inventory and comparative analysis of rice and arabidopsis atp-binding cassette (abc) systems

doi:10.1016/j.jmb.2004.07.093 J. Mol. Biol. (2004) 343, 249–265

Inventory and Comparative Analysis of Rice andArabidopsis ATP-binding Cassette (ABC) Systems

Olivier Garcia1, Philippe Bouige1, Cyrille Forestier2 and Elie Dassa1*

1Unite des MembranesBacteriennes CNRS URA2172Departement de MicrobiologieFondamentale et MedicaleSite Fernbach, Institut Pasteur25, Rue du Docteur Roux75724 Paris Cedex 15 France

2CEA CadaracheDSV-DEVM-LEMSUMR 6191CEA-CNRS-Aix-Marseille II13108, St Paul-lez-DuranceCedex, France

0022-2836/$ - see front matter q 2004 P

Abbreviations used: ABC, ATP-bneighbour-joining; CCM, cytochromgreen fluorescent protein; FAE, fattyE-mail address of the correspond

[email protected]

ATP-binding cassette (ABC) proteins constitute a large superfamily foundin all kingdoms of living organisms. The recent completion of two draftsequences of the rice (Oryza sativa) genome allowed us to analyze andclassify its ABC proteins and to compare to those in Arabidopsis thaliana.We identified a similar number of ABC proteins in rice and Arabidopsis(121 versus 120), despite the rice genome being more than three times thesize of Arabidopsis. Both Arabidopsis and rice have representativemembers in all seven major subfamilies of ABC ATPases (A to G)commonly found in eukaryotes. This comparative analysis allowed thedetection of 29 potential orthologous sequences in Arabidopsis and rice.However, plant share with prokaryotes a specific set of ABC systems that isnot detected in animals. These ABC systems might be inherited from thecyanobacterial ancestor of chloroplasts. The present work provides the firstcomplete inventory of rice ABC proteins and an updated inventory of thoseproteins in Arabidopsis.

q 2004 Published by Elsevier Ltd.

Keywords: ATP binding cassette; phylogeny; classification; Arabidopsisthaliana; Oryza sativa
*Corresponding author
Introduction

ABC proteins constitute one of the largestfamilies of paralogous sequences, and they arefound in all living organisms.1 They share a highlyconserved ATPase domain, the ATP-binding cas-sette or ABC, which has been demonstrated to bindand hydrolyze ATP, thereby providing energy for alarge number of fundamental biological processes.2

The amino acid sequence of this cassette displaysthree major conserved motifs, the Walker motifs Aand B commonly found in ATPases, and anadditional specific signature LSGGQ motif. MostABC proteins are transporters, which are composedof four structural domains: two hydrophobicintegral membrane domains (called thereafter IMdomains) and two hydrophilic domains containingthe ATP-binding cassette (called thereafter ABCdomains). ABC transporters are involved in theimport or the export of a wide variety of substances.Importers constitute mainly the prokaryotic trans-porters dependent upon an extracytoplasmic

ublished by Elsevier Ltd.

inding cassette; N-J,e c maturation; GFP,acid export.

ing author:

substrate-binding protein that provide bacteriawith essential nutrients. Usually, they carry IMand ABC domains on separate polypeptide chains.Such transporters have been called sometimesquarter transporters despite the fact that quartersare not equivalent (Figure 1). Exporters are found inboth prokaryotes and eukaryotes and are involvedin the extrusion of noxious substances and drugs,the secretion of extracellular toxins and the target-ing of membrane components.3 Generally, theirconstitutive IM or ABC domains are fused invarious combinations. Half-size transporters arecomposed of a single IM domain fused to an ABCdomain, a structural organization that could besymbolized as IM-ABC or ABC-IM depending onthe N- or C-terminal location of the IM domain.Full-size transporters are probably generated byduplication and fusion of “half-size” transportersand are symbolized as (IM-ABC)2 and (ABC-IM)2(Figure 1). Many ABC proteins are apparently notinvolved in transport but rather in cellular pro-cesses such as DNA repair,4 translation5 or regu-lation of gene expression.6 These proteins,symbolized as ABC2, do not have IM domainsand are composed of two ABC domains fusedtogether (Figure 1).We made previously a comparative analysis of

the sequences and the properties of the components

Figure 1. Diagrammatic view ofthe domain arrangements in ABCsystems. The structural organiz-ation of domains in the threeclasses of ABC systems is shown.The membrane is represented bythick blue horizontal line. Redrectangles represent Integral Mem-brane (IM) domains and greencircles ATP-binding Cassette(ABC) domains. N and C refer tothe N- and C termini of the pro-teins, when required. Within eachclass, from the top to the bottom,are represented: a schematic organ-ization, the families or subfamilieswhose members display such anorganization, the symbol used inthe text to represent it.

250 Rice ABC Proteins Inventory

of ABC systems and we derived a phylogenetic andfunctional classification.7 The classification wasused to build ABCISSE, a database of ABCproteins†. We found that ABC proteins segregatein three classes. Class 1 is comprised of the majorityof known exporters, class 2 contains all systemswith no known IM domain and involved in cellularprocesses different from transport, and class 3contains all binding protein-dependent importersand other less well-characterized systems. Withineach class, ABC proteins cluster according tofamilies and subfamilies of proteins that sharecommon functional features. Table 1 provides adescription of the families identified in plants, andthe correspondence between the nomenclature usedhere and that of eukaryote ABC proteins. Figure 1depicts the structural organization of proteins thatbelong to these families.

Plant ABC transporters have been analyzed inseveral publications.8–12 However, most of thesestudies were restricted to specific aspects of thephysiology of ABC transporters,11 to a specificfamily of transporters,10 or to full ABC transportersonly.11,12 Only one paper presents a complete

† http://www.pasteur.fr/recherche/unites/pmtg/abc/database.iphtml

inventory of ABC proteins in Arabidopsis thaliana.9

However, a few ORFs were miss-annotated hereand are corrected in the present report.

The completion of two shotgun sequences of thenuclear genome of Oryza sativa13,14 allowed us toinvestigate the relationships of its putative ABCsystems with well-characterized members in otherplants and organisms. Despite the large numberof ABC proteins identified in plants, the function ofmost of them remains unknown. Classification ofO. sativa ABC proteins into families may help toelucidate their functions.

A recent evolutionary analysis of the genome ofA. thaliana revealed that about 18% of nuclear genesderive from the cyanobacterial ancestor of chloro-plasts.15 The objective of our work is to place theanalysis of ABC proteins in the larger context oftheir evolution in living organisms, includingprokaryotes. The present inventory, based onsequence similarities with universally representedABC systems, indicates that the genome of O. sativacontains at least 116 ORFs corresponding to thesubset of ABC systems found generally in eukary-otes. In addition to these, 12 ORFs are stronglyrelated to prokaryote ABC accessory proteins,making a total of 128 ORFs as putative constituentsof ABC systems. We compared these ORFs to thoseof A. thaliana (127 ORFs) and we propose an

http://www.pasteur.fr/recherche/unites/pmtg/abc/database.iphtml

http://www.pasteur.fr/recherche/unites/pmtg/abc/database.iphtml

Table 1. Abbreviations used to qualify families and subfamilies of ABC systems

ABCISSE HGNC Description of the abbreviation

Family Subfamily Family

FAE ABCD Fatty acid exportDPL ABCB Drug, peptides and lipid export

HMT Similar to yeast heavy metal transporterLLP Lipid A-like exporters, putativeTAP Similar to the human transporter associated with antigen

presentationp-gP Similar to p-glycoprotein

OAD ABCC Organic anion and drug exportMRP Similar to multidrug resistance protein

EPD ABCG Eye pigment precursors and drug exportWHITE Similar to Drosophila white proteinPDR Similar to yeast pleiotropic drug resistance proteins

CCM Cytochrome c maturationRLI ABCE Similar to RNase L inhibitorART Antibiotic resistance and translation regulation

REG ABCF Regulation of gene expressionCBY Cobalt uptake and unknown function

Y179 Similar to cobalt uptake systems, unknown functionMKL Similar to the M. leprae mkl proteinABCX Similar to C. paradoxa abcx proteinDRA Drug resistance and abca

ABCA ABCA Similar to human abca1 proteinNO Unclassified systemsADT Unclassified systems

Only families found in plants are presented. In the ABCISSE database, the abbreviation of the families are derived from the function ofthe proteins of the family, the name of the best characterized protein, or for uncharacterized ABC proteins, from the name of the firstprotein that was identified in the family or subfamily. A correspondence with the seven families of human ABC proteins in given underthe column HGNC (Human Gene Nomenclature Committee).

Rice ABC Proteins Inventory 251

updated inventory of ABC proteins in this organ-ism. Despite of the difference in genome size(440 Mb versus 125 Mb) and gene content (up to65,000 versus 25,498), the two organisms appear tohave a very similar number of ABC-related pro-teins. Plant share with prokaryotes a specific set ofABC systems that have not been yet detected inanimals. It could be reminiscent of the endocytosisevent that led to the generation of chloroplasts.16,17

Results and Discussion

Classification of rice ABC proteins

The analysis of the genome sequence of O. sativarevealed that 128 ORFs, could be assigned to theABC superfamily. A detailed inventory of theseORFs, including alternative names, accession num-bers, chromosomal location, sizes, overall structuralorganization and most similar ORFs in A. thaliana ispresented in Table 2. Since the genome sequenceand the prediction of genes are not completed, it ispossible that this number slightly change in the nearfuture. 121 ORFs carry at least one ABC domain. Onthe basis of similarities with bacterial proteins, theseven non-ABC proteins are predicted to interactwith soluble ABC proteins to constitute multi-subunit transporters or enzymatic complexes.

The rice ABC ATPases segregate into 13 clusters(Figure 2), which parallel the families identified in

previous analyses of universally represented ABCproteins.7 Seven of these clusters are equivalent tothe seven families (ABCA to ABCG) of human ABCproteins.18 In most families with two ABC domains,the N-terminal and C-terminal domains clusterindependently. This confirms our previous obser-vations suggesting that the gene duplication eventthat probably gave rise to full transporters occurredbefore the specialization of the members of thefamily.19 We compared the ORFs identified in thisstudy to the ABC proteins of A. thaliana. Despite thefact that the rice genome is approximately 3.5 timesthe size of Arabidopsis, it encodes only oneadditional ABC protein but lacks an accessory orpartner protein.The rice ABC proteins display a similar organiz-

ation of ABC and IM domains and a similar size ascompared to well-characterized members of eachfamily or sub-family. Plants, like other eukaryotes,lack members of the prokaryotic binding protein-dependent ABC transporters involved in the uptakeof small nutrient molecules. In addition to the sevenfamilies commonly found in animals, plants havesix additional families. Four of them are sharedwithbacteria (ABCX, CCM, Y179, MKL), one (ADT) isfound also in fungi and the latter (NO) is not afamily in the normal meaning of the term but rathera collection of sequences that do not cluster with thedescribed families of ABC proteins. These proteinsare found on the tip of the longest branches on theneighbour-joining (N-J) tree (Figure 2).

Table 2. Detailed inventory of Oryza sativa ABC proteins

TIGR ORFname

TIGR synonym BAC Chr. Structure Size NCBI ORF name NCBI gi Jasinski’sname

Jasinski’s gi Best hit AT Orthology

Class 1 systemsDPL (ABCB) family, half transportersTAP subfamily (3/3)2880.m00093 2602.m00210 P0470A12 1 IM-ABC 548 P0470A12.8 20161359 At1g70610 O4620.m00053 5300.m00115 36I5 3 IM-ABC 611 36I5.4 12061244 At5g39040 O6098.m00123 4810.m00171 P0473C09 7 IM-ABC 710 At4g25450 OHMT subfamily (1/3)3024.m00134 P0538C01 6 IM-ABC 877 5734621 At5g58270LLP subfamily (1/1)5551.m00113 OJ000126_13 4 IM-ABC 682 OJ000126_13.6 32480256 At5g03910

DPL (ABCB) family, full transportersp-gP subfamily (24/22)2748.m00163 2750.m00175 P0706B05 1 (IM-ABC)2 1285 8468012 MDR4 27368863 At2g470003361.m00127 OJ1029_F04 1 (IM-ABC)2 1164 OJ1029_F04.23 20146377 MDR5 27368861 At2g470003361.m00121 OJ1029_F04 1 (IM-ABC)2 1282 OJ1029_F04.16 20146370 MDR6 27368871 At1g025202594.m00130 OJ1116_H09 1 (IM-ABC)2 1274 OJ1116_H09.9 22535563 MDR8 27368857 At1g025204739.m00134 P0431H09 1 (IM-ABC)2 1154 P0431H09.25 22535536 MDR9 27368855 At2g470004739.m00136 2594.m00125 P0431H09 1 ABC-IM-ABC 859 MDR7 27368859 At3g283602830.m00145 2576.m00137 P0459B04 1 (IM-ABC)2 1386 P0459B04.19 15290144 MDR10 27368853 At3g553204392.m00149 P0022F10 1 (IM-ABC)2 1203 P0022F10.15 20160550 MDR17 27368839 At3g283454300.m00147 5742.m00201 P0688H12 2 (IM-ABC)2 1239 At1g025206372.m00140 OSJNBb0031B09 2 ABC-IM-ABC 748 At3g283456372.m00142 OSJNBb0031B09 2 (IM-ABC)2 1235 At3g283456372.m00144 OSJNBb0031B09 2 (IM-ABC)2 1245 At3g283453858.m00172 4862.m00179 P0017H11 2 (IM-ABC)2 1123 MDR11 27368851 At4g259605155.m00097 OSJNBb0076N15 3 (IM-ABC)2 1451 OSJNBb0076N15.14 24960750 At3g288606665.m00117 OSJNBa0009C19 3 (IM-ABC)2 1410 At3g553205500.m00234 OSJNBa0036B21 4 (IM-ABC)2 1252 OSJNBa0036B21.21 21740907 MDR13 27368847 At3g288605504.m00124 3988.m00130 OSJNBb0079B02 4 (IM-ABC)2 1261 OSJNBb0079B02.16 21740420 MDR12 27368849 At3g288605579.m00151 OSJNBb0011N17 4 (IM-ABC)2 1279 OSJNBb0011N17.13 32480018 MDR1MDR2 2736886927368867 At3g283456396.m00179 5810.m00136 P0554F08 5 (IM-ABC)2 1213 At1g025206396.m00180 P0554F08 5 (IM-ABC)2 1218 At2g470002967.m00098 OJ1127B08 5 (IM-ABC)2 1276 MDR3 27368865 At2g470003917.m00147 3052.m00132 P0705A05 8 (IM-ABC)2 1363 P0705A05.10 28071306 MDR14 27368845 At2g36910 O5069.m00171 3089.m00172 OJ1349_D05 8 (IM-ABC)2 1141 OJ1066_B03.2 27817909 MDR15 27368843 At3g288605069.m00173 3089.m00174 OJ1349_D05 8 (IM-ABC)2 1242 OJ1066_B03.3 27817910 MDR16 27368841 At1g28010

OAD (ABCC) family, full transportersMRP subfamily (17/16)2817.m00139 OSJNBa0089K24 1 (IM-ABC)2 1493 OSJNBa0089K24.15 15128229 MRP3 27368887 At3g130802867.m00114 2698.m00134 B1157F09 1 ABC-IM-ABC 798 B1157F09.14 15408680 MRP8 27368877 At2g478004401.m00150 3799.m00138 B1065G12 1 (IM-ABC)2 1386 B1065G12.13 20161611 MRP2 27368889 At3g130802530.m00242 OJ1756_H07 2 (IM-ABC)2 1222 MRP5 27368883 At3g130802530.m00245 OJ1756_H07 2 (IM-ABC)2 1198 MRP4 27368885 At3g130804781.m00157 OSJNBa0083D24 3 (IM-ABC)2 1385 At1g04120 O5347.m00078 OSJNBb0022P19 4 (IM-ABC)2 1483 MRP10 27368873 At2g478005564.m00120 OSJNBa0035O13 4 (IM-ABC)2 1594 OSJNBa0035O13.21 32480153 MRP9 27368875 At3g62700

5388.m00143 5354.m00170 OSJNBb0016D16 4 (IM-ABC)2 1135 OSJNBb0016D16.21 32488777 MRP7 27368879 At3g601605388.m00144 5354.m00171 OSJNBb0016D16 4 (IM-ABC)2 1115 OSJNBb0016D16.20 32488776 MRP6 27368881 At3g601605470.m00141 OSJNBa0058K23 4 (IM-ABC)2 1650 OSJNBa0058K23.20 32482870 MRP1 27263148 At2g346606226.m00095 P0617H07 5 (IM-ABC)2 1474 At3g21250 O6273.m00143 P0702F05 6 (IM-ABC)2 1474 P0702F05.23 24060177 At3g59140 O6519.m00144 P0554A06 6 (IM-ABC)2 1204 At2g076801973.m00148 P0456F09 6 (IM-ABC)2 1196 MRP11 27368891 At3g212505260.m00121 OSJNBb0030I09 11 (IM-ABC)2 1464 At3g591406027.m00133 OSJNBb0076G11 12 (IM-ABC)2 1268 At3g59140

FAE (ABCD) family, half transporters (1/1)4993.m00055 2696.m00120 P0489G09 1 IM-ABC 1671 P0489G09.11 21327950 At1g54350 O

FAE (ABCD) family, full transporters (2/1)5523.m00189 P0458E05 1 (IM-ABC)2 1310 P0458E05.21 21902073 At4g398505723.m00197 OSJNBa0068N01 5 (IM-ABC)2 1368 28195114 PMP2 27368893 At4g39850

EPD (ABCG) family , half transportersWHITE subfamily (30/29)2701.m00089 P0410E01 1 ABC-IM 613 P0410E01.34 11034589 At1g53270 O2771.m00129 P0445D12 1 ABC-IM 674 P0445D12.3 13486800 At2g01320 O4448.m00155 P0506B12 1 ABC-IM 749 P0506B12.20 20160808 At3g550902188.m00162 OSJNBa0033P04 3 ABC-IM 687 OSJNBa0033P04.17 31193915 At2g28070 O4996.m00058 OSJNBa0011L14 3 ABC-IM 695 OSJNBa0011L14.6 21397268 At5g065305247.m00198 OSJNBa0044H10 3 ABC-IM 787 24796798 At3g550905247.m00200 OSJNBa0044H10 3 ABC-IM 765 24796803 At3g550905467.m00139 OSJNBa0074L08 4 ABC-IM 692 OSJNBa0074L08.3 21742080 At1g178405473.m00188 OSJNBa0040D17 4 ABC-IM 940 OSJNBa0040D17.18 32492268 At5G607403646.m00191 OJ1354D07 5 ABC-IM 658 At5g19410 O4754.m00144 6218.m00191 OJ1532D06 5 ABC-IM 700 At1g515002998.m00184 P0699E04 5 ABC-IM 680 8099135 At3g550902998.m00186 P0699E04 5 ABC-IM 699 8099136 At3g550901913.m00072 P0564B04 6 ABC-IM 624 At5g528604181.m00159 P0459E03 6 ABC-IM 1026 At5g607405130.m00142 3409.m00111 P0029C06 6 ABC-IM 687 At3g13220 O3433.m00103 4854.m00158 P0440B02 7 ABC-IM 694 At2g393506053.m00121 2043.m00141

5824.m00129B1126F07 7 ABC-IM 526 B1126F07.104-1 34395340 At1g17840

3506.m00216 5734.m00192 P0462E11 8 ABC-IM 635 At1g317705680.m00128 OJ1509_C06 9 ABC-IM 728 At1g178405680.m00129 OJ1509_C06 9 ABC-IM 767 At1g178405668.m00144 OJ1189_B05 9 ABC-IM 703 At1g178406497.m00158 B1080A02 9 ABC-IM 526 At1g178405959.m00179 OSJNBa0025H18 9 ABC-IM 740 At1g178404879.m00152 5012.m00058 P0650H04 9 ABC-IM 874 At3g551304843.m00141 3151.m00153 nbeb0015F09b 10 ABC-IM 792 OSJNBa0046P18.17 31432334 At5g607405192.m00132 OSJNBa0041P03 10 ABC-IM 723 OSJNBa0041P03.4 22128707 At1g178404144.m00165 OSJNBa0054A24 11 ABC-IM 612 At1g71960 O6476.m00122 OSJNBb0080P14 12 ABC-IM 572 At1g178406015.m00029 OSJNBb0092K12 12 ABC-IM 560 At1g17840

EPD (ABCG) family , full transportersPDR subfamily (21/15)2680.m00127 P0410E03 1 (ABC-IM)2 1479 P0410E03.4 12382004 PDR10 27368819 At1g155202680.m00129 P0410E03 1 (ABC-IM)2 1444 P0410E03.6 12382006 PDR11 27368817 At1g155202680.m00130 P0410E03 1 (ABC-IM)2 1457 P0410E03.7 12382007 PDR9 27368821 At1g15520

Table 2 (continued)

TIGR ORFname

TIGR synonym BAC Chr. Structure Size NCBI ORF name NCBI gi Jasinski’sname

Jasinski’s gi Best hit AT Orthology

2680.m00133 P0410E03 1 (ABC-IM)2 1450 P0410E03.10 12382010 PDR8 27368823 At1g155202832.m00074 2711.m00131 P0509B06 1 (ABC-IM)2 1353 PDR6 27368827 At2g26910 O4392.m00150 4389.m00142 P0022F10 1 (ABC-IM)2 1509 P0022F10.18 20160553 PDR15 27368811 At1g598704775.m00199 B1045F02 1 (ABC-IM)2 1491 B1045F02.11 21104703 At1g155202427.m00184 2430.m00177 OJ1003_F05 2 (ABC-IM)2 1444 PDR7 27368825 At1g15520 O5875.m00153 2448.m00170 OSJNBa0086N11 2 (ABC-IM)2 1123 PDR4 27368831 At4g152364282.m00175 3862.m00180 P0475F05 2 (ABC-IM)2 1404 PDR2 27368835 At3g534801988.m00113 4871.m00158 OJ1006_C01 6 (ABC-IM)2 1424 PDR12 27368815 At1g598702069.m00241 2009.m00158 OJ1372_D12 7 (ABC-IM)2 1315 OJ1372_D12.114 33146725 PDR5 27368829 At2g363803908.m00138 P0623F08 8 (ABC-IM)2 1324 PDR1 27368837 At1g669505010.m00032 6126.m00257 P0510C09 8 (ABC-IM)2 1507 At1g155204510.m00114 P0466E03 9 (ABC-IM)2 1343 At1g155205958.m00151 OSJNBa0017I18 9 (ABC-IM)2 1089 At1g155205958.m00155 5940.m00135 OSJNBa0017I18 9 (ABC-IM)2 1446 At1g155205958.m00159 OSJNBa0017I18 9 (ABC-IM)2 1120 At1g155204001.m00137 OSJNBb0048O22 10 (ABC-IM)2 1441 OSJNBb0048O22.8 20279475 PDR13 27368813 At2g29940 O3222.m00174 6376.m00141 P0704G09 11 (ABC-IM)2 1394 PDR3 27368833 At1g669504644.m00119 OSJNBa0010J13 12 (ABC-IM)2 1408 At3g53480

CCM family (2/2)5386.m00151 OSJNBb0060E08 4 ABC 295 OSJNBb0060E08.17 32488796 At1g63270 O5618.m00087 6359.m00061 OSJNBa0005K20 12 IM 369 At2g07681 O

m IM 240 ccmC 23495382 orf256 O

Class 2 systemsRLI (ABCE) family (2/3)4277.m00177 5933.m00052 P0462B05 2 ABC2 608 At4g192105030.m00198 6055.m00183 OSJNBa0038F07 11 ABC2 594 RLI 22074759 At4g19210 O

ART familyREG (ABCF) subfamily (5/5)4864.m00149 3848.m00194 OJ1375_D04 2 ABC2 691 At1g64550 O3998.m00221 OJ1004_D04 3 ABC2 710 OJ1004_D04.29 19697429 At3g54540 O5364.m00133 4207.m00141 24K23 4 ABC2 606 24K23.7 19387267 At3g545403917.m00149 3937.m00225

3052.m00134P0705A05 8 ABC2 592 P0705A05.9 28071305 At5g60790 O

3736.m00188 3848.m00194 unknown 11 ABC2 709 At5g64840

Class 3 systemsABCA (ABCA) family, half transporters (6/11)2430.m00156 3841.m00141 OJ1006_A02 2 IM-ABC 964 At3g477801947.m00136 1950.m00160 P0633E08 6 IM-ABC 950 At3g477803090.m00129 OJ1198_B10 8 IM-ABC 974 At3g477303090.m00132 3072.m00151 OJ1198_B10 8 IM-ABC 929 At3g477803090.m00133 3072.m00150 OJ1198_B10 8 IM-ABC 1001 At3g477805181.m00214 P0711F01 9 IM-ABC 875 At3g47780

ABCX family (3/4)2704.m00132 P0409B08 1 CYT 482 P0409B08.15 11275523 At1g32500 O4437.m00148 2849.m00176 P0446G04 1 CYT 544 P0446G04.9 20160641 At4g04770 O6582.m00136 OSJNBb0029K10 3 ABC 311 At3g10670 O

CBY family

Y179subfam

ily(3/3)

2718

.m0013

7P06

65A11

1ABC

225

P06

65A11.16

142095

74At4g33

460

O66

51.m

00117

OSJN

Bb00

47D03

11ABC

413

At5g14

100

O66

32.m

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162

13.m

0018

8P06

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322

At3g21

580

O

MKLfamily(3/3)

4400

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628

30.m

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6P0698

H10

1SSA

370

P06

98H10

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201609

39At3g20

320

O52

84.m

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5OSJN

Ba003

9O18

3ABC

281

OSJN

Ba003

9O18.1

3112

6727

At1g65

410

O55

02.m

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0OSJN

Ba001

0H02

4IM

344

OSJN

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010H

02.16

217408

42At1g19

800

O

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2489

.m0006

8OJ1116_

E04

2ABC

292

At1g03

905

6039

.m0013

6OSJN

Ba000

5J15

3ABC

388

At5g02

270

O

NO

family(2/2)

At2g37

330

P06

99E04

5IM

281

809912

2At2g37

330

O19

79.m

0019

166

58.m

0015

2P06

22F03

6ABC

331

At1g67

940

O

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(Jasinski’sgi).Column11

(BesthitAT)displaysthemost

closely

relatedArabidopsisprotein

(Besthit).AnO

incolumn12

(Orthology)indicates

that

rice

andArabidopsisproteinsarepotential

orthologues.


Class 1 ABC transporters

This class is comprised of all known ABCtransporters involved in the export of variousmolecules from the cytosol to the external mediumor to the matrix of organelles. They carry ABC andIM domains on the same polypeptide chain. Thesetransporters are composed either of one copy ofeach ABC and IM domains fused together (halftransporters) or alternately two tandemly repeatedcopies of such domains (full transporters). Fourclass 1 families have been described in eukaryotes,the DPL (ABCB), the OAD (ABCC), the FAE(ABCD) and the EPD (ABCG) families (see belowfor an explanation of these acronyms). In the DPL,OAD and FAE transporters, the IM domain issituated at the N terminus giving rise to trans-porters with the following organizations IM-ABC or(IM-ABC)2. Transporters of the EDP family has theopposite orientation ABC-IM and (ABC-IM)2 forhalf and full transporters, respectively (Figure 1).

The DPL (drug, peptides and lipid export) or ABCBfamily

Well characterized members of this family areinvolved in the export of a wide variety ofsubstances such as drugs, peptides, lipids, bacterio-cins, cell surface components, etc. Eukaryotespossess only four subfamilies: the HMT (similar toyeast heavy metal transporter), the MDL (similar toyeast MDL proteins) and the TAP (Similar to thehuman transporter associated with antigen presen-tation) subfamilies of half transporters and the p-gPsubfamily of full transporters. In addition, plantsshare with prokaryotes the LLP (lipid A-likeexporters, putative) subfamily and the CCM (cyto-chrome c maturation) family of proteins.The HMT subfamily is comprised of proteins

homologous to the Shizosaccharomyces pombe HMT1protein, a vacuolar phytochelatin transporterinvolved in heavy metal resistance by a sequestra-tion mechanism,20 and to the yeast ATM1 protein,which is essential for the transport of iron/sulphurclusters from the mitochondrial matrix to thecytosol.21 Only one ORF, 3024.m00134 was assignedto this family in rice. It is expected that this proteinwould homodimerize to form a functional trans-porter. The A. thaliana genome contains threehomologous genes encoding HMT subfamily pro-teins: AtATM1 (At4g28630), AtATM2 (At4g28620)and AtATM3 (At5g58270, STA1). This observationsuggests that a triplication event occurred in thecommon ancestor of rice and Arabidopsis. AtATM3is a functional orthologue of the yeast ABCtransporter Atm1p. A mutation in the gene encod-ing AtATM3 results in a light-dependent chlorosisand dwarfism of plants as well as alterations in thestructure of leaf and nuclei. The ectopic expressionof the AtATM2 homologue partially suppresses theAtATM3 mutant phenotype, indicating thatAtATM3, AtATM2, and possibly AtATM1 haveoverlapping functions.22 Like almost all members of

Figure 2.Unrooted N-J tree computed from the multiple alignments of rice ABC domains. ABC domains were alignedby using the ClustalW program and the Neighbour-Joining (N-J) treewas represented. For clarity, the names of rice ORFsand the bootstrap values were omitted. The family:subfamily name is given to highlight the clusters, which wererepresented by using different colors. ABC domains cluster according to families and subfamilies. In most cases, the N-terminal and C-terminal ABC domains (represented by -N and -C after the family name) cluster together. The scaleindicated in the top of the figure represents 20% divergence between sequences.


the HMT subfamily, ORF 3024.m00134 displays amitochondrion targeting signal sequence.

TAP1 and TAP2 proteins are essential for anti-genic peptide presentation to the major histocom-patibility complex (MHC) class I molecules on thecell surface and are necessary for T-cell recognitionin mammals.23 ORFs strongly similar to TAP pro-teins were detected in invertebrates and in plants.7,9

None has been characterized at the functional level.The TAP subfamily is represented in the ricegenome by three ORFs: 2880.m00093, 4620.m00053and 6098.m00123, which are orthologous to A. thali-ana At1g70610, At5g39040 and At4g25450 proteins,respectively. In barleys root cells, a half-ABC pro-tein IDI7, exhibiting a significant homology tohuman TAP1 and TAP2 has been the described.Transiently expressed in tobacco BY-2 cells, a fusionof IDI7 to green fluorescent protein (GFP) waslocalized to the tonoplast. Since the transcriptionlevel of its gene correlated with iron nutritionalstatus, it was proposed that the IDI7 protein isinvolved in the transport of compounds into the

vacuole, facilitating the synthesis/secretion ofphytosiderophores.24

LLP subfamily proteins are found mainly inprokaryotes, but their function is unknown. Mem-bers are similar to the MsbA protein, involved inLipid A trafficking in Gram-negative bacteria.25 Inthe rice genome, ORF 5551.m00113 belongs to thisfamily and is orthologous to A. thaliana ORFAt5g39010, named AtATH12 in.9

In animals, p-gP subfamily transporters areimplicated in several important biological processessuch as multidrug resistance, export of bile acidsand phosphatidyl choline. These full transportersdisplay an (ABC-IM)2 domain organization. Wedetected 24 members of this subfamily in the ricegenome and the A. thaliana genome contains 22.9

ORFs 6372.m00140 and 4739.m00136 seem to lackthe N-terminal IM domain. A corrected sequenceof 6372.m00140 was proposed in Ref. 12 On N-Jtrees computed on the basis of a multiple align-ment of ABC domains of A. thaliana and O. sativa,we distinguished three clusters of proteins (see


Supplementary Data). This suggests that p-gPsubfamily transporters arose by duplication of atleast three genes, which were probably present inthe common ancestor of these organisms. Withineach cluster, the N- and C-terminal ABC domainssegregate independently, suggesting that full trans-porters arose from the duplication and fusion of ahalf transporter.19

Two p-gP subfamily proteins have been function-ally characterized in A. thaliana. Protein At2g36910(AtPGP1, AtMDR1) was shown to be a plasmamembrane protein involved in light-dependenthypocotyl elongation.26 However, its mechanismof action remains unknown. Other groups reportedthat auxin transport activity was greatly impairedin amutant affected in the atmdr11 (At3g28860) geneand in the atmdr11 atpgp1 double mutant.27 Thesemutants exhibited faster and greater gravitropismand enhanced phototropism. These phenotypesresult from the disruption of the normal accumu-lation of the PIN1 auxin transporter, suggesting thatthese MDR proteins function in a mechanism thatconcentrates PIN1 to the basal end of stem cell.28

Recently, a homologue of Atmdr11 in Coptis japonicawas proposed to be involved in the translocation ofberberine from the root to the rhizome.29–31

Remarkably, this protein was shown to catalyzethe influx of berberine in transfected Xenopusoocytes, suggesting that CjMDR1 functioned as anABC importer. In wheat, another homologue wasinduced by aluminum and by inhibitors of calciumflux.32

The OAD (organic anion and drug export) or ABCCfamily

The OAD family is comprised of proteinsinvolved in ion channel regulation, ion channelformation and the efflux of organic anions acrosscellular membranes. Some proteins are linked toresistance to cytotoxic drugs but, in contrast withDPL family proteins described above, drug resist-ance is achieved by the efflux of drugs conjugated orassociated with anionic molecules such as glutha-tione or glucuronide derivatives. This family isfound exclusively in eukaryotes and the proteinshave an (IM-ABC)2 organization. Phylogenetic ana-lyses revealed the existence of three subfamilies.7

The CFTR subfamily, which is comprised ofproteins homologous to the cystic fibrosis trans-membrane conductance regulator in vertebrates,33

the SUR subfamily, which contains homologues ofhuman sulfonylurea receptors,34 and the MRP(similar to multidrug resistance-associated protein)subfamily of glutathione S-conjugate pumps.35

Plants appear to lack members of the two firstfamilies, which are characterized by their involve-ment in the regulation of ion channels. However, if adirect molecular demonstration that plant ABCproteins control ion channel is still lacking, strongevidence supporting this idea has been obtained.First, modulators of the human sulfonylurea recep-tor regulate stomatal movements and ion channels

in plant cells.36–38 Second, it has been suggested thatthe Arabidopsis MRP protein AtMRP5, might beinvolved of ion channel regulation.39 Third, it hasbeen recently shown by heterologous expression inHEK293 cells that AtMRP5 is indeed a sulfonylureareceptor, being moreover involved in the KChomeostasis in Arabidopsis.40

We detected 17 representatives of the MRPsubfamily in the rice genome, as compared to 16in A. thaliana. Only one cluster of MRP ABCdomains is observed onN-J trees (see SupplementaryData), suggesting that MRP proteins descend from asingle gene present in the common ancestor ofA. thaliana andO. sativa. Five members of this familyhave been studied in details in A. thaliana. AtMRP1(At1g30400), AtMRP2 (At2g34660, most similar to5470.m00141), AtMRP3 (At3g13080, most similar to2817.m00139) and AtMRP4 (At2g47800, most simi-lar to 5347.m00078) were demonstrated to partici-pate in the export of various compounds.41–44 Thetranscription of atmrp3 was increased in roots inresponse to a cadmium treatment.45 A null atmrp5mutant exhibited decreased root growth andincreased lateral root formation, and auxin levelsin roots of mutant were increased.39 Recently, it wasshown that AtMRP4 and AtMRP5 are involved inthe control of water use in guard cells.46 In Zea mays,ZmMRP3 expression is co-regulated with theanthocyanin pathway. This pigment acts as a UV-Bsunscreen when deposited into the vacuole afterconjugation with reduced gluthatione. The productof this gene is localized in the tonoplast and isinvolved in the transport of anthocyanin into thevacuole. This is the first report of a plant MRP in thein vivo transport of an endogenous substrate.47

The FAE (fatty acid export) or ABCD family

The FAE (ABCD) family is comprised of halftransporters with an IM-ABC organization. Thefunction of these transporters is poorly understood,but it has been proposed that they could export intoperoxisomes very long chain fatty acids or theenzyme(s) responsible for their degradation.48 Therice genome encodes three FAE transporters(4993.m00055, 5523.m00189 and 5723.m00197) ascompared to two (At1g54350 and At4g39850) inA. thaliana. 5523.m00189, 5723.m00197 andAt4g39850 are full-size transporters, a feature thatis exceptional in this family. It was shown that amutated form of At4g39850, also named PXA1 orPED3, determined in Arabidopsis seeds an impairedfatty acid catabolismand adefect in germination.49–52

4993.m00055 and At1g54350 are half-size trans-porters, and blast analyses show that there arestrongly related to cyanobacterial ABC transportersputatively involved in the export of toxic cyclicpeptide-polyketides such as microcystins.53

The EPD (eye pigment precursors and drug export)or ABCG family

The EPD family is subdivided into two


subfamilies: the WHITE (similar to DrosophilaWhite protein) subfamily of half transporters andthe PDR (similar to yeast pleiotropic drug resistanceproteins) subfamily of full transporters. WHITEsubfamily proteins were detected in the threekingdoms of life. In contrast, PDR subfamilyproteins are not represented in the genomes ofanimals and prokaryotes.

WHITE subfamily proteins are homologous theWhite, Brown and Scarlet proteins of diptera, whichare involved in the export a metabolic intermediate(such as 3-hydroxy kynurenine) from the cytoplasminto the pigment granules of the Drosophila eyecells.54 In mammals, ABCG2 (MXR, BCRP) isassociated with anthracyclin drug resistance whenoverexpressed in certain cell lines.55,56 Three otherhomologues have been shown to modulate choles-terol and lipid metabolism. ABCG1 (ABC8) ishighly induced in lipid-loaded macrophagessuggesting a role in cholesterol and phospholipidtrafficking.57,58 Mutations in the human ABCG5 andABCG8 transporters cause phytositosterolemia, aninheritable disease resulting in the elevation ofplasma levels of plant sterols.59 We identified 30members of this family in rice. 29 proteins arepresent in the thale cress genome, and among these,five are potential orthologues of rice proteins(Table 2 and Supplementary Data).

The PDR subfamily was first detected in yeastand is well represented in plants. In fungi, membersof this subfamily are involved in multiple drugresistance, mediating resistance to a wide varietyof structurally unrelated toxic products andsteroids,60,61 but also to weak organic acids62 andto oligomycin.63 In pathogenic fungi, PDR sub-family proteins participate to resistance againstantifungal agents64 and have been implicated inpathogenesis65 and virulence.66 In plants, theSpirodela polyrrhiza TUR2 gene is induced followingtreatment with the steroid-like hormone, abscisicacid, and that this induction can be repressed by theadenine-derived hormone kinetin. Furthermore,TUR2 gene expression is induced by environmentalstress treatments such as low temperature and high-salt.67 Recently, it was shown that the product ofthis gene is localized to the plasma membrane andthat expression of this protein in Arabidopsis leadsto the acquisition of resistance to the diterpenoidantifungal agent sclareol.68 A very similar gene(NpABC1) in Nicotiana plumbaginifolia was inducedby sclareol and sclareolide, leading cells to excrete alabeled synthetic sclareolide analogue.69 In Nicoti-ana tabacum, NtPDR1 might be involved in thegeneral defence response to microbial elicitors.70 Inrice, gene OsPDR9 is induced by heavy metals,hypoxic stress and redox perturbations.71 Weidentified 21 ORFs of this family in the ricegenome, as compared to 15 in A. thaliana, five ofwhich could be considered as potential orthologues(Table 2 and Supplementary Data). The rice ORF2427.m00184 (PDR7) and the A. thaliana At1g15520(PDR12) protein72 are most similar to spTUR2.

The CCM (cytochrome c maturation) family

a- and g-proteobacteria, deinococci and mito-chondria of plants and protozoa share a cytochromec biogenesis and maturation machinery that includeproteins that have been proposed to constitute anABC transporter: the CcmA ABC protein and theCcmB and CcmC IM proteins. In prokaryotes, theCcmA, CcmB and CcmC proteins are encoded in anoperon ccmABCDEFGH whose genes are all essen-tial for cytochrome c biogenesis.73 However, theinvolvement of CcmC in this ABC transporter isstill a matter of debate.74 In O. sativa, thenuclear genome encodes ORFs 5386.m00151 and5816.m00087 that are homologous to CcmA andCcmB, respectively. The mitochondrial genomeencodes a copy of CcmC named ORF240.75 Weidentified in the A. thaliana genome At1g63270 (aCcmA homologue) and At2g07681 (a CcmB homo-logue). These proteins may constitute an ABCtransporter together with the CcmC homologueORF204 encoded in the mitochondrial genome.76 Inaddition, the mitochondrial genome encodesORF256, which is identical with At2g07681. It wasshown recently that wheat CcmB is targeted to theinner membrane of mitochondria,77 therefore giv-ing credence to the hypothesis that plant andbacterial proteins play similar roles.

Class 2 ABC proteins

Class 2 proteins have a similar domain organiz-ation. They are constituted of two ABC domainsfused together and they apparently lack IMdomains. It is therefore very probable that theseproteins would not be transporters. Two class 2protein families have been detected in plants: theRLI (ABCE) family and the REG (ABCF) subfamily.On N-J trees, the N- and C-terminal ABC domainsof RLI family proteins segregate independently,suggesting that they arose from the duplication andfusion of a gene encoding the ABC domain in thecommon ancestor of Rice and Arabidopsis. Thesame feature is observed for ART:REG subfamilyproteins (see Supplementary Data).

The RLI (Rnase-L inhibitor) or ABCE family

RLI family proteins display a ferredoxin iron–sulphur centre in the N-terminal ABC domain. TheRNAse-L inhibitor (RLI) has been implicated inanimals in the regulation of the transcription ofsomemRNAs.78 Recently, human RLI was shown tobe essential for post-translational events in imma-ture HIV-1 capsid assembly.79 Two ORFs4277.m00177 and 5030.m00198, highly similar toRLI are encoded in the rice genome. Three RLIfamily proteins were found in the A. thalianagenome, one of these (At40300 or AtNAP15) lacksone of the two ABC domains, which are character-istic of this family. The role of RLI proteins was notinvestigated in plants.


The REG (regulation of gene expression) or ABCFsubfamily

The ART family of ABC proteins is subdividedinto two subfamilies: the ARE subfamily, which isexclusively found in eubacteria and involved in theresistance to antibiotics by an unknown mechan-ism; and the REG subfamily, which is found in botheubacteria and eukaryotes.7 Some REG subfamilyproteins have been implicated in the regulation ofgene expression, such as the Agrobacterium tume-faciens ChvD protein80,81 and the yeast GCN20protein.6 We detected five REG subfamily proteinsencoded in the genomes of rice and A. thaliana.

Class 3 ABC systems

Most class 3 systems are involved in the uptake ofsmall molecules in prokaryotes and they constitutethe so-called binding protein-dependent trans-porters. However, some class 3 systems seem todeviate from this rule, since they have beenreported to be involved in the resistance towardsdrugs. For instance, the DrrA–DrrB system medi-ates resistance to doxorubicin in Streptomycesfradiae.82 These systems belong to the DRR familyof ABC transporters.7 Noticeably, the ABC domainsof the ABCA subfamily, which is found exclusivelyin eukaryotes, cluster with ABC proteins of the DRRfamily.7,83

The ABCA (similar to human ABCA1 protein)subfamily

In animals, proteins of the ABCA subfamily havebeen implicated in the severe inherited Stargardt’sand Tangier’s diseases.84,85 The ABCA1 protein isinvolved in the modulation of cholesterol andlipoprotein metabolism, and the ABCA4 (ABCR)protein in retinol cycling. Transporters of thissubfamily are found in plants but their physio-logical role is presently unknown. We detected sixABCA transporters in rice, as compared to 12 inA. thaliana (Table 2 and Supplementary Data). Mostof the Arabidopsis proteins are strongly similar toeach other and appear to be clustered on chromo-somes 3 and 5. This may indicate that they arosefrom several duplication events after the separationof Arabidopsis (dicot) and rice (monocot) ancestors.In contrast to animal ABCA full transporters, plantABCA proteins are mostly half transporters, withan IM-ABC organization. They were proposed toconstitute a specific subfamily named ATH.9 How-ever, a full-length ABCA transporter At2g41700(AtABCA1) was detected in the A. thaliana genome,but without apparent counterpart in the yeast andin the O. sativa genomes. We failed to detect ahomologue of At2g41700 in rice by using tblastnsearches. In plants, where more than 60 differentsterols have been identified, each tissue is able toform its own sterols, regulating membrane fluidityand membrane-bound enzyme activities.86 A role ofAtABCA1 in sterol metabolism is an attractive

hypothesis. As revealed by expression profileanalysis, AtABCA1 is weakly but ubiquitouslyexpressed and induced in response to several planthormones. In addition, preliminary experimentswith knockout plants suggest a role of this trans-porter in lipid homeostasis (D. Pamard,N. Leonhardt and C. Forestier, unpublished results).

The ABCX (similar to C. paradoxa ABCX protein)family

These systems are found in the genomes ofseveral bacteria and archaea, and on the plastidgenome of red algae. They consist of one gene sufCencoding the ABC protein, which is almost alwaysassociated with two genes (sufB, sufD) encodingconserved soluble proteins (CYT). In most eubac-teria, these three genes are organized in an operon,which contains genes (sufA, sufE, sufS) encodinghomologues of enzymes known to be involved inthe biogenesis of iron/sulphur centres.87,88 Veryrecently, it was shown that bacterial CYT proteinsinteract with the ABC protein to form a complexnecessary for the activity of enzymes containingoxygen-labile iron/sulphur centres under oxidativestress conditions.89 This ABC systemmay constitutea minor pathway of iron/sulphur centre assemblyin bacteria.90 Three rice ORFs 2704.m00132 (CYT),4437.m00148 (CYT) and 6582.m00136 (ABC),encoded a canonical ABCX system. These are poten-tial orthologues of ORFs At1g32500 (AtNAP6),At4g04770 (LAF6, AtABC1, AtNAP1) andAt3g10670 (AtNAP7), respectively (Table 2). Arabi-dopsis contains an additional CYT proteinAt5g44316. A genetic screen identified the A. thali-ana CYT protein AtNAP1, whose inactivationdetermines a long hypocotyl phenotype and theaccumulation of protoporphyrin IX. It wassuggested that a functional AtABC1 is requiredfor the transport and correct distribution of thiscompound, which may act as a light-specificsignalling factor involved in coordinating inter-compartmental communication between plastidsand the nucleus.91 Recently, the same groupdemonstrated that plastid-localized AtNAP7could partially rescue growth defects in an Escher-ichia coli sufC mutant during oxidative stress.92

Moreover, AtNAP7 can interact with AtNAP6, aplastidic Arabidopsis SufD homologue. BecauseArabidopsis plastids also harbour SufA, SufB, SufS,and SufE homologs, plastids probably contain acomplete SUF system, which may be involved inplastidic Fe–S cluster maintenance and repairduring embryogenesis.92 Interestingly, in Plasmo-dium falciparum and related parasites, the vestigialbut biosynthetically active plastid named apico-plast contains also a copy of a gene encoding a CYTprotein.93

The MKL (similar to the M. leprae MKL protein)family

This family, which is found mainly in


Gram-negative bacteria, is comprised of systemswith unknown function. A typical system iscomposed of genes encoding one ABC, one IMand two SSA proteins displaying a putative signalsequence. In bacteria, we proposed that MKLsystems might be implicated in the maintenanceof bacterial outer cell surface integrity.7 MKLsystems are apparently not restricted to prokaryotessince we detected typical MKL systems on thenuclear genomes of O. sativa and A. thaliana. ORFs5284.m00165 and At1g65410 are homologous toMKL family ABC ATPases, ORFs 5502.m00180 andAt1g19800 are IM proteins, and ORFs 4400.m00136and At3g20320 are homologous to SSA proteins(Table 2 and Supplementary Data). Program PSORTpredicts that MKL family ORF’s display potentialmitochondrial addressing sequences.

The Y179 (similar to M. janaschii Y179 protein)subfamily

This subfamily of ABC systems is found mainlyin prokaryotes. A typical system consists of twoABC proteins and one IM protein. Although beingwell represented in bacterial genomes, thefunction of Y179 subfamily systems have not beenexperimentally investigated. In O. sativa, ORFs2718.m00137 (ABC), 6651.m00117 (ABC) and6632.m00181 (IM) are predicted to constitute acanonical system. These ORFs are putative ortho-logues of A. thaliana ORFs At4g33460, At5g14100and At3g21580 respectively (Table 2 and Supple-mentary Data). At4g33460 and At5g14100 areputatively located in the stroma of chloroplasts, asindicated by PSORT predictions.

Unclassified ABC systems

The NO family

During the classification of universally foundABC systems, we found that a number of solubleABC proteins with unknown function were appar-ently unrelated to existing families. These weregrouped into the so-called NO family. The O. sativagenome contains one member of these ABCATPases (ORF 1979.m00191) and that of A. thalianahas 2: At1g67940 and At5g14100.

The ADT subfamily

Some members of the NO family could begrouped in a homogeneous cluster according tosequence similarity. The size of this subfamily isvery limited since only six members, from Arabi-dopsis and fungi are deposited in the databases.The prototype is the yeast protein YFL028C orCAF16p, which is part of the CCR4–NOT complexknown to participate in both positive and negativeregulation of certain genes.94 The specific role ofCAF16 in this complex is presently unknown. TwoORFs belonging to this subfamily are found on therice genome: 2489.m00068 and 6039.m00136. They

are orthologous to A. thaliana proteins At1g03905and At5g02270, respectively. The genome ofA. thaliana encodes an additional protein At5g44110(see Supplementary Data). This gene, AtNAP2, ishighly similar to At1g03905 and induced by sugar.In roots, tissue expression of this gene is alsodependent on the light conditions suggesting itsinvolvement in the plant development (E. Marin,F. Divol, N. Bechtold, A. Vavasseur, L. Nussaumeand C. Forestier, submitted).

The A. thaliana ABC proteome revisited

In the course of the comparison of ABC proteinsbetween rice and thale cress, we corrected andupdated the inventory of A. thaliana ABC proteins(see Supplementary Data). We noticed thatsome A. thaliana proteins were mistakenly anno-tated as ABC transporters in a published paper9 andin the TAIR database. Proteins At2g39190,At2g40090, At4g01660 and At5g64940 were namedAtATH8, AtATH9, AtATH10 andAtATH13 in Ref. 9and annotated as ABC transporter-related in TAIR.In fact, these proteins are homologous to the yeastABC1 gene (for activity of cytochrome bc 1complex). When overexpressed, this gene sup-presses a cytochrome b mRNA translation defect.Inactivation of this gene led to a respiratory defectdue to the arrest of electron transfer in thecytochrome bc 1 complex.95 The Arabidopsis genesmentioned above are also homologous to the E. coliaarF gene, which is required for ubiquinoneproduction.96 ABC1, AarF and their homologuesin Arabidopsis, when compared to the ABCproteins present in the ABCISSE database, gavescores equal or lower than 32 bits with E valueshigher that 0.28, suggesting that they are totallyunrelated to ABC proteins. We assigned someproteins named NAP (for non-intrinsic ABCproteins) in9 into identified bacterial families. Forinstance, AtNAP1 (At4g04770), AtNAP6 (At1g32500)and AtNAP7 (At3g10670) belong to the ABCXfamily. Other NAP proteins were found to befragments of proteins of the MRP (AtNAP5,At1g71330), RLI (AtNAP15, At4g30300) and EPD(AtNAP12, At2g37010) families. Protein At5g03910,named AtATH12 in Ref. 9 belongs to the DPL:LLP(ABCB) subfamily. In summary, we identified inA. thaliana 120 proteins that carry a characteristicATP-binding cassette domain, and eight proteinsthat are predicted to be interacting partners of ABCproteins. We have not included in this inventory theproteins of the SMC family, since they contain alarge coiled-coiled domain between the Walkermotif A and the signature that render difficult thegeneration of multiple alignments and theinterpretation of trees.

Conclusions

Our work provides an overview of ABC proteinsof O. sativa and an updated comparison of the ABC


proteomes of rice and thale cress, the two plantswhose genome sequence is finished to date. Thegenomes of O. sativa and A. thaliana encode respec-tively 121 and 120 ABC proteins. Sequenced animalgenomes are comprised of about 50–60 ABCproteins.18,97 The difference is due to a significantincrease in the number of pg-P, MRP, and WHITEsubfamilies, and to the specific occurrence of PDRsubfamily transporters in plants. Since severalmembers of these subfamilies have been describedto participate in resistance towards xenobiotics, theincrease of ABC transporters might be related to thefact that plants are immobile organisms that couldnot escape environmental stresses.

The total number of genes encoding ABCproteins was nearly identical in the two species,despite of large differences in genome size and genecontent. A similar result was obtained from thecomparison of P-ATPases.98 However, the distri-bution of ABC proteins into families and sub-families is quite different: O. sativa has more PDRproteins and less ABCA proteins than A. thaliana.

Only about 30 ABC proteins are predicted toconstitute orthologous sequences in O. sativa andA. thaliana. Large p-gP and WHITE subfamilies arepopulated with a rather small number of putativeorthologues, leading to the notion that proteins ofthese subfamilies diverged by duplication afterspeciation events. In contrast, members of smallsubfamilies are highly conserved and constitutepotential orthologues, whose parents were prob-ably present in the common ancestor of dicots andmonocots. Moreover, all types of eukaryote ABCsystems are found in the two species, except the fulllength ABCA transporter we failed to detect in rice.

Plants share with bacteria, and most notablycyanobacteria, a specific set of ABC families, whichis not yet described in animal genomes: the ABCX,MKL, CCM and CBY families. ABCX and CBYproteins are located or predicted to be located inchloroplasts. CCM and MKL proteins have beenshown or predicted to be addressed to the mito-chondrion. Since it was proposed that several ricemitochondrial and nuclear sequences probablyoriginated from the plastid ancestor genome,75

this set of systems might be reminiscent of theendocytosis event that led to the generation ofchloroplasts.17

Materials and Methods

The Whole Rice Genome Automated AnnotationDatabase was downloaded from “The Institute ofGenomic Research” (TIGR) FTP site†. This databasecontains all rice BAC/PAC sequences (phase 2 andphase 3) in the HTGS division of GenBank as well as allfinished rice BACs in the PLANT division of GenBank.With the exception of PLANT division TIGR’s BACs,these sequences were processed through an automatedannotation pipeline. BAC clones are fromCUGI (Clemson

† ftp://ftp.tigr.org/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/BAC_PAC_clones

University Genomics Institute), Monsanto, or theJapanese Rice Genome Program. The sequences ofA. thaliana ABC systems were retrieved from theABCISSE database, which comprises 13,029 sequencesof ABC proteins and their partners.7

The rice predicted ORFs were compared to the wholeset of sequences in the ABCISSE database. Comparisonswere made using the Blastp program.99 Blast scoreshigher than 400, corresponding to a computed E value ofabout e-120 were considered as significant. This Blastsearch against a database of pre-classified ABC proteinsallowed to sort the ORFs according to families and sub-families of ABC proteins described in.7 Very short ORFs ofless than 150 residues and ORFs interrupted by atransposon were excluded from the analysis. Identicalsequences present on two different BACswere consideredas the same one by checking that the two BACs dooverlap. ABC domains were extracted from proteinsequences by performing Pfam HMM searches‡. Theclassification was refined by performing a multiplealignment of the ABC domains and by constructing aneighbour-joining (N-J) tree by using program ClustalWwith the default settings.100 Bootstrapping was doneusing ClustalW. Trees viewed with the TreeViewapplication.101

Detection of putative orthologous sequences was doneby performing two-way Blastp searches. These ortholo-gous genes may share a conserved function that waspresent in the common ancestor of the two organisms.Rice proteins were used to search for hits in a library ofArabidopsis proteins and, conversely, each Arabidopsisprotein was used to search for hits in a library of riceproteins. If a rice protein has the best score with a givenArabidopsis protein and vice versa, they may constitute acouple of orthologous sequences. Potential orthologyrelationships were also inferred from the examination ofN-J trees computed from multiple alignments of rice andArabidopsis ABC domains. Two sequences were assumedto be orthologous when they gave symmetrical best hits intwo-way Blastp searches and when they constitute a pairon the same branch on N-J trees.Organelle sorting signals were detected by using

programs PSORT and iPSORT.102

Acknowledgements

We are indebted to Arnould Savoure and MartineBoccara for critical reading of the manuscript. Thiswork was initiated in the Unite de Programmationmoleculaire and Toxicologie Genetique leaded bythe late Maurice Hofnung (Institut Pasteur).

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2004.07.093.

‡ http://pfam.wustl.edu

http://dx.doi.org/doi:10.1016/j.jmb.2004.07.093

http://dx.doi.org/doi:10.1016/j.jmb.2004.07.093

http://ftp://ftp.tigr.org/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/BAC_PAC_clones

http://ftp://ftp.tigr.org/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/BAC_PAC_clones

http://pfam.wustl.edu


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Edited by I. B. Holland

(Received 3 May 2004; received in revised form 23 July 2004; accepted 27 July 2004)

inventory and comparative analysis of rice and arabidopsis atp-binding cassette (abc) systems

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