Protoplasma (2003) 221: 41– 46 DOI 10.1007/s00709-002-0062-3 Summary. We examined the nature of the posttranslational modifi- cation 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 -proteolipo- somes with trypsin. One population, containing the posttranslation- ally 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 oc- curred only in the amino-terminal region. The amino-terminal peptide was isolated by tryptic peptide mapping followed by treat- ment 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. Keywords: Ascorbate; Cytochrome b 561 ; Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry; Acetylation; Posttranslational modification; Proteoliposome. Abbreviations: AsA ascorbate; MALDI-TOF matrix-assisted laser desorption and ionization time-of-flight; SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis; AARE acylamino-acid-releasing enzyme. Introduction Cytochrome b 561 is an electron transport protein of neuroendocrine secretory vesicles. The function of cytochrome b 561 is to supply electrons from cytosolic ascorbate (AsA) to support dopamine b-hydroxylase activity in the catecholamine-storing vesicles and peptidylglycine a-amidating monooxygenase activity in neurosecretory peptide-storing vesicles (Eipper et al. 1983, Kent and Fleming 1987, Asada et al. 2002). The functional transmembrane nature of cytochrome b 561 has been verified by the reconstitution of purified cytochrome b 561 in AsA-loaded vesicles (Srivastava et al. 1984), although its orientation in the proteolipo- somes was not examined. Transmembrane electron transfer was verified by addition of ferricytochrome c to the external medium, resulting in the reduction of the heme c center (Srivastava et al. 1984). The trans- membrane electron transfer could further support the enzymatic activities of dopamine b-hydroxylase and peptidylglycine a-amidating monooxygenase on the extravesicular side (Kent and Fleming 1987). We have clarified that cytochrome b 561 contained two heme b centers (Tsubaki et al. 1997), one for electron accep- tance from AsA on the extravesicular side and the other for electron donation to monodehydroascor- bate radical on the intravesicular side (Kobayashi et al. 1998, Okuyama et al. 1998, Tsubaki et al. 2000, Takeuchi et al. 2001), suitable for the transmembrane electron transfer reaction. Kent and Fleming (1990) reported that cytochrome b 561 was covalently fatty acylated through an ester linkage to Ser or Thr residues in the amino-terminal region. There is a possibility that the fatty acid group(s) in the cytoplasmic loops connecting the puta- tive transmembrane helices, together with an unknown PROTOPLASMA Printed in Austria 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 Mariko Nakamura, Fusako Takeuchi, and Motonari Tsubaki * Department of Life Science, Graduate School of Science, Himeji Institute of Technology,Akou-gun, Hyogo Received May 9, 2002; accepted July 26, 2002; published online May 21, 2003 © Springer-Verlag 2003 * Correspondence and reprints: Department of Molecular Science, Graduate School of Science and Technology, Kobe University, Rokkodai-cho 1-1, Nada-ku, Hyogo 657-8501, Japan.