose (white) of WT, ubp8Δ gcn5Δ, and ubp8Δ gcn5Δ strains. (C) Alcohol dehydrogenase activities of ADH1 and ADH2 stained by a native in-gel assay in the indicated strains grown in 2 and 0.2% glucose for 30, 60, and 90 h, respectively. G6PDHp staining shown as an internal standard. Schematics on the left show the compositions of Adh1p (gray) and Adh2p (orange) homo- and heterotetramers (44). (D) Glucose consumption (g) per 1 OD600 of cells of indicated strains.

  • FIG 5
    FIG 5

    Metabolic effects on glycolysis in the absence of Gcn5p and Ubp8p. The model shows enzymes whose ubiquitylation is altered in the absence of Gcn5p and Ubp8p and possible routes redirecting glycolytic flux toward fermentation, gluconeogenesis/anaplerotic routes, or a pentose phosphate shunt according to the altered redox balance of the mutants. The partial reoxidation of the cytosolic NADH by the mitochondrial transdehydrogenase Nde1p in gcn5Δ is deduced by the contemporary presence of Adh1p and Adh2p in high glucose medium.

Tables

  • TABLE 1

    Significant changes in S. cerevisiae Ub-proteome of the three mutant strains, as determined by proteomic analysisa

    TABLE 1

    • ↵a Differentially expressed proteins resulted from the MAProMa comparison of ubp8Δ, gcn5Δ, and ubp8Δ gcn5Δ strains versus the WT. In particular, each protein is marked by a color code, which is defined by the DAve value obtained in the three examined comparisons. The color is assigned according to a chromatic scale representing the confidence ranges of DAve values adopted (−2.00 to 0 [from blue to white] and +2.00 to 0 [from red to white]). Positive DAve values indicate proteins down-expressed in mutant strains, while negative DAve values indicate proteins up-expressed in mutant strains. The molecular function, gene name, known physical (P) and/or genetic (G) interactions with Ubp8p and/or Gcn5p, and the name of the human orthologues are indicated. Yeast genes corresponding to human pathologies are indicated in green. The complete list of the reported proteins was extracted from the differential lists shown in Table S2 in the supplemental material.

  • TABLE 2

    Saccharomyces cerevisiae strains derived from isogenic W303

    TABLE 2

Supplemental Material

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KEYWORDS

ubiquitylation
Gcn5p
Ubp8p
glycolytic flux
sugar utilization

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