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Science 325 (5942): 834-840

Copyright © 2009 by the American Association for the Advancement of Science

Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions

Chunaram Choudhary1,2, Chanchal Kumar1, Florian Gnad1, Michael L. Nielsen1,2, Michael Rehman3, Tobias C. Walther3, Jesper V. Olsen1,2, and Matthias Mann1,2,*

1 Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Martinsried, Germany.
2 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark.
3 Organelle Architecture and Dynamics, Max Planck Institute for Biochemistry, 82152 Martinsried, Germany.


Figure 1
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Fig. 1. Overview of in vivo acetylome analysis. (A) Independent validation of lysine acetylation of proteins. (B) Overlap of acetylated proteins and sites in three different cell lines. Ten different proteins from the acetylome data set were immunoprecipitated from GFP-tagged BAC transgenic cell lines and stained with antibody to acetyl-lysine. The bands marked with an asterisk indicate acetylated proteins.

 

Figure 2
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Fig. 2. Properties of acetylated proteins and sites. (A) Cellular distribution of acetylated proteins and sites. Proteins were assigned based on exclusive Gene Ontology (GO) annotations. (B) Distribution of all lysines and acetylated lysines in structured and unstructured regions of the proteins. (C) Sequence logo plots represent normalized amino acid frequencies for ±6 amino acids from the lysine acetylation site. (D) Domain architecture of acetylated proteins. The green bars indicate Pfam protein families and domains that are significantly overrepresented, and the red bars indicate underrepresented domains in the acetylome as compared with those in the entire proteome. The light green and orange striped bars represent cytoplasmic domains.

 

Figure 3
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Fig. 3. Acetylation-modulated functional networks. (A to G) Interaction networks of acetylated proteins in different cellular functions from STRING analysis of the acetylome. Individual networks were generated for each specific functional category (table S1). Gray nodes indicate proteins previously reported to be acetylated.

 

Figure 4
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Fig. 4. Acetylation of Cdc28 and 14-3-3 impair their functions. (A) Mutation of acetylation site K40 on Cdc28 impairs its function. Growth of haploid yeast strains harboring empty vector or plasmids expressing CDC28, cdc28-K40R, or cdc28-K40Q in a cdc28{Delta} strain were tested for growth on 5-FOA plates, which select against the wild-type copy of CDC28 on a separate URA3-marked plasmid. SC-Trp plates are shown as control. (B) Mutation of acetylated lysine on 14-3-3-{varepsilon} abolishes binding to RAF1 and KIF1c phosphopeptide ligands. Binding of recombinant GST–14-3-3 proteins to phosphopeptides was analyzed by means of peptide pull-down assays. (C to E) Mutation of K50Q and K118+K123Q impairs binding of 14-3-3 to full-length proteins. Proteins interacting with 14-3-3 wild type (WT) or mutants were identified and quantified by use of SILAC-based MS as described in fig. S9. Proteins in red are quantified in all six experiments, and proteins that were quantified in four or more experiments are blue. The x axis shows the relative binding of 14-3-3–WT compared with the indicated 14-3-3 mutants (log2 SILAC ratio for WT/mutant). The y axis shows the relative binding of the indicated 14-3-3 mutants compared with 14-3-3–WT (log2 SILAC ratio for mutant/WT).

 


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