ELECTRONIC SUPPLEMENTARY MATERIAL - Cellular and Molecular Life Sciences -

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1 ELECTRONIC SUPPLEMENTARY MATERIAL - Cellular and Molecular Life Sciences - Single nucleotide evolution quantifies the importance of each site along the structure of mitochondrial carriers Ciro Leonardo Pierri 1, Ferdinando Palmieri 1,2,*, Anna De Grassi 1,3,* 1 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via Orabona 4, 70125, Bari, Italy. 2 Center of Excellence in Comparative Genomics, University of Bari, Italy. 3 Département Systématique et Evolution, Ecole Pratique des Hautes Etudes, Muséum National d'histoire Naturelle, Paris, France. * corresponding authors, addresses: anna.degrassi@uniba.it, ferdinando.palmieri@uniba.it TABLE OF CONTENTS: Figure 1: Variability of strictly nonsynonymous phylop scores of each human MC amino acid Figure 2: Protein multi-alignment of the 53 human MCs Figure 3: Alignment of symmetry-related amino acid triplets of the 53 human MCs Figure 4: Cis/trans configuration of PG level glycines in the 53 human MCs Table 1: Genes, transcripts and proteins employed in this study Table 2: Sequence, evolutionary and structural information of the 255 MC sites analyzed

2 Fig. 1 Variability of strictly nonsynonymous phylop scores of each human MC amino acid. Distributions of phylop scores of strictly nonsynonymous sites are reported as kernel density estimates for each type of amino acid. The median value of each distribution is indicated (first value and red line) together with the total number of strictly nonsynonymous sites analyzed. Most distributions show peaks of density close to zero (neutral evolution) and five (extreme negative selection). Fig. 2 Protein multi-alignment of the 53 human MCs. The multi-alignment is shown for the odd-numbered transmembrane helices H1, H3 and H5 (a), matrix short helices h12, h34 and h56 (b), matrix loops b mlb12, mlb34 and mlb56 and even-numbered transmembrane helices H2, H4 and H6 (c) according to the BtAAC1 structure information. * indicates glycine residues of PG level 1 and PG level 2; # indicates the substrate-binding site common contact points on even transmembrane helices and the residues of odd transmembrane helices that are located at the same height in the carrier cavity as the contact points; indicates the charged residues of the c-gate and m-gate; indicates glutamine residues of triplet 37; ^ indicates proline residues of triplet 76; > < indicates the dipeptide DC together with the other residues ( V ) of the sequence motif in matrix short helices. Amino acids are colored according to their physico-chemical properties using the Zappo style in Jalview. It is to note that the present RefSeq protein sequences of SLC25A26 and SLC25A48 lack most of the matrix short helix h12 and the entire third repeat, respectively. Fig. 3 Alignment of symmetry-related amino acid triplets in the 53 human MCs. The triplets correspond to positions of the odd-numbered transmembrane helices (a), the matrix short helices (b) and the matrix loops b and even-numbered transmembrane helices (c). Each triplet consisting of the three aligned residues of each carrier derives from the inter-repeat multiple sequence alignment of the human MCs. The triplets are ordered horizontally according to the number of the first BtAAC1 repeat. Amino acids are colored according to their physico-chemical properties using the Zappo style in Jalview. See legend of Fig. 2 for symbol descriptions. Fig. 4 Cis/trans configuration of PG level glycines in the 53 human MCs. A cyan ribbon representation of the BtAAC1 structure residues (a) and residues (b) is shown. Residues are located at the end of the matrix loop (mlb12) and in transmembrane helix H2, and residues are located in transmembrane helix H3. P83, P133, G66, G73, G120 and G124 are in stick representation with the corresponding triplets reported in brackets.

3 Figure 1 Ala Arg Asn Asp Cys density PhyloP score Gln Glu Gly His Ile Leu Lys Inner Met Phe Pro Ser Thr Trp Tyr Val Start Met Stop

11 Table 1 Genes, transcripts and proteins employed in this study Gene Name Transcript Protein Common Protein Chrom Strand CDS Start CDS End RefSeq ID RefSeq ID Acronym Substrates Citrate, malate, isocitrate, SLC25A1 NM_ chr NP_ CIC cis-aconitate, phosphoenolpyruvate SLC25A2 NM_ chr NP_ ORC2 Ornithine, lysine, citrulline, arginine, histidine SLC25A3 NM_ chr NP_ PiC-A Phosphate SLC25A4 NM_ chr NP_ AAC1 ADP, ATP SLC25A5 NM_ chrx NP_ AAC2 ADP, ATP SLC25A6 NM_ chrx NP_ AAC3 ADP, ATP SLC25A7 NM_ chr NP_ UCP1 SLC25A8 NM_ chr NP_ UCP2 SLC25A9 NM_ chr NP_ UCP3 SLC25A10 NM_ chr NP_ DIC SLC25A11 NM_ chr NP_ OGC SLC25A12 NM_ chr NP_ AGC1 SLC25A13 NM_ chr NP_ AGC2 SLC25A14 NM_ chrx NP_ UCP5 H + SLC25A27 NM_ chr NP_ UCP4 H + SLC25A28 NM_ chr NP_ MFRN2 unknown SLC25A29 NM_ chr NP_ CACL Ornithine, palmitoylcarnitine SLC25A30 NM_ chr NP_ KMCP unknown SLC25A31 NM_ chr NP_ AAC4 ADP, ATP SLC25A32 NM_ chr NP_ MFT folates, FAD SLC25A33 NM_ chr NP_ PNC1 SLC25A34 NM_ chr NP_ unknown SLC25A35 NM_ chr NP_ unknown SLC25A36 NM_ chr NP_ PNC2 SLC25A37 NM_ chr NP_ MFRN1 unknown SLC25A38 NM_ chr NP_ Gly/δ-aminolevulinic acid SLC25A39 NM_ chr NP_ unknown SLC25A40 NM_ chr NP_ unknown SLC25A41 NM_ chr NP_ APC4 ATP-Mg, ATP, Pi, ADP, AMP CoA, adenosine SLC25A42 NM_ chr NP_ ',5'-diphosphate (PAP), dephospho-coa, ADP, ATP SLC25A43 NM_ chrx NP_ unknown SLC25A44 NM_ chr NP_ unknown SLC25A45 NM_ chr NP_ unknown SLC25A46 NM_ chr NP_ unknown SLC25A47 NM_ chr NP_ unknown SLC25A48 NM_ chr NP_ unknown SLC25A49 NM_ chr NP_ MTCH1 unknown SLC25A50 NM_ chr NP_ MTCH2 unknown SLC25A51 NM_ chr NP_ MCART1 unknown SLC25A52 NM_ chr NP_ MCART2 unknown SLC25A53 NM_ chrx NP_ MCART6 unknown H + H + H + Malate, succinate, phosphate, sulfate, thiosulfate 2-oxoglutarate, malate Aspartate, glutamate, cysteinesulfinate Aspartate, glutamate, cysteinesulfinate SLC25A15 NM_ chr NP_ ORC1 Ornithine, citrulline, arginine, lysine SLC25A16 NM_ chr NP_ unknown SLC25A17 NM_ chr NP_ PMP34 FAD, CoA, NAD + SLC25A18 NM_ chr NP_ GC2 Glutamate SLC25A19 NM_ chr NP_ TPC ThPP, ThMP, dndp, dntp, ADP, ATP SLC25A20 NM_ chr NP_ CAC carnitine, acylcarnitines SLC25A21 NM_ chr NP_ ODC 2-oxoadipate, 2-oxoglutarate SLC25A22 NM_ chr NP_ GC1 Glutamate SLC25A23 NM_ chr NP_ APC2 ATP-Mg, ATP, Pi, ADP, AMP SLC25A24 NM_ chr NP_ APC1 ATP-Mg, ATP, Pi, ADP, AMP SLC25A25 NM_ chr NP_ APC3 ATP-Mg, ATP, Pi, ADP, AMP SLC25A26 NM_ chr NP_ SAMC S-adensylmethionine, S-adenosylhomocysteine pyrimidine (deoxy)nucleotides pyrimidine (deoxy)nucleotides

12 Table2 Sequence, evolutionary and structural information of the 255 MC sites analyzed Inactivating Most RefTriplet RefPos RefAA RefStruct Side #paralogs %paralogs TS SD Evidence Role FreqAA 7 7 S H1 C S unknown Y H3 C S unknown H H5 C S unknown 8 8 F H1 C F unknown F H3 C L unknown I H5 C I unknown 9 9 L H1 C A unknown A H3 C L unknown SL525A4: A114P I H5 C V unknown K H1 C K unknown G H3 C A unknown V H5 C V unknown D H1 C D unknown N H3 C N unknown S H5 C H unknown F H1 C F unknown L H3 C L unknown W H5 C F unknown L H1 C L unknown SLC25A13: G333D A H3 C L unknown M H5 C V unknown A H1 C A unknown SLC25A15: A15E S H3 C A unknown I H5 C S unknown G H1 C G literature PG level 1 RnSLC25A20: G17A SLC25A3: G72E G H3 C G literature PG level A H5 C G literature PG level 1 SLC25A15: G216S G H1 C G unknown G H3 C S unknown SLC25A1: G130D Q H5 C A unknown V H1 C V unknown A H3 C L unknown T H5 C L unknown A H1 C A novel Substrate binding area A H3 C A novel Substrate binding area SLC25A4: A123D V H5 C A novel Substrate binding area A H1 C G literature PG level 1 RnSLC25A20: G21M G H3 C G literature PG level T H5 C G literature PG level A H1 C A unknown A H3 C A unknown A H5 C A unknown I H1 C V unknown SLC25A20: C23R T H3 C T unknown V H5 C C unknown S H1 C A novel Binding site in odd helices S H3 C A novel Binding site in odd helices ScCtp1p: E122C A H5 C A novel Binding site in odd helices K H1 M V novel Binding site in odd helices L H3 M V novel Binding site in odd helices G H5 M A novel Binding site in odd helices T H1 M T unknown C H3 M T unknown L H5 M L unknown A H1 M A unknown F H3 M V unknown V H5 M A unknown V H1 M T novel Substrate binding area site-directed mutations ScCtp1p: G119C; ScMir1p: E126Q HamsterSLC25A32: K235A ScAac2p: K38A; ScMir1p: H32A Human disease-related mutations SLC25A15: G113C; SLC25A19: G125S; SLC25A38: G130E SLC25A38: R134C; SLC25A38: R134H; SLC25A13: G531D; SLC25A15: G220R; SLC25A22: G236W; SLC25A38: G228V SLC25A20: R28C; SLC25A15: G27R; SLC25A15: G27E V H3 M T novel Substrate binding area RnSLC25A20: M128A SLC25A13: T446P

13 S H5 M S novel Substrate binding area ScMir1p: S232C A H1 M F novel Aromatic belts RnSLC25A20: H29A Y H3 M N novel Aromatic belts Y H5 M Y novel Aromatic belts P H1 M P literature PG level P H3 M P literature PG level 2 SLC25A15: P126R P H5 M P literature PG level I H1 M L unknown L H3 M L unknown F H5 M L unknown E H1 M D literature m-gate ScMir1p: D39N D H3 M E literature m-gate ScAac2p: D249S; D H5 M D literature m-gate SLC25A20: D231H ScMir1p: D236N R H1 M L novel m-gate area SLC25A15: T32R F H3 M V novel m-gate area SLC25A20: R133W T H5 M V novel m-gate area V H1 M V unknown A H3 M V unknown SLC25A38: I147N V H5 M V unknown K H1 M K literature m-gate ScAac2p: R152A; R H3 M K literature m-gate ScMir1p: R140A; SLC25A13: K453R HsSLC25A20: K35A R H5 M K literature m-gate L H1 M T novel m-gate area ScMir1p: T43A T H3 M T novel m-gate area R H5 M T novel m-gate area ScAac2p: R253I L H1 M R novel m-gate area R H3 M R novel m-gate area ScMir1p: R142A R H5 M R novel m-gate area ScAac2p: R254I L H1 M L novel m-gate area SLC25A15: M37R L H3 M L novel m-gate area M H5 M M novel m-gate area Q H1 M Q novel m-gate area SLC25A38: Q56K A H3 M Q novel m-gate area M H5 M Q novel m-gate area SLC25A20: Q238R V H1 M V novel m-gate area A H3 M A novel m-gate area M H5 M N novel m-gate area Q H1 M Q novel m-gate area D H3 M Q novel m-gate area Q H5 M Q novel m-gate area H H1 M G unknown V H3 M G unknown S H5 M G unknown A H1 M E unknown G H3 M K unknown G H5 M G unknown Q h12 M Q E h34 M K M h56 M Q Y h12 M Y F h34 M Y Y h56 M Y K h12 M K T h34 M R T h56 M K G h12 M G G h34 M G G h56 M G ScAac2p: D149S; ScMir1p: E137Q; HsSLC25A20: E132A; HamsterSLC25A32: W142A ScAac2p: K48A; ScCtp1p: K37C; ScMir1p :K42A ScAac2p: R252A; ScCtp1p: K239C; HamsterSLC25A32: R249A SLC25A20: P230R; SLC25A13: A541D SLC25A20: D32N; SLC25A13: D350N SLC25A13: T546R; SLC25A13: T546M

14 54 54 I h12 M I unknown SLC25A20: T55N L h34 M L unknown T h56 M T unknown I h12 M F unknown G h34 M L unknown V h56 M L unknown D h12 M D N h34 M D D h56 M D C h12 M C novel Cysteine rich triplet C h34 M C novel Cysteine rich triplet C h56 M C novel Cysteine rich triplet V h12 M L unknown I h34 M A unknown W h56 M L unknown V h12 M R T h34 M R R h56 M R R h12 M K K h34 M K K h56 M K I h12 M I unknown I h34 M I unknown I h56 M I unknown P h12 M L unknown F h34 M L unknown A h56 M L unknown K h12 M R unknown K h34 M R unknown K h56 M R unknown E h12 M E unknown S h34 M E unknown D h56 M E unknown Q mlb12 M E novel m-gate area D mlb34 M E novel m-gate area E mlb56 M E novel m-gate area G mlb12 M G literature PG level 2 SLC25A13: G386V G mlb34 M G literature PG level G mlb56 M G literature PG level F mlb12 M F unknown L mlb34 M L unknown P mlb56 M P unknown L mlb12 M R unknown R mlb34 M R unknown K mlb56 M K unknown S mlb12 M G literature PG level G mlb34 M G literature PG level A mlb56 M A literature PG level F mlb12 M L novel Aromatic belts L mlb34 M L novel Aromatic belts F mlb56 M L novel Aromatic belts W mlb12 M Y novel Aromatic belts Y mlb34 M Y novel Aromatic belts F mlb56 M Y novel Aromatic belts R mlb12 M R unknown Q mlb34 M R unknown K mlb56 M K unknown G mlb12 M G literature PG level 2 SLC25A13: G393S G mlb34 M G literature PG level 2 SLC25A19: G177A G mlb56 M G literature PG level N H2 M L unknown F H4 M L unknown A H6 M L unknown L H2 M G unknown N H4 M V unknown W H6 M S unknown A H2 M P novel Substrate binding area V H4 M P novel Substrate binding area

15 S H6 M P novel Substrate binding area N H2 M N novel Substrate binding area ScMir1p: T79A S H4 M T novel Substrate binding area SLC25A13: C489R; N H6 M N novel Substrate binding area V H2 M L unknown SLC25A15: L71Q V H4 M L unknown V H6 M L unknown SLC25A15: M273K I H2 M L unknown Q H4 M L unknown L H6 M L unknown ScAac2p: R96A; Binding site in even R H2 C R literature HamsterSLC25A32: SLC25A20: G81R helices G91L G H4 C R literature R H6 C R literature Binding site in even Y H2 C Q literature ScMir1p: Y83A helices Binding site in even HsSLC25A20: D179A; I H4 C D literature SLC25A15: E180K helices HsSLC25A15: E180A G H6 C L literature F H2 C A unknown I H4 C I unknown M H6 C G unknown P H2 C P literature PG level I H4 C P literature PG level 1 SLC25A38: P190R G H6 C P literature PG level 1 HsSLC25A20: P278V ScCtp1p: K83C; T H2 C E novel Substrate specificity HsSLC25A15: E77A Y H4 C F novel Substrate specificity gap gap H6 C A novel Substrate specificity SLC25A12: Q590R Q H2 C N novel Substrate specificity R H4 C S novel Substrate specificity ScAac2p: R204A G H6 C N novel Substrate specificity HsSLC25A20: N280G A H2 C A novel Substrate binding area SLC25A20: A87V A H4 C A novel Substrate binding area A H6 C A novel Substrate binding area SLC25A20: A281V L H2 C I unknown A H4 C I unknown F H6 C I unknown N H2 C K novel Substrate specificity Y H4 C Y novel Substrate specificity V H6 C M novel Substrate specificity SLC25A15: L283F F H2 C F novel Aromatic belts F H4 C F novel Aromatic belts HamsterSLC25A32: F200A SLC25A15: F188L L H6 C F novel Aromatic belts A H2 C G unknown SLC25A4: A90D G H4 C G unknown SLC25A13: P502L; SLC25A22: P206L V H6 C V unknown SLC25A13: L598R; HsSLC25A20: F284G SLC25A4: V289M F H2 C A unknown SLC25A13: V411M V H4 C T unknown SLC25A15: G190D L H6 C T unknown Binding site in even helices Binding site in even helices Binding site in even helices K H2 C Y novel Aromatic belts ScMir1p: Y94A Y H4 C Y novel Aromatic belts ScCtp1p: R276C; ScMir1p: R276A ScCtp1p: R181C; ScMir1p: K179A; HamsterSLC25A32: G192E; HsSLC25A15: R179A SLC25A13: R585H; SLC25A15: T272I; SLC25A38: R278G; SLC25A1: R282H SLC25A20: R178Q; SLC25A38: R187Q; SLC25A38: R187P ScAac2p: R294A; ScCtp1p: R279C; HsSLC25A20: R275A; SLC25A13: R588Q; HsSLC25A15: R275A; SLC25A15: R275Q HamsterSLC25A32: R288A HsSLC25A15: N78A; HamsterSLC25A32: W96A ScCtp1p: R87C; ScMir1p: K90A ScCtp1p: R189C; ScMir1p: K187A

16 Y H6 C Y novel Aromatic belts D H2 C E literature c-gate D H4 C E literature c-gate D H6 C E literature c-gate SLC25A13: E601K K H2 C F unknown T H4 C L unknown E H6 C F unknown Y H2 C L unknown A H4 C L unknown I H6 C L unknown K H2 C K literature c-gate ScMir1p: K98A K H4 C K literature c-gate K H6 C K literature c-gate ScMir1p: S195A Q H2 C Q unknown G H4 C E unknown K H6 C K unknown I H2 C L unknown SLC25A4: L98P M H4 C L unknown F H6 C L unknown F H2 C L unknown L H4 C L unknown V H6 C L unknown HamsterSLC25A32: Y300A Footnotes RefTriplet RefPos RefAA RefStructure Side MostFreqAA #paralogs %paralogs TS SD Evidence Role Inactivating site-directed mutations Human disease-related mutations Reference triplet (see Electronic Supplementary Material Fig. 3). Reference position in the BtAAC1 sequence. The amino acid present in the BtAAC1 sequence. Secondary structural element in the crystallized BtAAC1. Cytosolic (C) or matrix (M) half in the BtAAC1 structure. Most frequent amino acid in the 53 multialigned human MCs. Number of human MCs hosting the most frequent amino acid. Percentage of the human MCs hosting the most frequent amino acid. Transversal score. Standard deviation (inter-paralog variability). Evidence of structural and/or functional importance proposed by previous studies (literature), by the present study (novel) or no evidence (-). Known or predicted role. Site-directed mutations that inactivate the protein function (RTA<10%) are reported in the following organisms and genes: ScAac2p, S. cerevisiae ADP/ATP carrier isoform 2; ScCtp1p, S. cerevisiae Citrate transport protein; ScMir1p, S. cerevisiae Phosphate transport protein isoform 1; HsSLC25A20, H. sapiens SLC25A20; RnSLC25A20, R. norvegicus SLC25A20; HsSLC25A15, H. sapiens SLC25A15; HamsterSLC25A32, Cricetulus griseus SLC25A32. Pathological mutations detected in the indicated human genes and amino acids.

LAB#23: Biochemical Evidence of Evolution Name: Period Date :

LAB#23: Biochemical Evidence of Name: Period Date : Laboratory Experience #23 Bridge Worth 80 Lab Minutes If two organisms have similar portions of DNA (genes), these organisms will probably make similar