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1.
Fig 1

Fig 1. From: Oxidative Stress Responses Involve Oxidation of a Conserved Ubiquitin Pathway Enzyme.

The ubiquitin pathway. The transfer of ubiquitin to the target protein requires the sequential formation of an E1∼ and an E2∼ubiquitin thioester involving the catalytic cysteine residue of each enzyme. With the subsequent function of a specific E3, ubiquitin is finally transferred to the target protein. In S. cerevisiae, a single E1, Uba1, transfers ubiquitin to proteins via 11 E2 enzymes and multiple E3 enzymes, which provide target specificity.

Kathryn S. Doris, et al. Mol Cell Biol. 2012 November;32(21):4472-4481.
2.
Fig 8

Fig 8. From: Oxidative Stress Responses Involve Oxidation of a Conserved Ubiquitin Pathway Enzyme.

Model for the regulation of cellular responses to oxidative stress by Cdc34 oxidation. In our model, certain oxidative stress conditions promote thiol oxidation of a specific ubiquitin pathway E2 enzyme, Cdc34, which inhibits ubiquitinylation of downstream substrates (Sub). This inhibition results in cellular responses to oxidative stress, including, for example, a delay in cell cycle progression. In the model, reduced glutathione is coupled to Uba1 and Cdc34 oxidation via a negative feedback loop that reverses their oxidation, restoring normal regulation of downstream substrates when redox conditions have improved.

Kathryn S. Doris, et al. Mol Cell Biol. 2012 November;32(21):4472-4481.
3.
Fig 4

Fig 4. From: Oxidative Stress Responses Involve Oxidation of a Conserved Ubiquitin Pathway Enzyme.

Uba1 forms a disulfide complex with Cdc34. (A to D) Western blot analyses revealed that Uba1 and Cdc34 are components of the HMW disulfide complex induced by H2O2 and diamide. Extracts were prepared from cells expressing either Cdc34-Myc (KD79), Uba1-HA (KD102), or both Cdc34-Myc and Uba1-HA (KD103) from their normal chromosomal locus as indicated; cells were either not treated (C and D) or treated with 5 mM diamide for 30 min (A) or 2 mM H2O2 for 20 min (B to D). (B) Immunoprecipitated proteins were analyzed. (C and D) Whole-cell lysate (WCL) and immunopreciptiated were analyzed. (A to D) Proteins were prepared as described in Materials and Methods and separated by nonreducing SDS-PAGE. Antibodies used for immunoprecipitation (IP) or Western blotting (WB) were either anti-Myc or anti-HA antibodies as indicated. Cdc34-Myc, Uba1-HA, and HMW complexes containing both Cdc34 and Uba1 are indicated (arrows). Molecular masses (in kilodaltons) are shown to the left of panels. (C and D) For comparison, WCL equivalent to 7% (C) and 12% (D) of the lysates used for IP were analyzed.

Kathryn S. Doris, et al. Mol Cell Biol. 2012 November;32(21):4472-4481.
4.
Fig 7

Fig 7. From: Oxidative Stress Responses Involve Oxidation of a Conserved Ubiquitin Pathway Enzyme.

Inhibition of Gsh1 by BSO stimulates formation of the Uba1-Cdc34 disulfide complex. Western blot analysis of extracts prepared under nonreducing conditions from cells (KD79) expressing Cdc34-Myc revealed that treatment with 5 mM buthionine sulfoximine (BSO), an inhibitor of gamma glutamylcysteine synthetase (Gsh1 in S. cerevisiae), for 0, 2 and 4 h stimulated the formation of the Cdc34-Myc- and Uba1-containing HMW disulfide complexes (lanes 1 to 6). Lanes 7 and 8 contain extracts from cells that were exposed to BSO for 4 h (equivalent to lanes 5 and 6), except that 1 mM reduced glutathione (+GSH) was added 2 h after the addition of BSO. Thus, comparison of lanes 5 and 7 indicates that reduced GSH inhibits the formation of the disulfide complex. Proteins were separated by SDS-PAGE (with or without β-mercaptoethanol [βm] treatment prior to loading) and analyzed with anti-Myc antibodies. Cdc34-Myc and the Cdc34-Myc- and Uba1-containing HMW disulfide complexes are indicated (arrows). Molecular masses (in kilodaltons) are shown to the left.

Kathryn S. Doris, et al. Mol Cell Biol. 2012 November;32(21):4472-4481.
5.
Fig 3

Fig 3. From: Oxidative Stress Responses Involve Oxidation of a Conserved Ubiquitin Pathway Enzyme.

Cdc34 is more sensitive than Ubc1 to oxidation by oxidative stress. (A to F) Western blot analyses demonstrated that Cdc34 is more sensitive to oxidation than another abundant E2, Ubc1, forming a predominant HMW disulfide complex (*) after H2O2 and diamide treatment. (A and B) Western blot analyses of extracts prepared from cells expressing either Cdc34-Myc (KD79) or Ubc1-Myc (KD130) from their normal chromosomal locus; cells were treated with the indicated concentrations of either diamide for 30 min (A) or H2O2 for 20 min (B). HMW Cdc34-containing complexes (*) and HMW Ubc1-containing complexes formed under these stress conditions (* and **) are indicated. (C to E) Western blot analyses of samples not treated or treated prior to loading with either β-mercaptoethanol (βm) or TCEP revealed that the stress-induced HMW form of Cdc34 (*) is a disulfide complex and identified the E2∼ubiquitin thioester forms of Cdc34 (Cdc34-MycUb) and Ubc1 (Ubc1-MycUb). Extracts were prepared from cells expressing Cdc34-Myc (KD79) that were either not treated or treated with 5 mM diamide for 30 min (C) or 2 mM H2O2 for 20 min (C and D) or from untreated cells expressing Ubc1-Myc (KD130) (E). (F) Western blot analysis indicated that Cdc34∼ubiquitin thioester forms start to decrease with a timing similar to that of the formation of the HMW Cdc34-containing disulfide complex (*). Extracts were prepared from cells expressing Cdc34-Myc (KD79) that were treated with 2 mM H2O2 for the indicated times. (A to F) All extracts were prepared under nonreducing conditions, and proteins were separated by SDS-PAGE and analyzed by Western blotting with anti-Myc antibodies. Molecular masses (in kilodaltons) are shown to the left of panels. (A, B, and F) Lighter exposures are shown (bottom panels) to demonstrate the effects of diamide and H2O2 on the E2∼ubiquitin thioester forms.

Kathryn S. Doris, et al. Mol Cell Biol. 2012 November;32(21):4472-4481.
6.
Fig 5

Fig 5. From: Oxidative Stress Responses Involve Oxidation of a Conserved Ubiquitin Pathway Enzyme.

The catalytic cysteine residue of Uba1 is essential for disulfide complex formation with Cdc34. (A to C) Western blot analyses revealed that plasmid-expressed wild-type Uba1-HA but not Uba1C600S-HA can compete with chromosomally expressed untagged Uba1 for HMW complex formation with chromosomally expressed Cdc34-Myc. (A) Western blot analysis of extracts from wild-type (W303-1a) cells containing either YEplac112 (vector), pUba1-3HA, or pUba1C600S-3HA that were treated with 5 mM diamide for 0 (−) or 30 (+) min revealed that mutation of the catalytic cysteine residue, Cys600, of Uba1 did not decrease protein stability. Extracts were prepared under nonreducing conditions, and proteins were separated by nonreducing SDS-PAGE and analyzed with either anti-HA or anti-Skn7 (loading control) antibodies. (B and C) Immunoprecipitation (IP) and Western blot (WB) analyses of proteins extracted from cells (KD79) expressing Cdc34-Myc and untagged Uba1 from their normal chromosomal locus and containing either pUba1-3HA or pUba1C600S-3HA; cells were treated with 2 mM H2O2 for 0 (−) or 20 (+) min, and the results demonstrate that the catalytic cysteine residue of Uba1 (Cys600) is essential for HMW complex formation with Cdc34. Whole-cell lysate (WCL) and immunoprecipitated proteins, prepared as described in Materials and Methods, were separated by nonreducing SDS-PAGE and analyzed by Western blotting. Extracts from cells (KD102) expressing Uba1-HA and untagged Cdc34 from their normal chromosomal locus that were treated with 2 mM H2O2 for 20 min were included as a negative control (lane 1). The antibodies used for IP or WB were either anti-Myc or anti-HA antibody as indicated. (B) Two nonspecific bands (*) present in all IP lanes, including lane 1, which contains no Myc epitope-tagged proteins, are indicated. (A to C) Cdc34-Myc, Uba1-HA, Skn7, and HMW complexes containing both Cdc34-Myc and Uba1-HA are indicated (arrows). For comparison, WCL equivalent to 11% of the lysates used for IP were analyzed. Molecular masses (in kilodaltons) are shown to the left of panels.

Kathryn S. Doris, et al. Mol Cell Biol. 2012 November;32(21):4472-4481.
7.
Fig 6

Fig 6. From: Oxidative Stress Responses Involve Oxidation of a Conserved Ubiquitin Pathway Enzyme.

Oxidation of Cdc34 is associated with delays of cell cycle progression and stabilization of the Cdc34 substrate Sic1. (A to D) Sic1 is required for the response of cells to diamide. (A) DNA content analyses of asynchronous cultures of SIC1 wild-type (W303-1a) and sic1Δ (KD6) cells not treated or treated with 5 mM diamide for 2 h revealed that Sic1 is required for the accumulation of cells in G1 phase in response to diamide. (B) Cells lacking Sic1 display increased sensitivity to diamide. Tenfold serial dilutions of SIC1 wild-type (wt) cells (W303-1a) and cells lacking Sic1 (sic1Δ) (KD6) were spotted onto either YPD medium or YPD medium containing 1.75 mM diamide. Plates were incubated at 30°C for 2 days. (C) The Hog1 pathway is not activated by diamide-induced oxidative stress. Western blot analysis of protein extracts prepared from wild-type (W303-1a) cells treated with either 0.4 M NaCl or 1 mM diamide for the indicated times revealed that, in contrast to NaCl treatment, diamide treatment does not stimulate phosphorylation of Hog1. Anti-phospho-p38 antibodies were used to detect phosphorylation of Hog1 (Hog1-P), and anti-Skn7 (Skn7) antibodies were used to confirm loading. (D) Sic1 is essential for delay in G1 phase in response to diamide. DNA content analyses of SIC1 wild-type (W303-1a) and sic1Δ (KD6) cells synchronized in G1 phase with α-factor and then released into medium with or without 2 mM diamide is shown. (E and F) Western blot and DNA content analyses of cells expressing Cdc34-Myc and Sic1-Myc from their normal chromosomal locus that were synchronized in G1 phase with α-factor and then released into medium either with or without 2 mM diamide (E) or with or without 0.5 mM H2O2 (F) revealed diamide- and H2O2-induced formation of the HMW Cdc34-Myc-containing complex, delay of destabilization of Sic1-Myc, and transient delay of cell cycle progression. Bands with the expected mobilities of Cdc34-Myc, Sic1-Myc, and the Cdc34-Myc- and Uba1-containing HMW disulfide complex are indicated (arrows). Anti-Hog1 (Hog1) and anti-Skn7 (Skn7) antibodies confirmed loading. DNA content analyses performed at the indicated times on the cells used in the Western blot analyses revealed a diamide- and H2O2-induced delay in cell cycle progression that corresponded with the formation of the HMW Cdc34-Myc-containing complex and the persistence of Sic1-Myc. (C, E, and F) Molecular masses (in kilodaltons) are shown to the left of panels.

Kathryn S. Doris, et al. Mol Cell Biol. 2012 November;32(21):4472-4481.
8.
Fig 2

Fig 2. From: Oxidative Stress Responses Involve Oxidation of a Conserved Ubiquitin Pathway Enzyme.

Uba1 and Cdc34 form HMW complexes in response to oxidative stress conditions which do not decrease global levels of protein ubiquitinylation. (A to C) Western blot analyses detected an HMW Uba1-containing disulfide complex (*) formed in response to diamide or H2O2. Extracts were prepared under nonreducing conditions from cells (KD102) expressing 3HA epitope-tagged Uba1 (Uba1-HA) from the normal chromosomal locus that were either untreated or treated with the indicated concentrations of H2O2 or diamide (A and B) or 5 mM diamide or 2 mM H2O2 (C). In the experiment whose results are shown in panel C, samples were prepared with or without β-mercaptoethanol treatment (βm) prior to loading. (D) Western blot analyses indicated that Cdc34 but not other abundant ubiquitin pathway E2 enzymes forms a predominant HMW complex (*) after diamide treatment. Extracts were prepared under nonreducing conditions from BY4741-derived cells expressing TAP-tagged Ubc1, Ubc2/Rad6, Ubc3/Cdc34, Ubc4, Ubc6, or Ubc13 from their normal chromosomal locus; the cells were untreated or treated with 5 mM diamide. The mobility of each individual TAP-tagged E2 enzyme is indicated by an arrow and the labeling at the top of each panel. (E to G) Western blot analyses revealed that the levels of global protein ubiquitinylation are not decreased by treatment of cells with diamide or H2O2. (E) Protein-ubiquitin conjugates in cells expressing HA epitope-tagged ubiquitin were detected as a large collection of HMW bands. Extracts were prepared from wild-type (W303-1a) cells containing either p416-MET25 (vector) or pHA-Ubiquitin, expressing ubiquitin tagged with 3HA epitopes. (F and G) Extracts were prepared from wild-type (W303-1a) cells containing pHA-Ubiquitin treated with the indicated concentrations of H2O2 (F) or diamide (G). (E to G) Protein-ubiquitin conjugates and a band of the approximate size of epitope-tagged ubiquitin (HA-Ubi) are indicated. An example of ubiquitinylated protein whose levels were observed to decrease following exposure to oxidative stress (a) is indicated. (A to D, F, and G) Cells were treated with either diamide for 30 min or H2O2 for 20 min. (A to G) Western blots were probed with anti-HA (A to C and E to G), anti-CBP (D), and anti-Skn7 (loading control) (F and G) antibodies. Molecular masses (in kilodaltons) are shown to the left of panels.

Kathryn S. Doris, et al. Mol Cell Biol. 2012 November;32(21):4472-4481.

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