Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan adenosine triphosphate (ATP) synthases.
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.
Affiliation: From the ‡Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge, CB2 0XY, United Kingdom and.
- Conserved Sequence*
- Protein Subunits/chemistry/isolation & purification/metabolism*
- Proton-Translocating ATPases/chemistry/metabolism*
- Amino Acid Sequence
- Molecular Sequence Data
- Molecular Weight
- Protein Processing, Post-Translational
- Spectrometry, Mass, Electrospray Ionization
- Tandem Mass Spectrometry
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Figure 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).
The post-translational modification of c-subunits was localized to a specific region of the proteins by the MALDI-TOF-MS analysis of chymotryptic digests of the gel bands. In all but the c-subunits from the molluscs, C. gigas and M. edulis, a peptide with a mass in the range 1343.6–1343.9 Da was observed (Table II), corresponding to residues 37–47 (ARNPSLKQQLF) of almost all known vertebrate sequences (Fig. 3), and in many invertebrate sequences (Fig. 4), plus 42.0606–42.0657 Da). The location of the modified residue in the peptide was obtained by MALDI-TOF analysis of its fragment ions. In a typical example provided by the peptide from the Atlantic salmon, S. salar (Fig. 5), the fragment ion spectrum of the 1343.8 Da ion is dominated by a prominent ion with mass of 1284.6 Da. This ion corresponds to the loss of trimethylammonium (59 Da) from the peptide precursor, diagnostic of the presence of a trimethylated lysine (32, 33). In these, and also in other analyses conducted in an OrbiTrap mass spectrometer with fragmentation by higher energy collisions (not shown), there was no indication of any immonium ion (126.1 Da), which would arise if the peptide were acetylated. Therefore, in common with the human, bovine, and ovine c-subunits (17), the lysine-43 residues in the c-subunit of the salmon and the other species listed in Table I, are completely trimethylated on their ε-amino groups. In this spectrum, and those arising from the same peptide in other species, the presence of other fragment ions confirmed the sequence ARNPSLKQQLF, particularly in the N-terminal region, but usually these spectra did not contain sufficient information to allow the modification to be localized definitively, and therefore other analyses were conducted, as described below. Peptides with masses of 1315.75 and 1286.76 Da, were observed in the chymotryptic digests of the c-subunits from C. gigas and M. edulis, respectively, and their fragment ion spectra (supplemental Fig. S1) also contained abundant ions with masses 59 Da less than the parent ions, again providing evidence for trimethylation rather than acetylation of these peptides.