www.sciencesignaling.org/cgi/content/full/4/183/ra48/DC1 Supplementary Materials for Proteome-Wide Mapping of the Drosophila Acetylome Demonstrates a High Degree of Conservation of Lysine Acetylation Brian T. Weinert, Sebastian A. Wagner, Heiko Horn, Peter Henriksen, Wenshe R. Liu, Jesper V. Olsen, Lars J. Jensen, Chunaram Choudhary* *To whom correspondence should be addressed. E-mail: [email protected]Published 26 July 2011, Sci. Signal. 4, ra48 (2011) DOI: 10.1126/scisignal.2001902 This PDF file includes: 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 Lys 9 acetylation in S. cerevisiae. Fig. S6. Identification of in vivo UBE2D3 Lys 8 acetylation in human cells. Fig. S7. Confirmation of Lys 8 acetylation in recombinant UBE2D3 purified from E. coli. Fig. S8. Effect of UBE2D3 Lys 8 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 Other Supplementary Material for this manuscript includes the following: (available at www.sciencesignaling.org/cgi/content/full/4/183/ra48/DC1) Table S1 (Microsoft Excel format). List of Drosophila in vivo acetylation sites. Table S2 (Microsoft Excel format). Drosophila acetylated lysine conservation. Table S3 (Microsoft Excel format). Human acetylated lysine conservation.
15
Embed
Supplementary Materials for - Science Signalingstke.sciencemag.org/content/sigtrans/suppl/2011/07/22/4.183.ra48.DC1/4...S/T pS/pT Dm compared to Hs S/T pS/pT Hs compared to Dm Frequency
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Proteome-Wide Mapping of the Drosophila Acetylome Demonstrates a High Degree of Conservation of Lysine Acetylation
Brian T. Weinert, Sebastian A. Wagner, Heiko Horn, Peter Henriksen, Wenshe R. Liu,
Jesper V. Olsen, Lars J. Jensen, Chunaram Choudhary*
*To whom correspondence should be addressed. E-mail: [email protected]
Published 26 July 2011, Sci. Signal. 4, ra48 (2011) DOI: 10.1126/scisignal.2001902
This PDF file includes:
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
Other Supplementary Material for this manuscript includes the following: (available at www.sciencesignaling.org/cgi/content/full/4/183/ra48/DC1)
Table S1 (Microsoft Excel format). List of Drosophila in vivo acetylation sites. Table S2 (Microsoft Excel format). Drosophila acetylated lysine conservation. Table S3 (Microsoft Excel format). Human acetylated lysine conservation.
cytoskeleton organizationgeneration of precursor metabolites and energy
mitochondrionnucleotide binding
nucleusoxidation reduction
ribonucleoprotein complexribosome
RNA bindingspindle organization
transcriptiontranslation
# of genes % of total fold enrichment p-valueDm Hs Dm Hs Dm Hs Dm Hs
Fig. S1. Functional annotation of Drosophila and human acetylomes. Comparison of Gene Ontology (GO) term “Biological Process” enrichment for Drosophila (Dm) and human (Hs) genes encoding acetylated proteins. The number of genes enriched for each category (# of genes), the percentage of genes encoding acetylated proteins for each category (% of total), the fold enrichment of the genes relative to the genomic distribution (fold enrichment), and the significance (P value) of the enrichment are shown.
pS/pTS/T
Dm compared to Hs
pS/pTS/T
Hs compared to Dm
Freq
uenc
y of
am
ino
acid
con
serv
atio
n
0%
10%
20%
30%
40%
50%
60%
70%
p = 0.89 p = 0.12
Figure S2
Fig. S2. Conservation of serine and threonine phosphorylation sites to either serine or threonine. The frequency of serine and threonine (S/T) conservation to either serine or threonine is compared to the frequency of phosphoserine and phosphothreonine (pS/pT) conservation to either serine or threonine. Drosophila (Dm) sites are compared to human (Hs) orthologs and human sites are compared to Drosophila orthologs.
UBE2D2/eff Human-K8, K144 Fly-K8
UBE2D3/eff Human-K8, K144 Fly-K8
UBE2L3 Human-K9, K189, K196
UBE2K/UbcD4 Human-K14 Fly-K14
UBE2N/Ubc-E2H Human-K10, K82, K92, K94 Fly-K92
UBE2H Human-K60, K64
UBE2I/lwr (SUMO ligase) Human-K65 Fly-K65
UFC1/CG8386 (UFM1 ligase) Human-K122 Fly-K122
Human acetylationConserved acetylation
Figure S3
Fig. S3. Summary of acetylation sites identified in E2 conjugating enzymes. Acetylation sites identified only in humans (red) or conserved sites (green) are shown.
Fig. S5. Identification of UBC4 Lys9 acetylation in S. cerevisiae. MS2 spectra showing identification of acetylated lysine at Lys9 in S. cerevisiae UBC4.
Fig. S6. Identification of in vivo UBE2D3 Lys8 acetylation in human cells. MS2 spectra showing acetylation at Lys8 of UBE2D3 identified in peptides prepared from U2OS cells.
Fig. S7. Confirmation of Lys8 acetylation in recombinant UBE2D3 purified from E. coli. (A) MS2 spectra showing identification of acetylated lysine at Lys8 in human UBE2D3 purified from E. coli, which was used in the in vitro ubiquitylation assay shown in Fig. 6D. (B) Recombinant UBE2D3 purified from E. coli was immunoblotted with antibodies recognizing UBE2D3 or acetyllysine.
Figure S8
Time (min.)0 05 5U
BE
2D3
UB
E2D
3 K
8Ac
UB
E2D
3 K
8Ac
UB
E2D
3
UB
E2D
3
UB
E2D
3 K
8Ac
UB
E2D
3 K
8Ac
UB
E2D
3
0 05 5
- DTT + DTT
UBE2D3
UBE2D3-ubiquitin
Fig. S8. Effect of UBE2D3 Lys8 acetylation on ubiquitin thiolester formation. Ubiquitin thiolester formation was assayed by incubating purified recombinant UBE2D3 with E1 and ubiquitin for the indicated time. Ubiquitin thiolester formation results in a UBE2D3 mobility shift that was detected by immunoblot for UBE2D3. Treatment with the reducing agent dithiothreitol (DTT) demonstrated that the mobility shift is due to the formation of thiolester linkage with ubiquitin and is not due to auto-ubiquitylation of lysine residues in UBE2D3.
30 deg SC -URA
100µM Hygromycin B
15% ethanol
0.5µM cyclohexamide
1µM cycloheximide
empty vector
UBC4
UBC4 K9Q
empty vector
UBC4
UBC4 K9Q
empty vector
UBC4
UBC4 K9Q
empty vector
UBC4
UBC4 K9Q
empty vector
UBC4
UBC4 K9Q
Figure S9
Fig. S9. Rescue of growth sensitivity in S. cerevisiae ubc4 mutant cells. Rescue of growth sensitivity is compared for wild-type UBC4, UBC4 K9Q, and empty vector control. Growth on control media (SC -URA) is compared with growth on selective media containing hygromycin B, ethanol, or two different concentrations of cycloheximide. The mutant UBC4 K9Q showed impaired rescue in the presence of higher (1 µM) concentrations of cycloheximide. The black wedge indicates 5-fold serial dilution of the indicated yeast strains onto the selective media. The experiment was repeated 3 times.
Definitions of the olumns for Table S1 S3 c s to
Table S1. List of Drosophila in vivo acetylation sites
Proteins: Fly base protein identifiers that match peptide sequence
Protein: Single protein identifier
Position: Position of acetylated lysine in the single protein identifier
Protein Names: Protein names
Gene Names: Gene names
Protein Descriptions: Fly base protein descriptions
Uniprot: Uniprot protein identifiers
ENSEMBL: ENSEMBL protein identifiers
Number of Acetyl (K): Number of acetylated lysines on the peptide