Palmitoylation of the three isoforms of human endothelin-converting enzyme-1.
Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Here we have examined whether the three isoforms of human ECE-1 (ECE-1a, ECE-1b and ECE-1c) are modified by the covalent attachment of the fatty acid palmitate and have evaluated a potential functional role of this modification. To do this, wild-type and mutant enzymes were expressed and analysed by metabolic labelling with [3H]palmitate, immunoprecipitation and SDS/PAGE. All three ECE-1 isoforms were found to be palmitoylated via hydroxylamine-sensitive thioester bonds. In addition, the isoforms showed similar levels of acylation. Cys46 in ECE-1a, Cys58 in ECE-1b and Cys42 in ECE-1c were identified as sites of palmitoylation and each of these cysteines accounted for all the palmitoylation that occured in the corresponding isoform. Immunofluorescence analysis demonstrated further that palmitoylated and non-palmitoylated ECE-1 isoforms had the same subcellular localizations. Moreover, complete solubility of the three isoforms in Triton X-100 revealed that palmitoylation does not target ECE-1 to cholesterol and sphingolipid-rich membrane domains or caveolae. The enzymic activities of ECE-1a, ECE-1b and ECE-1c were also not significantly affected by the absence of palmitoylation.
Schweizer
A
A
F.Hoffmann-La Roche Ltd., Pharma Division, Preclinical Cardiovascular Research, Grenzacherstrasse 124, 4070 Basel, Switzerland.
Löffler
B M
BM
Rohrer
J
J
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D003545:Cysteine / Q000378:metabolism; D000072396:Endothelin-Converting Enzymes; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D019811:Hydroxylamine / Q000378:metabolism; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000032:analysis; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010169:Palmitic Acids / Q000032:analysis / Q000378:metabolism*; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D012995:Solubility; D014162:Transfection
D051242:Caveolin 1; D022461:Caveolins; D007527:Isoenzymes; D008565:Membrane Proteins; D010169:Palmitic Acids; D019811:Hydroxylamine; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; D003545:Cysteine