0

200

400

600

800

1000

1200

present only in

LOPIT1&2

present in

all experiments

present only in

LOPIT3&4

Nu

mb

er

of

pro

tein

s

no TMD

with TMD

Figure S1. Number of proteins with and without predicted TMDs identified and quantitated in

the four LOPIT experiments

For each protein the number of predicted TMDs were extracted from the TAIR server (based on

HMMTOP prediction) and the proteins separated into two groups: without any predicted TMD and

with one or more predicted TMDs. The 1385 proteins reported in Table S2 were categorised into three

groups: present only in LOPIT1 and 2, present in all LOPIT experiments and present only in LOPIT 3

and 4.

Figure S2. Positional analysis of amino acid composition of TMDs from different organellesThe position relative to the cytosolic edge of the TMDs is plotted on the horizontal axis and thenormalised proportion of each amino acid is plotted on the vertical axis.

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Cysteine composition

0 10.0 20.0 30.0

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Aspartic acid composition

0 10.0 20.0 30.0

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Glutamic acid composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Phenylalanine composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Glycine composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Histidine composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Isoleucine composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Lysine Composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Leucine composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Methionine composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Asparagine composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Proline composition

Pro

port

ion

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

ERGolgi/TGNPM

Glutamine composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Arginine composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Serine composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Threonine composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Tryptophan composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Tyrosine composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

ERGolgi/TGNPM

Alanine composition

0 10 20 30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Position

Pro

port

ion

Valine composition

ERGolgi/TGNPM

DUF579_consensus 78 PQQNLAEISISFNILKKLAPANFLVFGL--GRDSLMWASLNPRGKTLFLEEDLEWFQKVTKD-----SPFLRAHHVRY 148 (289aa)3cvo 14 LTMPPAEAEALRMAYEEA--EVILEYGS---GGSTVVAAELPGKHVTSVESDRAWARMMKAWLAANPPAEGTEVNIVW 86 (202aa)3ntv 53 PIVDRLTLDLIKQLIRMNNVKNILEIGTAIGYSSMQFASISDDIHVTTIERNETMIQYAKQNLATYHFENQVRIIEGN 130 (232aa)

2gpy 36 PIMDLLGMESLLHLLKMAAPARILEIGTAIGYSAIRMAQALPEATIVSIERDERRYEEAHKHVKALGLESRIELLFGD 113 (233aa)Secondary structure ZZZZZZZZZZZZZ 11111 AAAAAAAAA 222222 BBBBBBBBBBBBB 333333

A

B

Figure S3. HHpred alignment output of DUF579 query sequence against the PDB database

(A) Graph summarizing the alignment positions and color-coded significances of the database

sequence matches; (B) Multiple alignment of DUF579 and the top three HHpred targets, PDB entries

of MT structures (3CVO, 3NTV and 2GPY), showing the SAM binding domain. The topology of the

SAM-binding domain consists of alternating strands (1, 2, and 3) and helices (Z, A and B). Colors

indicate predicted secondary structure, red – helices, blue – strands.

β α

α β

A

Figure S4. HHpred alignment output of KOB1 query sequence against the PDB database.

(A) Graph summarizing the alignment positions and color-coded significance of the top three matches,

PDB entries of GT structures (1QG8, 3BCV and 3CKJ); (B) Multiple alignment of KOB1 and the

highest Arabidopsis database matches; sequences of the two KOB1 related proteins, and the DUF23-

containing GTNC and GT92. Colors indicate predicted secondary structure, red – helices, blue –

strands. The putative catalytic DxD motif is indicated in a rectangle.

α β

BAT3G08550 (KOB1) 139 GLDQILPWMFYH-KVLGVSTFFLFVEGKAATPSISKVLESI---PGVKVIYRTKELEEKQAKSRIWNETWLSSFFYKPCN 214 (533aa)

AT3G57200 125 GLEQTLPWIYFH-KVIGVSTFYLFVEGKAASPSISKVLESI---PGVKVIYRTKELEEKQAKSRIWNETWLSSFFYKPCN 200 (514aa)AT2G41451 106 GLEQTLPWIFYH-KVIGVETFYLFVEGTAASPNVSRVLETI---PGVNVIYRTRELEEEQAKSRIWNETWLEKFFYKPCN 181 (451aa)

AT1G27200 (GTNC) 302 QAPFLREWIMYH-SWLGVERWFIYDNN--SDDGIQEEIELLSSENYNVSRHVWPW----------IKT------------ 361 (575aa)

AT4G37420 (GTNC) 319 VAKYLREWVMYH-AAIGIQRFIIYDNG--SDDELNDVVKGLNSEKYDVIKVLWIW----------PKT------------ 378 (588aa)

AT5G40720 (GTNC) 286 AANVLREWVMYH-AGIGVQRWFIYDNN--SDDDIVSEIKNLENRGYNISRHFWPW----------IKT------------ 345 (583aa)

AT3G27330 (GTNC) 289 AAAVLREWVMYH-AGIGVQRWFIYDNN--SDDDIIAEIENLERRGYNISRHFWPW----------IKT------------ 348 (913aa)AT5G44670 (GT92) 270 SPQRIREWIAYHVRFFGERSHFVLHDAGGITEEVFEVLKPWIELGRVTVHD------------IREQER----------- 329 (519aa)

AT4G20170 (GT92) 255 SPQRVREWIAYHVRFFGERSHFVLHDAGGIHEEVFEVLKPWIELGRVTLHD------------IRDQER----------- 311 (504aa)

AT2G33570 (GT92) 245 SASRMREWMAYHAWFFGDKSHFVFHDAGGVSPEVRKVLEPWIRAGRVTVQN------------IRDQSQ----------- 309 (496aa)

AT3G08550 (KOB1) 215 Y-ELFVKQSLNMEMAIVMARDA-GMDWILHLDTDELIY-------PAGAREYSLRRLLLDV-----PPNVDMVIFPNYES 293 (533aa)AT3G57200 201 Y-ELFVKQSLNMEMAITMAQDA-GMEWIIHLDTDELIH-------PSGTHEYSLRKLLGNI-----SADVDVVIFPNYES 264 (514aa)

AT2G41451 182 Y-ELFVKQNLNMEMAITMARDA-GMDWILHLDTDELVH-------PSGTREYSLRNLLRDV-----PADVDEVIFTNYES 245 (451aa)

AT1G27200 (GTNC) 362 -------QEAGFSHCAVRAKEE--CNWVGFFDVDEFYYFPTHRSQGLPSKNALKSLVSNYTS----WDLVGEIRTDCHSY 421 (575aa)

AT4G37420 (GTNC) 379 -------QEAGFSHAAVYGND--TCTWMMYLDVDEFLFSPAWDKQSQPSDQMIRSLLPSD------QSMIGQVSFKSHEF 436 (588aa)

AT5G40720 (GTNC) 346 -------QEAGFANCAIRAK--SDCDWVAFIDVDEFFYI------PSGQ--TLTNVIRNHTTTPSSSGEIGEIRTPCHSF 401 (583aa)AT3G27330 (GTNC) 349 -------QEAGFSNCAIRAK--SDCDWIAFIDVDEFFYI------PS--GETLTSVIRNYTTT----DSIGEIRTPCHSF 400 (913aa)

AT5G44670 (GT92) 330 FDGYYHNQFMVVNDCLHRYRFMA--KWMFFFDVDEFIYV------P--AKSSISSVMVSL------EEYSQFTIEQMPMS 383 (519aa)

AT4G20170 (GT92) 312 FDGYYHNQFMIVNDCLHRYRFMT--KWMFFFDVDEFLHV------PVKET--ISSVMESL------EEYSQFTIEQMPMS 368 (504aa)

AT2G33570 (GT92) 310 YDGYYYNQFLIVNDCLHRYRYAA--NWTFFFDVDEYIYL------P--HGNTLESVLDEF------SVNTQFTIEQNPMS 361 (496aa)

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