Determination of the structural requirements for palmitoylation of p63.
Palmitoylation of p63, a type II membrane protein localized in the endoplasmic reticulum, is induced in a reversible manner by the drug brefeldin A. To study the requirements for palmitoylation, mutant forms of p63 were expressed in COS cells and analyzed by metabolic labeling with [3H]palmitate, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis. By investigating deletion and point mutations, Cys100 in the 106-amino acid cytoplasmic tail of p63 has been identified as the site of acylation. Site-directed mutagenesis of residues 99-105 together with cytoplasmic tail truncation mutants showed that the amino acids surrounding Cys100 are not critical for palmitoylation of this residue. Analysis of a chimeric construct between p63 and the plasma membrane protein dipeptidylpeptidase IV further revealed that p63 palmitoylation is not dependent on its transmembrane domain. In contrast, the six-amino acid distance between the end of the predicted transmembrane domain and the palmitoylation site was found to be essential for proper acylation of p63.
Schweizer
A
A
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Rohrer
J
J
Kornfeld
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020126:Brefeldin A; D002460:Cell Line; D002522:Chlorocebus aethiops; D003517:Cyclopentanes / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*
D003517:Cyclopentanes; D008565:Membrane Proteins; D010169:Palmitic Acids; D020126:Brefeldin A; D019308:Palmitic Acid; D003545:Cysteine