Cytochrome b561 is not fatty acylated but acetylated at amino terminus in chromaffin vesicle membranes: an approach for the identification of posttranslational modification of transmembrane proteins.
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.
Nakamura
Mariko
M
Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Akou-gun, Hyogo, Japan.
Takeuchi
Fusako
F
Tsubaki
Motonari
M
D000215:Acylation; D000818:Animals; D002417:Cattle; D019439:Chromaffin Cells / Q000378:metabolism*; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003573:Cytochrome b Group; D005227:Fatty Acids; C023646:cytochrome b561; D008715:Methionine