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The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf

The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding... Background: Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs) that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. Results: Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7). Conclusion: Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains. Page 1 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 Background Guanine Nucleotide Exchange Factors (GEFs) are obliga- Bacteria tory components of signaling cascades regulated by small GTP-binding proteins (called small G proteins hereafter). RalF Insects Their biochemical activity is to stimulate the dissociation Mammals Nematode of the tightly bound GDP nucleotide from the small G protein in response to cellular signals. Thereby, they favor FBS the binding of the more abundant cellular GTP, organiz- ing the active conformation of the small G protein which SYT1 BRAG can recruit its effectors (reviewed in [1]). Each small G SYT2 EFA6 protein family features its own ensemble of GEFs charac- CYH terized by a conserved catalytic domain responsible for GBG nucleotide exchange, which is generally combined with (GBF/BIG) non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of Fungi the Arf family, which are major regulators in membrane trafficking (reviewed in [2]), the exchange domain is a conserved module of ~200 amino acids called the Sec7 domain [3]. Its biochemical (reviewed in [4]) and struc- TBS Plants tural [5,6] mechanisms have been investigated in detail. Alveolates Remarkably, the Sec7 domain is the only domain that is conserved in all ArfGEFs (reviewed in [7,8]) and it is to some extent interchangeable between species [9]. In con- Venn diag according to found Figure 1 ram the of the nin species wh e Sec7 ere each -containi subfamily ng subfamili has been es sorted trast, little is known about the functions of the other Venn diagram of the nine Sec7-containing sub- domains, which are likely to determine intracellular local- families sorted according to the species where each subfamily has been found. The TBS subfamily was identi- ization of ArfGEFs and their responsiveness to specific fied in this study. The BIG and GBF subfamilies are merged in signals. a higher order subfamily (GBG), and are the only subfamily common to all eukaryotes. As for most small G proteins, Arf family members are out- numbered by ArfGEFs in many species. In humans for instance, 5 Arf proteins have been identified, and there are at least 13 proteins carrying a Sec7 domain, of which most have been characterized as bona fide ArfGEFs (reviewed in [7,8]). Thus an individual Arf protein may be activated by active on Arf6 at the plasma membrane where they may more than one GEF, emphasizing that essential aspects in function in the crosstalk of membrane traffic, cytoskele- building up the Arf responses may be encoded by the ton dynamics and signalling in endosomal pathways modular architecture of their GEFs. Sequence similarity in (reviewed in [17]). Most members of the BIG and GBF the non-catalytic regions forms the basis for the classifica- subfamilies characterized so far function in vesicular traf- tion of ArfGEFs into subfamilies. 8 subfamilies are cur- ficking at the Golgi [12,14,18], except for BIG2, which rently identified in eukaryotes with sizes ranging from also localizes on recycling endosomes [19], and GNOM small (~40–80 kD including CYH, EFA6 and FBS), to which acts in the endosomal recycling pathway [11]. medium (~100–150 kD, including BRAG/LONER, SYT1, SYT2) and large (~170–200 kD) ArfGEFs (reviewed in The domain architecture of non-catalytic regions of Arf- [7,8]). Large ArfGEFs comprise two subfamilies which we GEFs, hence their contribution to specific aspects of the will refer to as the BIG and GBF subfamilies after the name build-up of the Arf response, is essentially not established of their human representatives. The GBF subfamily except for those ArfGEFs with domains found in other includes human GBF1 [10], Arabidopsis GNOM [11] and classes of cellular regulators. The known domains include Saccharomyces Gea1 and Gea2 [12], the BIG subfamily membrane-interacting PH domains in the CYH (reviewed human BIG1 and BIG2 [13,14] and yeast Sec7p [15]. An in [20]), EFA6 [21] and possibly BRAG/LONER[22] sub- additional subfamily called RalF is found in Rickettsie and families, and a putative F-box in the FBS subfamily [23], a Legionella bacteria, likely acting on an host Arf pathway protein-protein interaction domain that has been [16]. Analysis of the CYH and EFA6 subfamilies, present involved in the recruitment of substrates to the SCF ubiq- only in multicellular animals, and that of the large Arf- uitination machinery. Coiled-coil structures have also GEFs, found in all eukaryotes, have yielded most of the been predicted in the N-terminus of the CYH subfamily functional data currently available. CYH and EFA6 are and in the C-terminus of the EFA6 subfamily. In CYH, Page 2 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 Table 1: BIG and GBF protein sequences used in this study. Species Protein name Accession Number Size in amino acids Metazoa Ag Q7PWN5 EAA14874 1522 Q7PXQ7 EAA00837 1285 Ce Q9XWG5 NP_493386 1628 Q9XTF0 NP_499522 1820 Dm Q9VJW1 AAF53331 1653 Q9V696 AAF58532 1983 Hs BIG1 Q9Y6D6 1849 BIG2 Q9Y6D5 1785 GBF1 Q92538 1859 Rn BIG1 XP_232614 1987 BIG2 Q7TSU1 1791 GBF1 XP_347197 1883 Fungi Ca EAL04295 EAL04295 1839 EAL02873 EAL02873 1015 Nc Q7SAX4 EAA33549 1940 Q7SAL8 EAA33457 1626 Sc SEC7 P11075 2009 GEA1 P47102 1408 GEA2 P39993 1459 Sp SC71 Q9UT02 1811 SC72 Q9P7V5 1822 Q9P7R8 NP_596613 1462 Viridiplantae At At1g01960 Q9LPC5 1750 At3g43300 NP_189916 1728 At3g60860 Q9LZX8 1793 At4g35380 O65490 1711 At4g38200 NP_195533 1698 GNOM Q42510, At1g13980 1451 GNL1 Q9FLY5, At5g39500 1443 GNL2 NP_197462, At5g19160 1375 Os 9631.m01366 Q8S565 1789 9630.m00920 Q9XGN9 1687 9634.m04029 - 1704 9635.m03752 - 1680 9631.m04495 - 1456 9630.m02122 - 1396 9632.m00175 Q7XT11 1407 Alveolata Pt GGG1 CR533425 1615 GGG2 CR533424 1628 GGG3 CR533423 1598 GGG4 CR533422 1599 GGG5 CR533421 1435 Unnamed sequences are designated by their NCBI accession number, AGI (Arabidopsis Genome Initiative) locus numbers for At and TIGR model temporary IDs for Os. BIG and GBF subfamily members are in normal and bold characters respectively, except for Pt members which have not been assigned to either subfamily (see also Figure 8). Species abbreviations are: Ag, Anopheles gambiae; Ce, Caenorhabditis. elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens; Rn, Rattus norvegicus; Ca, Candida albicans; Nc, Neurospora crassa; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; At, Arabidopsis thaliana; Os, Oryza sativa; Pt, Parameciumtetraurelia). they are involved in dimerization [3], recruitment of part- been the subject of many investigations, their architecture ners [24] and Golgi targeting [25], and in actin remode- is barely described, making it difficult to associate bio- ling functions in the case of EFA6 [21]. On the other hand, chemical activities with their molecular structure. although the functions of BIG and GBF subfamilies have Page 3 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 Here we investigate the domain architecture in the BIG GNOM Gea2 Gea1-2 GBF1 2 4 5 7 and GBF subfamilies, including all sequences from fully Cyp5 Drsp2 Gmh1p p115 annotated eukaryotic genomes and our novel annotation of Sec7-containing proteins from the Paramecium tetraure- HUS HDS1 HDS2 HDS3 DCB Sec7 DCB HUS Sec7 HDS1 HDS2 HDS3 lia alveolate. Sequence comparisons combined with sec- ondary structures and splicing patterns analysis identifies BIG1 BIG1-2 BIG1-2 BIG2 five novel domains that are conserved between BIG and 8 1 3 MyosinIXB 6 GABA Rβ FKBP13 PKA GBF subfamilies, thus unifying them as a higher order subfamily with a probable common ancestor. Our analy- The com s Figure 2 ubfamilies mon domain architecture of the BIG and GBF sis of Sec7-domain containing sequences from Para- The common domain architecture of the BIG and mecium also introduces a novel subfamily of ArfGEFs GBF subfamilies. From N- to C-terminus : DCB , HUS, unique to alveolates, which we call TBS (TBC-Sec7). Sec7, HDS1, HDS2, HDS3. Linker regions of variable length and sequence are shown in grey, with alternate splicing sites in human GBF1, BIG1 and BIG2 in black, white and grey dia- Results and discussion mond shapes respectively. Interactions reported in the litter- A conserved domain architecture in BIG and GBF ature are indicated in boxes of width corresponding to the subfamilies mapped regions, except for myosin IXb interaction which The BIG and GBF subfamilies are the only ArfGEFs sub- was studied only with full-length BIG1. Arrows indicate pre- families common to all eukaryotes [8] and the sole Arf- 1 2 3 dicted Protein kinase A-anchoring motifs. [45]; [27]; [46]; GEFs present in plants [26] (Figure 1). They are therefore 4 5 6 7 8 [47]; [48]; [49]; [50]; [51]. possible representatives of ancestral ArfGEF functions and may provide a model to understand the nature and imple- mentation of activities associated with the exchange func- tion carried by the conserved Sec7 domain. However, domain 'hunting' in BIG and GBF subfamilies was com- plicated by the facts that the Sec7 domain is their only which is also found in the yeast protein Ysl2p [28], all of domain that could be identified from known domain rep- them are unique to these two ArfGEFs subfamilies within ertoires, and that their poorly characterized non-catalytic the detection limits of the BLAST search. The HUS domain regions were not found outside these ArfGEF subfamilies. features a remarkably conserved N(Y/F)DC(D/N) motif, Alternatively, we based our search of candidate structural which we call the HUS box, which is predicted to locate in domains in BIGs and GBFs on the bioinformatics analysis a loop where it may be available for functional interac- of their own sequences, taking advantage of the growing tions (Figure 4). The N- and C-terminal ends of BIGs and number of sequences from fully annotated genomes from GBFs are more variable, including an unusual enrichment mammals, insects, plants, nematode, and fungi, to which in Asp/Glu or Pro residues in some members. A specific we included our annotation of Sec7-containing proteins feature of BIG members is that their C-terminus is in gen- from the newly sequenced genome of Paramecium. eral less variable than that of GBFs, and is predicted with a significant amount of secondary structures. In contrast Multiple alignments of 42 sequences (listed in Table 1) to the predicted structural domains, the intervening revealed that the BIG and GBF subfamilies share an unex- regions are highly variable in length and do not yield pected conserved architecture (schematized in Figure 2). aligned sequences. Analysis of their amino-acid composi- Two homology domains are located in N-terminus of the tion reveals a paucity of hydrophobic residues which is Sec7 domain – the DCB (~150 aa) and HUS (Homology predicted to associate with an essentially unfolded Upstream of Sec7, ~170 aa) domains – and three in its C- conformation, suggesting that they act as linkers to tether terminus -the HDS1 (Homology Downstream of Sec7, the functional domains together. ~130 aa), HDS2 (~160 aa) and HDS3 (~120 aa) domains (Figure 3,4,5,6,7). In Arabidopsis GNOM, the DCB To further investigate the predicted organization of BIGs domain is included in an N-terminal region of ~250 resi- and GBFs in 6 conserved helical domains connected by dues involved in dimerization and possibly binding to variable linkers, splicing patterns of human BIGs and cyclophilin5 and called the Dimerization/Cyclophilin GBFs were analyzed in the large number of cDNAs and Binding region [27], after which the new domain was ESTs in the databases that correspond to GBF/BIG tran- named (Figure 3). All domains are predicted to have a scripts. This revealed the use of alternate splice donor and high content of α-helices that co-align in the multiple acceptor sites predicted to yield proteins with insertions sequence alignments, reinforcing the prediction of and deletions ranging from 1 to 38 residues, and a sequence similarities and suggesting that these domains number of splice variants arising from exon skipping form folded structural units that may share common func- (Table 2). Strikingly, all observed sequence variations tional features. Except for the N-terminal DCB domain occur in regions identified as linkers between conserved Page 4 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 1 1020 3040 50 BIG1_HUMAN/70-228 SKTNFIEADKYFLPFELACQS..KCPRIVSTSLDCLQKLIAYGHLTGNAPD...................STTPG BIG2_HUMAN/58-216 PKANFIEADKYFLPFELACQS..KSPRVVSTSLDCLQKLIAYGHITGNAPD...................SGAPG Q9VJW1_DROME/72-230 DAASIINAETYFLPFELACKS..RSPRIVVTALDCLQKLIAYGHLTGSIQD...................SANPG Q9XWG5_CAEEL/69-227 AGGTAVEADRYFLPFELACNS..KSPKIVITALDCLQKLIAYGHLTGRGAD...................ISNPE 3g60860_ARATH/77-233 IEYSLADSELIFSPLINACGT..GLAKIIEPAIDCIQKLIAHGYIRGESDP...................SGGAE 1g01960_ARATH/72-228 AEYSLAESEIILSPLINASST..GVLKIVDPAVDCIQKLIAHGYVRGEADP...................TGGPE 4g35380_ARATH/62-214 SGLAASDADSVLQPFLLSLET..AYSKVVEPSLDCAFKLFSLSILRGEIQS.....................SKQ 4g38200_ARATH/61-215 FGLTTSDADAVLQPLLLSLDT..GYAKVIEPALDCSFKLFSLSLLRGEVCS.....................SSP 3g43300_ARATH/97-252 HTLGGAEVELVLKPLRLAFET..KNLKIFDAALDCLHKLIAYDHLEGDPGL...................DGGKN SEC7_YEAST/267-445 NNPHYVDSILVFEALRASCRT..KSSKVQSLALDCLSKLFSFRSLDETLLVNPPDSLASNDQRQDAADGITPPPK GGG2_PARTE/36-189 QIKDFYDANHLLKVYQQCIES..KQAKLIELALFDIKNIVDQGYLAGEQII......................GE GBF1_HUMAN/54-215 TELSEIEPNVFLRPFLEVIRSEDTTGPITGLALTSVNKFLSYALIDPTHEG.......................T Q9V696_DROME/56-217 EDLRQIEPQVFLAPFLEVIRTADATGPLTSLALASVNKLLSYGLIDPTSPN.......................L Q9XTF0_CAEEL/56-248 ADLADMNPQTYLSPFLDVIKAQNTNGPITEAALAAVAKFLNYGLIDASSIK.......................A GNOM_ARATH/83-245 QPWHTISPMLYLQPFLDVIRSDETGAPITSIALSSVYKILNLNVIDQNTAN.......................I GNL1_ARATH/81-243 SNWQYVDPRLYIQPFLDVILSDETGAPITGVALSSVYKILTLEVFTLETVN.......................V GNL2_ARATH/67-228 QDWRTIDPSVYLSPFLEVIQSDEIPASATAVALSSILKILKIEIFDEKTPG.......................A GEA2_YEAST/87-248 KNLDNIDSLTILQPFLLIVSTSSISGYITSLALDSLQKFFTLNIINESSQN.......................Y GEA1_YEAST/90-251 KGLDSLNALELLKPFLEIVSASSVSGYTTSLALDSLQKVFTLKIINKTFND.......................I .............................. 60 70 80 90 BIG1_HUMAN/70-228 KKLIDRIIIC ET CG F...QGPQ.TDEGVQLQ..............................IIL KA LTAVTS..Q BIG2_HUMAN/58-216 KRLIDRIVIC ET CS F...QGPQ.TDEGVQLQ..............................IIL KA LTAVTS..P Q9VJW1_DROME/72-230 HLLIDRIVIC VT YG F...SGPQ.TDEAVQLQ..............................IIL KA LTVVTS..Q Q9XWG5_CAEEL/69-227 RKLIDRIVIP EA CA F...LGQG.TDETVLLQ..............................LIV KA LAVVLS..T 3g60860_ARATH/77-233 SLLLFKLIVC DS CK H...D..L.GDESIELP..............................VLL KT LSAINS..I 1g01960_ARATH/72-228 ALLLSKLIIC ET CK H...E..L.DDEGLELL..............................VLL KT LTAVTS..I 4g35380_ARATH/62-214 DSILFKLVVV NA SK G.....AI.AEEPIQLA..............................VLL RV LAAVRS..P 4g38200_ARATH/61-215 DSLLYKLIIV HA CK C.....GI.GEESIELA..............................VLL RV LAAVRS..P 3g43300_ARATH/97-252 SAPFTDILVC NM CS V...DNS..SPDSTVLQ..............................VLL KV LTAVAS..G SEC7_YEAST/267-445 QKIIDAAIIC DT SD F...QGEG.TDDRVELQ..............................IVL RA SSCILEEDS GGG2_PARTE/36-189 KRAIEIALVT DL MQ Q.....LE.KEETVQIH..............................MII KA QAIMTN..K GBF1_HUMAN/54-215 AEGMENMAVA DA TH RFVGTDPA.SDEVVLMK..............................ILL QV RTLLLT..P Q9V696_DROME/56-217 ADIVERIAVA DA TH RFMGTDQS.SDGVTFMR..............................VIL EV HTLIRS..P Q9XTF0_CAEEL/56-248 ANAVESIAVT YA VH KFIGGKSTGSDECLFLRNSRKKVNFHKKSTQNLLEMCQNFIRSANLDEFL YV RSLLLS..P GNOM_ARATH/83-245 EDAMHLVVVC DS TS RFEVTDPA.SEEVVLMK..............................ILL QV LACMKN..K GNL1_ARATH/81-243 GEAMHIIVVC DA KS RFEVTDPA.SEEVVLMK..............................ILL QV LACVKS..K GNL2_ARATH/67-228 KDAMNSIVIC SG TS RLEKTDLV.SEDAVMMR..............................ILL QV TGIMKH..P GEA2_YEAST/87-248 IGAHRATVLC NA TH RFEGSQQL.SDDSVLLK..............................VVL FL RSIVDS..P GEA1_YEAST/90-251 QIAVRETVLC VA TH RFEASKQI.SDDSVLLK..............................VVL TL RDIITS..S 100 110 120 130 140 150 BIG1_HUMAN/70-228 HI.EIHEGTVLQAVRTCYNIYLAS..KNLINQTTAKATLTQMLNVIFARMENQALQEAKQMEKERHRQH BIG2_HUMAN/58-216 HI.EIHEGTILQTVRTCYNIYLAS..KNLINQTTAKATLTQMLNVIFTRMENQVLQEARRLEKPIQSKP Q9VJW1_DROME/72-230 HV.EIHEFTLLQAVRTCYDIYLSS..KNLVNQTTARATLTQMLNVIFARMENQVYELPPPNSNPTNGSI Q9XWG5_CAEEL/69-227 HC.EVHGASLILAVRTCFNIYLTS..KSPINQATAKGTLTQVINTVFGNMEKFGNIKDDETIVREVVEV 3g60860_ARATH/77-233 SL.RIHGKCLLLVVRTCYDIYLGS..KNVVNQTTAKASLIQILVIVFRRMEADSSTVPIQPIVVAELME 1g01960_ARATH/72-228 SL.RIHGDSLLQIVRTCYGIYLGS..RNVVNQATAKASLVQMSVIVFRRMEADSSTVPIQPIVVAELME 4g35380_ARATH/62-214 CI.LIRGDCLLHVVKTCYNIYLGG..LSGTTQICAKSVLAQMMLVIFTRSEEDSLDVSVKTIYVNEL.. 4g38200_ARATH/61-215 RI.LIRGDCLLHLVRTCYNVYLGG..FNGTNQICAKSVLAQIMLIVFTRSEANSMDASLKTVNVNDLLA 3g43300_ARATH/97-252 KF.KVHGEPLLGVIRVCYNIALNS....PINQATSKAMLTQMISIVFRRMETDIVSASSTVSQEEHVSG SEC7_YEAST/267-445 SS.LCHGASLLKAIRTIYNVFVFS..LNPSNQGIAQATLTQIISSVYDKIDLKQSTSSAVSLSTKNHQQ GGG2_PARTE/36-189 KH.HIYGESVTRVFSLLINLHSVS..KIVAIINASKEACQKIVSTYFARLEDYGILAEDEYQLAIQQQG GBF1_HUMAN/54-215 VGAHLTNESVCEIMQSCFRICFEMR.LSELLRKSAEHTLVDMVQLLFTRLPQFKEEPKNYVGTNMKKLK Q9V696_DROME/56-217 EGAAVSNVSMCEVMLSCFKISFEPR.LSELLRRSAEKSLKDMVLLFFMRLPQFAEERSDTMLQKRFTIG Q9XTF0_CAEEL/56-248 PGILLSNEAVCDMMQSCFRIVFEQN.LSLLLRKAAESTLADMTQLIFTRLPTFVEDTRHPYIRQLVNPT GNOM_ARATH/83-245 ASVMLSNQHVCTVVNTCFRVVHQAGMKGELLQRVARHTMHELVRCIFSHLPDVERTETTLVNRAGSIKQ GNL1_ARATH/81-243 ASNGLSNQDICTIVNTCLRVVHQSSSKSELLQRIARHTMHELIRCIFSQLPFISPLANECELHVDNKVG GNL2_ARATH/67-228 SSELLEDQAVCTIVNTCFQVVQQSTGRGDLLQRNGRYTMHELIQIIFSRLPDFEVRGDEGGEDSESDT. GEA2_YEAST/87-248 YGDLLSNSIIYDVLQTILSLACNNR.RSEVLRNAAQSTMIAVTVKIFSKLKTIEPVNVNQIYINDESYT GEA1_YEAST/90-251 FGDYLSDTIIYDVLQTTLSLACNTQ.RSEVLRKTAEVTIAGITVKLFTKLKLLDPPTKTEKYINDESYT Th Figure 3 e conserved domains of the BIG/GBF subfamily: DCB domain The conserved domains of the BIG/GBF subfamily: DCB domain. Multiple sequence alignement of the conserved domains from BIG and GBF representative sequences showing secondary structure predictions that co-align in all sequences. Colour coding is red for invariant residues, yellow for a sequence similarity score threshold of 0.15 using the BLOSUM62 matrix. The gap in helix 4 is due to an insert in the drosophila Q9V696 sequence, and may be resulting from a sequence anno- tation error. Page 5 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 1 102030 405060 BIG1_HUMAN/411-593 PGAKFSHILQKDAFLVFRSLCKLSMKPLSDG...PPDPKSHELRSKILSLQLLLSILQNAGPIFRTNEM...... BIG2_HUMAN/362-544 VAARFSHVLQKDAFLVFRSLCKLSMKPLGEG...PPDPKSHELRSKVVSLQLLLSVLQNAGPVFRTHEM...... Q9VJW1_DROME/304-486 VTAKFTHILQKDAFLVFRALCKLSMKPLPDG...HPDPKSHELRSKVLSLHLLLLILQNAGPVFRSNEM...... Q9XWG5_CAEEL/264-443 DQFTFMNAYQKDAFLVFRALCILAQKEE.GG...A..SNEMSLRSKLLALEMLLLVLQNSSSILQSSQP...... 3g60860_ARATH/337-519 LEVQIENKLRRDACLVFRALCKLSMKAPPKE...SS.ADPQSMRGKILALELLKILLENAGAVFRTSEK...... 1g01960_ARATH/328-507 SEVQIGNKLRRDAFLVFRALCKLSMKTPPKE.......DPELMRGKIVALELLKILLENAGAVFRTSDR...... 4g35380_ARATH/284-466 SETGDMSKVRQDAFLLFKNLCKLSMRFSSKE...NN.DDQIMVRGKTLSLELLKVIIDNGGSVWRTNES...... 4g38200_ARATH/263-456 EDEGTGSKIREDGFLLFKNLCKLSMKFSSQE...NT.DDQILVRGKTLSLELLKVIIDNGGPIWLSDER.QLTLP 3g43300_ARATH/324-509 IELESMSIGQRDALLVFRTLCKMGMKEDS.........DEVTTKTRILSLELLQGMLEGVSHSFTKNFH...... SEC7_YEAST/488-678 IAITNQDLAVKDAFLVFRVMAKICAKPLETE....LDMRSHAVRSKLLSLHIIYSIIKDHIDVFLSHNI.FL..P GGG2_PARTE/318-500 SHSTFSEQYVKDAYEILEMLCQLSQRDPQN.....PQLAQMIIKCKVLSLELIYEALAQSDTTILQHKPK..... GBF1_HUMAN/392-566 EGTALVPYGLPCIRELFRFLISLTNPHDR..........HNSEVMIHMGLHLLTVALESAP..VAQCQT...... Q9V696_DROME/356-532 DVTSLSPYGLPFIQELFRFLIILCNPLDK..........QNSDSMMHTGLSLLTVAFEVAADNIGKYEG...... Q9XTF0_CAEEL/377-558 GGEEKMPYGLPCCRELLRFLITMTNPVDR..........HNTESMVILGLNLLIVALEAIADFLPNYDI...... GNOM_ARATH/305-491 LHIMTEPYGVPSMVEIFHFLCSLLNVVEHVGMGSRSNTIAFDEDVPLFALNLINSAIELGGSSIRHHPR...... GNL1_ARATH/306-492 ENAMMAPYGIPCMVEIFHFLCTLLNVGENGEVNSRSNPIAFDEDVPLFALGLINSAIELGGPSFREHPK...... GNL2_ARATH/233-414 ...MSGGYGIRCCIDIFHFLCSLLNVVEVVENLEGTNVHTADEDVQIFALVLINSAIELSGDAIGQHPK...... GEA2_YEAST/320-508 QAYADDNYGLPVVRQYLNLLLSLIAPE.........NELKHSYSTRIFGLELIQTALEISGDRLQLYPR...... GEA1_YEAST/305-490 AENVEPNYGITVIKDYLGLLLSLVMPE.........NRMKHTTSAMKLSLQLINAAIEISGDKFPLYPR...... 70 80 90 100 110 120 BIG1_HUMAN/411-593 ......FINAIKQYLCVALSKNG.VSSVPEVFELSLSIFLTLLSNFKTHLKMQIEVFFKEIFLYILET....... BIG2_HUMAN/362-544 ......FINAIKQYLCVALSKNG.VSSVPDVFELSLAIFLTLLSNFKMHLKMQIEVFFKEIFLNILET....... Q9VJW1_DROME/304-486 ......FIMAIKQYLCVALSNNG.VSLVPEVFELSLSIFVALLSNFKVHLKRQIEVFFKEIFLNILEA....... Q9XWG5_CAEEL/264-443 ......CIIVIKRTLCMALTRNA.VSNNIQVFEKSLAIFVELLDKFKTHLKASIEVFFNSVILPMLDS....... 3g60860_ARATH/337-519 ......FSADIKQFLCLSLLKNS.ASTLMIIFQLSCSIFISLVARFRAGLKAEIGVFFPMIVLRVVEN....... 1g01960_ARATH/328-507 ......FLGAIKQYLCLSLLKNS.ASNLMIIFQLSCSILLSLVSRFRAGLKAEIGVFFPMIVLRVLEN....... 4g35380_ARATH/284-466 ......FINAVKQYLCLSLLKNS.AVSIMSIFQLQCAIFMSLLSKLRSVLKAEIGIFFPMIVLRVLEN....... 4g38200_ARATH/263-456 PQKICRFLNAIKQLLCLSLLKNS.ALSVMSIFQLQCAIFTTLLRKYRSGMKSEVGIFFPMLVLRVLEN....... 3g43300_ARATH/324-509 ......FIDSVKAYLSYALLRAS.VSQSSVIFQYASGIFSVLLLRFRDSLKGEIGIFFPIIVLRSLDN....... SEC7_YEAST/488-678 GKERVCFIDSIRQYLRLVLSRNA.ASPLAPVFEVTLEIMWLLIANLRADFVKEIPVFLTEIYFPISEL....... GGG2_PARTE/318-500 ......LISILKEQLLESLLKNS.LSAEKQLLILTLNIFIQLIWRVRSHLKKELEALIENVYFKFLES....... GBF1_HUMAN/392-566 ......LLGLIKDEMCRHLFQLL.SIERLNLYAASLRVCFLLFESMREHLKFQMEMYIKKLMEIITVE....... Q9V696_DROME/356-532 ......LLELVKDDLCRNLISLL.SSERLSIFAADLQLCFLLFESLRGHLKFQLEAYLRKLSEIIASD....... Q9XTF0_CAEEL/377-558 ......LMPLIKNELCRNLLQLL.DTNRLPVLAATNRCCFLLFESMRMHMKFQLESYLKKLQSIVLTEEKQHE.. GNOM_ARATH/305-491 ......LLSLIQDELFRNLMQFG.LSMSPLILSMVCSIVLNLYQHLRTELKLQLEAFFSCVILRLAQG....... GNL1_ARATH/306-492 ......LLTLIQDDLFCNLMQFG.MSMSPLILSTVCSIVLNLYLNLRTELKVQLEAFFSYVLLRIAQS....... GNL2_ARATH/233-414 ......LLRMVQDDLFHHLIHYG.ASSSPLVLSMICSCILNIYHFLRKFMRLQLEAFFSFVLLRVTAF....... GEA2_YEAST/320-508 ......LFTLISDPIFKSILFIIQNTTKLSLLQATLQLFTTLVVILGNNLQLQIELTLTRIFSILLDDGTANNSS GEA1_YEAST/305-490 ......LFSLISDPIFKSVLFIIQSSTQYSLLQATLQLFTSLVVILGDYLPMQIELTLRRIFEILEDT...TISG ......... 130 140 150 160 170 180 BIG1_HUMAN/411-593 ...STS.SFDHKWMVIQ.........TLTRIC.ADAQSVVDIYVNYDCDLNAANIFERLVNDLSKIAQGR BIG2_HUMAN/362-544 ...STS.SFEHRWMVIQ.........TLTRIC.ADAQCVVDIYVNYDCDLNAANIFERLVNDLSKIAQGR Q9VJW1_DROME/304-486 ...NSS.SFEHKWMVIQ.........ALTRIC.ADAQSVVDIYVNYDCDFSAANLFERLVNDLSKIAQGR Q9XWG5_CAEEL/264-443 ...NTC.AFEQKWIVLN.........TIGKIL.ANPQSVVDMFVNYDCDMTSPNLFKSIVEVVSKTTRTT 3g60860_ARATH/337-519 ...VAQPNFQQKMIVLR.........FLDKLC.LDSQILVDIFLNYDCDVNSSNIFERMVNGLLKTAQGV 1g01960_ARATH/328-507 ...VAQPDFQQKMIVLR.........FLDKLC.VDSQILVDIFINYDCDVNSSNIFERMVNGLLKTAQGV 4g35380_ARATH/284-466 ...VLQPSYLQKMTVLN.........LLDKMS.QDPQLMVDIFVNYDCDVESSNILERIVNGLLKTALGP 4g38200_ARATH/263-456 ...VLQPSFVQKMTVLS.........LLENIC.HDPNLIIDIFVNFDCDVESPNIFERIVNGLLKTALGP 3g43300_ARATH/324-509 ...SECPN.DQKMGVLRYNIFLLVQMMLEKVC.KDPQMLVDVYVNYDCDLEAPNLFERMVTTLSKIAQGS SEC7_YEAST/488-678 ...TTS.TSQQKRYFLS.........VIQRIC.NDPRTLVEFYLNYDCNPGMPNVMEITVDYLTRLALTR GGG2_PARTE/318-500 ...SNS.SFDHKQYTLK.........VFNKIL.TRPKVVIEIFVNYDCSVGQNNLLKKILDMQCRIIQGR GBF1_HUMAN/392-566 ...NPKMPYEMKEMALE.........AIVQLW.RIPSFVTELYINYDCDYYCSNLFEELTKLLSKNAFPV Q9V696_DROME/356-532 ...NPKTPYEMRELALD.........NLLQLW.RIPGFVTELYINYDCDLYCTDMFESLTNLLSKYTLSA Q9XTF0_CAEEL/377-558 ...NGGGGTEQKEMALE.........SLVQLW.RIPGLVTEMYLNFDCDLYCGNIFEDLTKLLVENSFPT GNOM_ARATH/305-491 ...KYGPSYQQQEVAME.........ALVNFC.RQKSFMVEMYANLDCDITCSNVFEELSNLLSKSTFPV GNL1_ARATH/306-492 ...KHGSSYQQQEVAME.........ALVDLC.RQHTFIAEVFANFDCDITCSNVFEDVSNLLSKNAFPV GNL2_ARATH/233-414 .....TGFLPLQEVALE.........GLINFC.RQPAFIVEAYVNYDCDPMCRNIFEETGKVLCRHTFPT GEA2_YEAST/320-508 SENKNKPS.IIKELLIE.........QISILWTRSPSFFTSTFINFDCNLDRADVSINFLKALTKLALPE GEA1_YEAST/305-490 DVSKQKPP.AIRELIIE.........QLSILWIHSPAFFLQLFVNFDCNLDRSDLSIDFIKELTKFSLPA Th Figure 4 e conserved domains of the BIG/GBF subfamily: HUS domain The conserved domains of the BIG/GBF subfamily: HUS domain. See Figure 3 legend for alignment details. The highly conserved HUS motif is boxed in blue. The gap in helix 5 domain is due to an insert in the Arabidopsis 3g43300 sequence, and may be resulting from a sequence annotation error. domains (Figure 2). Together with our domain analysis, where the impact upon folding of domains with essential this suggests that splicing at non-canonical exon/intron function would be minimal. boundaries is only tolerated in regions of the protein Page 6 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 1 10203040506070 BIG1_HUMAN/915-1083 MEQMAKTAKALMEAVSHVQAPFTSATHLEHVRPMFKLAWTPFLAAFSVGLQDCDDTEVASLCLEGIRCAIRIACI BIG2_HUMAN/860-1028 MEQMAKTAKALMEAVSHAKAPFTSATHLDHVRPMFKLVWTPLLAAYSIGLQNCDDTEVASLCLEGIRCAIRIACI Q9VJW1_DROME/801-969 MEVISLTATNLMQSVSHVKSPFTSAKHLEHVRPMFKMAWTPFLAAFSVGLQDCDDPEIATLCLDGIRCAIRIACI Q9XWG5_CAEEL/767-934 .FFRTSKKLALMESASDADAYFTPAQHQHHVKPMFKICWTPCLAAFSVGVQMSDDEEEWSLCLRGFRLGVRAACV 3g60860_ARATH/843-1008 DDLMKHMQEQFKEKARKSESTYYAATDVVILRFMIEACWAPMLAAFSVPLDQSDDLIVINICLEGFHHAIHATSL 1g01960_ARATH/835-999 .DLIRHMQERFKEKARKSESVYYAASDVIILRFMVEVCWAPMLAAFSVPLDQSDDAVITTLCLEGFHHAIHVTSV 4g35380_ARATH/793-958 GRLIRDIQEQFQAKPEKSESVYHTVTDISILRFILEVSWGPMLAAFSVTIDQSDDRLATSLCLQGFRYAVHVTAV 4g38200_ARATH/776-942 GLLIKDIQEKFRSKSGKSESAYHVVTDVAILRFMVEVSWGPMLAAFSVTLDQSDDRLAAVECLRGFRYAVHVTAV 3g43300_ARATH/796-957 TEDIVRKTQEIFRKHGVKRGVFHTVEQVDIIRPMVEAVGWPLLAAFSVTMEVGDNKPRILLCMEGFKAGIHIAYV SEC7_YEAST/1055-1220 ISSKTELVFKNLNKNKGGPDVYYAASHVEHVKSIFETLWMSFLAALTPPFKDYDDIDTTNKCLEGLKISIKIAST GGG2_PARTE/791-943 ...EDSLKKWFKEHP..NSDAFCYVNSIEHMKSLLQQTWSVIFASISVFLEQSEDQQQILLCFETIQAFIQLMGR GBF1_HUMAN/893-1066 LVRENYVWNVLLHRGATPEGIFLRVPTASYDLDLFTMTWGPTIAALSYVFDKSLEETIIQKAISGFRKCAMISAH Q9V696_DROME/839-1021 LVRENYQWKVLLRRGDTHDGHFHYVHDASYDVEIFNIVWGASLSALSFMFDKST.ETGYQRTLAGFSKSAAISAH Q9XTF0_CAEEL/854-1051 .VKEDYMWKVLLRRGETAEGSFYHAPTGWNDHDLFAVCWGPAVAALSYVFDKSEHEQILQKALTGYRKCAKIAAY GNOM_ARATH/756-930 PEMTPSRWIDLMHKSKKTAPYILADSRAYLDHDMFAIMSGPTIAAISVVFDHAEHEDVYQTCIDGFLAIAKISAC GNL1_ARATH/758-930 ..MTASRWISVIYKSKETSPYIQCDAASHLDRDMFYIVSGPTIAATSVVFEQAEQEDVLRRCIDGLLAIAKLSAY GNL2_ARATH/688-861 .EMNPNRWIELMNRTKTTQPFSLCQFDRRIGRDMFATIAGPSIAAVSAFFEHSDDDEVLHECVDAMISIARV.AQ GEA2_YEAST/777-982 .ISSTTVITEIKKDTQSVMDKLTPLELLNFDRAIFKQVGPSIVSTLFNIYVVASDDHISTRMITSLDKCSYISAF GEA1_YEAST/773-972 .....SVMTEMQRDFTNPISKLAQIDILQYEKAIFSNVRDIILKTLFKIFTVASSDQISLRILDAISKCTFINYY 80 90 100 110 BIG1_HUMAN/915-1083 FI S QLERDY A VQALR A FTLLTVSSGITE...................................MKQKT NID IKTL BIG2_HUMAN/860-1028 FM G QLERDY A VQALR A FSLLTASSSITK...................................MKQKT NID IKTL Q9VJW1_DROME/801-969 FM H SLERDY A VQALR A FTLLNANSPINE...................................MKAKT NID IKTL Q9XWG5_CAEEL/767-934 LA Q TLERNF A IQALR A FTLLTAKNSLGE...................................MRVKA NIE IKLL 3g60860_ARATH/843-1008 MM S KTHRDF A VTSLK A FTSLHSPA...D...................................IKQRA NIE IKAI 1g01960_ARATH/835-999 ML S KTHRDF A VTSLK A FTSLHSPA...D...................................IKQKA NIE IKAI 4g35380_ARATH/793-958 MM G QTQRDF A VTSMK A FTNLHCAA...D...................................MKQKA NVD VKAI 4g38200_ARATH/776-942 MM G QTQRDF A VTSMK A FTNLHCAG...D...................................MKQKA NVD VKAI 3g43300_ARATH/796-957 LM G DTMRYF A LTSLR V FTFLHAPK...E...................................MRSKA NVE LRIL SEC7_YEAST/1055-1220 FI R NDARTF S VGALQ V FCNLQNLE...E...................................IKVKA NVN MVIL GGG2_PARTE/791-943 FL D DEEKDF T ISFLR Y YCTNI.PS........................................NYKG QIL VQTL GBF1_HUMAN/893-1066 YL G SDVFDL N IISLK C FTALSSE..............................SIENLPSVFGSNPKA AHI AKTV Q9V696_DROME/839-1021 YL N HSDFDL A VLTLK C FTTLLSSVEQHEPAPANNE...TQ.................QAVNFGLNGKA AQA MRTV Q9XTF0_CAEEL/854-1051 YM G KEVFDL N CIHLK C FTTLTSMRDGGAGGGADED...VDLSAAALLSHS..SSPEAVALAFGENHKA AQL TRTL GNOM_ARATH/756-930 HL H EDVLDL D VVSLK C FTTLLNPSSV.............................DEPVLAFGDDAKA ARM TITI GNL1_ARATH/758-930 YL H NSVLDL D VVSLK C FTPFFAPLSA.............................DEAVLVLGEDARA ARM TEAV GNL2_ARATH/688-861 YL G EDILDL E IASFK C FTTLLNPYTT............................PEETLFAFSHDMKA PRM TLAV GEA2_YEAST/777-982 FF D KDLFNI D LNSIK A GTTLINSSHDDELSTLAFEYGPMPLVQIKFEDTNTEIPVSTDAVRFGRSFKN GQL TVVF GEA1_YEAST/773-972 FF S DQSYNT D VLHLE G MTTLAQSSA....KAVELDVDSIPLVEIFVEDTGSKISVSNQSIRLGQNFKC AQL TVLY 120 130 140 150 160 BIG1_HUMAN/915-1083 IV T AHTDGN...YLGNSWHEILC K ISQLKLAQLIGTGVKP..RYISGTVRGREGSLTGT BIG2_HUMAN/860-1028 IV T AHTDGN...YLGNSWHEILC K ISQLELAQLIGTGVKT..RYLSGSGREREGSLKGH Q9VJW1_DROME/801-969 IV M AHTDGN...YLGSSWLDIVC K ISQLELAQLIGTGVRP..QFLSGAQTTLKDSLNPS Q9XWG5_CAEEL/767-934 LI L GDEDGE...YLEENWVDVMC K MSSLELVQLIGTGLNS..AMSHDTDSSRQYVMKAT 3g60860_ARATH/843-1008 LL R ADEEGN...YLQDAWEHILC T VSRFEQLHLLGEGAPP..DATFFASKQNESEKSKQ 1g01960_ARATH/835-999 VL K AEEEGN...YLQDAWEHILC T VSRFEHLHLLGEGAPP..DATFFAFPQTESGNSPL 4g35380_ARATH/793-958 II T AIEDGN...HLHGSWEHILC T LSRIEHLQLLGEVSPS..EKRYVPTKKAEVDDKKA 4g38200_ARATH/776-942 II S AIEDGN...HLQDAWEHILC T LSRIEHLQLLGEGAPS..DASYFASTETEEKKALG 3g43300_ARATH/796-957 LL G CDSEPD...TLQDTWNAVLC E VSRLEFII.STPGIAA..TVMHGSNQISRDGV... SEC7_YEAST/1055-1220 LV E ALSEGN...YLEGSWKDILV L VSQMERLQLISKGIDR..DTVPDVAQARVANPRVS GGG2_PARTE/791-943 IV K ILQSGQ...YLRKSWKVALL Q ISRLEQLHQVVKKIKV..DSPYKENYNQED..... GBF1_HUMAN/893-1066 FL H AHRHGD...ILREGWKNIMA E MLQLFRAQLLPKAMIE..VEDFVDPNGKISLQREE Q9V696_DROME/839-1021 FL L VHDYGD...CLRESWKHILL D YLQLFRLKLLPKSLIE..VEDFCEANGKAMLILEK Q9XTF0_CAEEL/854-1051 FL Y VHENGN...ILREGWRNLFA E LLQLFRARLLPAELTE..VEDYVDEKGWVNIQRVH GNOM_ARATH/756-930 FI T ANKYGD...YIRTGWRNILC D ILRLHKLGLLPARVAS..DAADESEHSSEQGQGKP GNL1_ARATH/758-930 FI L ANKYGD...YISAGWKNILC E VLSLNKLHILPDHIAS..DAADDPELSTSNLEQEK GNL2_ARATH/688-861 FL T ANTFGD...SIRGGWRNIVC D LLKLRKLQLLPQSVIE..FEINEENGGSESDMNNV GEA2_YEAST/777-982 FI R IRRNKDPKIFSKELWLNIVI N ILTLYEDLILSPDI..FPDLQKRLKLSNLPKPSPE GEA1_YEAST/773-972 FI Q IKEISDPSIVSTRLWNQIVL Q ILKLFENLLMEPNLPFFTNFHSLLKLPELPLPDPD Th Figure 5 e conserved domains of the BIG/GBF subfamily: HDS1 domain The conserved domains of the BIG/GBF subfamily: HDS1 domain. See Figure 3 legend for alignment details. Evolution of BIGs and GFBs from a common ancestor unified as a higher order ArfGEF subfamily (called below Combined, our analysis reveals that the BIG and GBF sub- GBG for GBF/BIG GEFs), from which unrooted phyloge- families share the same overall domain organization, and netic trees can be built (Figure 8). Unlike previous phylo- are likely to descend from a common ancestor gene that genetic analysis which compared ArfGEFs based on their duplicated first to form the BIG and GBF groups, and Sec7 domains after diverging non-catalytic regions have again within these groups to yield species-specific BIG and been trimmed [8], our trees were established from the GBF members. These two subfamilies can therefore be simultaneous alignment of all 6 conserved domains Page 7 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 1 10 20304050 BIG1_HUMAN/1107-1289 ASIQESIGETSSQSVV..VA.VDI R FTGSTRLDGNAIVDFVRWLCAVSMDELLST..........TH...PRMFS BIG2_HUMAN/1054-1236 ASFQESVGETSSQSVV..VA.VDI R FTGSTRLDGNAIVDFVRWLCAVSMDELASP..........HH...PRMFS Q9VJW1_DROME/968-1149 PSVKEHIGETSSQSVV..VA.VDI R FTGSMRLDGDAIVDFVKALCQVSVDELQQ...........QQ...PRMFS Q9XWG5_CAEEL/943-1125 HSLQDALGETSSQSVV..VA.IDI R FNGSARLSAEAIVYFVRALCAVSREELSHP..........AA...PRMFL 3g60860_ARATH/1053-1235 EQMSSIVSNLNLLEQVG..E.MNV Q FSQSQKLNSEAIIDFVKALCKVSMDELRSP..........SN...PRVFS 1g01960_ARATH/1044-1226 EQMNNLISNLNLLEQVG..D.MSI R FTRSQRLNSEAIIDFVKALCKVSMDELRSP..........SD...PRVFS 4g35380_ARATH/1000-1184 EQIKSFIANLNLLDQIGNFE.LNV H YANSQRLNSEAIVSFVKALCKVSMSELQSP..........TD...PRVFS 4g38200_ARATH/982-1166 DQINNFIANLNLLDQIGSFQ.LNV N YAHSQRLKTEAIVAFVKALCKVSMSELQSP..........TD...PRVFS 3g43300_ARATH/951-1132 QISRDGVVQSLKEL..AGRP.AEV Q FVNSVKLPSESVVEFFTALCGVSAEELKQ...........SP...ARVFS SEC7_YEAST/1253-1438 TLSPEISKFISSSELV..VL.MDI N FTKSSELSGNAIVDFIKALTAVSLEEIESS..........ENASTPRMFS GGG2_PARTE/945-1125 ....ISIERLFQQI..QYDQ.IDI K FNSSINLDSNSILEFIRALCELSKEEIKY................NRLFL GBF1_HUMAN/1098-1277 TENQEAKRVALECI..KQCD.PEM K ITESKFLQLESLQELMKALVSVTPD.....E........ETYDEEDAAFC Q9V696_DROME/1048-1236 YEEQDFIKLGRKCI..KECQ.LDM Q LQESKFVQLESLQELLKCVLALLKA.....PQGH.KSIGLPYAEDQTVFW Q9XTF0_CAEEL/1084-1278 QEQLSSMKLASQVI..SECR.PSI Q VADSKYLTSTSLAELLSSIAANSAQIVEQAEPQQKTASLSGEDEDALVFY GNOM_ARATH/973-1158 EQQLAAHQRTLQTI..QKCH.IDI S FTESKFLQAESLLQLARALIWAAGR........PQKGTSSPEDEDTAVFC GNL1_ARATH/973-1149 EEELAAYKHARGIV..KDCH.IDI S FSDSKFLQAESLQQLVNSLIRASGK.................DEASSVFC GNL2_ARATH/894-1084 ALGMSEFEQNLKVI..KQCR.IGI Q FSKSSVLPDVAVLNLGRSLIYAAAGKGQ.......KFSTAIEEEETVKFC GEA2_YEAST/1011-1194 .EEIKSSKKAMECI..KSSNIAAV S FGNESNITADLIKTLLDSAKTE............KNADNSRYFEAELLFI GEA1_YEAST/1012-1184 ............CV..KASHPLSV S FENNQLVSPKMIETLLSSLVIE............KTSENSPYFEQELLFL 60 70 80 90 100 110 120 130 BIG1_HUMAN/1107-1289 LI QK VEISYYNR MG IRLQWI SR WI EV GDHFNKVGCNPNE.DVAIFAVDSLRQLK SM FLEKG..ELANFD FR QK FL BIG2_HUMAN/1054-1236 LI QK VEISYYNR MN IRLQWI SR WI HV GDHFNKVGCNPNE.DVAIFAVDSLRQLK SM FLEKG..ELANFD FR QK FL Q9VJW1_DROME/968-1149 LI QK VEISYYNR ME IRLQWI SR WL QV GEHFNAVGCNSNE.EISFFALDSLRQLK SM FMEKG..EFSNFD FR QK FL Q9XWG5_CAEEL/943-1125 LV GK VEVAFYNR MN IRLEWI SR WI NV GEHFNAAGCNSNE.AVAYFSVDALRQLK SI FLEKG..ELPNFD FR QK FL 3g60860_ARATH/1053-1235 LI TK VEIAHYNR MN IRLVWI SS WL QV SGFFVTIGCSENL.SIAIFAMDSLRQLK SM FLERE..ELANFE YN QN FM 1g01960_ARATH/1044-1226 LI TK VEIAHYNR MN IRLVWI SS WL HV SDFFVTIGCSDNL.SIAIFAMDSLRQLK SM FLERE..ELANFE YN QN FM 4g35380_ARATH/1000-1184 LL TK VETAHYNR MN IRLVWI SR WL NV SDFFVSVGLSENL.SVAIFVMDSLRQLK SM FLERE..ELANFE YH QH FL 4g38200_ARATH/982-1166 LL TK VEIAHYNR MN IRLVWI SR WL SI SDFFVSVGLSENL.SVAIFVMDSLRQLK SM FLERE..ELANFE YN QN FL 3g43300_ARATH/951-1132 LL QK VEISYYNR IA IRMVWI AR WL SV AEHFVSAGSHHDE.KIAMYAIDSLRQLK GM YLERA..ELTNFD FT QN IL SEC7_YEAST/1253-1438 LM QK VDVCYYNR MD IKLEWL TP WM AV GKAFNKIATNSNL.AVVFFAIDSLRQLR SM FLDIE..ELSGFD FE QH FL GGG2_PARTE/945-1125 LV SR IDVAEFNR MN IKIIWM SR WM EI REHFLEVGCLKNV.DVAIYAIDQLKQLK SC FLQQP..ELTNFE YY QK FL GBF1_HUMAN/1098-1277 LL EM LRIVLENR RD VGCVWV QT RL DH YHLCVQAQD..FC.FLVERAVVGLLRLR AI LL.......RRIQ EE SA VL Q9V696_DROME/1048-1236 ML EF VKIVVHNR RD MIPLWV PA RM DQ YLLLMGSASCGYD.YLLNRCIVAVLKLY AI LM.......RNLI EE CP VL Q9XTF0_CAEEL/1084-1278 LI EL VAITLENR KD LPLVWV PH RL RH EWLLSPRFG..RCPVLVERAVVGLLRVR AN NLF......RDVD NT SD VL GNOM_ARATH/973-1158 LL EL IAITLNNR RD IVLLWV QG YI EH ATIAQSTVM..PC.NLVDKAIFGLLRIR CQ LLP......YKLE ES AD LL GNL1_ARATH/973-1149 LL EL IAVTLNNR RD ILLIWV PT YI EH LGIVQLTLT..PC.TLVEKAVFGVLKIR CQ LLP......YKLE EN TD LL GNL2_ARATH/894-1084 WI DL ITIALSNR VH FNMFWY PS HL EY LNVANFPL.FSPI.PFVEKGLPGLFRVK CI ILASN....LQLE DH PE LI GEA2_YEAST/1011-1194 IT EL IALFL.FE CK EKELGI KF LV QK FQLSHTKG.....LTKRTVRRMLTYKII LL SLCADQTEYLSIE KL ND LL GEA1_YEAST/1012-1184 LS EI IILIS.EY AS GQEFGI AL AM DH INISNLDG.....LSKEAIARLASYKMV FL SRFDNPRDILSID DL EH FL 140 150 160 170 180 BIG1_HUMAN/1107-1289 .RPFEHIMK.....RNRSPTIRDV M VRCIAQMVNSQAANIS R .....GKI W N FV S FHLAASDQ BIG2_HUMAN/1054-1236 .RPFEHIMK.....KNRSPTIRDA M IRCIAQMVNSQAANIS R .....GKI W N FV A FHQAASDH Q9VJW1_DROME/968-1149 .RPFEHIMK.....KNASPAIRDV M VRCIAQMVNSQAHNIS R .....GKI W N FI S FHLAAGDN Q9XWG5_CAEEL/943-1125 .RPFEVIMV.....RNGSAQTRDV L VRCCAHLVEAHSSRLS K .....GQL W N FV S WTIAAGDP 3g60860_ARATH/1053-1235 .TPFVIVMR.....RSNDVEIREI L IRCVSQMVLSRVNNVS K .....GKM W S FV M FTTAAYDD 1g01960_ARATH/1044-1226 .KPFVVVMR.....KSGAVEIREI L IRCVSQMVLSRVDNVS K .....GKM W S FI M FTTAAHDA 4g35380_ARATH/1000-1184 .RPFVVVMQ.....KSSSAEIREI L VRCVSQMVLSRVSNVS K .....GKV W N FV T FTTAALDE 4g38200_ARATH/982-1166 .RPFVIVMQ.....KSSSAEIREI L VRCISQMVLSRVSNVS K .....GKV W S FV K FTTAAADE 3g43300_ARATH/951-1132 .KPFVIIMR.....NTQSQTIRSI L VDCIVQMIKSKVGSIS K .....GRV W S FI M FTAAADDE SEC7_YEAST/1253-1438 .KPFEYTVQ.....NSGNTEVQEI M IECFRNFILTKSESIS K .....GKI W P LS E LQYTARSS GGG2_PARTE/945-1125 .LPFEQIFSHTQAQQQNKIQLREL F LSCMCMITNICFNSIS K .....GKI W I MI S VNQALQDD GBF1_HUMAN/1098-1277 .LSLRILLLMK...PSVLSRVSHV Q AYGLHELLKTNAANIS H ...GDDAL W T FL T LECIGSGV Q9V696_DROME/1048-1236 .QSLKMLLMLK...PALLLRISKI Q SIGIYELLKTSAQNIS H ...EQDQI W I FL N LECVGAGA Q9XTF0_CAEEL/1084-1278 .HSLSMLLRLS...PKALFIFSRI Q AFGLYELIRANAANVK H ...KEHAL W V FL A LEAAGAAV GNOM_ARATH/973-1158 .RSLQLVLKLD...ARVADAYCEI Q AIEVSRLVKANANHIS R ...QAGRI W T TL S LSITARHP GNL1_ARATH/973-1149 .KSLQLVLKLK...AKVADAYCEI R AQEVVRLVKANASHVS R ...RTGRI W T IL S LSITARHP GNL2_ARATH/894-1084 FRSLTIMWKID...KEIIETCYDI T TEFVSKIIIDYSANLT H ...NIGKV W S LL Q LSLCGRHP GEA2_YEAST/1011-1194 .KKGDIFTQ.....KFFATNQGKF E LKRLFSLTESEFYRGL F LGNENFKL W F RV K TAMKEQ.. GEA1_YEAST/1012-1184 .VKNEIFNT.....KYYESEWGKV Q INDLFTHLNDVKYNEA R LKNVKFNL W F RL I ISAKDR.. Th Figure 6 e conserved domains of the BIG/GBF subfamily: HDS2 domain The conserved domains of the BIG/GBF subfamily: HDS2 domain. See Figure 3 legend for alignment details. (DCB, HUS, Sec7, HDS1, HDS2 and HDS3), excluding sis strongly supported this topology for most branches. variable linkers. The same tree topology was obtained Only a few small branches located at the base of the with both neighbor-joining and maximum likelihood groups were found in less than 60% of the trials in one of methods, and was retained using any one of the new con- the two methods, but this never occurred with both meth- served domains alone (data not shown). Bootstrap analy- ods simultaneously. Page 8 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 110 20 30 40 50 60 BIG1_HUMAN/1372-1489 .APEDRVWVRGWFPILFELSCIINRCK.LDVRTRGLTVMFEIMK..TYGHTYEKH...WWQDLFRIVFRIFDNMK BIG2_HUMAN/1319-1436 .APGDRVWVRGWFPILFELSCIINRCK.LDVRTRGLTVMFEIMK..SYGHTFEKH...WWQDLFRIVFRIFDNMK Q9VJW1_DROME/1233-1350 .AEEDRVWVRGWFPMLFSLSCVVNRCK.LDVRTRALTVLFEIVK..TYGESFKPH...WWKDLFNVIFRIFDNMK Q9XWG5_CAEEL/1212-1329 .TADQHVWLRGWFPIFFELSCIINRCK.LDVRTRSLTVMFEIMK..HHGSDFRPE...WWKDLLEIVFRIFDPSK 3g60860_ARATH/1337-1472 EIV.NNNHLYFWFPLLSGLSELSFDPR.PEIRKSALQIMFDTLR..NHGHLFSLP..LWEKVFESVLFPIFDYVR 1g01960_ARATH/1321-1452 .FLESDEHLYSWFPLLAGLSELSFDPR.AEIRKVALKVLFDTLR..NHGDHFSLA..LWERVFESVLFRIFDYVR 4g35380_ARATH/1278-1410 .FTDLDEQVSYWIPLLTGLCKQVSDPR.PAIRKRSIEVLFHILM..DHGHLFTRP..FWTGIFSSIILPVFNNIR 4g38200_ARATH/1262-1394 .FMDADENISYWVPLLTGLSKLTSDSR.SAIRKSSLEVLFNILK..DHGHIFSRT..FWIGVFSSVIYPIFNSVW 3g43300_ARATH/1212-1331 DET.FDVTEHYWFPMLAGLSDLTSDYR.PEVRNCALEVLFDLLN..ERGNKFSTP..FWESIFHRILFPIFDHVS SEC7_YEAST/1525-1649 .LRGKDIFQDVWFPMLFCFNDTIMTAEDLEVRSRALNYMFDALV..AYGGKFNDD..FWEKICKKLLFPIFGVLS GGG2_PARTE/1195-1313 ....NKYLDQLWIPVLSALSVLYSDER.GVVQQQSVNTLFELLK..VHGEQQSNE..FWKIILRGVIRPLFDEIQ GBF1_HUMAN/1532-1644 .ADSRTLWAHCWCPLLQGIACLCCDAR.RQVRMQALTYLQRALL.VHDLQKLDAL..EWESCFNKVLFPLLTKLL Q9V696_DROME/1475-1592 .PQSAALWSPGWCPLLQGIARLAMDRR.REVRTHAISCLQQRALLVHDLQTLSGT..EWCSCFHQVLFPLLNELL Q9XTF0_CAEEL/1555-1667 .ATVAFIWTDIWRPLLQAIGRLSCDCR.RGVRAAALTHLQRAFL.PANMATLGAA..EWQSCFGEVLFPLLTKLL GNOM_ARATH/1235-1349 .GKMSQDIGEMWLRLVQGLRKVCLDQR.EDVRNHALQSLQKCLG.GVDGINLAHS..MWSQCFDKVIFTVLDDLL GNL1_ARATH/1227-1343 .MKLSEDIGKMWLKLVKNLKKVCLDQR.DEVRNHAISMLQRAIA.GADGIMLPQP..LWFQCFDSAVFILLDDVL GNL2_ARATH/1177-1298 .LRGVNFVHHLFLKLSEAFRKTTLARR.EEIRNRAVTSLEKSFTMGHEDLGFTPS..GCIYCIDHVIFPTIDDLH GEA2_YEAST/1277-1381 .EDNNDLRKNEIFAIIQALAHQCINPC.KQISEFAVVTLEQTLI..NKIEIPTNEMESVEELIEGGLLPLLNSSE GEA1_YEAST/1271-1375 .RSNSALNKNEIIAAIQGLAHQCLNPC.DELGMQALQALENILL..SRASQLRTEKVAVDNLLETGLLPIFELDE 70 80 90 100 110 BIG1_HUMAN/1372-1489 LPEQQ..................TEKAEWMTTTAI CNH LYA CDVFTQYLEVLSDVLLDDIFAQLYWCV BIG2_HUMAN/1319-1436 LPEQL..................SEKSEWMTTTAI CNH LYA CDVFTQFYEALNEVLLSDVFAQLQWCV Q9VJW1_DROME/1233-1350 LPEHV..................TEKSEWMTTTAI CNH LYA IDVFTQYFDVLGHLLLEELFAQLHWCV Q9XWG5_CAEEL/1212-1329 MDDHR..................SDKREWMSTTAV CNH MLS VEVFTQFYTQLSVYALPMIYRQFGIFI 3g60860_ARATH/1337-1472 HSIDPSGEDESADQGSSGGEVDELDHDAWLYETAV CTL LQL VDLFVKFYTTVNP.LLEKVLMLLVSFI 1g01960_ARATH/1321-1452 QDVDPSE.DDSTDQRGYNGE...VDQESWLYETAV CSL LQL VDLFVNFYKTVNP.LLKKVLMLFVSLI 4g35380_ARATH/1278-1410 SKTDMLFEESVDS.PSSAS..LDTEETTWDVETAL STL LQL VDLLVKFFRSVRS.QLPSVVSIIVGFI 4g38200_ARATH/1262-1394 GENDLLSKDEHSSFPSTFS..SHPSEVSWDAETAL SAM AQY VDLFVSFFTVIRS.QLSSVVSLLAGLI 3g43300_ARATH/1212-1331 HAGKESL.ISS..............GDVKFRETSL SIH LQL CNLFNTFYKEVCF.MLPPLLSLLLDCA SEC7_YEAST/1525-1649 KHWEVNQFN............SHDDLSVWLSTTAL LIQ LRN IALFTHYFESLNR.MLDGFLGLLVSCI GGG2_PARTE/1195-1313 ISKLQ..FA............KQSQSKQQVIQNTF CKM FYL TDLVVLYIQQMQP.CLNDLIDIYIQLV GBF1_HUMAN/1532-1644 ENI...................SPADVGGMEETAL RMR STL SKVFLQHLSPLLSL...STFAALW... Q9V696_DROME/1475-1592 PES.....NAA...........GQLDAALLEESTM RIR ATI SKVFLQHLTTLIEL..GNAFNELW... Q9XTF0_CAEEL/1555-1667 EPF...................SQMDPIGMEDTTV RVR LQI AKTLLNHLSALSAL...DSFPDLW... GNOM_ARATH/1235-1349 EIA....A..G...........SQKDYRNMEGTAL LLL IKL SKVFLQQLQELSQL...STFCKLW... GNL1_ARATH/1227-1343 TFS....IENS...........RKTLKKTVEETAM LVL TKL SKAFLQSLQDISQQ...PSFCRLW... GNL2_ARATH/1177-1298 EKLLDYSRREN...........AEREMRSMEGTAL LKI MKV MNVFLVYLEQIVES...AEFRTFW... GEA2_YEAST/1277-1381 TQ.........................EDQKILII ISS LTI SNVYLHYLKL......GKTSNETF... GEA1_YEAST/1271-1375 IQ.........................DVKMKRIL ITS LSV SKIFLGQLVE......GVTSNETF... Th Figure 7 e conserved domains of the BIG/GBF subfamily: HDS3 domain The conserved domains of the BIG/GBF subfamily: HDS3 domain. See Figure 3 legend for alignment details. The tree topology strongly suggests that in most organ- trafficking at the Golgi within each group appear largely isms, GBG members sort in separate branches, overlapping [12,14]. In contrast, plants encode a large corresponding to their classification in BIGs and GBFs. number of GBGs in both the BIG and GBF branches but Remarkably, our annotation of Sec7-containing proteins lack other ArfGEFs (Figures 1, 8). In Arabidopsis, none of in the genome of Paramecium reveals the first departure the GBGs map to duplicated chromosomes where identi- from this distribution, as all GBGs in this species are cal functions may be encoded [26,29]. In addition, com- located in a single branch, which is closer to the BIGs. This parative analysis with the rice genome nearing unexpected tree topology may indicate that alveolates completion identifies at least five branches each repre- diverged from animals/fungi and plants before the dupli- sented by one rice and one or two Arabidopsis homologs cation of an ancestral GBG into the BIG and GBF, and that (Figure 8). This correspondence between two highly diver- GBGs in that organism are representative of this ancestral gent plant species indicates that GBGs diversified early gene. Alternatively, duplication may have been followed during plant evolution, probably reflecting functional by loss of GBF genes. Current knowledge of the phyloge- specialization along with the establishment of plant mul- netic branching of alveolates relative to the plants and ani- ticellularity. While GNOM has a plant-specific function in mal/fungi branches does not permit resolution between recycling plasma-membrane proteins needed for cell-cell those two possibilities. communication and cell polarity establishment [11], pos- sibly closer to the function of EFA6 or CYH subfamilies in GBGs in plants: refining the functional evolution of BIGs metazoans, other plant GBGs are expected to fulfill the and GBFs presumed ancient function of regulating Golgi trafficking In fungi and mammals, BIGs and GBFs are represented by exemplified by mammalian and yeast GBGs. Comparison only one or two members, whose functions in vesicular of orthologous pairs in plants further reveals that they Page 9 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 a,b Table 2: Alternate splice variants of human GBF1, BIG1 and BIG2 Change in protein Apparent cause of variation in transcript GBF1 Extra Q at 337, 55 residues upstream of HUS domain Insertion of 3 nucleotides (nt) resulting from use of alternate 3' acceptor site within intron during splicing of exons 10 and 11 New Ser and loss of 14 residues at 613, between HUS and Sec7 Loss of 36 nt resulting from use of alternate 5' donor site within domains exon 15 during splicing with exon 16 Loss of VSQD at 1494, 38 residues upstream of HDS3 Loss of 12 nt resulting from use of alternate 5' donor site within exon 33 during splicing with exon 34 Frame-shift at 1625 causing loss of last 19 residues of HDS3 Intron retention between exons 36 and 37 leading to frame shift and premature termination Loss of 38 residues starting at 1784, near C-terminus Loss of 114 nt resulting from use of novel cryptic splice donor and acceptor sites within exon 40. BIG1 Frame-shift at 1340, 32 residues upstream of HDS3 Loss of 59 nt resulting from use of alternate 5' donor site within exon 28 during splicing with exon 29 Loss of VSEKPL at 1557, 68 residues downstream of HDS3 Loss of 18 nt resulting from use of alternate 5' donor site within exon 33 during splicing to exon 34 New T and loss of 33 residues at 1607, 118 residues downstream of Loss of 96 nt resulting from use of alternate 3' acceptor site within HDS3 exon 35 during splicing with exon 34 BIG2 Frame-shift at 1542, 106 residues downstream of HDS3 Loss of exon 35 resulting from splicing of 5'donor site of exon 34 with 3' acceptor site of exon 36 All changes were expressed relative to the reference sequence stored under accession number NM_004193 (hGBF1), NM_006421 (hBIG1) and NM_006420.1 (hBIG2). All variants are supported by one or more cDNA/ESTs as detailed in the Aceview for each gene that can be obtained at [38]. have different sensitivities to Brefeldin A (a widely used ure 9), which was found only in the protists kingdom. The fungal inhibitor of Golgi traffic) as predicted from the TBC domain is predicted to carry a GAP (GTPase activat- sequences of the binding site of the drug carried by the ing protein) activity towards small G proteins of the Rab Sec7 domain [6]. This observation clearly illustrates that family [30], suggesting a potential crosstalk between Rab differences in outcome following BrefeldinA treatment and Arf pathways. Such a relationship between these two may not reflect differences in underlying molecular mech- small G proteins families, which are major regulators of anisms, but instead simply reflect neutral sequence differ- membrane traffic, would not be unprecedented, as for ences at the Sec7 domains between species. In particular, example the SYT1 ArfGEF gene was identified in yeast by not all BIGs may be BFA-sensitive or GBFs BFA-resistant, its genetic interactions with Rab proteins in the exocytic unlike suggested by their original nomenclature. pathway [31]. Interestingly, alveolates have specialized exocytic pathways based on a membrane organelle lying A novel ArfGEF subfamily in alveolates beneath the plasma membrane, the trichocyst, where this A remarkable evolutionary feature of ArfGEFs is that while unique ArfGEF family may potentially function. GBGs seem to be ubiquitous to all eukaryotes, fungi and animals kingdoms evolved their own ArfGEFs subfamilies Conclusion A conserved scenario for the activation of Arf proteins by unrelated to those of the other kingdoms. We thus addressed the question of whether Paramecium, which their GEFs? has a large number of GBGs (at least five, of which four The identification of a conserved modular architecture in are present as pairs as the result of recent duplications) but all GBG subfamily members suggests that the mechanistic appears to lack the specialization into the BIG and GBF basis for their activation of Arf is likely to follow a similar subgroups, has the same ArfGEF distribution as plants or scenario. Candidate functions for the conserved domains features a second ArfGEF subfamily. We thus searched the include oligomerization, the collection of input signals, newly sequenced genome from Paramecium tetraurelia and membrane localization, regulation of the exchange activ- the available alveolate genomes from Cryptosporidium ity, scaffolding of Arf proteins to their downstream effec- parvum and Tetrahymena thermophila for additional Sec7- tors, not excluding signaling to partners outside the Arf containing proteins. This identified a novel putative Arf- pathways. Dimerization has been reported in the BIG sub- GEF subfamily characterized by the association of the group for BIG1, which forms heterodimers with the Sec7 domain with a TBC (Tre/Bub2/Cdc16) domain (Fig- highly homologous BIG2 ArfGEF [14], and in the GBF Page 10 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 Os 9635.m03752 At 3g43300 Os 9631.m01366 At 1g01960 At 3g60860 Ag Q7PWN5 Os 9634.m04029 Dm Q9VJW1 Ce Q9XWG5 Os 9630.m00920 Sc SEC7 At 4g38200 Ca EAL04295 At 4g35380 Nc Q7SAX4 Pt GGG2 Sp SC72 Sp SC71 Pt GGG1 Os 9631.m04495 Pt GGG5 At GNOM Os 9630.m02122 Pt GGG4 Pt GGG3 At GNL1 Nc Q7SAL8 Os 9632.m00175 At GNL2 Sp Q9P7R8 Hs GBF1 Ca EAL02873 Rn GBF1 Ag Q7PXQ7 Sc GEA1 Dm Q9V696 Sc GEA2 Ce Q9XTF0 Unro Figure 8 oted neighbor-joining phylogenetic tree of the BIG/GBF subfamily Unrooted neighbour-joining phylogenetic tree of the BIG/GBF subfamily. Colour coding for the main groups is green for plants, marine blue for fungi, orange and red for animals, cyan for protists. Branches found in less than 60% bootstrap trials by either the neighbor-joining or the maximum likelihood method are in dotted lines. Species abbreviations are as in Table 1. subgroup for GNOM, which forms homodimers [27]. The an almost invariant motif in the HUS domain argues in conservation of the DCB domain in GBGs, which is favor of this domain interacting with a conserved partner. responsible for the dimerization of GNOM, suggests that However, the ancient divergence into the BIG and GBF such a dimerization function may be general to this groups and their subsequent divergence into species-spe- domain in GBGs. Another unresolved issue is the conser- cific members suggest that specialized requirements are vation of the cellular partners effecting the functions asso- likely to have evolved in most organisms, possibly yield- ciated with the conserved domains. Our identification of ing less conserved partners outside the Sec7 and HUS Page 11 of 14 (page number not for citation purposes) Hs BIG1 Hs BIG2 Rn BIG1 Rn BIG2 BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 Sec7 TBC Sec7 TBC TB Figure 9 S: a novel ArfGEF subfamily in alveolates TBS: a novel ArfGEF subfamily in alveolates. Top: Domain structure of the TBS subfamily. Below: Sequences of the TBC domain from Paramecium TBS aligned with TBC domains from known RabGAPs. Secondary structures are from the crystal structure of yeast GYP1 [30]. domains. Finally, whereas in plants all ArfGEFs are pre- Methods dicted to function according to the scheme defined by the Protein sequence databases were searched with amino conserved domains, other species have additional ArfGEF acid sequences from human BIG1, human GBF1 and Ara- subfamilies with a modular architecture unrelated to that bidopsis GNOM using the BLAST algorithm [32]. Para- of the GBG subfamily. It is not known to what extent the mecium tetraurelia genes were identified with the BLAST GBG's scenario for Arf activation will also apply to non- algorithm using genome sequence data from Genoscope GBGs ArfGEFs, acting alone or in association with protein [33] and manually annotated using Artemis [34]. partners. In the case of the GBGs, our definition of the Tetrahymena sequences were retrieved from the Tetrahy- structural homology domains as reported here should mena thermophila genome sequencing project server [35]. now provide a robust background for future investiga- Arabidopsis sequences were retrieved from the tions of their interactions and functions. Arabidopsis Genome Initiative database [36], rice sequences from the TIGR Rice annotation project [37]. Page 12 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 2. 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Average sequence mechanisms of guanine nucleotide exchange and GTP-myr- istoyl switching. Cell 1998, 95:237-248. identities were respectively 24 % (DCB domain), 26 % 6. Renault L, Guibert B, Cherfils J: Structural snapshots of the (HUS domain), 44% (Sec7 domain), 26% (HDS1 mechanism and inhibition of a guanine nucleotide exchange domain), 28% (HDS2 domain) and 21% (HDS3 factor. Nature 2003, 426:525-530. 7. Jackson CL, Casanova JE: Turning on ARF: the Sec7 family of domain). Aligned sequences were displayed with ESPript guanine-nucleotide-exchange factors. Trends Cell Biol 2000, [42] using a similarity global score of 0.15 calculated 10:60-67. 8. Cox R, Mason-Gamer RJ, Jackson CL, Segev N: Phylogenetic anal- using the BLOSUM62 matrix. Unrooted phylogenetic ysis of Sec7-domain-containing Arf nucleotide exchangers. trees were generated using the neighbor-joining algorithm Mol Biol Cell 2004, 15:1487-1505. of ClustalW excluding gapped regions, and with a maxi- 9. Peyroche A, Paris S, Jackson CL: Nucleotide exchange on ARF mediated by yeast Gea1 protein. Nature 1996, 384:479-481. mum likelihood method using the PHYML package [43]. 10. Claude A, Zhao BP, Kuziemsky CE, Dahan S, Berger SJ, Yan JP, Phylogenetic trees for individual domains was performed Armold AD, Sullivan EM, Melancon P: GBF1: A novel Golgi-asso- ciated BFA-resistant guanine nucleotide exchange factor on the subset of sequences used in Figure 3. The reliability that displays specificity for ADP-ribosylation factor 5. J Cell of the trees was assessed by a bootstrap analysis (1000 Biol 1999, 146:71-84. replicates). Trees were drawn with TreeView version 1.6.6. 11. Geldner N, Anders N, Wolters H, Keicher J, Kornberger W, Muller P, Delbarre A, Ueda T, Nakano A, Jürgens G: The Arabidopsis Secondary structure predictions on aligned sequences GNOM ARF-GEF mediates endosomal recycling, auxin were carried out with the PHD program along with the transport, and auxin-dependent plant growth. Cell 2003, ClustalW multiple alignment [39]. Non-structured linkers 112:219-230. 12. Peyroche A, Courbeyrette R, Rambourg A, Jackson CL: The ARF poor in hydrophobic residues were predicted with the exchange factors Gea1p and Gea2p regulate Golgi structure PONDR algorithm [44]. and function in yeast. J Cell Sci 2001, 114:2241-2253. 13. Mansour SJ, Skaug J, Zhao XH, Giordano J, Scherer SW, Melançon P: p200 ARF-GEP1: a Golgi-localized guanine nucleotide Abbreviations exchange protein whose Sec7 domain is targeted by the drug GEF: Guanine nucleotide exchange factor. CYH: cytohes- brefeldin A. Proc Natl Acad Sci U S A 1999, 96:7968-7973. 14. Yamaji R, Adamik R, Takeda K, Togawa A, Pacheco-Rodriguez G, Fer- ins/ARNO; EFA: Exchange Factor for Arf6; FBS: F-Box/ rans VJ, Moss J, Vaughan M: Identification and localization of two Sec7; TBS: TBC/Sec7; GBF: Golgi-associated BFA-resistant brefeldin A-inhibited guanine nucleotide-exchange proteins guanine nucleotide exchange Factor; BIG: BFA-Inhibited for ADP-ribosylation factors in a macromolecular complex. Proc Natl Acad Sci U S A 2000, 97:2567-2572. Guanine nucleotide exchange factor; GBG: GBF/BIG Gefs; 15. Achstetter T, Franzusoff A, Field C, Schekman R: SEC7 encodes an SYT1: Suppressor of ypt. DCB: Dimerization/Cyclophilin unusual high molecular weight protein required fir mem- brane traffic from the yeast Golgi apparatus. J Biol Chem 1988, Binding; HUS: Homology Upstream of Sec7; HDS: 263:11711-11717. Homology Downstream of Sec7; TBC: Tre/Bub2/Cdc16; 16. Nagai H, Kagan JC, Zhu X, Kahn RA, Roy CR: A bacterial guanine SCF: Skp1/Cull1/F box. nucleotide exchange factor activates ARF on Legionella phagosomes. Science 2002, 295:679-682. 17. Donaldson JG: Multiple roles for Arf6: sorting, structuring, and Authors' contributions signaling at the plasma membrane. J Biol Chem 2003, B.M. and V.B. carried out sequence and phylogenetic anal- 278:41573-41576. 18. Zhao X, Lasell TK, Melançon P: Localization of large ADP-ribo- ysis. A.J. participated in the domain analysis. J.Co. anno- sylation factor-guanine nucleotide exchange factors to dif- tated Paramecium sequences. D.S. and P.M. performed ferent Golgi compartments: evidence for distinct functions in protein traffic. Mol Biol Cell 2002, 13:119-133. splicing pattern analysis. N.K. and G.J. analyzed the distri- 19. Shin HW, Morinaga N, Noda M, Nakayama K: BIG2, a guanine bution of large ArfGEFs in plants. J.Ch. conceived and nucleotide exchange factor for ADP-ribosylation factors: its coordinated the study and wrote the manuscript. localization to recycling endosomes and implication in the endosome integrity. Mol Biol Cell 2004, 15:5283-5294. 20. Cullen PJ, Chardin P: Membrane targeting: what a difference a Acknowledgements G makes. Curr Biol 2000, 10:R876-8. This work was supported by a Human Frontiers in Science Program grant 21. Derrien V, Couillault C, Franco M, Martineau S, Montcourrier P, Houlgatte R, Chavrier P: A conserved C-terminal domain of to G.J., P.M. and J.Ch. We thank Genoscope for access to Paramecium EFA6-family ARF6-guanine nucleotide exchange factors whole genome shotgun primary data and Linda Sperling (CNRS, Gif-sur- induces lengthening of microvilli-like membrane Yvette) for help with annotations. protrusions. J Cell Sci 2002, 115:2867-2879. 22. Chen EH, Pryce BA, Tzeng JA, Gonzalez GA, Olson EN: Control of myoblast fusion by a guanine nucleotide exchange factor, References loner, and its effector ARF6. Cell 2003, 114:751-762. 1. Cherfils J, Chardin P: GEFs: structural basis for their activation 23. Ilyin GP, Rialland M, Pigeon C, Guguen-Guillouzo C: cDNA cloning of small GTP-binding proteins. Trends Biochem Sci 1999, and expression analysis of new members of the mammalian 24:306-311. F-box protein family. Genomics 2000, 67:40-47. Page 13 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 24. Mansour M, Lee SY, Pohajdak B: The N-terminal coiled coil 51. Saeki N, Tokuo H, Ikebe M: BIG1 is a binding partner of myosin domain of the cytohesin/ARNO family of guanine nucleotide IXB and regulates its Rho gap activity. J Biol Chem 2005. exchange factors interacts with the scaffolding protein CASP. J Biol Chem 2002, 277:32302-32309. 25. 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Charych EI, Yu W, Miralles CP, Serwanski DR, Li X, Rubio M, De Blas Your research papers will be: AL: The brefeldin A-inhibited GDP/GTP exchange factor 2, a available free of charge to the entire biomedical community protein involved in vesicular trafficking, interacts with the beta subunits of the GABA receptors. J Neurochem 2004, peer reviewed and published immediately upon acceptance 90:173-189. cited in PubMed and archived on PubMed Central 50. Garcia-Mata R, Sztul E: The membrane-tethering protein p115 interacts with GBF1, an ARF guanine-nucleotide-exchange yours — you keep the copyright factor. EMBO Rep 2003, 4:320-325. BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 14 of 14 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Genomics Springer Journals

The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf

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Springer Journals
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Copyright © 2005 by Mouratou et al; licensee BioMed Central Ltd.
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Life Sciences; Life Sciences, general; Microarrays; Proteomics; Animal Genetics and Genomics; Microbial Genetics and Genomics; Plant Genetics & Genomics
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10.1186/1471-2164-6-20
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Abstract

Background: Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs) that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. Results: Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7). Conclusion: Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains. Page 1 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 Background Guanine Nucleotide Exchange Factors (GEFs) are obliga- Bacteria tory components of signaling cascades regulated by small GTP-binding proteins (called small G proteins hereafter). RalF Insects Their biochemical activity is to stimulate the dissociation Mammals Nematode of the tightly bound GDP nucleotide from the small G protein in response to cellular signals. Thereby, they favor FBS the binding of the more abundant cellular GTP, organiz- ing the active conformation of the small G protein which SYT1 BRAG can recruit its effectors (reviewed in [1]). Each small G SYT2 EFA6 protein family features its own ensemble of GEFs charac- CYH terized by a conserved catalytic domain responsible for GBG nucleotide exchange, which is generally combined with (GBF/BIG) non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of Fungi the Arf family, which are major regulators in membrane trafficking (reviewed in [2]), the exchange domain is a conserved module of ~200 amino acids called the Sec7 domain [3]. Its biochemical (reviewed in [4]) and struc- TBS Plants tural [5,6] mechanisms have been investigated in detail. Alveolates Remarkably, the Sec7 domain is the only domain that is conserved in all ArfGEFs (reviewed in [7,8]) and it is to some extent interchangeable between species [9]. In con- Venn diag according to found Figure 1 ram the of the nin species wh e Sec7 ere each -containi subfamily ng subfamili has been es sorted trast, little is known about the functions of the other Venn diagram of the nine Sec7-containing sub- domains, which are likely to determine intracellular local- families sorted according to the species where each subfamily has been found. The TBS subfamily was identi- ization of ArfGEFs and their responsiveness to specific fied in this study. The BIG and GBF subfamilies are merged in signals. a higher order subfamily (GBG), and are the only subfamily common to all eukaryotes. As for most small G proteins, Arf family members are out- numbered by ArfGEFs in many species. In humans for instance, 5 Arf proteins have been identified, and there are at least 13 proteins carrying a Sec7 domain, of which most have been characterized as bona fide ArfGEFs (reviewed in [7,8]). Thus an individual Arf protein may be activated by active on Arf6 at the plasma membrane where they may more than one GEF, emphasizing that essential aspects in function in the crosstalk of membrane traffic, cytoskele- building up the Arf responses may be encoded by the ton dynamics and signalling in endosomal pathways modular architecture of their GEFs. Sequence similarity in (reviewed in [17]). Most members of the BIG and GBF the non-catalytic regions forms the basis for the classifica- subfamilies characterized so far function in vesicular traf- tion of ArfGEFs into subfamilies. 8 subfamilies are cur- ficking at the Golgi [12,14,18], except for BIG2, which rently identified in eukaryotes with sizes ranging from also localizes on recycling endosomes [19], and GNOM small (~40–80 kD including CYH, EFA6 and FBS), to which acts in the endosomal recycling pathway [11]. medium (~100–150 kD, including BRAG/LONER, SYT1, SYT2) and large (~170–200 kD) ArfGEFs (reviewed in The domain architecture of non-catalytic regions of Arf- [7,8]). Large ArfGEFs comprise two subfamilies which we GEFs, hence their contribution to specific aspects of the will refer to as the BIG and GBF subfamilies after the name build-up of the Arf response, is essentially not established of their human representatives. The GBF subfamily except for those ArfGEFs with domains found in other includes human GBF1 [10], Arabidopsis GNOM [11] and classes of cellular regulators. The known domains include Saccharomyces Gea1 and Gea2 [12], the BIG subfamily membrane-interacting PH domains in the CYH (reviewed human BIG1 and BIG2 [13,14] and yeast Sec7p [15]. An in [20]), EFA6 [21] and possibly BRAG/LONER[22] sub- additional subfamily called RalF is found in Rickettsie and families, and a putative F-box in the FBS subfamily [23], a Legionella bacteria, likely acting on an host Arf pathway protein-protein interaction domain that has been [16]. Analysis of the CYH and EFA6 subfamilies, present involved in the recruitment of substrates to the SCF ubiq- only in multicellular animals, and that of the large Arf- uitination machinery. Coiled-coil structures have also GEFs, found in all eukaryotes, have yielded most of the been predicted in the N-terminus of the CYH subfamily functional data currently available. CYH and EFA6 are and in the C-terminus of the EFA6 subfamily. In CYH, Page 2 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 Table 1: BIG and GBF protein sequences used in this study. Species Protein name Accession Number Size in amino acids Metazoa Ag Q7PWN5 EAA14874 1522 Q7PXQ7 EAA00837 1285 Ce Q9XWG5 NP_493386 1628 Q9XTF0 NP_499522 1820 Dm Q9VJW1 AAF53331 1653 Q9V696 AAF58532 1983 Hs BIG1 Q9Y6D6 1849 BIG2 Q9Y6D5 1785 GBF1 Q92538 1859 Rn BIG1 XP_232614 1987 BIG2 Q7TSU1 1791 GBF1 XP_347197 1883 Fungi Ca EAL04295 EAL04295 1839 EAL02873 EAL02873 1015 Nc Q7SAX4 EAA33549 1940 Q7SAL8 EAA33457 1626 Sc SEC7 P11075 2009 GEA1 P47102 1408 GEA2 P39993 1459 Sp SC71 Q9UT02 1811 SC72 Q9P7V5 1822 Q9P7R8 NP_596613 1462 Viridiplantae At At1g01960 Q9LPC5 1750 At3g43300 NP_189916 1728 At3g60860 Q9LZX8 1793 At4g35380 O65490 1711 At4g38200 NP_195533 1698 GNOM Q42510, At1g13980 1451 GNL1 Q9FLY5, At5g39500 1443 GNL2 NP_197462, At5g19160 1375 Os 9631.m01366 Q8S565 1789 9630.m00920 Q9XGN9 1687 9634.m04029 - 1704 9635.m03752 - 1680 9631.m04495 - 1456 9630.m02122 - 1396 9632.m00175 Q7XT11 1407 Alveolata Pt GGG1 CR533425 1615 GGG2 CR533424 1628 GGG3 CR533423 1598 GGG4 CR533422 1599 GGG5 CR533421 1435 Unnamed sequences are designated by their NCBI accession number, AGI (Arabidopsis Genome Initiative) locus numbers for At and TIGR model temporary IDs for Os. BIG and GBF subfamily members are in normal and bold characters respectively, except for Pt members which have not been assigned to either subfamily (see also Figure 8). Species abbreviations are: Ag, Anopheles gambiae; Ce, Caenorhabditis. elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens; Rn, Rattus norvegicus; Ca, Candida albicans; Nc, Neurospora crassa; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; At, Arabidopsis thaliana; Os, Oryza sativa; Pt, Parameciumtetraurelia). they are involved in dimerization [3], recruitment of part- been the subject of many investigations, their architecture ners [24] and Golgi targeting [25], and in actin remode- is barely described, making it difficult to associate bio- ling functions in the case of EFA6 [21]. On the other hand, chemical activities with their molecular structure. although the functions of BIG and GBF subfamilies have Page 3 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 Here we investigate the domain architecture in the BIG GNOM Gea2 Gea1-2 GBF1 2 4 5 7 and GBF subfamilies, including all sequences from fully Cyp5 Drsp2 Gmh1p p115 annotated eukaryotic genomes and our novel annotation of Sec7-containing proteins from the Paramecium tetraure- HUS HDS1 HDS2 HDS3 DCB Sec7 DCB HUS Sec7 HDS1 HDS2 HDS3 lia alveolate. Sequence comparisons combined with sec- ondary structures and splicing patterns analysis identifies BIG1 BIG1-2 BIG1-2 BIG2 five novel domains that are conserved between BIG and 8 1 3 MyosinIXB 6 GABA Rβ FKBP13 PKA GBF subfamilies, thus unifying them as a higher order subfamily with a probable common ancestor. Our analy- The com s Figure 2 ubfamilies mon domain architecture of the BIG and GBF sis of Sec7-domain containing sequences from Para- The common domain architecture of the BIG and mecium also introduces a novel subfamily of ArfGEFs GBF subfamilies. From N- to C-terminus : DCB , HUS, unique to alveolates, which we call TBS (TBC-Sec7). Sec7, HDS1, HDS2, HDS3. Linker regions of variable length and sequence are shown in grey, with alternate splicing sites in human GBF1, BIG1 and BIG2 in black, white and grey dia- Results and discussion mond shapes respectively. Interactions reported in the litter- A conserved domain architecture in BIG and GBF ature are indicated in boxes of width corresponding to the subfamilies mapped regions, except for myosin IXb interaction which The BIG and GBF subfamilies are the only ArfGEFs sub- was studied only with full-length BIG1. Arrows indicate pre- families common to all eukaryotes [8] and the sole Arf- 1 2 3 dicted Protein kinase A-anchoring motifs. [45]; [27]; [46]; GEFs present in plants [26] (Figure 1). They are therefore 4 5 6 7 8 [47]; [48]; [49]; [50]; [51]. possible representatives of ancestral ArfGEF functions and may provide a model to understand the nature and imple- mentation of activities associated with the exchange func- tion carried by the conserved Sec7 domain. However, domain 'hunting' in BIG and GBF subfamilies was com- plicated by the facts that the Sec7 domain is their only which is also found in the yeast protein Ysl2p [28], all of domain that could be identified from known domain rep- them are unique to these two ArfGEFs subfamilies within ertoires, and that their poorly characterized non-catalytic the detection limits of the BLAST search. The HUS domain regions were not found outside these ArfGEF subfamilies. features a remarkably conserved N(Y/F)DC(D/N) motif, Alternatively, we based our search of candidate structural which we call the HUS box, which is predicted to locate in domains in BIGs and GBFs on the bioinformatics analysis a loop where it may be available for functional interac- of their own sequences, taking advantage of the growing tions (Figure 4). The N- and C-terminal ends of BIGs and number of sequences from fully annotated genomes from GBFs are more variable, including an unusual enrichment mammals, insects, plants, nematode, and fungi, to which in Asp/Glu or Pro residues in some members. A specific we included our annotation of Sec7-containing proteins feature of BIG members is that their C-terminus is in gen- from the newly sequenced genome of Paramecium. eral less variable than that of GBFs, and is predicted with a significant amount of secondary structures. In contrast Multiple alignments of 42 sequences (listed in Table 1) to the predicted structural domains, the intervening revealed that the BIG and GBF subfamilies share an unex- regions are highly variable in length and do not yield pected conserved architecture (schematized in Figure 2). aligned sequences. Analysis of their amino-acid composi- Two homology domains are located in N-terminus of the tion reveals a paucity of hydrophobic residues which is Sec7 domain – the DCB (~150 aa) and HUS (Homology predicted to associate with an essentially unfolded Upstream of Sec7, ~170 aa) domains – and three in its C- conformation, suggesting that they act as linkers to tether terminus -the HDS1 (Homology Downstream of Sec7, the functional domains together. ~130 aa), HDS2 (~160 aa) and HDS3 (~120 aa) domains (Figure 3,4,5,6,7). In Arabidopsis GNOM, the DCB To further investigate the predicted organization of BIGs domain is included in an N-terminal region of ~250 resi- and GBFs in 6 conserved helical domains connected by dues involved in dimerization and possibly binding to variable linkers, splicing patterns of human BIGs and cyclophilin5 and called the Dimerization/Cyclophilin GBFs were analyzed in the large number of cDNAs and Binding region [27], after which the new domain was ESTs in the databases that correspond to GBF/BIG tran- named (Figure 3). All domains are predicted to have a scripts. This revealed the use of alternate splice donor and high content of α-helices that co-align in the multiple acceptor sites predicted to yield proteins with insertions sequence alignments, reinforcing the prediction of and deletions ranging from 1 to 38 residues, and a sequence similarities and suggesting that these domains number of splice variants arising from exon skipping form folded structural units that may share common func- (Table 2). Strikingly, all observed sequence variations tional features. Except for the N-terminal DCB domain occur in regions identified as linkers between conserved Page 4 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 1 1020 3040 50 BIG1_HUMAN/70-228 SKTNFIEADKYFLPFELACQS..KCPRIVSTSLDCLQKLIAYGHLTGNAPD...................STTPG BIG2_HUMAN/58-216 PKANFIEADKYFLPFELACQS..KSPRVVSTSLDCLQKLIAYGHITGNAPD...................SGAPG Q9VJW1_DROME/72-230 DAASIINAETYFLPFELACKS..RSPRIVVTALDCLQKLIAYGHLTGSIQD...................SANPG Q9XWG5_CAEEL/69-227 AGGTAVEADRYFLPFELACNS..KSPKIVITALDCLQKLIAYGHLTGRGAD...................ISNPE 3g60860_ARATH/77-233 IEYSLADSELIFSPLINACGT..GLAKIIEPAIDCIQKLIAHGYIRGESDP...................SGGAE 1g01960_ARATH/72-228 AEYSLAESEIILSPLINASST..GVLKIVDPAVDCIQKLIAHGYVRGEADP...................TGGPE 4g35380_ARATH/62-214 SGLAASDADSVLQPFLLSLET..AYSKVVEPSLDCAFKLFSLSILRGEIQS.....................SKQ 4g38200_ARATH/61-215 FGLTTSDADAVLQPLLLSLDT..GYAKVIEPALDCSFKLFSLSLLRGEVCS.....................SSP 3g43300_ARATH/97-252 HTLGGAEVELVLKPLRLAFET..KNLKIFDAALDCLHKLIAYDHLEGDPGL...................DGGKN SEC7_YEAST/267-445 NNPHYVDSILVFEALRASCRT..KSSKVQSLALDCLSKLFSFRSLDETLLVNPPDSLASNDQRQDAADGITPPPK GGG2_PARTE/36-189 QIKDFYDANHLLKVYQQCIES..KQAKLIELALFDIKNIVDQGYLAGEQII......................GE GBF1_HUMAN/54-215 TELSEIEPNVFLRPFLEVIRSEDTTGPITGLALTSVNKFLSYALIDPTHEG.......................T Q9V696_DROME/56-217 EDLRQIEPQVFLAPFLEVIRTADATGPLTSLALASVNKLLSYGLIDPTSPN.......................L Q9XTF0_CAEEL/56-248 ADLADMNPQTYLSPFLDVIKAQNTNGPITEAALAAVAKFLNYGLIDASSIK.......................A GNOM_ARATH/83-245 QPWHTISPMLYLQPFLDVIRSDETGAPITSIALSSVYKILNLNVIDQNTAN.......................I GNL1_ARATH/81-243 SNWQYVDPRLYIQPFLDVILSDETGAPITGVALSSVYKILTLEVFTLETVN.......................V GNL2_ARATH/67-228 QDWRTIDPSVYLSPFLEVIQSDEIPASATAVALSSILKILKIEIFDEKTPG.......................A GEA2_YEAST/87-248 KNLDNIDSLTILQPFLLIVSTSSISGYITSLALDSLQKFFTLNIINESSQN.......................Y GEA1_YEAST/90-251 KGLDSLNALELLKPFLEIVSASSVSGYTTSLALDSLQKVFTLKIINKTFND.......................I .............................. 60 70 80 90 BIG1_HUMAN/70-228 KKLIDRIIIC ET CG F...QGPQ.TDEGVQLQ..............................IIL KA LTAVTS..Q BIG2_HUMAN/58-216 KRLIDRIVIC ET CS F...QGPQ.TDEGVQLQ..............................IIL KA LTAVTS..P Q9VJW1_DROME/72-230 HLLIDRIVIC VT YG F...SGPQ.TDEAVQLQ..............................IIL KA LTVVTS..Q Q9XWG5_CAEEL/69-227 RKLIDRIVIP EA CA F...LGQG.TDETVLLQ..............................LIV KA LAVVLS..T 3g60860_ARATH/77-233 SLLLFKLIVC DS CK H...D..L.GDESIELP..............................VLL KT LSAINS..I 1g01960_ARATH/72-228 ALLLSKLIIC ET CK H...E..L.DDEGLELL..............................VLL KT LTAVTS..I 4g35380_ARATH/62-214 DSILFKLVVV NA SK G.....AI.AEEPIQLA..............................VLL RV LAAVRS..P 4g38200_ARATH/61-215 DSLLYKLIIV HA CK C.....GI.GEESIELA..............................VLL RV LAAVRS..P 3g43300_ARATH/97-252 SAPFTDILVC NM CS V...DNS..SPDSTVLQ..............................VLL KV LTAVAS..G SEC7_YEAST/267-445 QKIIDAAIIC DT SD F...QGEG.TDDRVELQ..............................IVL RA SSCILEEDS GGG2_PARTE/36-189 KRAIEIALVT DL MQ Q.....LE.KEETVQIH..............................MII KA QAIMTN..K GBF1_HUMAN/54-215 AEGMENMAVA DA TH RFVGTDPA.SDEVVLMK..............................ILL QV RTLLLT..P Q9V696_DROME/56-217 ADIVERIAVA DA TH RFMGTDQS.SDGVTFMR..............................VIL EV HTLIRS..P Q9XTF0_CAEEL/56-248 ANAVESIAVT YA VH KFIGGKSTGSDECLFLRNSRKKVNFHKKSTQNLLEMCQNFIRSANLDEFL YV RSLLLS..P GNOM_ARATH/83-245 EDAMHLVVVC DS TS RFEVTDPA.SEEVVLMK..............................ILL QV LACMKN..K GNL1_ARATH/81-243 GEAMHIIVVC DA KS RFEVTDPA.SEEVVLMK..............................ILL QV LACVKS..K GNL2_ARATH/67-228 KDAMNSIVIC SG TS RLEKTDLV.SEDAVMMR..............................ILL QV TGIMKH..P GEA2_YEAST/87-248 IGAHRATVLC NA TH RFEGSQQL.SDDSVLLK..............................VVL FL RSIVDS..P GEA1_YEAST/90-251 QIAVRETVLC VA TH RFEASKQI.SDDSVLLK..............................VVL TL RDIITS..S 100 110 120 130 140 150 BIG1_HUMAN/70-228 HI.EIHEGTVLQAVRTCYNIYLAS..KNLINQTTAKATLTQMLNVIFARMENQALQEAKQMEKERHRQH BIG2_HUMAN/58-216 HI.EIHEGTILQTVRTCYNIYLAS..KNLINQTTAKATLTQMLNVIFTRMENQVLQEARRLEKPIQSKP Q9VJW1_DROME/72-230 HV.EIHEFTLLQAVRTCYDIYLSS..KNLVNQTTARATLTQMLNVIFARMENQVYELPPPNSNPTNGSI Q9XWG5_CAEEL/69-227 HC.EVHGASLILAVRTCFNIYLTS..KSPINQATAKGTLTQVINTVFGNMEKFGNIKDDETIVREVVEV 3g60860_ARATH/77-233 SL.RIHGKCLLLVVRTCYDIYLGS..KNVVNQTTAKASLIQILVIVFRRMEADSSTVPIQPIVVAELME 1g01960_ARATH/72-228 SL.RIHGDSLLQIVRTCYGIYLGS..RNVVNQATAKASLVQMSVIVFRRMEADSSTVPIQPIVVAELME 4g35380_ARATH/62-214 CI.LIRGDCLLHVVKTCYNIYLGG..LSGTTQICAKSVLAQMMLVIFTRSEEDSLDVSVKTIYVNEL.. 4g38200_ARATH/61-215 RI.LIRGDCLLHLVRTCYNVYLGG..FNGTNQICAKSVLAQIMLIVFTRSEANSMDASLKTVNVNDLLA 3g43300_ARATH/97-252 KF.KVHGEPLLGVIRVCYNIALNS....PINQATSKAMLTQMISIVFRRMETDIVSASSTVSQEEHVSG SEC7_YEAST/267-445 SS.LCHGASLLKAIRTIYNVFVFS..LNPSNQGIAQATLTQIISSVYDKIDLKQSTSSAVSLSTKNHQQ GGG2_PARTE/36-189 KH.HIYGESVTRVFSLLINLHSVS..KIVAIINASKEACQKIVSTYFARLEDYGILAEDEYQLAIQQQG GBF1_HUMAN/54-215 VGAHLTNESVCEIMQSCFRICFEMR.LSELLRKSAEHTLVDMVQLLFTRLPQFKEEPKNYVGTNMKKLK Q9V696_DROME/56-217 EGAAVSNVSMCEVMLSCFKISFEPR.LSELLRRSAEKSLKDMVLLFFMRLPQFAEERSDTMLQKRFTIG Q9XTF0_CAEEL/56-248 PGILLSNEAVCDMMQSCFRIVFEQN.LSLLLRKAAESTLADMTQLIFTRLPTFVEDTRHPYIRQLVNPT GNOM_ARATH/83-245 ASVMLSNQHVCTVVNTCFRVVHQAGMKGELLQRVARHTMHELVRCIFSHLPDVERTETTLVNRAGSIKQ GNL1_ARATH/81-243 ASNGLSNQDICTIVNTCLRVVHQSSSKSELLQRIARHTMHELIRCIFSQLPFISPLANECELHVDNKVG GNL2_ARATH/67-228 SSELLEDQAVCTIVNTCFQVVQQSTGRGDLLQRNGRYTMHELIQIIFSRLPDFEVRGDEGGEDSESDT. GEA2_YEAST/87-248 YGDLLSNSIIYDVLQTILSLACNNR.RSEVLRNAAQSTMIAVTVKIFSKLKTIEPVNVNQIYINDESYT GEA1_YEAST/90-251 FGDYLSDTIIYDVLQTTLSLACNTQ.RSEVLRKTAEVTIAGITVKLFTKLKLLDPPTKTEKYINDESYT Th Figure 3 e conserved domains of the BIG/GBF subfamily: DCB domain The conserved domains of the BIG/GBF subfamily: DCB domain. Multiple sequence alignement of the conserved domains from BIG and GBF representative sequences showing secondary structure predictions that co-align in all sequences. Colour coding is red for invariant residues, yellow for a sequence similarity score threshold of 0.15 using the BLOSUM62 matrix. The gap in helix 4 is due to an insert in the drosophila Q9V696 sequence, and may be resulting from a sequence anno- tation error. Page 5 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 1 102030 405060 BIG1_HUMAN/411-593 PGAKFSHILQKDAFLVFRSLCKLSMKPLSDG...PPDPKSHELRSKILSLQLLLSILQNAGPIFRTNEM...... BIG2_HUMAN/362-544 VAARFSHVLQKDAFLVFRSLCKLSMKPLGEG...PPDPKSHELRSKVVSLQLLLSVLQNAGPVFRTHEM...... Q9VJW1_DROME/304-486 VTAKFTHILQKDAFLVFRALCKLSMKPLPDG...HPDPKSHELRSKVLSLHLLLLILQNAGPVFRSNEM...... Q9XWG5_CAEEL/264-443 DQFTFMNAYQKDAFLVFRALCILAQKEE.GG...A..SNEMSLRSKLLALEMLLLVLQNSSSILQSSQP...... 3g60860_ARATH/337-519 LEVQIENKLRRDACLVFRALCKLSMKAPPKE...SS.ADPQSMRGKILALELLKILLENAGAVFRTSEK...... 1g01960_ARATH/328-507 SEVQIGNKLRRDAFLVFRALCKLSMKTPPKE.......DPELMRGKIVALELLKILLENAGAVFRTSDR...... 4g35380_ARATH/284-466 SETGDMSKVRQDAFLLFKNLCKLSMRFSSKE...NN.DDQIMVRGKTLSLELLKVIIDNGGSVWRTNES...... 4g38200_ARATH/263-456 EDEGTGSKIREDGFLLFKNLCKLSMKFSSQE...NT.DDQILVRGKTLSLELLKVIIDNGGPIWLSDER.QLTLP 3g43300_ARATH/324-509 IELESMSIGQRDALLVFRTLCKMGMKEDS.........DEVTTKTRILSLELLQGMLEGVSHSFTKNFH...... SEC7_YEAST/488-678 IAITNQDLAVKDAFLVFRVMAKICAKPLETE....LDMRSHAVRSKLLSLHIIYSIIKDHIDVFLSHNI.FL..P GGG2_PARTE/318-500 SHSTFSEQYVKDAYEILEMLCQLSQRDPQN.....PQLAQMIIKCKVLSLELIYEALAQSDTTILQHKPK..... GBF1_HUMAN/392-566 EGTALVPYGLPCIRELFRFLISLTNPHDR..........HNSEVMIHMGLHLLTVALESAP..VAQCQT...... Q9V696_DROME/356-532 DVTSLSPYGLPFIQELFRFLIILCNPLDK..........QNSDSMMHTGLSLLTVAFEVAADNIGKYEG...... Q9XTF0_CAEEL/377-558 GGEEKMPYGLPCCRELLRFLITMTNPVDR..........HNTESMVILGLNLLIVALEAIADFLPNYDI...... GNOM_ARATH/305-491 LHIMTEPYGVPSMVEIFHFLCSLLNVVEHVGMGSRSNTIAFDEDVPLFALNLINSAIELGGSSIRHHPR...... GNL1_ARATH/306-492 ENAMMAPYGIPCMVEIFHFLCTLLNVGENGEVNSRSNPIAFDEDVPLFALGLINSAIELGGPSFREHPK...... GNL2_ARATH/233-414 ...MSGGYGIRCCIDIFHFLCSLLNVVEVVENLEGTNVHTADEDVQIFALVLINSAIELSGDAIGQHPK...... GEA2_YEAST/320-508 QAYADDNYGLPVVRQYLNLLLSLIAPE.........NELKHSYSTRIFGLELIQTALEISGDRLQLYPR...... GEA1_YEAST/305-490 AENVEPNYGITVIKDYLGLLLSLVMPE.........NRMKHTTSAMKLSLQLINAAIEISGDKFPLYPR...... 70 80 90 100 110 120 BIG1_HUMAN/411-593 ......FINAIKQYLCVALSKNG.VSSVPEVFELSLSIFLTLLSNFKTHLKMQIEVFFKEIFLYILET....... BIG2_HUMAN/362-544 ......FINAIKQYLCVALSKNG.VSSVPDVFELSLAIFLTLLSNFKMHLKMQIEVFFKEIFLNILET....... Q9VJW1_DROME/304-486 ......FIMAIKQYLCVALSNNG.VSLVPEVFELSLSIFVALLSNFKVHLKRQIEVFFKEIFLNILEA....... Q9XWG5_CAEEL/264-443 ......CIIVIKRTLCMALTRNA.VSNNIQVFEKSLAIFVELLDKFKTHLKASIEVFFNSVILPMLDS....... 3g60860_ARATH/337-519 ......FSADIKQFLCLSLLKNS.ASTLMIIFQLSCSIFISLVARFRAGLKAEIGVFFPMIVLRVVEN....... 1g01960_ARATH/328-507 ......FLGAIKQYLCLSLLKNS.ASNLMIIFQLSCSILLSLVSRFRAGLKAEIGVFFPMIVLRVLEN....... 4g35380_ARATH/284-466 ......FINAVKQYLCLSLLKNS.AVSIMSIFQLQCAIFMSLLSKLRSVLKAEIGIFFPMIVLRVLEN....... 4g38200_ARATH/263-456 PQKICRFLNAIKQLLCLSLLKNS.ALSVMSIFQLQCAIFTTLLRKYRSGMKSEVGIFFPMLVLRVLEN....... 3g43300_ARATH/324-509 ......FIDSVKAYLSYALLRAS.VSQSSVIFQYASGIFSVLLLRFRDSLKGEIGIFFPIIVLRSLDN....... SEC7_YEAST/488-678 GKERVCFIDSIRQYLRLVLSRNA.ASPLAPVFEVTLEIMWLLIANLRADFVKEIPVFLTEIYFPISEL....... GGG2_PARTE/318-500 ......LISILKEQLLESLLKNS.LSAEKQLLILTLNIFIQLIWRVRSHLKKELEALIENVYFKFLES....... GBF1_HUMAN/392-566 ......LLGLIKDEMCRHLFQLL.SIERLNLYAASLRVCFLLFESMREHLKFQMEMYIKKLMEIITVE....... Q9V696_DROME/356-532 ......LLELVKDDLCRNLISLL.SSERLSIFAADLQLCFLLFESLRGHLKFQLEAYLRKLSEIIASD....... Q9XTF0_CAEEL/377-558 ......LMPLIKNELCRNLLQLL.DTNRLPVLAATNRCCFLLFESMRMHMKFQLESYLKKLQSIVLTEEKQHE.. GNOM_ARATH/305-491 ......LLSLIQDELFRNLMQFG.LSMSPLILSMVCSIVLNLYQHLRTELKLQLEAFFSCVILRLAQG....... GNL1_ARATH/306-492 ......LLTLIQDDLFCNLMQFG.MSMSPLILSTVCSIVLNLYLNLRTELKVQLEAFFSYVLLRIAQS....... GNL2_ARATH/233-414 ......LLRMVQDDLFHHLIHYG.ASSSPLVLSMICSCILNIYHFLRKFMRLQLEAFFSFVLLRVTAF....... GEA2_YEAST/320-508 ......LFTLISDPIFKSILFIIQNTTKLSLLQATLQLFTTLVVILGNNLQLQIELTLTRIFSILLDDGTANNSS GEA1_YEAST/305-490 ......LFSLISDPIFKSVLFIIQSSTQYSLLQATLQLFTSLVVILGDYLPMQIELTLRRIFEILEDT...TISG ......... 130 140 150 160 170 180 BIG1_HUMAN/411-593 ...STS.SFDHKWMVIQ.........TLTRIC.ADAQSVVDIYVNYDCDLNAANIFERLVNDLSKIAQGR BIG2_HUMAN/362-544 ...STS.SFEHRWMVIQ.........TLTRIC.ADAQCVVDIYVNYDCDLNAANIFERLVNDLSKIAQGR Q9VJW1_DROME/304-486 ...NSS.SFEHKWMVIQ.........ALTRIC.ADAQSVVDIYVNYDCDFSAANLFERLVNDLSKIAQGR Q9XWG5_CAEEL/264-443 ...NTC.AFEQKWIVLN.........TIGKIL.ANPQSVVDMFVNYDCDMTSPNLFKSIVEVVSKTTRTT 3g60860_ARATH/337-519 ...VAQPNFQQKMIVLR.........FLDKLC.LDSQILVDIFLNYDCDVNSSNIFERMVNGLLKTAQGV 1g01960_ARATH/328-507 ...VAQPDFQQKMIVLR.........FLDKLC.VDSQILVDIFINYDCDVNSSNIFERMVNGLLKTAQGV 4g35380_ARATH/284-466 ...VLQPSYLQKMTVLN.........LLDKMS.QDPQLMVDIFVNYDCDVESSNILERIVNGLLKTALGP 4g38200_ARATH/263-456 ...VLQPSFVQKMTVLS.........LLENIC.HDPNLIIDIFVNFDCDVESPNIFERIVNGLLKTALGP 3g43300_ARATH/324-509 ...SECPN.DQKMGVLRYNIFLLVQMMLEKVC.KDPQMLVDVYVNYDCDLEAPNLFERMVTTLSKIAQGS SEC7_YEAST/488-678 ...TTS.TSQQKRYFLS.........VIQRIC.NDPRTLVEFYLNYDCNPGMPNVMEITVDYLTRLALTR GGG2_PARTE/318-500 ...SNS.SFDHKQYTLK.........VFNKIL.TRPKVVIEIFVNYDCSVGQNNLLKKILDMQCRIIQGR GBF1_HUMAN/392-566 ...NPKMPYEMKEMALE.........AIVQLW.RIPSFVTELYINYDCDYYCSNLFEELTKLLSKNAFPV Q9V696_DROME/356-532 ...NPKTPYEMRELALD.........NLLQLW.RIPGFVTELYINYDCDLYCTDMFESLTNLLSKYTLSA Q9XTF0_CAEEL/377-558 ...NGGGGTEQKEMALE.........SLVQLW.RIPGLVTEMYLNFDCDLYCGNIFEDLTKLLVENSFPT GNOM_ARATH/305-491 ...KYGPSYQQQEVAME.........ALVNFC.RQKSFMVEMYANLDCDITCSNVFEELSNLLSKSTFPV GNL1_ARATH/306-492 ...KHGSSYQQQEVAME.........ALVDLC.RQHTFIAEVFANFDCDITCSNVFEDVSNLLSKNAFPV GNL2_ARATH/233-414 .....TGFLPLQEVALE.........GLINFC.RQPAFIVEAYVNYDCDPMCRNIFEETGKVLCRHTFPT GEA2_YEAST/320-508 SENKNKPS.IIKELLIE.........QISILWTRSPSFFTSTFINFDCNLDRADVSINFLKALTKLALPE GEA1_YEAST/305-490 DVSKQKPP.AIRELIIE.........QLSILWIHSPAFFLQLFVNFDCNLDRSDLSIDFIKELTKFSLPA Th Figure 4 e conserved domains of the BIG/GBF subfamily: HUS domain The conserved domains of the BIG/GBF subfamily: HUS domain. See Figure 3 legend for alignment details. The highly conserved HUS motif is boxed in blue. The gap in helix 5 domain is due to an insert in the Arabidopsis 3g43300 sequence, and may be resulting from a sequence annotation error. domains (Figure 2). Together with our domain analysis, where the impact upon folding of domains with essential this suggests that splicing at non-canonical exon/intron function would be minimal. boundaries is only tolerated in regions of the protein Page 6 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 1 10203040506070 BIG1_HUMAN/915-1083 MEQMAKTAKALMEAVSHVQAPFTSATHLEHVRPMFKLAWTPFLAAFSVGLQDCDDTEVASLCLEGIRCAIRIACI BIG2_HUMAN/860-1028 MEQMAKTAKALMEAVSHAKAPFTSATHLDHVRPMFKLVWTPLLAAYSIGLQNCDDTEVASLCLEGIRCAIRIACI Q9VJW1_DROME/801-969 MEVISLTATNLMQSVSHVKSPFTSAKHLEHVRPMFKMAWTPFLAAFSVGLQDCDDPEIATLCLDGIRCAIRIACI Q9XWG5_CAEEL/767-934 .FFRTSKKLALMESASDADAYFTPAQHQHHVKPMFKICWTPCLAAFSVGVQMSDDEEEWSLCLRGFRLGVRAACV 3g60860_ARATH/843-1008 DDLMKHMQEQFKEKARKSESTYYAATDVVILRFMIEACWAPMLAAFSVPLDQSDDLIVINICLEGFHHAIHATSL 1g01960_ARATH/835-999 .DLIRHMQERFKEKARKSESVYYAASDVIILRFMVEVCWAPMLAAFSVPLDQSDDAVITTLCLEGFHHAIHVTSV 4g35380_ARATH/793-958 GRLIRDIQEQFQAKPEKSESVYHTVTDISILRFILEVSWGPMLAAFSVTIDQSDDRLATSLCLQGFRYAVHVTAV 4g38200_ARATH/776-942 GLLIKDIQEKFRSKSGKSESAYHVVTDVAILRFMVEVSWGPMLAAFSVTLDQSDDRLAAVECLRGFRYAVHVTAV 3g43300_ARATH/796-957 TEDIVRKTQEIFRKHGVKRGVFHTVEQVDIIRPMVEAVGWPLLAAFSVTMEVGDNKPRILLCMEGFKAGIHIAYV SEC7_YEAST/1055-1220 ISSKTELVFKNLNKNKGGPDVYYAASHVEHVKSIFETLWMSFLAALTPPFKDYDDIDTTNKCLEGLKISIKIAST GGG2_PARTE/791-943 ...EDSLKKWFKEHP..NSDAFCYVNSIEHMKSLLQQTWSVIFASISVFLEQSEDQQQILLCFETIQAFIQLMGR GBF1_HUMAN/893-1066 LVRENYVWNVLLHRGATPEGIFLRVPTASYDLDLFTMTWGPTIAALSYVFDKSLEETIIQKAISGFRKCAMISAH Q9V696_DROME/839-1021 LVRENYQWKVLLRRGDTHDGHFHYVHDASYDVEIFNIVWGASLSALSFMFDKST.ETGYQRTLAGFSKSAAISAH Q9XTF0_CAEEL/854-1051 .VKEDYMWKVLLRRGETAEGSFYHAPTGWNDHDLFAVCWGPAVAALSYVFDKSEHEQILQKALTGYRKCAKIAAY GNOM_ARATH/756-930 PEMTPSRWIDLMHKSKKTAPYILADSRAYLDHDMFAIMSGPTIAAISVVFDHAEHEDVYQTCIDGFLAIAKISAC GNL1_ARATH/758-930 ..MTASRWISVIYKSKETSPYIQCDAASHLDRDMFYIVSGPTIAATSVVFEQAEQEDVLRRCIDGLLAIAKLSAY GNL2_ARATH/688-861 .EMNPNRWIELMNRTKTTQPFSLCQFDRRIGRDMFATIAGPSIAAVSAFFEHSDDDEVLHECVDAMISIARV.AQ GEA2_YEAST/777-982 .ISSTTVITEIKKDTQSVMDKLTPLELLNFDRAIFKQVGPSIVSTLFNIYVVASDDHISTRMITSLDKCSYISAF GEA1_YEAST/773-972 .....SVMTEMQRDFTNPISKLAQIDILQYEKAIFSNVRDIILKTLFKIFTVASSDQISLRILDAISKCTFINYY 80 90 100 110 BIG1_HUMAN/915-1083 FI S QLERDY A VQALR A FTLLTVSSGITE...................................MKQKT NID IKTL BIG2_HUMAN/860-1028 FM G QLERDY A VQALR A FSLLTASSSITK...................................MKQKT NID IKTL Q9VJW1_DROME/801-969 FM H SLERDY A VQALR A FTLLNANSPINE...................................MKAKT NID IKTL Q9XWG5_CAEEL/767-934 LA Q TLERNF A IQALR A FTLLTAKNSLGE...................................MRVKA NIE IKLL 3g60860_ARATH/843-1008 MM S KTHRDF A VTSLK A FTSLHSPA...D...................................IKQRA NIE IKAI 1g01960_ARATH/835-999 ML S KTHRDF A VTSLK A FTSLHSPA...D...................................IKQKA NIE IKAI 4g35380_ARATH/793-958 MM G QTQRDF A VTSMK A FTNLHCAA...D...................................MKQKA NVD VKAI 4g38200_ARATH/776-942 MM G QTQRDF A VTSMK A FTNLHCAG...D...................................MKQKA NVD VKAI 3g43300_ARATH/796-957 LM G DTMRYF A LTSLR V FTFLHAPK...E...................................MRSKA NVE LRIL SEC7_YEAST/1055-1220 FI R NDARTF S VGALQ V FCNLQNLE...E...................................IKVKA NVN MVIL GGG2_PARTE/791-943 FL D DEEKDF T ISFLR Y YCTNI.PS........................................NYKG QIL VQTL GBF1_HUMAN/893-1066 YL G SDVFDL N IISLK C FTALSSE..............................SIENLPSVFGSNPKA AHI AKTV Q9V696_DROME/839-1021 YL N HSDFDL A VLTLK C FTTLLSSVEQHEPAPANNE...TQ.................QAVNFGLNGKA AQA MRTV Q9XTF0_CAEEL/854-1051 YM G KEVFDL N CIHLK C FTTLTSMRDGGAGGGADED...VDLSAAALLSHS..SSPEAVALAFGENHKA AQL TRTL GNOM_ARATH/756-930 HL H EDVLDL D VVSLK C FTTLLNPSSV.............................DEPVLAFGDDAKA ARM TITI GNL1_ARATH/758-930 YL H NSVLDL D VVSLK C FTPFFAPLSA.............................DEAVLVLGEDARA ARM TEAV GNL2_ARATH/688-861 YL G EDILDL E IASFK C FTTLLNPYTT............................PEETLFAFSHDMKA PRM TLAV GEA2_YEAST/777-982 FF D KDLFNI D LNSIK A GTTLINSSHDDELSTLAFEYGPMPLVQIKFEDTNTEIPVSTDAVRFGRSFKN GQL TVVF GEA1_YEAST/773-972 FF S DQSYNT D VLHLE G MTTLAQSSA....KAVELDVDSIPLVEIFVEDTGSKISVSNQSIRLGQNFKC AQL TVLY 120 130 140 150 160 BIG1_HUMAN/915-1083 IV T AHTDGN...YLGNSWHEILC K ISQLKLAQLIGTGVKP..RYISGTVRGREGSLTGT BIG2_HUMAN/860-1028 IV T AHTDGN...YLGNSWHEILC K ISQLELAQLIGTGVKT..RYLSGSGREREGSLKGH Q9VJW1_DROME/801-969 IV M AHTDGN...YLGSSWLDIVC K ISQLELAQLIGTGVRP..QFLSGAQTTLKDSLNPS Q9XWG5_CAEEL/767-934 LI L GDEDGE...YLEENWVDVMC K MSSLELVQLIGTGLNS..AMSHDTDSSRQYVMKAT 3g60860_ARATH/843-1008 LL R ADEEGN...YLQDAWEHILC T VSRFEQLHLLGEGAPP..DATFFASKQNESEKSKQ 1g01960_ARATH/835-999 VL K AEEEGN...YLQDAWEHILC T VSRFEHLHLLGEGAPP..DATFFAFPQTESGNSPL 4g35380_ARATH/793-958 II T AIEDGN...HLHGSWEHILC T LSRIEHLQLLGEVSPS..EKRYVPTKKAEVDDKKA 4g38200_ARATH/776-942 II S AIEDGN...HLQDAWEHILC T LSRIEHLQLLGEGAPS..DASYFASTETEEKKALG 3g43300_ARATH/796-957 LL G CDSEPD...TLQDTWNAVLC E VSRLEFII.STPGIAA..TVMHGSNQISRDGV... SEC7_YEAST/1055-1220 LV E ALSEGN...YLEGSWKDILV L VSQMERLQLISKGIDR..DTVPDVAQARVANPRVS GGG2_PARTE/791-943 IV K ILQSGQ...YLRKSWKVALL Q ISRLEQLHQVVKKIKV..DSPYKENYNQED..... GBF1_HUMAN/893-1066 FL H AHRHGD...ILREGWKNIMA E MLQLFRAQLLPKAMIE..VEDFVDPNGKISLQREE Q9V696_DROME/839-1021 FL L VHDYGD...CLRESWKHILL D YLQLFRLKLLPKSLIE..VEDFCEANGKAMLILEK Q9XTF0_CAEEL/854-1051 FL Y VHENGN...ILREGWRNLFA E LLQLFRARLLPAELTE..VEDYVDEKGWVNIQRVH GNOM_ARATH/756-930 FI T ANKYGD...YIRTGWRNILC D ILRLHKLGLLPARVAS..DAADESEHSSEQGQGKP GNL1_ARATH/758-930 FI L ANKYGD...YISAGWKNILC E VLSLNKLHILPDHIAS..DAADDPELSTSNLEQEK GNL2_ARATH/688-861 FL T ANTFGD...SIRGGWRNIVC D LLKLRKLQLLPQSVIE..FEINEENGGSESDMNNV GEA2_YEAST/777-982 FI R IRRNKDPKIFSKELWLNIVI N ILTLYEDLILSPDI..FPDLQKRLKLSNLPKPSPE GEA1_YEAST/773-972 FI Q IKEISDPSIVSTRLWNQIVL Q ILKLFENLLMEPNLPFFTNFHSLLKLPELPLPDPD Th Figure 5 e conserved domains of the BIG/GBF subfamily: HDS1 domain The conserved domains of the BIG/GBF subfamily: HDS1 domain. See Figure 3 legend for alignment details. Evolution of BIGs and GFBs from a common ancestor unified as a higher order ArfGEF subfamily (called below Combined, our analysis reveals that the BIG and GBF sub- GBG for GBF/BIG GEFs), from which unrooted phyloge- families share the same overall domain organization, and netic trees can be built (Figure 8). Unlike previous phylo- are likely to descend from a common ancestor gene that genetic analysis which compared ArfGEFs based on their duplicated first to form the BIG and GBF groups, and Sec7 domains after diverging non-catalytic regions have again within these groups to yield species-specific BIG and been trimmed [8], our trees were established from the GBF members. These two subfamilies can therefore be simultaneous alignment of all 6 conserved domains Page 7 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 1 10 20304050 BIG1_HUMAN/1107-1289 ASIQESIGETSSQSVV..VA.VDI R FTGSTRLDGNAIVDFVRWLCAVSMDELLST..........TH...PRMFS BIG2_HUMAN/1054-1236 ASFQESVGETSSQSVV..VA.VDI R FTGSTRLDGNAIVDFVRWLCAVSMDELASP..........HH...PRMFS Q9VJW1_DROME/968-1149 PSVKEHIGETSSQSVV..VA.VDI R FTGSMRLDGDAIVDFVKALCQVSVDELQQ...........QQ...PRMFS Q9XWG5_CAEEL/943-1125 HSLQDALGETSSQSVV..VA.IDI R FNGSARLSAEAIVYFVRALCAVSREELSHP..........AA...PRMFL 3g60860_ARATH/1053-1235 EQMSSIVSNLNLLEQVG..E.MNV Q FSQSQKLNSEAIIDFVKALCKVSMDELRSP..........SN...PRVFS 1g01960_ARATH/1044-1226 EQMNNLISNLNLLEQVG..D.MSI R FTRSQRLNSEAIIDFVKALCKVSMDELRSP..........SD...PRVFS 4g35380_ARATH/1000-1184 EQIKSFIANLNLLDQIGNFE.LNV H YANSQRLNSEAIVSFVKALCKVSMSELQSP..........TD...PRVFS 4g38200_ARATH/982-1166 DQINNFIANLNLLDQIGSFQ.LNV N YAHSQRLKTEAIVAFVKALCKVSMSELQSP..........TD...PRVFS 3g43300_ARATH/951-1132 QISRDGVVQSLKEL..AGRP.AEV Q FVNSVKLPSESVVEFFTALCGVSAEELKQ...........SP...ARVFS SEC7_YEAST/1253-1438 TLSPEISKFISSSELV..VL.MDI N FTKSSELSGNAIVDFIKALTAVSLEEIESS..........ENASTPRMFS GGG2_PARTE/945-1125 ....ISIERLFQQI..QYDQ.IDI K FNSSINLDSNSILEFIRALCELSKEEIKY................NRLFL GBF1_HUMAN/1098-1277 TENQEAKRVALECI..KQCD.PEM K ITESKFLQLESLQELMKALVSVTPD.....E........ETYDEEDAAFC Q9V696_DROME/1048-1236 YEEQDFIKLGRKCI..KECQ.LDM Q LQESKFVQLESLQELLKCVLALLKA.....PQGH.KSIGLPYAEDQTVFW Q9XTF0_CAEEL/1084-1278 QEQLSSMKLASQVI..SECR.PSI Q VADSKYLTSTSLAELLSSIAANSAQIVEQAEPQQKTASLSGEDEDALVFY GNOM_ARATH/973-1158 EQQLAAHQRTLQTI..QKCH.IDI S FTESKFLQAESLLQLARALIWAAGR........PQKGTSSPEDEDTAVFC GNL1_ARATH/973-1149 EEELAAYKHARGIV..KDCH.IDI S FSDSKFLQAESLQQLVNSLIRASGK.................DEASSVFC GNL2_ARATH/894-1084 ALGMSEFEQNLKVI..KQCR.IGI Q FSKSSVLPDVAVLNLGRSLIYAAAGKGQ.......KFSTAIEEEETVKFC GEA2_YEAST/1011-1194 .EEIKSSKKAMECI..KSSNIAAV S FGNESNITADLIKTLLDSAKTE............KNADNSRYFEAELLFI GEA1_YEAST/1012-1184 ............CV..KASHPLSV S FENNQLVSPKMIETLLSSLVIE............KTSENSPYFEQELLFL 60 70 80 90 100 110 120 130 BIG1_HUMAN/1107-1289 LI QK VEISYYNR MG IRLQWI SR WI EV GDHFNKVGCNPNE.DVAIFAVDSLRQLK SM FLEKG..ELANFD FR QK FL BIG2_HUMAN/1054-1236 LI QK VEISYYNR MN IRLQWI SR WI HV GDHFNKVGCNPNE.DVAIFAVDSLRQLK SM FLEKG..ELANFD FR QK FL Q9VJW1_DROME/968-1149 LI QK VEISYYNR ME IRLQWI SR WL QV GEHFNAVGCNSNE.EISFFALDSLRQLK SM FMEKG..EFSNFD FR QK FL Q9XWG5_CAEEL/943-1125 LV GK VEVAFYNR MN IRLEWI SR WI NV GEHFNAAGCNSNE.AVAYFSVDALRQLK SI FLEKG..ELPNFD FR QK FL 3g60860_ARATH/1053-1235 LI TK VEIAHYNR MN IRLVWI SS WL QV SGFFVTIGCSENL.SIAIFAMDSLRQLK SM FLERE..ELANFE YN QN FM 1g01960_ARATH/1044-1226 LI TK VEIAHYNR MN IRLVWI SS WL HV SDFFVTIGCSDNL.SIAIFAMDSLRQLK SM FLERE..ELANFE YN QN FM 4g35380_ARATH/1000-1184 LL TK VETAHYNR MN IRLVWI SR WL NV SDFFVSVGLSENL.SVAIFVMDSLRQLK SM FLERE..ELANFE YH QH FL 4g38200_ARATH/982-1166 LL TK VEIAHYNR MN IRLVWI SR WL SI SDFFVSVGLSENL.SVAIFVMDSLRQLK SM FLERE..ELANFE YN QN FL 3g43300_ARATH/951-1132 LL QK VEISYYNR IA IRMVWI AR WL SV AEHFVSAGSHHDE.KIAMYAIDSLRQLK GM YLERA..ELTNFD FT QN IL SEC7_YEAST/1253-1438 LM QK VDVCYYNR MD IKLEWL TP WM AV GKAFNKIATNSNL.AVVFFAIDSLRQLR SM FLDIE..ELSGFD FE QH FL GGG2_PARTE/945-1125 LV SR IDVAEFNR MN IKIIWM SR WM EI REHFLEVGCLKNV.DVAIYAIDQLKQLK SC FLQQP..ELTNFE YY QK FL GBF1_HUMAN/1098-1277 LL EM LRIVLENR RD VGCVWV QT RL DH YHLCVQAQD..FC.FLVERAVVGLLRLR AI LL.......RRIQ EE SA VL Q9V696_DROME/1048-1236 ML EF VKIVVHNR RD MIPLWV PA RM DQ YLLLMGSASCGYD.YLLNRCIVAVLKLY AI LM.......RNLI EE CP VL Q9XTF0_CAEEL/1084-1278 LI EL VAITLENR KD LPLVWV PH RL RH EWLLSPRFG..RCPVLVERAVVGLLRVR AN NLF......RDVD NT SD VL GNOM_ARATH/973-1158 LL EL IAITLNNR RD IVLLWV QG YI EH ATIAQSTVM..PC.NLVDKAIFGLLRIR CQ LLP......YKLE ES AD LL GNL1_ARATH/973-1149 LL EL IAVTLNNR RD ILLIWV PT YI EH LGIVQLTLT..PC.TLVEKAVFGVLKIR CQ LLP......YKLE EN TD LL GNL2_ARATH/894-1084 WI DL ITIALSNR VH FNMFWY PS HL EY LNVANFPL.FSPI.PFVEKGLPGLFRVK CI ILASN....LQLE DH PE LI GEA2_YEAST/1011-1194 IT EL IALFL.FE CK EKELGI KF LV QK FQLSHTKG.....LTKRTVRRMLTYKII LL SLCADQTEYLSIE KL ND LL GEA1_YEAST/1012-1184 LS EI IILIS.EY AS GQEFGI AL AM DH INISNLDG.....LSKEAIARLASYKMV FL SRFDNPRDILSID DL EH FL 140 150 160 170 180 BIG1_HUMAN/1107-1289 .RPFEHIMK.....RNRSPTIRDV M VRCIAQMVNSQAANIS R .....GKI W N FV S FHLAASDQ BIG2_HUMAN/1054-1236 .RPFEHIMK.....KNRSPTIRDA M IRCIAQMVNSQAANIS R .....GKI W N FV A FHQAASDH Q9VJW1_DROME/968-1149 .RPFEHIMK.....KNASPAIRDV M VRCIAQMVNSQAHNIS R .....GKI W N FI S FHLAAGDN Q9XWG5_CAEEL/943-1125 .RPFEVIMV.....RNGSAQTRDV L VRCCAHLVEAHSSRLS K .....GQL W N FV S WTIAAGDP 3g60860_ARATH/1053-1235 .TPFVIVMR.....RSNDVEIREI L IRCVSQMVLSRVNNVS K .....GKM W S FV M FTTAAYDD 1g01960_ARATH/1044-1226 .KPFVVVMR.....KSGAVEIREI L IRCVSQMVLSRVDNVS K .....GKM W S FI M FTTAAHDA 4g35380_ARATH/1000-1184 .RPFVVVMQ.....KSSSAEIREI L VRCVSQMVLSRVSNVS K .....GKV W N FV T FTTAALDE 4g38200_ARATH/982-1166 .RPFVIVMQ.....KSSSAEIREI L VRCISQMVLSRVSNVS K .....GKV W S FV K FTTAAADE 3g43300_ARATH/951-1132 .KPFVIIMR.....NTQSQTIRSI L VDCIVQMIKSKVGSIS K .....GRV W S FI M FTAAADDE SEC7_YEAST/1253-1438 .KPFEYTVQ.....NSGNTEVQEI M IECFRNFILTKSESIS K .....GKI W P LS E LQYTARSS GGG2_PARTE/945-1125 .LPFEQIFSHTQAQQQNKIQLREL F LSCMCMITNICFNSIS K .....GKI W I MI S VNQALQDD GBF1_HUMAN/1098-1277 .LSLRILLLMK...PSVLSRVSHV Q AYGLHELLKTNAANIS H ...GDDAL W T FL T LECIGSGV Q9V696_DROME/1048-1236 .QSLKMLLMLK...PALLLRISKI Q SIGIYELLKTSAQNIS H ...EQDQI W I FL N LECVGAGA Q9XTF0_CAEEL/1084-1278 .HSLSMLLRLS...PKALFIFSRI Q AFGLYELIRANAANVK H ...KEHAL W V FL A LEAAGAAV GNOM_ARATH/973-1158 .RSLQLVLKLD...ARVADAYCEI Q AIEVSRLVKANANHIS R ...QAGRI W T TL S LSITARHP GNL1_ARATH/973-1149 .KSLQLVLKLK...AKVADAYCEI R AQEVVRLVKANASHVS R ...RTGRI W T IL S LSITARHP GNL2_ARATH/894-1084 FRSLTIMWKID...KEIIETCYDI T TEFVSKIIIDYSANLT H ...NIGKV W S LL Q LSLCGRHP GEA2_YEAST/1011-1194 .KKGDIFTQ.....KFFATNQGKF E LKRLFSLTESEFYRGL F LGNENFKL W F RV K TAMKEQ.. GEA1_YEAST/1012-1184 .VKNEIFNT.....KYYESEWGKV Q INDLFTHLNDVKYNEA R LKNVKFNL W F RL I ISAKDR.. Th Figure 6 e conserved domains of the BIG/GBF subfamily: HDS2 domain The conserved domains of the BIG/GBF subfamily: HDS2 domain. See Figure 3 legend for alignment details. (DCB, HUS, Sec7, HDS1, HDS2 and HDS3), excluding sis strongly supported this topology for most branches. variable linkers. The same tree topology was obtained Only a few small branches located at the base of the with both neighbor-joining and maximum likelihood groups were found in less than 60% of the trials in one of methods, and was retained using any one of the new con- the two methods, but this never occurred with both meth- served domains alone (data not shown). Bootstrap analy- ods simultaneously. Page 8 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 110 20 30 40 50 60 BIG1_HUMAN/1372-1489 .APEDRVWVRGWFPILFELSCIINRCK.LDVRTRGLTVMFEIMK..TYGHTYEKH...WWQDLFRIVFRIFDNMK BIG2_HUMAN/1319-1436 .APGDRVWVRGWFPILFELSCIINRCK.LDVRTRGLTVMFEIMK..SYGHTFEKH...WWQDLFRIVFRIFDNMK Q9VJW1_DROME/1233-1350 .AEEDRVWVRGWFPMLFSLSCVVNRCK.LDVRTRALTVLFEIVK..TYGESFKPH...WWKDLFNVIFRIFDNMK Q9XWG5_CAEEL/1212-1329 .TADQHVWLRGWFPIFFELSCIINRCK.LDVRTRSLTVMFEIMK..HHGSDFRPE...WWKDLLEIVFRIFDPSK 3g60860_ARATH/1337-1472 EIV.NNNHLYFWFPLLSGLSELSFDPR.PEIRKSALQIMFDTLR..NHGHLFSLP..LWEKVFESVLFPIFDYVR 1g01960_ARATH/1321-1452 .FLESDEHLYSWFPLLAGLSELSFDPR.AEIRKVALKVLFDTLR..NHGDHFSLA..LWERVFESVLFRIFDYVR 4g35380_ARATH/1278-1410 .FTDLDEQVSYWIPLLTGLCKQVSDPR.PAIRKRSIEVLFHILM..DHGHLFTRP..FWTGIFSSIILPVFNNIR 4g38200_ARATH/1262-1394 .FMDADENISYWVPLLTGLSKLTSDSR.SAIRKSSLEVLFNILK..DHGHIFSRT..FWIGVFSSVIYPIFNSVW 3g43300_ARATH/1212-1331 DET.FDVTEHYWFPMLAGLSDLTSDYR.PEVRNCALEVLFDLLN..ERGNKFSTP..FWESIFHRILFPIFDHVS SEC7_YEAST/1525-1649 .LRGKDIFQDVWFPMLFCFNDTIMTAEDLEVRSRALNYMFDALV..AYGGKFNDD..FWEKICKKLLFPIFGVLS GGG2_PARTE/1195-1313 ....NKYLDQLWIPVLSALSVLYSDER.GVVQQQSVNTLFELLK..VHGEQQSNE..FWKIILRGVIRPLFDEIQ GBF1_HUMAN/1532-1644 .ADSRTLWAHCWCPLLQGIACLCCDAR.RQVRMQALTYLQRALL.VHDLQKLDAL..EWESCFNKVLFPLLTKLL Q9V696_DROME/1475-1592 .PQSAALWSPGWCPLLQGIARLAMDRR.REVRTHAISCLQQRALLVHDLQTLSGT..EWCSCFHQVLFPLLNELL Q9XTF0_CAEEL/1555-1667 .ATVAFIWTDIWRPLLQAIGRLSCDCR.RGVRAAALTHLQRAFL.PANMATLGAA..EWQSCFGEVLFPLLTKLL GNOM_ARATH/1235-1349 .GKMSQDIGEMWLRLVQGLRKVCLDQR.EDVRNHALQSLQKCLG.GVDGINLAHS..MWSQCFDKVIFTVLDDLL GNL1_ARATH/1227-1343 .MKLSEDIGKMWLKLVKNLKKVCLDQR.DEVRNHAISMLQRAIA.GADGIMLPQP..LWFQCFDSAVFILLDDVL GNL2_ARATH/1177-1298 .LRGVNFVHHLFLKLSEAFRKTTLARR.EEIRNRAVTSLEKSFTMGHEDLGFTPS..GCIYCIDHVIFPTIDDLH GEA2_YEAST/1277-1381 .EDNNDLRKNEIFAIIQALAHQCINPC.KQISEFAVVTLEQTLI..NKIEIPTNEMESVEELIEGGLLPLLNSSE GEA1_YEAST/1271-1375 .RSNSALNKNEIIAAIQGLAHQCLNPC.DELGMQALQALENILL..SRASQLRTEKVAVDNLLETGLLPIFELDE 70 80 90 100 110 BIG1_HUMAN/1372-1489 LPEQQ..................TEKAEWMTTTAI CNH LYA CDVFTQYLEVLSDVLLDDIFAQLYWCV BIG2_HUMAN/1319-1436 LPEQL..................SEKSEWMTTTAI CNH LYA CDVFTQFYEALNEVLLSDVFAQLQWCV Q9VJW1_DROME/1233-1350 LPEHV..................TEKSEWMTTTAI CNH LYA IDVFTQYFDVLGHLLLEELFAQLHWCV Q9XWG5_CAEEL/1212-1329 MDDHR..................SDKREWMSTTAV CNH MLS VEVFTQFYTQLSVYALPMIYRQFGIFI 3g60860_ARATH/1337-1472 HSIDPSGEDESADQGSSGGEVDELDHDAWLYETAV CTL LQL VDLFVKFYTTVNP.LLEKVLMLLVSFI 1g01960_ARATH/1321-1452 QDVDPSE.DDSTDQRGYNGE...VDQESWLYETAV CSL LQL VDLFVNFYKTVNP.LLKKVLMLFVSLI 4g35380_ARATH/1278-1410 SKTDMLFEESVDS.PSSAS..LDTEETTWDVETAL STL LQL VDLLVKFFRSVRS.QLPSVVSIIVGFI 4g38200_ARATH/1262-1394 GENDLLSKDEHSSFPSTFS..SHPSEVSWDAETAL SAM AQY VDLFVSFFTVIRS.QLSSVVSLLAGLI 3g43300_ARATH/1212-1331 HAGKESL.ISS..............GDVKFRETSL SIH LQL CNLFNTFYKEVCF.MLPPLLSLLLDCA SEC7_YEAST/1525-1649 KHWEVNQFN............SHDDLSVWLSTTAL LIQ LRN IALFTHYFESLNR.MLDGFLGLLVSCI GGG2_PARTE/1195-1313 ISKLQ..FA............KQSQSKQQVIQNTF CKM FYL TDLVVLYIQQMQP.CLNDLIDIYIQLV GBF1_HUMAN/1532-1644 ENI...................SPADVGGMEETAL RMR STL SKVFLQHLSPLLSL...STFAALW... Q9V696_DROME/1475-1592 PES.....NAA...........GQLDAALLEESTM RIR ATI SKVFLQHLTTLIEL..GNAFNELW... Q9XTF0_CAEEL/1555-1667 EPF...................SQMDPIGMEDTTV RVR LQI AKTLLNHLSALSAL...DSFPDLW... GNOM_ARATH/1235-1349 EIA....A..G...........SQKDYRNMEGTAL LLL IKL SKVFLQQLQELSQL...STFCKLW... GNL1_ARATH/1227-1343 TFS....IENS...........RKTLKKTVEETAM LVL TKL SKAFLQSLQDISQQ...PSFCRLW... GNL2_ARATH/1177-1298 EKLLDYSRREN...........AEREMRSMEGTAL LKI MKV MNVFLVYLEQIVES...AEFRTFW... GEA2_YEAST/1277-1381 TQ.........................EDQKILII ISS LTI SNVYLHYLKL......GKTSNETF... GEA1_YEAST/1271-1375 IQ.........................DVKMKRIL ITS LSV SKIFLGQLVE......GVTSNETF... Th Figure 7 e conserved domains of the BIG/GBF subfamily: HDS3 domain The conserved domains of the BIG/GBF subfamily: HDS3 domain. See Figure 3 legend for alignment details. The tree topology strongly suggests that in most organ- trafficking at the Golgi within each group appear largely isms, GBG members sort in separate branches, overlapping [12,14]. In contrast, plants encode a large corresponding to their classification in BIGs and GBFs. number of GBGs in both the BIG and GBF branches but Remarkably, our annotation of Sec7-containing proteins lack other ArfGEFs (Figures 1, 8). In Arabidopsis, none of in the genome of Paramecium reveals the first departure the GBGs map to duplicated chromosomes where identi- from this distribution, as all GBGs in this species are cal functions may be encoded [26,29]. In addition, com- located in a single branch, which is closer to the BIGs. This parative analysis with the rice genome nearing unexpected tree topology may indicate that alveolates completion identifies at least five branches each repre- diverged from animals/fungi and plants before the dupli- sented by one rice and one or two Arabidopsis homologs cation of an ancestral GBG into the BIG and GBF, and that (Figure 8). This correspondence between two highly diver- GBGs in that organism are representative of this ancestral gent plant species indicates that GBGs diversified early gene. Alternatively, duplication may have been followed during plant evolution, probably reflecting functional by loss of GBF genes. Current knowledge of the phyloge- specialization along with the establishment of plant mul- netic branching of alveolates relative to the plants and ani- ticellularity. While GNOM has a plant-specific function in mal/fungi branches does not permit resolution between recycling plasma-membrane proteins needed for cell-cell those two possibilities. communication and cell polarity establishment [11], pos- sibly closer to the function of EFA6 or CYH subfamilies in GBGs in plants: refining the functional evolution of BIGs metazoans, other plant GBGs are expected to fulfill the and GBFs presumed ancient function of regulating Golgi trafficking In fungi and mammals, BIGs and GBFs are represented by exemplified by mammalian and yeast GBGs. Comparison only one or two members, whose functions in vesicular of orthologous pairs in plants further reveals that they Page 9 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 a,b Table 2: Alternate splice variants of human GBF1, BIG1 and BIG2 Change in protein Apparent cause of variation in transcript GBF1 Extra Q at 337, 55 residues upstream of HUS domain Insertion of 3 nucleotides (nt) resulting from use of alternate 3' acceptor site within intron during splicing of exons 10 and 11 New Ser and loss of 14 residues at 613, between HUS and Sec7 Loss of 36 nt resulting from use of alternate 5' donor site within domains exon 15 during splicing with exon 16 Loss of VSQD at 1494, 38 residues upstream of HDS3 Loss of 12 nt resulting from use of alternate 5' donor site within exon 33 during splicing with exon 34 Frame-shift at 1625 causing loss of last 19 residues of HDS3 Intron retention between exons 36 and 37 leading to frame shift and premature termination Loss of 38 residues starting at 1784, near C-terminus Loss of 114 nt resulting from use of novel cryptic splice donor and acceptor sites within exon 40. BIG1 Frame-shift at 1340, 32 residues upstream of HDS3 Loss of 59 nt resulting from use of alternate 5' donor site within exon 28 during splicing with exon 29 Loss of VSEKPL at 1557, 68 residues downstream of HDS3 Loss of 18 nt resulting from use of alternate 5' donor site within exon 33 during splicing to exon 34 New T and loss of 33 residues at 1607, 118 residues downstream of Loss of 96 nt resulting from use of alternate 3' acceptor site within HDS3 exon 35 during splicing with exon 34 BIG2 Frame-shift at 1542, 106 residues downstream of HDS3 Loss of exon 35 resulting from splicing of 5'donor site of exon 34 with 3' acceptor site of exon 36 All changes were expressed relative to the reference sequence stored under accession number NM_004193 (hGBF1), NM_006421 (hBIG1) and NM_006420.1 (hBIG2). All variants are supported by one or more cDNA/ESTs as detailed in the Aceview for each gene that can be obtained at [38]. have different sensitivities to Brefeldin A (a widely used ure 9), which was found only in the protists kingdom. The fungal inhibitor of Golgi traffic) as predicted from the TBC domain is predicted to carry a GAP (GTPase activat- sequences of the binding site of the drug carried by the ing protein) activity towards small G proteins of the Rab Sec7 domain [6]. This observation clearly illustrates that family [30], suggesting a potential crosstalk between Rab differences in outcome following BrefeldinA treatment and Arf pathways. Such a relationship between these two may not reflect differences in underlying molecular mech- small G proteins families, which are major regulators of anisms, but instead simply reflect neutral sequence differ- membrane traffic, would not be unprecedented, as for ences at the Sec7 domains between species. In particular, example the SYT1 ArfGEF gene was identified in yeast by not all BIGs may be BFA-sensitive or GBFs BFA-resistant, its genetic interactions with Rab proteins in the exocytic unlike suggested by their original nomenclature. pathway [31]. Interestingly, alveolates have specialized exocytic pathways based on a membrane organelle lying A novel ArfGEF subfamily in alveolates beneath the plasma membrane, the trichocyst, where this A remarkable evolutionary feature of ArfGEFs is that while unique ArfGEF family may potentially function. GBGs seem to be ubiquitous to all eukaryotes, fungi and animals kingdoms evolved their own ArfGEFs subfamilies Conclusion A conserved scenario for the activation of Arf proteins by unrelated to those of the other kingdoms. We thus addressed the question of whether Paramecium, which their GEFs? has a large number of GBGs (at least five, of which four The identification of a conserved modular architecture in are present as pairs as the result of recent duplications) but all GBG subfamily members suggests that the mechanistic appears to lack the specialization into the BIG and GBF basis for their activation of Arf is likely to follow a similar subgroups, has the same ArfGEF distribution as plants or scenario. Candidate functions for the conserved domains features a second ArfGEF subfamily. We thus searched the include oligomerization, the collection of input signals, newly sequenced genome from Paramecium tetraurelia and membrane localization, regulation of the exchange activ- the available alveolate genomes from Cryptosporidium ity, scaffolding of Arf proteins to their downstream effec- parvum and Tetrahymena thermophila for additional Sec7- tors, not excluding signaling to partners outside the Arf containing proteins. This identified a novel putative Arf- pathways. Dimerization has been reported in the BIG sub- GEF subfamily characterized by the association of the group for BIG1, which forms heterodimers with the Sec7 domain with a TBC (Tre/Bub2/Cdc16) domain (Fig- highly homologous BIG2 ArfGEF [14], and in the GBF Page 10 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 Os 9635.m03752 At 3g43300 Os 9631.m01366 At 1g01960 At 3g60860 Ag Q7PWN5 Os 9634.m04029 Dm Q9VJW1 Ce Q9XWG5 Os 9630.m00920 Sc SEC7 At 4g38200 Ca EAL04295 At 4g35380 Nc Q7SAX4 Pt GGG2 Sp SC72 Sp SC71 Pt GGG1 Os 9631.m04495 Pt GGG5 At GNOM Os 9630.m02122 Pt GGG4 Pt GGG3 At GNL1 Nc Q7SAL8 Os 9632.m00175 At GNL2 Sp Q9P7R8 Hs GBF1 Ca EAL02873 Rn GBF1 Ag Q7PXQ7 Sc GEA1 Dm Q9V696 Sc GEA2 Ce Q9XTF0 Unro Figure 8 oted neighbor-joining phylogenetic tree of the BIG/GBF subfamily Unrooted neighbour-joining phylogenetic tree of the BIG/GBF subfamily. Colour coding for the main groups is green for plants, marine blue for fungi, orange and red for animals, cyan for protists. Branches found in less than 60% bootstrap trials by either the neighbor-joining or the maximum likelihood method are in dotted lines. Species abbreviations are as in Table 1. subgroup for GNOM, which forms homodimers [27]. The an almost invariant motif in the HUS domain argues in conservation of the DCB domain in GBGs, which is favor of this domain interacting with a conserved partner. responsible for the dimerization of GNOM, suggests that However, the ancient divergence into the BIG and GBF such a dimerization function may be general to this groups and their subsequent divergence into species-spe- domain in GBGs. Another unresolved issue is the conser- cific members suggest that specialized requirements are vation of the cellular partners effecting the functions asso- likely to have evolved in most organisms, possibly yield- ciated with the conserved domains. Our identification of ing less conserved partners outside the Sec7 and HUS Page 11 of 14 (page number not for citation purposes) Hs BIG1 Hs BIG2 Rn BIG1 Rn BIG2 BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 Sec7 TBC Sec7 TBC TB Figure 9 S: a novel ArfGEF subfamily in alveolates TBS: a novel ArfGEF subfamily in alveolates. Top: Domain structure of the TBS subfamily. Below: Sequences of the TBC domain from Paramecium TBS aligned with TBC domains from known RabGAPs. Secondary structures are from the crystal structure of yeast GYP1 [30]. domains. Finally, whereas in plants all ArfGEFs are pre- Methods dicted to function according to the scheme defined by the Protein sequence databases were searched with amino conserved domains, other species have additional ArfGEF acid sequences from human BIG1, human GBF1 and Ara- subfamilies with a modular architecture unrelated to that bidopsis GNOM using the BLAST algorithm [32]. Para- of the GBG subfamily. It is not known to what extent the mecium tetraurelia genes were identified with the BLAST GBG's scenario for Arf activation will also apply to non- algorithm using genome sequence data from Genoscope GBGs ArfGEFs, acting alone or in association with protein [33] and manually annotated using Artemis [34]. partners. In the case of the GBGs, our definition of the Tetrahymena sequences were retrieved from the Tetrahy- structural homology domains as reported here should mena thermophila genome sequencing project server [35]. now provide a robust background for future investiga- Arabidopsis sequences were retrieved from the tions of their interactions and functions. Arabidopsis Genome Initiative database [36], rice sequences from the TIGR Rice annotation project [37]. Page 12 of 14 (page number not for citation purposes) BMC Genomics 2005, 6:20 http://www.biomedcentral.com/1471-2164/6/20 2. 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Charych EI, Yu W, Miralles CP, Serwanski DR, Li X, Rubio M, De Blas Your research papers will be: AL: The brefeldin A-inhibited GDP/GTP exchange factor 2, a available free of charge to the entire biomedical community protein involved in vesicular trafficking, interacts with the beta subunits of the GABA receptors. J Neurochem 2004, peer reviewed and published immediately upon acceptance 90:173-189. cited in PubMed and archived on PubMed Central 50. Garcia-Mata R, Sztul E: The membrane-tethering protein p115 interacts with GBF1, an ARF guanine-nucleotide-exchange yours — you keep the copyright factor. EMBO Rep 2003, 4:320-325. BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 14 of 14 (page number not for citation purposes)

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