Supplementary Figure 1. CFTR protein structure and domain architecture.
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1 A Plasma Membrane NH ₂ COOH Supplementary Figure. CFT protein structure and domain architecture. (A) Open state CFT homology model, ribbon representation from Serohijos et al. 8 PNAS 5:356. CFT domains are color coded as shown in the legend. () Schematic representation of CFT domain organization in and around the plasma membrane.
2 A LAST -log E-value ACC AC ACA ACG ACD ACE ACF CFT ACC AC ACA ACG ACD ACE ACF Supplementary Figure. Sequence similarity and domain architecture of CFT and related AC transporters. (A) Statistical significance of LASTP hits of the human CFT protein sequence against representative human AC family members. () Dendogram showing the relationship between CFT and representative AC family members along with their domain architectures.
3 CFT MQSPLEKASVVSKLFFSWTPILKGYQLELSDIYQIPSVDSADNLSEKLEEWDELASKKN----PKLINALC ACC4 MLPVYQEVKPNPLQDANLCSVFFWWLNPLFKIGHKLEEDDMYSVLPEDSQHLGEELQGFWDKEVLAENDAQKPSLTAIIKC CFT FFWFMFYGIFLYLGEVTKAVQPLLLGII---ASYDPDNKEESIAIYLGIGLCLLFIVTLLLHPAIFGLHHIGMQMIAMFSLI ACC4 YWKSYLVLGIFTLIEESAKVIQPIFLGKIINYFENYDPMDSVALNTAYAYATVLTFCTLILAILHHLYFYHVQCAGMLVAMCHMI CFT YKKTLKLSSVLDKISIGQLVSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGMMMKY ACC4 YKALLSNMAMGKTTTGQIVNLLSNDVNKFDQVTVFLHFLWAGPLQAIAVTALLWMEIGISCLAGMAVLIILLPLQSCFGKLFSSL CFT DQAGKISELVITSEMIENIQSVKAYCWEEAMEKMIENLQTELKLTKAAYVYFNSSAFFFSGFFVVFLSVLPYALIKGIIL- ACC4 SKTATFTDAITMNEVITGIIIKMYAWEKSFSNLITNLKKEISKILSSCLGMNLASFFSASKIIVFVTFTTYVLLGSVITA CFT KIFTTISFCIVLMAVTQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTT---TEVVMENVTAFWEEGFGELFEKAKQNNN ACC4 SVFVAVTLYGAVLTVTLFFPSAIEVSEAIVSIIQTFLLLDEISQNQLPSDGKKMVHVQDFTAFWDK CFT NKTSNGDDSLFFSNFSLLGTPVLKDINFKIEGQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGISFCSQFSWIMPGTIKE ACC ASETPTLQGLSFTVPGELLAVVGPVGAGKSSLLSAVLGELAPSHGLVSVHGIAYVSQQPWVFSGTLS CFT NIIFGVSYDEYYSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQAISLAAVYKDADLYLLDSPFGYLDVLTEKEIFESCV ACC4 NILFGKKYEKEYEKVIKACALKKDLQLLEDGDLTVIGDGTTLSGGQKAVNLAAVYQDADIYLLDDPLSAVDAEVSHLFELCI CFT CKLMANKTILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAENSILTETLHFSLEGDAPVS ACC4 CQILHEKITILVTHQLQYLKAASQILILKD CFT WTETKKQSFKQTGEFGEKKNSILNPINSIKFSIVQKTPLQMNGIEEDSDEPLELSLVPDSEQGEAILPISVISTGPTLQA ACC CFT QSVLNLMTHSVNQGQNIHKTTASTKVSLAPQANLTELDIYSLSQETGLEISEEINEEDLKECFFDDMESIPAVTTWNTYLY ACC CFT ITVHKSLIFVLIWCLVIFLAE ACC GKMVQKGTYTEFLKSGIDFGSLLKKDNEESEQPPVPGTPTLNTFSESVWSQQSSPSLKDGALE CFT VAASLVVLWLLGN TPLQDKGNSTHSN-N ACC4 SQDTENVPVTLSEENSEGKVGFQAYKNYFAGAHWIVFIFLILLNTAAQVAYVLQDWWLSYWANKQSMLNVTVNGGGNVTEKLDLN CFT SYAVIITSTSSYYVFYIYVGVADTLLAMGFFGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNFSKDIAILDDLLP ACC4 WY LGIYSGLTVATVLFGIASLLVFYVLVNSSQTLHNKMFESILKAPVLFFDNPIGILNFSKDIGHLDDLLP CFT LTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLAYFLQTSQQLKQLESEGSPIFTHLVTSLKGLWTLAFGQPYFE ACC4 LTFLDFIQTLLQVVGVVSVAVAVIPWIAIPLVPLGIIFIFLYFLETSDVKLESTTSPVFSHLSSSLQGLWTIAYKAEECQ CFT TLFHKALNLHTANWFLYLSTLWFQMIEMIFVIFFIAVTFISILTTG-EGEGVGIILTLAMNIMSTLQWAVNSSIDVDSLMSVS ACC4 ELFDAHQDLHSEAWFLFLTTSWFAVLDAICAMFVIIVAFGSLILAKTLDAGQVGLALSYALTLMGMFQWCVQSAEVENMMISVE CFT VFKFIDMPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAILENISFSISPGQVGLLGTGSGK ACC4 VIEYTDLEKEAPWEYQKPPPAWPHEGVIIFDNVNF----MYSPGGPLVLKHLTA LIKSQEKVGIVGTGAGK CFT STLLSAFLLLNTEGEIQIDGVSWDSITLQQWKAFGVIPQKVFIFSGTFKNLDPYEQWSDQEIWKVADEVGLSVIEQFPGKLDFV ACC4 SSLISALFLSEPEGKIWIDKILTTEIGLHDLKKMSIIPQEPVLFTGTMKNLDPFNEHTDEELWNALQEVQLKETIEDLPGKMDTE CFT LVDGGCVLSHGHKQLMCLASVLSKAKILLLDEPSAHLDPVTYQIITLKQAFADCTVILCEHIEAMLECQQFL ACC4 LAESGSNFSVGQQLVCLAAILKNQILIIDEATANVDPTDELIQKKIEKFAHCTVLTIAHLNTIIDSDKIMVLDSGLKEYDE CFT VIEENKVQYDSIQKLLNE ACC4 PYVLLQNKESLFYKMVQQLGKAEAAALTETAKQVYFKNYPHIGHTDHMVTNTSNGQPSTLTIFETAL CFT -SLFQAISPSDVKLFPHNSSKCKSKPQIAALKEETEEEVQDTL ACC Supplementary Figure 3. Pairwise alignment of human CFT and ACC4 proteins shown along with locations of the protein coding sequence exons. The CFT exon number is shown first followed by the corresponding ACC4 exon. The locations of CFT domains are indicated with colors as in Supplementary Figure.
4 A CFT ACC Conserved CFT exons /7.8 Conserved ACC4 exons 7/3.87 Average exon conservation.84 CFT ACC CFT ACC Conserved CFT exons 8/7.3 Conserved ACC5 exons 5/9.7 Average exon conservation.3 C.9 Average exon conservation Supplementary Figure 4. Comparison of exon-intron structure between CFT and ACC subfamily members. Correspondence of exons between CFT-ACC4 (A) and CFT-ACC5 (). Corresponding exonic regions are placed in the same column, and exons (or exonic regions) that do not have corresponding sequences are marked with. Locations of CFT domains are indicated above. Fractions of conserved exons are shown below. (C) Average exon conservation (i.e. fractions of conserved exons) are shown between CFT and members of the ACC subfamily.
5 Supplementary Figure 5. Comparison of gene structures between CFT and closely related ACC subfamily members. The CFT exon-intron structure is schematically represented in comparison to those of the most closely related ACC subfamily members: ACC4 & ACC5. Exons are represented as black boxes and vertical blue lines between gene models connect corresponding (i.e. homologous) exons. For purposes of visual clarity, exons are drawn to scale, whereas introns are arbitrarily truncated.
6 CTG AGTA T CTA C A C E PhyloP score +/ 5bp of 5' SS Exon PhyloP score +/ 5bp of 5' SS p =.933 domain Others Exon Group Exon 4 Acceptor Exon 5 Acceptor Exon 6 Acceptor AG CATCAAGT ACTGCTC GATG CTTCAGCGCAA GCTCAGGTAGGC Intron Exon D F PhyloP score +/ 5bp of 3' SS Exon PhyloP score +/ 5bp of 3' SS p =.364 domain Others Exon Group Exon 4 Donor Exon 5 Donor Exon 6 Donor CGTAG TTGAGAGAGGCTCG AA ATCGAGGTGATAAG C T CAT CCGT GAAGCTAGGTT Supplementary Figure 6. Comparative splice site analysis for CFT-specific domain splice sites versus the splice sites of the remaining CFT exons. (A-) ates of sequence conservation (PhyloP scores) are compared for 5 splice acceptor sites and 3 splice donor sites for CFT domain encoding exons (red) versus the remaining CFT exons (blue). (C-D) Average ± SE rates of splice site sequence conservation are shown along with the P-values for the differences. (E-F) Splice acceptor and donor sites are shown for CFT domain encoding (and proximal) exons 4, 5 & 6.
7 A p =.667 Cell type-specificity Mean cell type-specificity domain Others Exon Exon Group Supplementary Figure 7. Comparison of cell type-specificity for CFT domain encoding exons (red) versus the remaining CFT exons (blue). Cell type-specifity values are shown for individual CFT exons (A) along with average ± SE vales and () the P-values for the differences.
8 A Pufferfish(ayfish) hesus abbit Pufferfish(ayfish) hesus abbit at at.5. C CFT D hesus abbit Pufferfish(ayfish) hesus abbit Pufferfish(ayfish) at. at. E Pufferfish(ayfish) hesus abbit F Pufferfish(ayfish) hesus abbit at. at. Supplementary Figure 8. Phylogenies of CFT and individual CFT domains. Phylogenies show two main groups: fish and other vertebrates. For each domain, the ratio /C shown in Figure 6 are calculated as the between group branch length (shown in red) divided by the average within group branch lengths (shown in black).
9 Supplementary Table. CFT and AC transporter sequences used in the analyses reported here. CFT and ACC subfamily protein & gene sequences Sequence Name efseq Accession Gene ID CFT (ACC7) NP_483 8 ACC NP_ ACC NP_ ACC3 NP_ ACC4 NP_ ACC5 NP_ ACC6 NP_6 368 ACC8 NP_ ACC9 NP_568, NP_ ACC NP_ ACC NP_ ACC NP_ Vertebrate CFT protein and coding nucleotide sequences Species Protein Accession Nucleotide Accession (Homo Sapiens) NP_483 NM_49 (Pan troglodytes) NP_73386 NM_7997 Orangutan (Pongo abelii) NP_67 NM_68545 hesus (Macaca mulatta) NP_8 NM_3938 abbit (Oryctolagus cuniculus) NP_7685 NM_876 (Canis lupus familiaris) NP_744 NM_743 (os taurus) NP_ NM_748 (Sus scrofa) NP_984 NM_495 (Monodelphis domestica) XP_36845 XM_36843 at (attus norvegicus) NP_3694 NM_356 (Mus musculus) NP_66388 NM_5 (Gallus gallus) NP_9936 NM_5666 (Xenopus laevis) AAC63 U69 (Danio rerio) NP_38348 NM_44883 (Salmo salar) NP_75 NM_3533 (Fundulus heteroclitus) AAC47 AF7 ( rubripes) NP_455 NM_484 Pufferfish (Tetraodon nigroviridis) AA633 AE79
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0.0389 0.0927 0.2641 0.4115 0.3630 0.3618 0.5153 0.3840 0.0357 1.026 0.0866 0.0423 0.1296 0.2052 0.2869 0.0948 0.6285 0.0835 0.0543 0.1135 0.1445 0.1501 0.3272 0.0418 0.4993 0.0624 0.0938 0.1189 0.0768
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