title: Palmitoylation of carboxypeptidase D. Implications for intracellular trafficking.
abstract: Covalent lipid modifications mediate protein-membrane and protein-protein interactions and are often essential for function. The purposes of this study were to examine the Cys residues of the transmembrane domain of metallocarboxypeptidase D (CPD) that could be a target for palmitoylation and to clarify the function of this modification. CPD is an integral membrane protein that cycles between the trans Golgi network and the plasma membrane. We constructed AtT-20 cells stably expressing various constructs carrying a reporter protein (albumin) fused to a transmembrane domain and the CPD cytoplasmic tail. Some of the constructs contained the three Cys residues present in the CPD transmembrane region, while other constructs contained Ala in place of the Cys. Constructs carrying Cys residues were palmitoylated, while those constructs lacking the Cys residues were not. Because palmitoylation of several proteins affects their association with cholesterol and sphingolipid-rich membrane domains or caveolae, we tested endogenous CPD and several of the reporter constructs for resistance to extraction with Triton X-100. A construct containing the Cys residues of the CPD transmembrane domain was soluble in Triton X-100 as was endogenous palmitoylated CPD, indicating that palmitoylation does not target CPD to detergent-resistant membrane rafts. Interestingly, constructs of CPD that lack palmitoylation sites have an increased half-life, a slightly more diffuse steady-state localization, and a slower rate of exit from the Golgi as compared with constructs containing palmitoylation sites. Thus, the covalent attachment of palmitic acid to the Cys residues of CPD has a functional significance in the trafficking of the protein.
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Kalinina
authors.forename: Lloyd D
authors.initials: LD
authors.lastname: Fricker
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002268:Carboxypeptidases / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D017830:Octoxynol / Q000494:pharmacology; D019308:Palmitic Acid / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection
chemical_list: D019308:Palmitic Acid; D003553:Cystine; D017830:Octoxynol; D002268:Carboxypeptidases; C078874:carboxypeptidase D; D003545:Cysteine