Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan adenosine triphosphate (ATP) synthases.
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.
Affiliation: From the ‡Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge, CB2 0XY, United Kingdom and.Show MeSH
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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.
Affiliation: From the ‡Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge, CB2 0XY, United Kingdom and.