title: Identification of dually acylated proteins from complementary DNA resources by cell-free and cellular metabolic labeling.
abstract: To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP were generated and their susceptibility to protein N-myristoylation and S-palmitoylation were evaluated by metabolic labeling with [(3)H]myristic acid or [(3)H]palmitic acid either in an insect cell-free protein synthesis system or in transfected mammalian cells. As a result, EEPD1, one of five proteins (RFTN1, EEPD1, GNAI1, PDE2A, RNF11) found to be dually acylated, was shown to be a novel dually fatty acylated protein. Metabolic labeling experiments using G2A and C7S mutants of EEPD1-EGFP revealed that the palmitoylation site of EEPD1 is Cys at position 7. Analysis of the intracellular localization of EEPD1 C-terminally tagged with FLAG tag or EGFP and its G2A and C7S mutants revealed that the dual acylation directs EEPD1 to localize to the plasma membrane. Thus, dually fatty acylated proteins can be identified from cDNA resources by cell-free and cellular metabolic labeling of N-myristoylated proteins with Cys residue(s) close to the N-myristoylated N-terminus.
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Koko
authors.initials: K
authors.lastname: Moriya
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Kimoto
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Kanako
authors.initials: K
authors.lastname: Matsuzaki
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Kiwado
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Emi
authors.initials: E
authors.lastname: Takamitsu
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. Electronic address: utsumi@yamaguchi-u.ac.jp.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Utsumi
mesh_terms: D000215:Acylation; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry; D002474:Cell-Free System; D002522:Chlorocebus aethiops; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2 / Q000096:biosynthesis* / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases / Q000096:biosynthesis* / Q000737:chemistry; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000096:biosynthesis* / Q000737:chemistry; D006801:Humans; D054878:Lipoylation; D019308:Palmitic Acid / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C404508:RNF11 protein, human; D019308:Palmitic Acid; C000608105:EEPD1 protein, human; D004706:Endodeoxyribonucleases; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; C517275:PDE2A protein, human; C584114:GNAI1 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go
keywords: Cell-free protein synthesis system; EEPD1; Metabolic labeling; N-myristoylation; Protein acylation; S-palmitoylation