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. 2004 Jul 30;279(31):32262-8.
doi: 10.1074/jbc.M404173200. Epub 2004 May 27.

Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses

Weidong Zhou  1 , Jennifer J Ryan, Huilin Zhou
Affiliations

Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses

Weidong Zhou et al. J Biol Chem. .

Abstract

We have undertaken a global analysis of sumoylated proteins in Saccharomyces cerevisiae by tandem mass spectrometry. Exposure of cells to oxidative and ethanol stresses caused large increases in protein sumoylation. A large number of new sumoylated proteins were identified in untreated, hydrogen peroxide-treated, and ethanol-treated cells. These proteins are known to be involved in diverse cellular processes, including gene transcription, protein translation, DNA replication, chromosome segregation, metabolic processes, and stress responses. Additionally, the known enzymes, including E1, E2, and E3 of the sumoylation cascade were found to be auto-sumoylated. Taken together, these results show that protein sumoylation is broadly involved in many cellular functions and this mass spectrometry-based proteomic approach is useful in studying the regulation of protein sumoylation in the cells.

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Figures

Fig. 1
Fig. 1. Sumoylation pattern in the HZY1017 yeast strain
HZY1017 cells were grown to log phase in YPD media. 15 μg of whole cell lysate was separated by 4 –12% SDS-PAGE and detected with anti-FLAG antibody. Monomer Smt3 is indicated.
Fig. 2
Fig. 2. Silver staining and Western blot of the purified sumoylated proteins from HZY1017 cells
A, silver staining of the purified proteins. The sumoylated proteins were purified by tandem affinity purification as described in the text. The majority of the sample (90%) was visualized by silver staining. B, Western blot of the purified proteins. The rest of the sample (10%) was detected by anti-FLAG antibody. Anti-mouse secondary antibody-alkaline phosphatase conjugate was used, and the membrane was developed in 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. The arrows indicate the positions of the thirteen positive bands, which were subjected to in-gel digestion and mass spectrometric analysis.
Fig. 3
Fig. 3. Confirmation of sumoylation of Elp5 and Sod1
A, Western blot of the purified Elp5 by anti-FLAG antibody. B, Western blot of the purified Elp5 by anti-HA antibody. C, Western blot of the purified Sod1 by anti-FLAG antibody. D, Western blot of the purified Sod1 by anti-HA antibody. C-terminal tagged Elp5 and Sod1 were purified as described in the text.
Fig. 4
Fig. 4. Purification of the sumoylated Sod1
A, silver staining of the sumoylated Sod1. B, Western blot of sumoylated Sod1 by anti-HA antibody (see text for details).
Fig. 5
Fig. 5. Sumoylation patterns in HZY1017 cells under various stress conditions
HZY1017 cells were grown to the log phase in YPD media and treated with either 1 mM H2O2 or 10% ethanol for 1 h before harvesting. 15 μg of each whole cell lysate from H2O2 treatment (lane 1), ethanol treatment (lane 2), and without treatment (lane 3) was analyzed by 4 –12% SDS-PAGE and immunoblotting with anti-FLAG antibody. Monomer Smt3 is indicated.
Fig. 6
Fig. 6. Silver staining of the purified sumoylated proteins from HZY1017 cells following hydrogen peroxide and ethanol treatments
Twenty equal segments were sliced from the gel and subjected to in-gel digestion and mass spectrometry analysis. The arrows indicate the positions of the excised gel slices.
Fig. 7
Fig. 7. Western blot analysis of the purified Pol30
6HisFLAG-tagged Pol30 was purified from various conditions: without stress treatment (lane 1), with 1 mM H2O2 (lane 2), and 10% ethanol (lane 3), then analyzed by SDS-PAGE and detected with anti-HA antibody.
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References

    1. Hochstrasser M. Annu Rev Genet. 1996;30:405–439. - PubMed
    1. Pickart CM. Annu Rev Biochem. 2001;70:503–533. - PubMed
    1. Weissman AM. Nat Rev Mol Cell Biol. 2001;2:169–178. - PubMed
    1. Melchior F. Annu Rev Cell Dev Biol. 2000;16:591–626. - PubMed
    1. Schwartz DC, Hochstrasser M. Trends Biochem Sci. 2003;28:321–328. - PubMed

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