Toc33G

Toc159A

Toc132A

Toc120A

Toc159G

Figure. S1. Prediction of CK2 phosphorylation sites. NetPhosK 1.0 has been used to predict CK2 phosphorylation sites for the A-domains of Arabidopis Toc120, Toc132, and Toc159, and the G-domains of Toc33 and Toc159. Serine phosphoryla-tion sites are indicated in red and threonine phosphorylation sites in gray. Sequences used for the in silico prediction are identical to the sequences of the construction used in the in vitro phosphorylation assay, see Figure 7 B.

1- -265

727- -1093

1- -343

1- -740

1- -431

Toc159 full lengthA-dom fragment

2001169766

45

31

proto

plasts

P1 1.50

0xg

SN1 1.50

0xg

116

116976645

31

kDa

BSA (breakage buffer)LSU

CAB

1 2 3 4 5 6

IVTIVT -TL +TL+TL-TL

2001169766

45

31

kDa kDa

A

B

protoplastsrupturing device

broken protoplasts1.500 x g, 4 min

P1 SN1

Toc15GM

Toc159M

G MA G M1 1503 728 1503

Toc159

Toc15GM

Toc159M

1 2 3

Toc159 Toc15GM

C

anti-Toc159A

Phosphor-imager

Amido Black

Supplemental Figure S2. Toc159 cleavage. (A) A Toc159 processing activity in isolated chlo-roplasts. [35S]-methionin-labelled, in vitro translated (IVT) Toc159 or Toc159GM were incubated with isolated chloroplasts. Chloroplasts were re-isolated and incubated with (+TL) or without (-TL) thermolysin. Samples were analyzed by SDS-PAGE and PhosphorImaging. (B) Occurrence of the A-domain fragment after gentle cell disruption. Protoplasts of wild-type Arabidopsis plants were disrupted with a rupturing device employing 23 and 18 µm nylon mesh in two layers (Smith et al., 2002). Organelles were separated by centrifugation. 50 µg of protoplast, pellet and superna-tant protein were analyzed by Western Blotting with anti-Toc159A. (C) An annotated semi-tryptic peptide identified in the SN100 soluble fraction. To identify the most C-terminal amino acid of the soluble A-domain, MS/MS spectra were searched by Mascot for peptides that show tryptic specifi-city at one terminus only, and where the other terminus may be the result of non-tryptic cleavage. The semi-tryptic peptide mapping to amino acids 733-744 of atToc159 was identified in the SN100 fraction of three independent purifications with ion scores of 37, 42 and 47.

locus peptide sequence title query number AT4G02510 VGADDLpSDSEK F059194 q801_p1 AT4G02510 VVEGDpS AEEDENKLPVEDIVSSR F075163 q1354_p1 AT4G02510 KVVEGDpSAEEDENKLPVEDIVSSR F075163 q1360_p1 AT4G02510 FDQIGDDDpSGEFEPVSDK F059194 q1585_p1 AT4G02510 VGADDLSDSEKEKPNLVGDGK F050642 q1602_p1 AT4G02510 ASSGIEAHSDEANISNNMSDR F059194 q1640_p1 AT4G02510 ASSGIEAHSDEAN ISNNMSDR F059194 q1641_p1 AT4G02510 VVEGDpSAEEDENKLPVEDIVSSR F059194 q1672_p1

Supplemental Table S1: List of phosphorylated peptides presented in the main text (Tab.I) and the respective query number as its spectra identifier.

Supplemental Spectra S1: Pages 3 to 10Spectra of phosphorylated peptides as listed before. Unambiguous phosphorylation sites are assigned by " p " prior to the phosphorylated aminoacid. The p-site assign-ment " * " in the peptide sequence on each spectra page was automatically annotated without validation.

m/z

Rel

ativ

e In

tens

ity

228 406 584 761 939 1117

0

20

40

60

80

100

0

6156

12312

18468

24624

30780

● ●●

● ●

●

●

● ●●

●

●

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●

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●

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●

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●

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●

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●

●

●

●● ●

●

●

●●

●

●

y(3)

y*−98(4)

y(6)

y*−98(7)

y−98(7)

y−98(8)

y(8)y−98(6)

y(7)

y*−98(8)

y(5)

y*(11)++

y_0(11)++

y*−98(11)++

y_0−98(11)++

y−98(11)++

363.

4570

936

3.45

709 75

8.63

591

758.

6359

1

775.

7818

6

890.

8259

4

988.

7398

660.

6883

7

873.

8252

887

3.82

528

645.

6223

599.

5510

859

9.55

108

550.

803

550.

803

559.

7746

6

a(6)

b_0(6)

b(10)

a_0(6) b−98(10)

a(10)++

b_0(5)

b−98(8)

b(8)b(6)

b(4)

b(5)

b*(11)++

b_0(11)++

b(11)++

b−98(11)++

543.

5436

5

553.

6396

3

1069

.971

2

525.

4536

8

971.

9331

6

521.

4496

4

440.

3908

8

755.

7938

4

853.

7042

6

571.

5636

9

343.

4491

6 458.

5234

1

590.

5979

359

0.59

793

599.

5510

8

550.

803

title: F059194query number: q832_p1mascot Score: 85.73precursormass/charge: 608.242810,2+delta score; pep rank1 − pep rank2: 33.9peptide sequence: VGADDL*SDSEK

y−98(5) b−98(7)y(5)

●●

●

[M+2H]−98

m/z

Rel

ativ

e In

tens

ity

338 590 841 1093 1344 1596

0

20

40

60

80

100

0

1219.6

2439.2

3658.8

4878.4

6098

●●

●

●

●

●●

●

●

●●

●

●

●●

●

●

●●

●

●● ●

●

●

●

●

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●

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●

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●

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●

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●

●

●

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●

● ●

●

●

●

●

●

●

●

●

●

●

●

●

●

● ●

●

●

●

●

●

●

●

●

●

●

●

y_0(13)++

y*−98(21)++

y_0−98(21)++y*−98(8)

y(21)++y−98(22)++

y−98(21)++y(7)

y(20)++

y(9)++

y(14)++

y*(21)++

y*−98(22)++

y_0(21)++y_0−98(22)++

y(9)734.

6896

5

1141

.867

511

41.8

675

789.

6618

4

1199

.466

311

99.4

663

1150

.874

5

805.

5226

8

1135

.231

4

501.

5168

800.

2479

2

1190

.854

411

90.8

544

1190

.854

411

90.8

544

1001

.638

8

b_0−98(11)

b−98(8)

b(19)++b−98(20)++

b_0(20)++

a*(15)++

a_0(15)++

b−98(11)

b*(14)++

b*−98(15)++

b_0(14)++

b(14)

b−98(14)++

a(14)++

b(14)++

b−98(15)++

1124

.984

9

769.

4314

1

1075

.264

210

75.2

642

1114

.699

6

824.

3531

282

4.35

312

1141

.867

5

789.

6618

478

9.66

184

789.

6618

4

1595

.677

2

749.

2216

8

784.

6995

7

798.

5633

279

8.56

332

title: F075163query number: q1354_p1mascot Score: 76.76precursormass/charge: 866.061710,3+delta score; pep rank1 − pep rank2: 63.39peptide sequence: VVEGD*SAEEDENKLPVEDIVSSR

y(18)++●

[M+3H]−98

m/z

Rel

ativ

e In

tens

ity

309 589 868 1147 1426 1706

0

20

40

60

80

100

0

8384

16768

25152

33536

41920

●●

●

●●

●

● ● ●●

●

●● ● ●

●

●

●●

●

●

●

●

● ●

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●

●

●

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●

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●

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● ● ●

●

● ●

●

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● ●

●

●

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●

●

●●

● ●●

● ●

●

●●

●

●

●●

●

●

●

●

●●

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●

●●

●

●●

●

●

●●

●

●

●●

●●

●

●●●

●

●

●

●●

y(23)++

y(5)

y*−98(6)

y_0(22)++y(17)++

y(18)++

y*−98(19)++

y_0−98(19)++

y(21)++

y−98(22)++

y(20)++

y(22)++

y−98(23)++

y(7)

y(10)

y(9)

1299

.015

5

561.

4644

356

1.46

443

1239

.524

8

965.

5858 10

49.0

353

1049

.035

310

49.0

353

1199

.910

811

99.9

108

1135

.398

8

1249

.234

1249

.234

805.

2995

7

1114

.714

3

1001

.576

6

a(15)++

b*(8)

b_0(8)

b(6)

b_0−98(15)++b(12)

b_0−98(11)++

b(11)

b(14)++

b*−98(15)++

b*(15)++

b*−98(16)++

b_0(15)++

b−98(15)++

b(15)++

a_0(18)++

848.

7045

484

8.70

454

848.

7045

4

628.

4139

2

804.

4290

8

1368

.533

1

561.

4644

3

1239

.524

8

805.

2995

780

5.29

957

853.

7804

185

3.78

041

853.

7804

1

813.

7185

3

862.

7162

8

1001

.576

6

title: F075163query number: q1360_p1mascot Score: 89.63precursormass/charge: 908.759280,3+delta score; pep rank1 − pep rank2: 72.73peptide sequence: KVVEGD*SAEEDENKLPVEDIVSSR

y(18)++

b−98(7)++ b−98(7)●

● ●

[M+3H]−98

m/z

Rel

ativ

e In

tens

ity

350 623 896 1168 1441 1714

0

20

40

60

80

100

0

385

770

1155

1540

1925

● ●●

● ●

●

●

●

●●

●●

●

●

●●

●

●

●

● ●

●

●

● ●

●

●

●

● ● ●

●

●

●

● ●

●●

●

●

●

●

●

● ●

●

●

●

●

●

●

● ●● ● ●

●

●

●

●

●

●

●

●

y−98(9)

y*(11)

y(9)++

y*(16)++y_0(16)++

y(9)

y*(6)

y_0(6)

y(6)

y(12)

y*−98(13)

y(7)

y*−98(18)++

y_0−98(18)++

y(5)

y−98(18)++

910.

0043

8

1272

.522

9

504.

6275

5

900.

8336

890

0.83

368

1008

.044

5

656.

7902

765

6.79

027

674.

7612

3

1404

.415

714

04.4

157

821.

9768

7

982.

9261

498

2.92

614

545.

6616

6

992.

0103

8

b(3)

b(11)

b*(16)++

b_0(16)++

a_0(15)++

a_0(18)++

b(8)b(7)

b*−98(8)

b_0−98(12)

b*−98(13)

b_0−98(13)

b−98(12)

b_0−98(7)

b*(13)b−98(18)++

391.

4979 12

59.9

282

900.

8336

890

0.83

368

843.

0711

5

1008

.044

5

906.

3201

8

791.

7043

279

1.70

432

1290

.120

8

1420

.058

1420

.058

1309

.044

3

674.

7612

3

1518

.219

6

982.

9261

4

title: F059194query number: q1585_p1mascot Score: 37.48precursormass/charge: 1040.407300,2+delta score; pep rank1 − pep rank2: 23.68peptide sequence: FDQIGDDD*SGEFEPVSDK

y−98(10)●

[M+2H]−98

m/z

Rel

ativ

e In

tens

ity

228 501 774 1046 1319 1592

0

20

40

60

80

100

0

26940

53880

80820

107760

134700

●

●● ●● ●● ●●

●● ●●

●● ●

●

●●

●

●

●

●●● ● ● ● ●●

●

● ●

●

●●

●●

● ● ●● ●

● ●●

●

●

● ●●●

●● ● ●

●

●● ●● ●●

●●

●● ● ●● ●●

●

●

●

●

●●

●

● ●● ● ● ●

●

●

●●● ● ● ●

●●

● ● ● ●● ●

● ●●●

●●

● ● ●●

● ●● ●● ●

●

●●

●

●

●

●●

●

● ● ● ●●

●●

●● ●● ● ●●

●

● ●

● ● ●● ●● ●

y−98(8)

y−98(15)++

y*−98(15)++

y_0−98(15)++

y−98(19)++

y(16)++

y*−98(17)++

y_0−98(17)++

y(17)++

y*−98(18)++

y−98(20)++

y−98(18)++

y−98(17)++

y(8)

y−98(16)++

y_0−98(14)++

701.

2635

5

793.

2822

378

4.42

358

784.

4235

8

999.

9456

6

898.

5214

889

8.52

148

898.

5214

8

956.

2191

495

6.21

914

1028

.897

9

964.

7992

6

907.

3411

2

799.

5572

9

849.

8199

7

700.

0958

9

b_0−98(13)++

b(16)++

b−98(17)++

b*(19)++

b_0(19)++b(18)++

b*−98(19)++

b_0−98(19)++

b−98(9)

b−98(15)++

b(13)++b−98(13)++b(19)++

b−98(19)++a(13)++b*(13)++

669.

6380

1

889.

9852

988

9.98

529

1016

.802

810

16.8

028

967.

7959

896

7.79

598

967.

7959

8

841.

9421

4

784.

4235

8

728.

1546

8

679.

1576

9

1025

.826

8

976.

6219

1

713.

5780

371

9.52

607

title: F050642query number: q1602_p1mascot Score: 68.72precursormass/charge: 752.015930,3+delta score; pep rank1 − pep rank2: 6.05peptide sequence: VGADDL*SDSEKEKPNLVGDGK

b−98(7)y−98(15)++y(15)++

●● ●

[M+3H]−98

m/z

Rel

ativ

e In

tens

ity

290 525 760 994 1229 1463

0

20

40

60

80

100

0

12766

25532

38298

51064

63830

●●

●

●

● ●

●

●

●

● ●●●

●

●●

●

●

●●

●

●

●

●

●

●

●●

●

●

● ●

●

●

●

●

●

●

●● ●

●● ●

●

●

●

●

●●

●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

● ●

●

●

●

● ●●

●

●●

●

●

y*(10)++

y(7)++

y(4)

y_0(9)++

y−98(7)

y(8)++

y*(16)++y*(8)

y(10)++

y(5)y*(12)++

y(8)

y*(6)y(6)

y_0−98(7) y(7)

560.

8954

5

420.

6601

3

524.

6162

852

4.61

628

740.

8134

9

477.

1507

5

935.

1783

293

5.17

832

569.

5786

8

638.

7220

9

683.

1845

6

952.

9550

9

735.

5824

8

752.

7161

4

723.

4491

1

839.

7779

6

b−98(12)++

b(12)++ b(20)++

b*(13)++b_0(13)++

b−98(14)++

a*(14)++a(14)++

b*−98(15)++

b_0(8)a*(15)++

a_0(15)++

b(13)++

b*(14)++

b−98(17)++b(14)++

569.

5786

8

618.

3541

1

1064

.146

666.

5826

666.

5826

683.

1845

6

709.

6528

3

718.

1283

718.

1283

735.

5824

8

752.

7161

475

2.71

614

675.

6338

6

723.

4491

1 839.

7779

6

732.

3936

9

title: F059194query number: q1640_p1mascot Score: 49.02precursormass/charge: 767.640560,3+delta score; pep rank1 − pep rank2: 7.88peptide sequence: ASSGIEAH*SDEANISNN*MSDR

b−98(9)++●

[M+3H]−98

m/z

Rel

ativ

e In

tens

ity

326 554 781 1009 1236 1463

0

20

40

60

80

100

0

9346

18692

28038

37384

46730

●

●

●

●●

●

●

●

●

● ●

●

●

●

●

●●

●

●

●

●

●

●

●

●

● ●

● ●

●●

●

●

●

● ● ●

●

●

●●

●

● ●●

●

●●

●●

● ●

●

●

●

●●●

●

●

● ●

●●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●●●

●

●

●

●

●

●

●

●

●

●

●

●

y*(10)++

y*(16)++

y(3) y(8)++

y(9)

y−98(20)++

y*(9)++

y_0(9)++

y*(12)++

y*(7)y*(6)

y(5)

y(8)

y(6)

y_0−98(7) y(7)

560.

8504

2

895.

1857

5

377.

4264

4

477.

1096

6

1066

.985

410

66.9

85452

4.85

287

524.

8528

7

683.

0506

4

822.

7598

735.

6313

8

638.

6735

4

953.

0109

5

752.

7654

8

723.

5759

3

839.

7541

5

b_0−98(11)++

b(12)++

b*−98(13)++

b_0−98(13)++

b−98(14)++

b−98(9)

b_0(16)++

b−98(8)

a*(14)++a_0(14)++

b(13)++

a(14)++

b*−98(15)++

a*(15)++

a_0(15)++

b*(14)++

524.

8528

7

617.

8261

361

7.82

613

617.

8261

3

683.

0506

4

822.

7598

822.

7598

735.

6313

8

709.

1449

709.

1449

675.

8296

7 718.

2354

471

8.23

544

752.

7654

875

2.76

548

723.

5759

3

title: F059194query number: q1641_p1mascot Score: 52.90precursormass/charge: 767.642180,3+delta score; pep rank1 − pep rank2: 0peptide sequence: AS*SGIEAHSDEANISNN*MSDR

b(3)●

[M+3H]−98

m/z

Rel

ativ

e In

tens

ity

328 582 835 1089 1343 1596

0

20

40

60

80

100

0

656.4

1312.8

1969.2

2625.6

3282

●

●●

●

●●

●

●

●

●

●

●

●

●

●●

●

●

●

●

●

●

●●

●

●

●

●

●

●●

●

●

●

●

●

●

●

●●

●

●

●

● ● ●●

●

●

●●

●

●●

●

●

● ●●

●●

●

●

y*−98(6)

y(11)++

y(4)

y(18)++

y*−98(19)++

y(16)++

y(6)

y(7)

y*−98(8)

y−98(21)++

y(9)++

y(14)++

y*(21)++

y*−98(22)++

y_0−98(22)++

y(9)

561.

7605

622.

2318

4

448.

5624 10

49.5

244

1049

.524

4

930.

3890

7

676.

8874

805.

8002

5

789.

9749

1151

.020

2

501.

7309

9

800.

9749

4

1191

.441

411

91.4

414

1191

.441

4

1002

.066

6

b−98(23)++b(12)

b−98(11)

b*(20)++b_0−98(7)

b(13)++b−98(20)++

b*(14)b*−98(15)

b_0(14)

b(14)

b(20)++

b_0−98(11)b*(14)++b−98(14)++ b−98(16)++ 12

40.8

145

1355

.033

3

1142

.735

6

1116

.339

5

622.

2318

4

742.

2049

3

1075

.606

5

1578

.218

615

78.2

186

1578

.218

6

1596

.240

3

1124

.960

211

24.9

602

789.

9749

749.

8348

1

847.

5266

2

title: F059194query number: q1672_p1mascot Score: 36.89precursormass/charge: 866.059720,3+delta score; pep rank1 − pep rank2: 25.44peptide sequence: VVEGD*SAEEDENKLPVEDIVSSR

y(18)++●

[M+3H]−98