Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan adenosine triphosphate (ATP) synthases

Abstract

The rotors of ATP synthases turn about 100 times every second. One essential component of the rotor is a ring of hydrophobic c-subunits in the membrane domain of the enzyme. The rotation of these c-rings is driven by a transmembrane proton-motive force, and they turn against a surface provided by another membrane protein, known as subunit a. Together, the rotating c-ring and the static subunit a provide a pathway for protons through the membrane in which the c-ring and subunit a are embedded. Vertebrate and invertebrate c-subunits are well conserved. In the structure of the bovine F1-ATPase-c-ring subcomplex, the 75 amino acid c-subunit is folded into two transmembrane α-helices linked by a short loop. Each bovine rotor-ring consists of eight c-subunits with the N- and C-terminal α-helices forming concentric inner and outer rings, with the loop regions exposed to the phospholipid head-group region on the matrix side of the inner membrane. Lysine-43 is in the loop region and its ε-amino group is completely trimethylated. The role of this modification is unknown. If the trimethylated lysine-43 plays some important role in the functioning, assembly or degradation of the c-ring, it would be expected to persist throughout vertebrates and possibly invertebrates also. Therefore, we have carried out a proteomic analysis of c-subunits across representative species from different classes of vertebrates and from invertebrate phyla. In the twenty-nine metazoan species that have been examined, the complete methylation of lysine-43 is conserved, and it is likely to be conserved throughout the more than two million extant metazoan species. In unicellular eukaryotes and prokaryotes, when the lysine is conserved it is unmethylated, and the stoichiometries of c-subunits vary from 9-15. One possible role for the trimethylated residue is to provide a site for the specific binding of cardiolipin, an essential component of ATP synthases in mitochondria.

Fig. 1.

Isolation of the c-subunits of F-ATP synthases. In parts A and B, respectively, the subunits of F-ATPases isolated by affinity chromatography and the hydrophobic proteins obtained by extraction of mitochondrial membranes with organic solvents were fractionated by SDS-PAGE, and stained with Coomassie blue dye. The tracks in parts A and B have been assembled from many different gels, as is evident. The c-subunit in each sample was identified by peptide mass fingerprinting of a chymotryptic digest. In part A, the positions of the subunits in the human enzyme are indicated on the left. Track a, Homo sapiens; b, Bos taurus; c, Ovis aries; d, Sus scrofa; e, Cervus elaphus; f, Mus musculus; g, Rattus norvegicus; h, Oryctolagus cuniculus; i, Gallus gallus; j, Anas platyrhynchos; k, Testudo graeca; l, Boa constrictor; m, Xenopus laevis n, Salmo salar; o, Onchorhynchus mykiss; p, Dicentrachus labrax; q, Squalus acanthias; r, Lumbricus terrestris; s, Calliphora vomitoria; t, Cancer pagurus; u, Homarus gammarus; v, Artemia salina. Part B, Analysis by SDS-PAGE of chloroform:methanol extracts from metazoan and plant species. The c-subunit was identified by peptide mass fingerprinting of chymotryptic peptides. The positions of the bovine c-subunit and the adenine nucleotide translocase (ANT) are indicated. Track a, Bos taurus; b, Trichosurus vulpecula c, Mytilus edulis; d, Crassostrea gigas; e, Caenorhabditis elegans; f, Drosophila melanogaster; g, Trichoplusia ni; h, Evechinus chloroticus; i, Australostichopus mollis; j, Crella incrustans; k, Solanum tuberosum.

Fig. 2.

Mass spectral analysis of the intact c-subunit of the F-ATPase from Salmo salar by ESI-MS. A series of multiply charged ions from the c-subunit are indicated. The insert contains a mathematical transformation of these data to a molecular mass scale.

Fig. 3.

Sequences of c-subunits from vertebrate F-ATPases. The secondary structure of the bovine protein is depicted above the aligned sequences. Where sequence data are available a representative species from each vertebrate order is shown. Alanine residues 13, 19, and 23, required for the formation of a c₈-ring, trimethylated lysine-43 and glutamate-58, which is essential for proton translocation, and are green, purple, and blue, respectively. Amino acid substitutions are red. The five letter UNIPROT codes for species are on the left; bold codes denote species where lysine-43 has been demonstrated experimentally to be trimethylated. HOMSA, Homo sapiens; BOVIN, Bos taurus (cow); CANFA, Canis lupus familiaris (dog); DASNO, Dasypus novemcinctus (armadillo); EQUCA, Equus caballus (horse); OVIAR, Ovis aries (sheep); SUSSC, Sus scrofa (pig); CEREL, Cervus elaphus (red deer); MONDE, Monodelphis domestica (gray short tailed opossum); MYOLU, Myotis lucifugus (bat); ORNAN, Ornithorhynchus anatinus (duckbill platypus); TUPCH, Tupaia chinensis (Chinese tree shrew); GALVA, Galeopterus variegatus (flying lemur); TRIMA, Trichechus manatus latirostris (Florida manatee); LOXAF, Loxodonta africana (African elephant); ELEED, Elephantulus edwardii (Cape elephant shrew); SARHA, Sarcophilus harrisii (Tasmanian devil); CHRAS, Chrysochloris asiatica (Cape golden mole); MUSMU, Mus musculus (mouse); ORYCU, Oryctolagus cuniculus (rabbit); RATNO, Rattus norvegicus (rat); TURTR, Tursiops truncates (bottle nosed dolphin); ANOCA, Anolis carolinesis (green anole); PELSI, Pelodiscus sinensis (Chinese softshell turtle); PYTBI, Python bivittatus (Burmese python); ANAPL, Anas platyrhynchus (wild duck); GALGA, Gallus gallus (chicken); TAEGU, Taenio guttat (zebrafinch); CALAN, Calypte anna (Anna's hummingbird); FALPE, Falco peregrinus (peregrine falcon); MELUN, Melopsittacus undulatus (budgerigar); COLLI, Columba livia (rock pigeon); APTFO, Aptenodytes forsteri (emperor penguin); XENLA, Xenopus laevis (West African clawed toad); AMBME, Ambystoma mexicanum (axolotl); DANRE, Danio rerio (zebrafish); ONCMY, Onchorhynchus mykiss (rainbow trout); SALSA, Salmo salar (salmon); TAKRU, Takifugu rubripes (pufferfish); CYNSE, Cynoglossus semilaevis (tongue sole); ORYLA, Oryzias latipes (Japanese medaka); POERE, Poecilia reticulata (guppy); ANOFI, Anoplopoma fimbria (sablefish); ORENI, Oreochromis niloticus (Nile tilapia); PERFL, Perca flavescens (yellow perch); OSSMO, Osmerus mordax (rainbow smelt); ESOLU, Esox lucius (northern pike); ASTME, Astyanax mexicanus (Mexican tetra); CYPCA, Cyprinus carpio (common carp); ICTPU, Ictalurus punctatus (channel catfish); LEPOC, Lepisosteus oculatus (spotted gar); LATCH, Latimeria chalumnae (coelocanth); CALMI, Callorhinchus milii (elephant shark).

Fig. 4.

Sequences of c-subunits from invertebrate F-ATPases. Where data are available sequences for all known invertebrate species are shown, with the exception of the arthropods and poriferans, where representative species were selected from each class when available. †, a related species was studied (L. terrestris, T. ni, E. chloroticus, and M. edulis, respectively). For the significance of the colors, and the five letter codes on the left, see the legend to Fig. 6. BRABE, Branchiostoma belcheri (Japanese lancelet); CIOIN, Ciona intestinalis (vase tunicate); CIOSA, Ciona savignyi (solitary sea squirt); STRPU, Stronglyocentrotus purpuratus (purple sea urchin); HELRO, Helobdella robusta (Californian leech); GLYTR, Glycera tridactyla; PLADU, Platynereis dumerilii (Dumeril's clam worm); CAPTE, Capitella teleta; HYDEL, Hydroides elegans; RIFPA, Riftia pachyptila (giant tube worm); LUMRU, Lumbricus rubellus (red earthworm); ACYPI, Acyrthosiphon pisum (pea aphid); APIME, Apis mellifera (honeybee); CULEX, Culex pipiens (common house mosquito) DROME, Drosophila melanogaster (fruit fly); CALVO, Calliphora vomitoria (blue bottle fly); GLOMM, Glossina morsitans (Savannah tsetse fly); IXOSC, Ixodes scapularis (black legged tick); LITVA, Litopenaeus vannamei (white leg shrimp); MANSE, Manduca sexta (tobacco hawkmoth); NASVI, Nasonia vitripennis (jewel wasp); OPICA, Opisthacanthus cayaporum (South American scorpion); STRMA, Strigamia maritima (European centipede); PEDHU, Pediculus humanus (head louse); PENJP, Peneus japonica (Kuruma prawn); APACA, Acartia pacifica (copepod); SIMVI, Simulium vittatum (black fly); SPOFR, Spodoptera frugiperda (fall armyworm); STEMI, Stegodyphus mimosarum (social spider); STOCA, Stomoxys calcitrans (stable fly); TRICA, Tribolium castaneum (red flour beetle); APLCA, Aplysia californica (California sea hare); HALDI, Haliotis diversicolor (variously colored abalone); LOTGA, Lottia gigantea (owl limpet); SINCO, Sinonovacula constricta (Chinese razor clam); MYTGA, Mytilus galloprovincalis (Mediterranean mussel); CRAGI, Crassostrea gigas (Pacific oyster); ECHGR, Echinococcus granulosus (hydatid worm); OPIVI, Opisthorchis viverrini (Southeast Asian liver fluke); CLOSI, Clonorchis sinensis (Chinese liver fluke); HYMMI, Hymenolepis microstoma (rodent tapeworm); SCHMA, Schistosoma mansoni (blood fluke); BRUMA, Brugia malayi; CAEEL, Caenorhabditis elegans; MELHA, Meloidogyne hapla (northern root knot nematode); NECAM, Necator americanus (New World hookworm); HAECO, Hemonchus contortus (barber pole worm); ANCCE, Ancylostoma ceylanicum (hookworm); LOALO, Loa loa (eye worm); ASCSU, Ascaris suum (pig roundworm); TRIAD, Trichoplax adhaerans ; CARBA, Carukia barnesi (Irukandji jellyfish); NEMVE, Nematostella vectensis (starlet sea anenome); HYDMA, Hydra magnipapillata (hydra); PLEBA, Pleurobrachia bachei (sea gooseberry); MNELE, Mnemiopsis leidyi (sea walnut); AGESC, Agelas schmitdi (brown tubular sponge); APHVA, Aphrocallistes vastus (cloud sponge); APLFU, Aplysina fulva (rope sponge); ECTFE, Ectyoplasia ferox (brown encrusting octopus sponge); HALDU, Halisarca dujardini; HIPLA, Hippospongia lachne (sheepswool sponge); IGENO, Igernella notablis; IRCST, Ircinia strobilina (black ball sponge); OSCCA, Oscarella carmela; SUBDO, Suberites domuncula; TOPOP, Topsentia ophiraphidites; CLACL, Clathrina clathrus (Mediterranean sponge); VALSP. Vaceletia sp.; AXICO, Axinella corrugate (marine sponge); GEONE, Geodia neptuni (leathery barrel sponge).

Fig. 5.

Tandem-MS analysis of a chymotryptic peptide from the c-subunit of the F-ATPase from Salmo salar. The singly charged chymotryptic peptide (MH⁺ 1343.69) corresponds to residues 37–47 of the protein. The prominent ion at m/z 1284.63 arises by loss of a trimethylammonium ion and is diagnostic of a trimethylated peptide. The inset contains the MALDI-TOF-MS spectrum of the chymotryptic digest. The residue numbers of the peptides from the salmon c-subunit are given in parentheses. The ion at m/z 1060.0598 is derived from the matrix, and the ion at m/z 1523.8110 is a fragment of chymotrypsin.

Fig. 6.

Tandem MS of a chymotryptic peptide from the c-subunit of Salmo salar. The peptide represents residues 37–47 of the protein. A triply charged version (m/z 448.60) was fragmented by ETD, and the fragments were analyzed in an OrbiTrap instrument. The upper and lower panels contain ions with m/z values of 100–700 and 700–1370, respectively. In the upper panel, the c- and z-ions are mapped onto the amino acid sequence of the peptide. The mass difference of 170.15 Da between the c6 and c7 ions shows that lysine-7 is trimethylated.

Fig. 7.

Metazoan tree of life. Part A, the vertebrate tree. Part B, the major metazoan phyla. The red branches contain species where lysine-43 in the c-subunit of mitochondrial F-ATP synthase has been demonstrated to be trimethylated. In part A, the analyzed examples are as follows: Mammalia, Homo sapiens, Bos taurus, Ovis aries, Sus scrofa, Oryctolagus cuniculus, Mus musculus, Rattus norvegicus, Cervus elaphus, Trichosurus vulpecula; Aves, Gallus gallus, Anas platyrhynchos; Reptilia, Testudo gracea, Boa constrictor; Amphibia, Xenopus laevis; Actinopterygii, Salmo salar, Onchorhynchus mykiss, Dicentrachus labrax; Chondrichthyes, Squalus acanthias. In part B, the analyzed examples are: Echinodermata, Evechinus chloroticus, Australostichopus mollis; Chordata, see Part A; Annelida, Lumbricus terrestris; Mollusca, Crassostrea gigas, Mytilus edulis; Crustacea, Homarus gammarus, Cancer pagurus, Artemia salina; Hexapoda, Drosophila melanogaster, Trichoplusia ni, Calliphora vomitoria; Nematode worms, Caenorhabditis elegans; Porifera, Crella incrustans.