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: chuna.choudhary@cpr.ku.dk

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.

Figure S1

134 170 12.8 9.0 2.2 1.9 2.63E-20 2.16E-1680 160 7.6 8.5 2.1 3.1 8.23E-12 2.07E-42121 95 11.5 5.0 2.7 1.9 2.79E-25 2.20E-0954 82 5.2 4.3 2.5 2.2 1.48E-10 2.43E-12116 277 11.1 14.7 1.7 2.3 5.35E-09 4.38E-44222 495 21.2 26.2 1.8 1.8 2.35E-20 9.93E-49175 711 16.7 37.7 2.6 1.7 7.38E-33 1.02E-6091 115 8.7 6.1 1.5 1.5 5.35E-05 2.28E-06116 201 11.1 10.7 2.5 3.5 3.47E-23 2.91E-6377 90 7.3 4.8 3.4 3.8 3.54E-24 1.12E-30103 254 9.8 13.5 1.6 2.9 3.71E-07 4.00E-6284 0 8.0 0.0 3.8 0.0 4.79E-29 N.D.69 0 6.6 0.0 1.5 0.0 2.54E-04 N.D.118 150 11.3 7.9 2.3 3.9 2.33E-19 1.33E-53

cell cyclechromosome

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.

-

UBE2D2 ------------------------------------------------------------

UBE2D3 ------------------------------------------------------------

UBE2D4 ------------------------------------------------------------

UBE2D1 ------------------------------------------------------------

UBE2E2 MSTEAQRVDD-SPSTSGGSSDGDQRESVQQEPE-REQVQP----KKKEGKIS-SKTAAKL 53

UBE2E3 MSSDRQRSDDESPSTSSGSSDADQRDPAAPEPEEQEERKPSATQQKKNTKLS-SKTTAKL 59

UBE2E1 MSDDDSRAST---SSSSSSSSNQQTEKETNTPK------------KKESKVSMSKNSKLL 45

UBE2W -----------------------------------------------------------M 1

UBE2K -------------------------------------------------------MANIA 5

UBE2N ----------------------------------------------------------MA 2

UBE2L3 ------------------------------------------------------------

UBE2L6 ------------------------------------------------------------

UBE2J1 ----------------------------------------------------METRYNLK 8

UBE2D2 -MALKRIHKELNDLARDPPAQ---CSAGPV--GDDMFHWQATIMGPNDSPYQGGVFFLTI 54

UBE2D3 -MALKRINKELSDLARDPPAQ---CSAGPV--GDDMFHWQATIMGPNDSPYQGGVFFLTI 54

UBE2D4 -MALKRIQKELTDLQRDPPAQ---CSAGPV--GDDLFHWQATIMGPNDSPYQGGVFFLTI 54

UBE2D1 -MALKRIQKELSDLQRDPPAH---CSAGPV--GDDLFHWQATIMGPPDSAYQGGVFFLTV 54

UBE2E2 STSAKRIQKELAEITLDPPPN---CSAGPK--GDNIYEWRSTILGPPGSVYEGGVFFLDI 108

UBE2E3 STSAKRIQKELAEITLDPPPN---CSAGPK--GDNIYEWRSTILGPPGSVYEGGVFFLDI 114

UBE2E1 STSAKRIQKELADITLDPPPN---CSAGPK--GDNIYEWRSTILGPPGSVYEGGVFFLDI 100

UBE2W ASMQKRLQKELLALQNDPPPG---MTLNEKSVQNSITQWIVDMEGAPGTLYEGEKFQLLF 58

UBE2K VQRIKREFKEVLKSEETSKNQ---IKVDLV-DE-NFTELRGEIAGPPDTPYEGGRYQLEI 60

UBE2N GLP-RRIIKETQRLLAEPVPG---IKAEPD--ESNARYFHVVIAGPQDSPFEGGTFKLEL 56

UBE2L3 MAASRRLMKELEEIRKCGMKN----FRNIQVDEANLLTWQGLIV-PDNPPYDKGAFRIEI 55

UBE2L6 MMASMRVVKELEDLQKKPPPY----LRNLSSDDANVLVWHALLL-PDQPPYHLKAFNLRI 55

UBE2J1 SPAVKRLMKEAAELKDPTDHY------HAQPLEDNLFEWHFTVRGPPDSDFDGGVYHGRI 62

:*: ** : . : . . :. : .

UBE2D2 HFPTDYPFKPPKVAFTTR--IYHPNINSN-GSICLDILR---SQWSPALTISKVLLSICS 108

UBE2D3 HFPTDYPFKPPKVAFTTR--IYHPNINSN-GSICLDILR---SQWSPALTISKVLLSICS 108

UBE2D4 HFPTDYPFKPPKVAFTTK--IYHPNINSN-GSICLDILR---SQWSPALTVSKVLLSICS 108

UBE2D1 HFPTDYPFKPPKIAFTTK--IYHPNINSN-GSICLDILR---SQWSPALTVSKVLLSICS 108

UBE2E2 TFSPDYPFKPPKVTFRTR--IYHCNINSQ-GVICLDILK---DNWSPALTISKVLLSICS 162

UBE2E3 TFSSDYPFKPPKVTFRTR--IYHCNINSQ-GVICLDILK---DNWSPALTISKVLLSICS 168

UBE2E1 TFTPEYPFKPPKVTFRTR--IYHCNINSQ-GVICLDILK---DNWSPALTISKVLLSICS 154

UBE2W KFSSRYPFDSPQVMFTGENIPVHPHVYSN-GHICLSILT---EDWSPALSVQSVCLSIIS 114

UBE2K KIPETYPFNPPKVRFITK--IWHPNISSVTGAICLDILK---DQWAAAMTLRTVLLSLQA 115

UBE2N FLPEEYPMAAPKVRFMTK--IYHPNVDKL-GRICLDILK---DKWSPALQIRTVLLSIQA 110

UBE2L3 NFPAEYPFKPPKITFKTK--IYHPNIDEK-GQVCLPVISA--ENWKPATKTDQVIQSLIA 110

UBE2L6 SFPPEYPFKPPMIKFTTK--IYHPNVDEN-GQICLPIISS--ENWKPCTKTCQVLEALNV 110

UBE2J1 VLPPEYPMKPPSIILLTA----NGRFEVG-KKICLSISGHHPETWQPSWSIRTALLAIIG 117

:. **: .* : : : .. :** : . * .. . ::

UBE2D2 LLC------------DPNPDDPLVPEIARIYKT-----------------DREKYNRIAR 139

UBE2D3 LLC------------DPNPDDPLVPEIARIYKT-----------------DRDKYNRISR 139

UBE2D4 LLC------------DPNPDDPLVPEIAHTYKA-----------------DREKYNRLAR 139

UBE2D1 LLC------------DPNPDDPLVPDIAQIYKS-----------------DKEKYNRHAR 139

UBE2E2 LLT------------DCNPADPLVGSIATQYMT-----------------NRAEHDRMAR 193

UBE2E3 LLT------------DCNPADPLVGSIATQYLT-----------------NRAEHDRIAR 199

UBE2E1 LLT------------DCNPADPLVGSIATQYMT-----------------NRAEHDRMAR 185

UBE2W MLS------------SCK--EKRRPPDNSFYVR-----------------TCNKNPKKTK 143

UBE2K LLA------------AAEPDDPQDAVVANQYKQ-----------------NPEMFKQTAR 146

UBE2N LLS------------APNPDDPLANDVAEQWKT-----------------NEAQAIETAR 141

UBE2L3 LVN------------DPQPEHPLRADLAEEYSK-----------------DRKKFCKNAE 141

UBE2L6 LVN------------RPNIREPLRMDLADLLTQ-----------------NPELFRKNAE 141

UBE2J1 FMPTKGEGAIGSLDYTPEERRALAKKSQDFCCEGCGSAMKDVLLPLKSGSDSSQADQEAK 177

:: : . :.

UBE2D2 EWTQKYAM---------------------------------------------------- 147

UBE2D3 EWTQKYAM---------------------------------------------------- 147

UBE2D4 EWTQKYAM---------------------------------------------------- 147

UBE2D1 EWTQKYAM---------------------------------------------------- 147

UBE2E2 QWTKRYAT---------------------------------------------------- 201

UBE2E3 QWTKRYAT---------------------------------------------------- 207

UBE2E1 QWTKRYAT---------------------------------------------------- 193

UBE2W WWYHDDTC---------------------------------------------------- 151

UBE2K LWAHVYAGAPVSSPEYTKKIENLCAMGFDRNAVIVALSSKSWDVETATELLLSN------ 200

UBE2N AWTRLYA------------MNNI------------------------------------- 152

UBE2L3 EFTKKYGEKRPVD----------------------------------------------- 154

UBE2L6 EFTLRFGVDRPS------------------------------------------------ 153

UBE2J1 ELARQISFKAEVNSSGKTISESDLNHSFSLTDLQDDIPTTFQGATASTSYGLQNSSAASF 237

Figure S4 Alignment of 13 E2 Ubiquitin Ligases

Ac

catalytic cysteine residue

Fig. S4. Sequence alignment of human E2 ubiquitin-conjugating enzymes. Protein sequences of 13 E2s with the

N-terminal RXXKE motif were aligned using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Invariant

amino acids within the conserved N-terminal domain are highlighted in red and the catalytic cysteine residue is

highlighted in yellow. Gene names for proteins for which acetylation was identified within the N-terminal domain

are underlined. Asterisks below the sequences indicate identical amino acid residues; double dots indicate

conserved amino acid substitutions; single dots indicate semiconserved substitutions.

Figure S5

_

y₉

I

y₈

b₂

A

y₇

b₃

ac

K

y₆

E

y₅

L

y₄

S

y₃

D

y₂

L

y₁

E R _

acK*126.0913

a₂157.1335

y₁-NH₃158.0924

y₁175.119

b₂185.1285

y₂304.1615

b₃355.234

y₃417.2456

y₄532.2726

y₅619.3046

y₆732.3886

y₇861.4312

y₈1031.537 y₉

1102.574

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z [amu]

020

4060

8010

012

0

Rel

ativ

e A

bund

ance

00.2

0.40.6

0.81

1.2

Intensity [10e4]

Fig. S5. Identification of UBC4 Lys9 acetylation in S. cerevisiae. MS2 spectra showing identification of acetylated lysine at Lys9 in S. cerevisiae UBC4.

Figure S6

_ I

b₂

N

y₇

b₃

ac

K

y₆

b₄ b5

E

y₅

L

y₄

S

y₃

D

y₂

L

y₁

200 300 400 500 600 700 800 900 1000 1100 1200 1300

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

elat

ive

Abu

ndan

ce561.22

175.27246.25

y3

228.15

527.00

639.88

398.85

y4

y5

y6

y7

359.07

474.26

674.38

803.50

b₂

b₃

b₄b5

y₂y₁

A R _

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.

A

B

Figure S7

_

y₉

I

y₈

b₂

N

y₇

b₃

ac

K

y₆

b₄

E

y₅

L

y₄

S

y₃

D

y₂

b₈

L

y₁

A R _

acK*126.0913

acK143.1179

y₁-NH₃158.0924

y₁175.119

a₂200.1394

b₂-NH₃211.1077

b₂228.1343

y₂246.1561

y₃-NH₃342.2136

y₃359.2401

b₃-NH₃381.2132

b₃398.2398

y₄-NH₃457.2405

y₄474.2671

b₄-NH₃510.2558

b₄527.2824

y₉²⁺544.2907

y₅561.2991

y₆-NH₃657.3566

y₆674.3832

y₇803.4258

b₈-H₂O937.4989

b₈955.5095

y₈973.5313

y₉-NH₃1070.548

y₉1087.574

020

4060

8010

012

0

Rel

ativ

e A

bund

ance

01

23

45

Intensity [10e6]

100 200 300 400 500 600 700 800 900 1000 1100 1200

m/z [amu]

UB

E2D

3

UB

E2D

3 K

8Ac

UB

E2D

3

UB

E2D

3 K

8Ac

anti-UBE2D3 anti-acetyllysine

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

Sequence Window: Amino acid sequence (+/- 6) surrounding the acetylated lysine

PEP: Posterior error probablility of the peptide identification, see (J. Cox et al, Nature

biotechnology 26, 1367 (2008))

Mascot Score: Peptide ion MS/MS Probability-based Mowse score from Matrix Science:

Mowse score = -10xLog10(p), where p is the likelihood that the identification is a random event

PTM Score: Probability-based phosphosite localization score based on the binominal

distribution score: PTM score = -10xLog10(p), where p is the likelihood that the identification is

a random event

Localization Probability: Localization probability of acetylation site assignment to a specific

lysine in the identified peptide based on the PTM score

Modified Sequence: Acetylated peptide sequence and modifications of amino acids on this

peptide sequence that were identified by mass spectrometer; M(ox) = oxidized methionine, ac =

N-Protein acetylated residue, K(ac) = acetylated residue.

Acetyl (K) Probabilities: Localization probability of acetylation site assignment to specific

lysines in the identified peptide based on the PTM score

Position in peptide: Position of the acetylated lysine in the peptide sequence

Charge: Peptide charge state

m/z: Peptide mass/charge ratio

Mass Error [ppm]: Peptide mass error (observed - expected) in parts per million

Table S2. Drosophila acetylated lysine conservation

and

Table S3. Human acetylated lysine conservation

Ensembl gene: Ensembl gene identifier

Ensembl protein: Ensemble protein identifier

Protein description: Protein description

Gene Name: Human Genome Nomenclature Committee gene identifier

Protein (Genbank) ID: Genbank protein identifier

Position in Protein: Position of acetylated lysine in the protein

Modified Peptide Sequence: Acetylated peptide sequence and modifications of amino acids on

this peptide sequence that were identified by mass spectrometer; M(ox) = oxidized methionine,

ac = N-Protein acetylated residue, K(ac) = acetylated residue.

Position in Peptide: Position of the acetylated lysine in the peptide sequence

Unique acK site ID: Modified peptide sequence + position in peptide defines a unique identifier

for each acetylation site

Has Ortholog in humans/Drosophila: Ortholog exists in the human/Drosophila genome

Ortholog in human/Drosophila acetylome: Ortholog identified as acetylated in

human/Drosophila dataset used in this study

Lysine conserved in human/Drosophila: Lysine conserved at the corresponding position in the

human/Drosophila ortholog

Acetylation conserved in human/Drosophila: Acetylation identified at the corresponding

position in the human/Drosophila ortholog

Lysine conserved in zebrafish: Lysine conserved at the corresponding position in the D. rerio

(zebrafish) ortholog

Lysine conserved in nematodes: Lysine conserved at the corresponding position in the C.

elegans (nematode) ortholog

Details egarding ata vailability r d a

The data associated with this manuscript may be downloaded from ProteomeCommons.org

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