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Research Article
Posttranslational Modification

Proteome-Wide Mapping of the Drosophila Acetylome Demonstrates a High Degree of Conservation of Lysine Acetylation

Science Signaling
26 Jul 2011
Vol 4, Issue 183
p. ra48

Age of the Acetylome

Acetylation and phosphorylation are regulatory posttranslational modifications that occur on proteins. With proteome-wide data in divergent species, insights regarding the evolution of these two regulatory processes can be revealed. Weinert et al. report the proteome-wide analysis of acetylated proteins in the fruit fly. Comparing the data on acetylated proteins in humans and flies with proteome sequences of nematodes and zebrafish indicated that acetylated sites were more conserved than were nonacetylated sites, and comparison of the human and fly acetylomes with their phosphoproteomes indicated that acetylation sites were more conserved than were phosphorylation sites. Acetylation intersected with another posttranslational modification, ubiquitylation: Acetylation occurred on one-third of human ubiquitin-conjugating E2 enzymes and influenced the activity of these enzymes, suggesting that acetylation provides another regulatory layer for this other type of posttranslational modification.

Abstract

Posttranslational modification of proteins by acetylation and phosphorylation regulates most cellular processes in living organisms. Surprisingly, the evolutionary conservation of phosphorylated serine and threonine residues is only marginally higher than that of unmodified serines and threonines. With high-resolution mass spectrometry, we identified 1981 lysine acetylation sites in the proteome of Drosophila melanogaster. We used data sets of experimentally identified acetylation and phosphorylation sites in Drosophila and humans to analyze the evolutionary conservation of these modification sites between flies and humans. Site-level conservation analysis revealed that acetylation sites are highly conserved, significantly more so than phosphorylation sites. Furthermore, comparison of lysine conservation in Drosophila and humans with that in nematodes and zebrafish revealed that acetylated lysines were significantly more conserved than were nonacetylated lysines. Bioinformatics analysis using Gene Ontology terms suggested that the proteins with conserved acetylation control cellular processes such as protein translation, protein folding, DNA packaging, and mitochondrial metabolism. We found that acetylation of ubiquitin-conjugating E2 enzymes was evolutionarily conserved, and mutation of a conserved acetylation site impaired the function of the human E2 enzyme UBE2D3. This systems-level analysis of comparative posttranslational modification showed that acetylation is an anciently conserved modification and suggests that phosphorylation sites may have evolved faster than acetylation sites.

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Supplementary Material

Summary

Fig. S1. Functional annotation of Drosophila and human acetylomes.
Fig. S2. Conservation of serine and threonine phosphorylation sites to either serine or threonine.
Fig. S3. Summary of acetylation sites identified in E2-conjugating enzymes.
Fig. S4. Sequence alignment of human E2 ubiquitin-conjugating enzymes.
Fig. S5. Identification of UBC4 Lys9 acetylation in S. cerevisiae.
Fig. S6. Identification of in vivo UBE2D3 Lys8 acetylation in human cells.
Fig. S7. Confirmation of Lys8 acetylation in recombinant UBE2D3 purified from E. coli.
Fig. S8. Effect of UBE2D3 Lys8 acetylation on ubiquitin thiolester formation.
Fig. S9. Rescue of growth sensitivity in S. cerevisiae ubc4 mutant cells.
Definitions of the columns for Tables S1 to S3
Details regarding data availability
Table S1. List of Drosophila in vivo acetylation sites.
Table S2. Drosophila acetylated lysine conservation.
Table S3. Human acetylated lysine conservation.

Resources

File (4_ra48_sm.pdf)
File (4_ra48_tables_s1_to_s3.zip)

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Published In

Science Signaling
Volume 4 | Issue 183
July 2011

Submission history

Received: 9 February 2011
Accepted: 7 July 2011

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Acknowledgments

Acknowledgments: We thank the members of the Departments of Proteomics and Disease Systems Biology at the Center for Protein Research (CPR) for their helpful discussions and the Protein Science and Technology unit at the CPR for providing recombinant RNF8. Funding: The CPR is funded by a grant from the Novo Nordisk Foundation. This work was funded by the European Commission’s 7th Framework Programme grants Proteomics Research Infrastructure Maximizing knowledge EXchange and access (XS) (INFRASTRUCTURES-F7-2010-262067/PRIME-XS) and by the Lundbeck Foundation (R48-A4649). S.A.W. is supported by a postdoctoral grant from the Danish Council for Independent Research (FSS: 10-083519). Author contributions: B.T.W. and C.C. conceived the project; B.T.W., S.A.W., and P.H. performed the experiments and analyzed the data; H.H. performed PTM conservation analysis; W.R.L. provided the plasmid vectors for expression of recombinant acetylated UBE2D3; J.V.O. provided the idea of comparing peptide length distributions; L.J.J. supervised the PTM site conservation analysis; and C.C. supervised the entire project. B.T.W. and C.C. wrote the manuscript. S.A.W., H.H., P.H., J.V.O., and L.J.J. read and commented on the manuscript. Competing interests: L.J.J. is a cofounder and scientific adviser of Intomics A/S; however, this company was not involved in this study. The other authors declare that they have no competing interests. Data availability: The data associated with this manuscript may be downloaded from ProteomeCommons.org (see Supplementary Materials for hashes).

Authors

Affiliations

Brian T. Weinert
Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.
Sebastian A. Wagner
Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.
Heiko Horn
Department of Disease Systems Biology, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Peter Henriksen
Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.
Wenshe R. Liu
Department of Chemistry, Texas A&M University, College Station, TX 77843, USA.
Jesper V. Olsen
Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.
Lars J. Jensen
Department of Disease Systems Biology, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Chunaram Choudhary* [email protected]
Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.

Notes

*
To whom correspondence should be addressed: E-mail: [email protected]

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