Post‐translational Modifications of the Mitochondrial Outer Membrane Proteins: Carnitine Palmitoyltransferase‐I, Long‐chain acyl‐CoA synthetase, and Voltage Dependent Anion Channel

Abstract

Long-chain fatty acids require activation and transport into the mitochondrial matrix before â-oxidation can occur. Carnitine palmitoyltransferase-I (CPT-I), long-chain acyl-CoA synthetase (LCAS), and voltage dependent anion channel (VDAC), localized in the mitochondrial outer membrane play a critical role in this transport process and are the focus of our study. We have developed a method for analysis of hydrophobic membrane proteins based on isolation of resident membranes, separation of proteins by gel electrophoresis, electroelution of the proteins, and enzymatic protein digestion by trypsin and proteinase K. The resulting peptides from the digestions were analyzed by MALDI-TOF and HPLC ESI-MS/MS. With this method, we have achieved 75% to 99% sequence coverage for the mentioned transmembrane proteins. This method provides a targeted approach for examining membrane proteins in detail including post-translational modifications and identification of less abundant isoforms. In summary using our method: (a) we documented phosphorylation sites on CPT-I, VDAC, and LCAS using peptide mass fingerprinting and ESI-MS/MS, (b) on CPT-I, we identified a nitrated tyrosine in position 589, (c) we provided evidence for acetylation of the N-terminal alanine in VDAC-1, and (d) we found that in the mature rat liver CPT-I the cDNA deduced N-terminal methionine is removed and alanine is acetylated. Studies regarding the functional/regulatory impact of these modifications are currently being addressed. Supported by DK-066107, PO1 AG15885, and VA Medical Research Service