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

Walpole TB, Palmer DN, Jiang H, Ding S, Fearnley IM, Walker JE - Mol. Cell Proteomics (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge, CB2 0XY, United Kingdom and.

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Related in: MedlinePlus

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).
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Figure 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).

Mentions: 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.


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

Walpole TB, Palmer DN, Jiang H, Ding S, Fearnley IM, Walker JE - Mol. Cell Proteomics (2015)

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).
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Related In: Results  -  Collection

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Figure 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).
Mentions: 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.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge, CB2 0XY, United Kingdom and.

Show MeSH
Related in: MedlinePlus