Additional file 1
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1 Additional File 1 2 ApST1 ACYPI001780 ApST4 ACYPI001980 ApST9 ACYPI001077 (+) Control (-) Control 66 63.4 kDa 45 53.7 kDa 53.4 kDa 57.6 kDa 36 29 24 20 14 3 Mr, kDa 4 Figure S1. Western blot analysis of recombinant A. pisum sugar transporters 5 expressed in yeast. Western blot analysis of total membranes (5 g membrane 6 protein/lane) from Saccharomyces cerevisiae hexose transport mutant EBY.VW4000 7 expressing ApST1, ApST4, ApST9. Positive control cells (+) were expressing ApST3 8 (previously reported in [1]); and negative control cells (-) were transformed with 9 empty pDR195 expression vector. Yeast membrane proteins were separated on a 10 12.5% polyacrylamide gel, transferred to nitrocellulose, probed with anti-c-Myc 11 antibodies and exposed to film. The signal intensities from each lane are directly 12 comparable. Calculated molecular weight (kDa) for each transporter protein is 13 shown in boxes. 1 Helix 1 HuGLUT1 HuGLUT4 HuGLUT3 HuGLUT2 ACYPI005975 DmGLUT1 ACYPI001980 AB550003 AB549996 ------------------------------------MEPSSKKLTGRLMLAVGGAVLG-SLQFGYNTGVINAPQKVIEEFYNQTW----VHRYG-------------------------- 53 ------------------------MPSGFQQIGSEDGEPPQQRVTGTLVLAVFSAVLG-SLQFGYNIGVINAPQKVIEQSYNETW----LGRQGP------------------------- 66 --------------------------------------MGTQKVTPALIFAITVATIG-SFQFGYNTGVINAPEKIIKEFINKTL----TDKGN-------------------------- 51 --------------------------------------MTEDKVTGTLVFTVITAVLG-SFQFGYDIGVINAPQQVIISHYRHVLGVPLDDRKAINNYVINSTDELPTISYSMNPKPTPW 81 MVAIKNELDTVQQVKEEIEELREYVDRLELQSHRRKLRLLEQGLTFFLSYTILASMLG-MLQFGYNTGVINAPEGNIEKFIKDVF----EDRYK-------------------------- 89 -----------------------------------MAFLCAPGLTFFLTYSIFSAVLG-MLQFGYNTGVINAPEKNIENFMKDVY----KDRYG-------------------------- 54 ----------------------MTEKQMVKDAEKQAPTPQNQSLNGRLLFAIIASAFGSAFQHGYNTGVVNAPQALIEKWISGVI----SGRNDGKP----------------------- 71 -----------MSKEGEDIDEHQTKEELTKQNGVSGPDHATKGLNPRLAFAIAAAAVGSAFQHGYNLGVVNAPQKLIEEWILGVI----KNQTDASPP---------------------- 83 ---------------------------------------MNPDFSINLVFACMVAAIGAGFQHGYNTGVVNAPQNVIEKWMSDVS----QERHGMPP----------------------- 54 .. * : : .* :*.**: **:***: * . . : HuGLUT1 HuGLUT4 HuGLUT3 HuGLUT2 ACYPI005975 DmGLUT1 ACYPI001980 AB550003 AB549996 ----ESILPTTLTTLWSLSVAIFSVGGMIGSFSVGLFVNRFGRRNSMLMMNLLAFVSAVLMGFSKLGKSFEMLILGRFIIGVYCGLTTGFVPMYVGEVSPTAFRGALGTLH QLGIVVGIL -EGPSSIPPGTLTTLWALSVAIFSVGGMISSFLIGIISQWLGRKRAMLVNNVLAVLGGSLMGLANAAASYEMLILGRFLIGAYSGLTSGLVPMYVGEIAPTHLRGALGTLN QLAIVIGIL ----APPSEVLLTSLWSLSVAIFSVGGMIGSFSVGLFVNRFGRRNSMLIVNLLAVTGGCFMGLCKVAKSVEMLILGRLVIGLFCGLCTGFVPMYIGEISPTALRGAFGTLN QLGIVVGIL AEEETVAAAQLITMLWSLSVSSFAVGGMTASFFGGWLGDTLGRIKAMLVANILSLVGALLMGFSKLGPSHILIIAGRSISGLYCGLISGLVPMYIGEIAPTALRGALGTFH QLAIVTGIL ----ENMDHGQAELLYSFAVSIFAIGGMLGGFSGGIIANRFGRKGGLLLNSFVGIGGACLMGLTKYFNSYEVLFIGRFIIGVNCGLNTSLVPMYISEIAPLNLRGGLGTVN QLAVTTGLL ----EDISEEFIQQLYSVAVSIFAIGGMLGGFSGGWMANRFGRKGGLLLNNVLGIAGACLMGFTKVSHSYEMLFLGRFIIGVNCGLNTSLVPMYISEIAPLNLRGGLGTVN QLAVTVGLL ------TDQTQVTLIWAVAVSIFCVGGMVGGLSTGFVAEKFGRKGGLLVSNALVILSAALQGVSKMYSSYELIIIGRFIIGINSGLNAGLTPMYLAEISPMNLRGSVGTVY QLVVTISIL ----SDANQTKVTMIFSIAVSIYCVGGMLGGAITGLVAEKYGRKGGLLFNNIFIVIAAALLGFSKAMNSYYMIIVGRFLLGINSGLNAGLTPMYLSEIAPVQLRGAVGTVY QLVLTISIL -------DKNDITFLFSLAVSIYCAGGIVGGLLTSTFAIHIGRRGGLFVNNLFALIAAAMMGLSKMAGSFELLIAGRCFSGLNSGLNSGLAGMYLVEVSPRSMRGALGSMY QLIITISIL :::.:*: :. **: .. . . ** .::. . . . .. : *. : * ::: ** . * .** :.:. **: *::* :**..*:. ** :. .:* HuGLUT1 HuGLUT4 HuGLUT3 HuGLUT2 ACYPI005975 DmGLUT1 ACYPI001980 AB550003 AB549996 IAQVFGLDSIMGNKDLWPLLLSIIFIPALLQCIVLPFCPESPRFLLINRNEENRAKSVLKKLRGTADVTHDLQEMKEESRQMMREKKVTILELFRSPAYRQPILIAVVL IAQVLGLESLLGTASLWPLLLGLTVLPALLQLVLLPFCPESPRYLYIIQNLEGPARKSLKRLTGWADVSGVLAELKDEKRKLERERPLSLLQLLGSRTHRQPLIIAVVL VAQIFGLEFILGSEELWPLLLGFTILPAILQSAALPFCPESPRFLLINRKEEENAKQILQRLWGTQDVSQDIQEMKDESARMSQEKQVTVLELFRVSSYRQPIIISIVL ISQIIGLEFILGNYDLWHILLGLSGVRAILQSLLLFFCPESPRYLYIKLDEEVKAKQSLKRLRGYDDVTKDINEMRKEREEASSEQKVSIIQLFTNSSYRQPILVALML ISQILGIEQILGTDEGWPLLLGLAICPAILQLILLPVCPESPRYLLITKQWEEEARKALRRLRATNQIEEDIEEMRAEERAQQSEATISMMELVCSPTLRQPLIISVVM LSQVLGIEQILGTNEGWPILLGLAICPAILQLILLPVCPESPRYLLITKQWEEEARKALRRLRASGSVEEDIEEMRAEERAQQSESHISTMELICSPTLRPPLIIGIVM ISQILGLDYILGTAELWPVLLALIIAPAIFMFATLPFCPESPKYTLINKKKDIEAERGLQWLRGTIEVHDEMDEMRAENEAMKVIPKVTLREMLSNPMLKTPLGISVMI ISQILGLNFILGTAELWPILLSLTIVPTIFQLITLPMCPESPKYLLITKGQEIESQRAVTWFRGTIEVHDEMDEMRREYESMKLVPKVTLREMLVNSALRIPLFISLVV VSQILGSQSIFGTDDLWPVLFGLTGIMALAQMLFLPCCPETPKH-IFNKGNKERAQKSLKWLRKREDVSAEMSEIQTEAEQEKSIGKASFQQFIQNPSLRKPLIIAIVI ::*::* : ::*. . * :*:.: :: * ***:*:. : . :. : : .: : *:: * : ::. : *: :.::: HuGLUT1 HuGLUT4 HuGLUT3 HuGLUT2 ACYPI005975 DmGLUT1 ACYPI001980 AB550003 AB549996 VFYYSTSIFEKAGVQQP--VYATIGSGIVNTAFTVVSLFVVERAGRRTLHLIGLAGMAGCAILMTIALALLEQLPWMSYLSIVAIFGFVAFFEVGPGPIPWFIVAELFSQGPRPAAIAVA VFYYSTSIFETAGVGQP--AYATIGAGVVNTVFTLVSVLLVERAGRRTLHLLGLAGMCGCAILMTVALLLLERVPAMSYVSIVAIFGFVAFFEIGPGPIPWFIVAELFSQGPRPAAMAVA VFYYSTGIFKDAGVQEP--IYATIGAGVVNTIFTVVSLFLVERAGRRTLHMIGLGGMAFCSTLMTVSLLLKDNYNGMSFVCIGAILVFVAFFEIGPGPIPWFIVAELFSQGPRPAAMAVA IFYYSTSIFQTAGISKP--VYATIGVGAVNMVFTAVSVFLVEKAGRRSLFLIGMSGMFVCAIFMSVGLVLLNKFSWMSYVSMIAIFLFVSFFEIGPGPIPWFMVAEFFSQGPRPAALAIA VFYYSTSLFITAGLAENVAKFVTIGIGVIMVNMTLVTMPLMDKTGRRTLHLYGLGGMFIFSIFITISLLITEMIDWMSYLAVVSILGFVVFFAVGPGSIPWMITAELFSQGPRPAAMSIA VFYYSTSLFMSSGLTEESAKFATIGIGAIMVVMTLVSIPLMDRTGRRTLHLYGLGGMFIFSIFITISFLIKEMIDWMSYLSVVATLGFVVFFAVGPGSIPWMITAELFSQGPRPSAMAIA VMFFSTKIFNMAGMSNDGAKYATLGMGSLNVIMTLISLFLVELTGRKTLLMIGFSSMFVVTVMLTIALMFVNVSSIVSGLAVVLVMAFVIAFAVGPGSIPWFLVSELFNSSARPLATSIA VIFFSTSIFQLASLG-DSAQLATLAMGAMNVLMTVISLVLVERVGRKVLLLVGFSGMFVITCLLAVALAYVKSNKWLPYVCILLVIAFVVMFAVGPGSIPWFLVSELFNQSALPLATSLA VIYYSTQIFQKAGMSQQEAQLATMIMGTVNIIMTVISVFLVEIAGRKTLLLIGFGLMFIVTALLAVLLEFIQYD-FASYMCVALVVLFIVCFATGPGSIPWFLVAELFGQDARPLAASIS ::::** :* :.: .*: * : :* ::: ::: .**: * : *:. * : :::: : . . :.: . *: * ***.***::.:*:*.... * * ::: HuGLUT1 HuGLUT4 HuGLUT3 HuGLUT2 ACYPI005975 DmGLUT1 ACYPI001980 AB550003 AB549996 GFSNWTSNFIVGMCFQYVEQLCGPYVFIIFTVLLVLFFIFTYFKVPETKGRTFDEIASGFRQG--GASQSDKTPEELFHPLGADSQV--------------------------GFSNWTSNFIIGMGFQYVAEAMGPYVFLLFAVLLLGFFIFTFLRVPETRGRTFDQISAAFHRTP-SLLEQEVKPSTELEYLGPDEND--------------------------GCSNWTSNFLVGLLFPSAAHYLGAYVFIIFTGFLITFLAFTFFKVPETRGRTFEDITRAFEGQAHGADRSGKDGVMEMNSIEPAKETTTNV----------------------AFSNWTCNFIVALCFQYIADFCGPYVFFLFAGVLLAFTLFTFFKVPETKGKSFEEIAAEFQKKSGSAHRP--KAAVEMKFLGATETV--------------------------VLINWVANFAVGIGFQPLKTALDNYTFLPFSVLLAIFWIFTYKKVPETKNKTFEEILALFRQNGRGSVLESSRLYGTSTTLSDGPGGVCCMRQHWQFPHDDVSERNSPVESHAQ VLVNWMANFVVGIGFPSMKTALENYTFLPFSVFLAIFWIFTYKKVPETKNKTFEEILALFRHN------NGSRA---------------------------------------VGVNWTANFVVGLGFLPLQEMLQSNVFLIFVVLLALFVLYVYKKVPETKNKTLEEIQMGFRQESYK-----------------------------------------------VGTNWTANFFVGLGFLPLQQLLGGHVFFIFAILQALFIVFIYKKVPETKNKTLEEISTMFKQISYT-----------------------------------------------IGCNWTANFLVGLFFLPLQELIGPKVFIIFAVLQLIFTIFIFFKVPETKNKSLDEVLKYFK----------------------------------------------------** .** :.: * .*: * . * : : :****:.:::::: *. Helix 2 Helix 5 Helix 7 Helix 11 14 Helix 3 Helix 4 Helix 5 Helix 6 Helix 8 169 185 167 201 205 170 185 199 167 Helix 7 Helix 9 QLSQQLSGINA QLSQQLSGINA QLSQQLSGINA HVAQQFSGING QLSQQLSGINA QLSQQFSGINA MLCQQLSGINA MIAQQLSGINA MIAQQLSGINA :.**:****. Helix 10 289 305 287 321 325 290 305 319 286 Helix 11 407 423 405 439 445 410 425 438 405 Helix 12 492 509 496 524 559 478 491 504 466 15 Figure S2. Sequence alignment and transmembrane predictions for GLUT 16 class I transporters. Human GLUT class I transporters (GLUTs 1 - 4) and related 17 transporters from: Acyrthosiphon pisum, Drosophila melanogaster and Nilaparvata 18 lugens are shown. Sequences were aligned using the ClustalX program, and ordered 19 according to their similarity. Membrane spanning regions (Helix 1 – 12) for each 20 transporter were predicted using the TMHMM program 21 (http://www.cbs.dtu.dk/services/TMHMM/) and shaded grey. Boxes highlight 22 conserved Gln177 and the 295MLC motif of ApST4 (ACYPI001980). Asterisks, 23 identical residues in all sequences; colon, conservative amino acid substitution; dot, 24 semi-conservative amino acid substitution. 2 s frc ga gl l c m a m n yo -in Helix 7 25 GLUT1 GLUT2 GLUT3 GLUT4 GLUT14 ACYPI005975 TC002929 PHUM563670 CG1086_GLUT1 GB13516 TC013487 TC014313 AB549996 TC013486 ACYPI001980 PHUM247290 AB550003 LOC409424 ACYPI009181 TC012857 GB18232 CG17976_sut3 CG17975_sut2 CG8714_sut1 CG7882 GLUT5 GLUT7 GLUT9 GLUT11 AYRQPILIAVVLQLSQQLSGINAVFYYST SYRQPILVALMLHVAQQFSGINGIFYYST SYRQPIIISIVLQLSQQLSGINAVFYYST THRQPLIIAVVLQLSQQLSGINAVFYYST SYRQPIIISIVLQLSQQLSGINAVFYYST TLRQPLIISVVMQLSQQLSGINAVFYYST TLRAPLIIGVVMQLSQQLSGINAVFYYST TLRAPLIIGVVMQLSQQLSGINAVFYYST TLRPPLIIGIVMQLSQQFSGINAVFYYST TLRAPLIIGVVMQLSQQLSGINAVFYYST VMSNVLICCCIINVANQLSGKNALTYFSV SLKIPLFIAMLVMVAQQFSGINIVIFYST SLRKPLIIAIVIMIAQQLSGINAVIYYST ALRIPLIICLCVMIAQQLSGINAVIFFST MLKTPLGISVMIMLCQQLSGINAVMFFST ALRIPLIIAIVVMIGQQLSGINAVMFFST ALRIPLFISLVVMIAQQLSGINAVIFFST TLRIPLIIALMVMFAQQLSGINAVMFFST SSRKAIIITCIIMLGQQLSGINAVFYYST SLLLPLLLVCSLQAGQQFSGINAVFYYSV NLKLPIFLVCIIQFGQQMSGINVVFYYSN KLKLPLFIVCSFHFVQQMSGISAIWFYSI ELWLPLLLVCSFQATQQLSGINAIFFYSL RLTLPLIIVCCFHGGQQLSGINAIFYYSV QLRLPLIIVCAFLGGQQLSGINAIFYYSV SLRWQLLSIIVLMGGQQLSGVNAIYYYAD SLRWQLLSIIVLMAGQQLSGINAINYYAD YVRWQVVTVIVTMACYQLCGLNAIWFYTN ALRRQVTSLVVLGSAMELCGNDSVYAYAS : ::.* . : :: 295 327 293 311 317 331 494 509 296 383 289 291 292 319 311 289 325 384 340 306 324 293 306 324 326 501 307 340 304 26 Figure S3. Sequence alignment of TM7 of human GLUT class I and II 27 transporters and insect orthologs. TM7 from human GLUT class I transporters 28 (GLUTs 1-4 and 14); GLUT class II transporters (GLUTs 5, 7, 9 and 11) and all insect 29 orthologs are shown. Sequences from TM7 of all transporters were aligned using 30 the ClustalX program. Boxed regions indicate location of the QLS substrate binding 31 motifs. Functionally characterized transporters are shaded grey and substrate 32 specificities are shown. Transported sugars are abbreviated: frc, fructose; gal, 33 galactose; glc, glucose; man, mannose; myo-ins, myo-inositol. Uncharacterized 34 insect transporters, predicted to be specific for glucose (based on the presence of a 35 QLS motif) are shown in red text. Asterisks indicate identical residues in all 36 sequences; colon indicates conservative amino acid substitution; dot marks semi- 37 conservative amino acid substitution. 3 38 39 Table S1. Summary of annotated A. pisum sugar porter family transporters. ST ID1 ACYPI ID Gene Identification ApST3 ApST1 ApST4 ApST7 ApST7 ApST26 ApST26 ApST5 ApST6 ApST27 ApST9 ApST9 ApST21 ApST16 ApST28 ApST11 ApST11 ApST43 ApST43 ApST43 ApST43 ApST43 ApST17 ApST17 ApST22 ApST23 ApST24 ApST25 ACYPI004204 ACYPI001780 ACYPI001980 ACYPI006139 ACYPI006139 ACYPI005083 ACYPI005083 ACYPI003008 ACYPI009181 ACYPI009304 ACYPI001077 ACYPI001077 ACYPI006232 ACYPI006113 ACYPI004302 ACYPI000812 ACYPI000812 ACYPI005975 ACYPI005975 ACYPI005975 ACYPI005975 ACYPI005975 ACYPI006604 ACYPI006604 ACYPI007333 ACYPI009298 ACYPI004901 ACYPI006799 LOC100163094 LOC100160486 LOC100160702 LOC100165178 LOC100165178 LOC100164041 LOC100164041 LOC100161811 LOC100168486 LOC100168622 LOC100159728 LOC100159728 LOC100165275 LOC100165150 LOC100163202 LOC100159441 LOC100159441 LOC100165001 LOC100165001 LOC100165001 LOC100165001 LOC100165001 LOC100165672 LOC100165672 LOC100166465 LOC100168616 LOC100163845 LOC100165882 Nucleotide Scaffold ID2 accession 1342 2 735 384 384 1482 1482 231 349 287 384 384 1171 1 1171 1211 1211 137 137 137 137 137 2 2 384 384 384 384 Protein accession XM_001950662 XM_001942918 XM_001950928 XM_001950568 XM_003245156 XM_001949779 XM_003248105 XM_001949885 XM_001951653 XM_001948294 XM_001946395 XM_003245157 XM_001952605 XM_001943769 XM_001952608 XM_001950955 XM_003247820 XM_001947713 XM_003242843 XM_003242844 XM_003242845 XM_003242846 XM_001949996 XM_003240048 XM_001946496 XM_001946444 XM_001946310 XM_001946266 4 XP_001950697 XP_001942953 XP_001950963 XP_001950603 XP_003245204 XP_001949814 XP_003248153 XP_001949920 XP_001951688 XP_001948329 XP_001946430 XP_003245205 XP_001952640 XP_001943804 XP_001952643 XP_001950990 XP_003247868 XP_001947748 XP_003242891 XP_003242892 XP_003242893 XP_003242894 XP_001950031 XP_003240096 XP_001946531 XP_001946479 XP_001946345 XP_001946301 # TM Helicies3 TIGR00879 score 4 (e-value) 12 12 12 12 12 12 12 10 12 12 12 13 10 12 10 12 12 12 12 12 12 12 10 10 10 12 10 12 5.6E-81 1.9E-89 3.0E-105 6.9E-65 x 5.8E-92 x 2.8E-62 6.6E-80 2.2E-68 2.2E-77 x 5.7E-66 1.8E-84 8.3E-64 1.1E-90 x 8.3E-112 x x x x 1.4E-88 x 2.6E-62 5.9E-73 4.6E-78 1.6E-78 40 Notes: 1A. pisum sugar transporter (ApST) identification number as described in [1]; 2A. pisum genomic scaffold identification 41 number from assembly Acyr_2.0 (NCBI accession number: ABLF00000000.2); 3Transmembrane (TM) helices predicted by 42 TMHMM (available at, http://www.cbs.dtu.dk/services/TMHMM/); 4TIGRFAMS major facilitator superfamily (MFS) 43 transporter, sugar porter (SP) family signature (TIGR00879) e-value scores [2]. Alternative splice forms of ApST genes are 44 shown in light grey. 5 45 Table S2. de novo identification of sugar porter (SP) family transporters 46 [transporter classification number (T.C #) 2.A.1.1] in insect genomes 47 Organism Taxonomy ID Assembly Genebuild # of MFS transporter, sugar porter family (TIGR00879) transporters Acyrthosiphon pisum Anopheles gambiae Apis mellifera Drosophila melanogaster Nasonia vitripennis Pediculus humanus Tribolium castaneum 7029 7165 7460 7227 7425 121224 7070 Acyr_2.0 AgamP3 Amel4.0 BDGP 5 Nvit_2.1 PhumU1 Tcas3 2013-07-AphidBase 2012-10-VectorBase 2013-07-BeeBase 2012-04-FlyBase 2010-12-NasoniaBase 2009-07-VectorBase 2012-09-24 19 16 8 14 11 10 26 48 Notes: Sugar porter (SP) family transporters (TIGRFAMS motif: TIGR00879 [2]) 49 were identified in insect reference protein datasets (available from 50 http://ensemblgenomes.org/) using HMMER 3.0 motif searches [3]. All identified 51 transporters have a sequence score >237.80 (trusted cutoff). 6 52 53 Table S3. Summary of annotated A. pisum solute:sodium symporter (SSS) family transporters. ACYPI ID Gene Identification Nucleotide accession Protein accession ACYPI009891 ACYPI009891 ACYPI007408 ACYPI000102 ACYPI002047 ACYPI003487 ACYPI001053 ACYPI001053 ACYPI009916 LOC100169257 LOC100169257 LOC100166547 LOC100158662 LOC100160776 LOC100162329 LOC100159703 LOC100159703 LOC100169284 XM_003244407 XM_001949169 XM_001951323 XM_001944878 XM_001943548 XM_001949016 XM_003241448 XM_001943453 XM_001944561 XP_003244455 XP_001949204 XP_001951358 XP_001944913 XP_001943583 XP_001949051 XP_003241496 XP_001943488 XP_001944596 TM PF00474 Helicies 1 score2 Species TCDB top Blast hit3 Gene symbol Gene name 13 13 13 13 13 13 13 13 13 4.5e-46 4.5e-46 1.2e-45 6.5e-38 1e-36 2.7e-36 3.8e-35 3.8e-35 3.9e-33 H. sapiens H. sapiens H. sapiens H. sapiens H. sapiens H. sapiens H. sapiens H. sapiens Rattus norvegicus SLC5A8 SLC5A8 SLC5A8 SLC5A12 SLC5A12 SLC5A12 SLC5A8 SLC5A8 SLC5A7 sodium/monocarboxylate cotransporter sodium/monocarboxylate cotransporter sodium/monocarboxylate cotransporter sodium/monocarboxylate cotransporter sodium/monocarboxylate cotransporter sodium/monocarboxylate cotransporter sodium/monocarboxylate cotransporter sodium/monocarboxylate cotransporter sodium/choline cotransporter e-value e-112 e-112 e-101 e-66 e-69 e-66 e-90 e-90 e-151 54 1Transmembrane (TM) helices predicted by TMHMM (available at, http://www.cbs.dtu.dk/services/TMHMM/); 2Pfam E-value 55 score for sodium:solute symporter signature PF00474; 3 Sequence information of top BLASTP hit, aphid transporter versus 56 transporter classification database (TCDB). Alternative splice forms are shown in light grey. 57 58 59 60 61 62 63 7 64 Table S4. Quantitative PCR primers for Acyrthosiphon pisum sugar 65 transporters and housekeeping gene glyceraldehyde-3-phosphate 66 dehydrogenase (GAPDH). Gene Identification Primer Primer sequence (5’ - 3’) ApST1 (ACYPI001780, LOC100160486) fwd rev fwd rev fwd rev fwd rev fwd rev fwd rev fwd rev fwd rev fwd rev fwd rev fwd rev fwd rev fwd rev fwd rev fwd rev fwd rev fwd rev CCCGGCACAAATCAAAGGT CAATGAAAGCGAAGAACCAGTTG CGCTGTTGTTTACGGCGTTT CGTGCAATATGTCCTGGATTTC GCGGTGTGATATCTGGCAGAA CGGCCCATATCAATGTCACTT GCTATCGTCGGATGGTTGATG CCAGGAATCTTCCCACGTAAAT AACAATGGTGTAATGAAACGATCATAC CCACCAATCAAGAATACGGAAAC CCGCATGTTCGCGTTCA GCATGCGCCAGAAAAGGA CGGACATATTCGGCAGGAA TCATTGCCCAGCTGATGATG TGCTCAATGGTAGTTGGATTCG GCCCGGTCTGTTCATCGA GAGAAAAAGTTCCAATCATGAAAGC AACCATAACCCCAAGGCCTAA GAGAAAAAGTTCCAATCATGAAAGC AACCATAACCCCAAGGCCTAA CGCTCATACTGCCGGATAATG GACCATAGTGACGCCGAACA CGCTCATACTGCCGGATAATG GACCATAGTGACGCCGAACA TTGGCGAAGGTGACGATAATT CCCAGCGCCGGACAT CAGCTTGGGCGTTTACATCA AAGAGCTCTCCGACCATGACA TCCGCGATCGCATCGT ATCGCGTACGTGGCCATAAG TCGGCCTGCGGCTATG ATGCCGACGGCGAAGTT CAATGGAAACAAGATCAAGGTGTT ACCAGCAGATCCCCATTGG ApST3 (ACYPI004204, LOC100163094) ApST4 (ACYPI001980, LOC100160702) ApST5 (ACYPI003008, LOC100161811) ApST6 (ACYPI009181, LOC100168486) ApST7 (ACYPI006139, LOC100165178) ApST9 (ACYPI001077, LOC100159728) ApST11 (ACYPI000812, LOC100159441) 1 ApST16 (ACYPI006113, LOC100165150) 1 ApST17 (ACYPI006604, LOC100165672) 2 ApST21 (ACYPI006232, LOC100165275) 2 ApST28 (ACYPI004302, LOC100163202) ApST22 (ACYPI007333, LOC100166465) ApST23 (ACYPI009298, LOC100168616) ApST27 (ACYPI009304, LOC100168622) ApST43 (ACYPI005975, LOC100165001) 67 GAPDH (ACYPI009769, LOC100169122) Amplicon (bp) QPCR efficiency (%) 68 98 75 93 69 93 70 104 106 91 65 97 69 96 69 99 80 99 80 94 74 98 74 91 63 99 75 92 68 95 63 94 67 97 68 Notes: Due to high sequence similarity between closely related sugar transporters 69 (shaded light grey) it was not possible to design gene specific primers. Primers 70 were designed to co-amplify 1ApST16 and 1ApST17 and co-ampify 2ApST21 and 71 2ApST28. 8 72 Table S5. A. pisum sugar transporter coding sequence primers for 73 Saccharomyces cerevisiae expression constructs. Gene Identification Primer RE site ApST1 (ACYPI001780, LOC100160486) fwd rev fwd rev fwd rev XhoI NotI XhoI NotI EcoRV NotI ApST4 (ACYPI001980, LOC100160702) 74 ApST9 (ACYPI001077, LOC100159728) Primer sequence (5’ - 3’) AACTCGAGATAATGGCTTCAGAAAAAATCGCC AAGCGGCCGCGTATTTTTCTTTGGTGTCGATGG ATCTCGAGATAATGGCCGAAAAACAAATGGTG ATGCGGCCGCTTTGTACGATTCCTGTCTGAAACC ATGATATCATAATGGACCAGGGCGTGTT AAGCGGCCGCGACGATCGCGGCGTG Amplicon (bp) 1488 1494 1777 75 Notes: Restriction enzyme (RE) sites for forward (fwd) and reverse (rev) primers 76 are underlined in primer sequence and Kozak translation initiation sequences [4] 77 are shown in bold. 9 78 References 79 1. Price DRG, Tibbles K, Shigenobu S, Smertenko A, Russell CW, Douglas AE, 80 Fitches E, Gatehouse AMR, Gatehouse JA: Sugar transporters of the major 81 facilitator superfamily in aphids; from gene prediction to functional 82 characterization. Insect Molecular Biology 2010, 19:97-112. 83 2. Haft DH, Loftus BJ, Richardson DL, Yang F, Eisen JA, Paulsen IT, White O: 84 TIGRFAMs: a protein family resource for the functional identification of 85 proteins. Nucleic Acids Research 2001, 29(1):41-43. 86 3. 87 88 Eddy SR: Profile hidden Markov models. Bioinformatics 1998, 14(9):755763. 4. Kozak M: Structural features in eukaryotic mRNAs that modulate the 89 initiation of translation. Journal of Biological Chemistry 1991, 90 266(30):19867-19870. 91 92 93 10
