Cytochrome b ₅₆₁ is not fatty acylated but acetylated at amino terminus in chromaffin vesicle membranes: an approach for the identification of posttranslational modification of transmembrane proteins

Summary.

 We examined the nature of the posttranslational modification of bovine cytochrome b ₅₆₁, a membrane-spanning protein and an essential component of neuroendocrine secretory vesicles. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) showed two populations in the partially digested fragments of cytochrome b ₅₆₁, which were obtained by controlled treatment of cytochrome b ₅₆₁-proteoliposomes with trypsin. One population, containing the posttranslationally modified amino-terminal region, showed molecular masses which were by about 40 Da larger than the theoretical molecular masses. The other population, without the modified amino-terminal region, showed a reasonable matching with the theoretical masses. This result suggested that the posttranslational modification occurred only in the amino-terminal region. The amino-terminal peptide was isolated by tryptic peptide mapping followed by treatment with acylamino-acid-releasing enzyme. Amino acid sequence and MALDI-TOF-MS analyses of the amino-terminal peptide showed that the initial Met residue was acetylated. There was no other posttranslational modification in the amino-terminal region, such as covalent fatty acylation through an ester linkage to Ser or Thr residues.