Identification of dually acylated proteins from complementary DNA resources by cell-free and cellular metabolic labeling
| Autor: | Toshihiko Utsumi, Emi Takamitsu, Mayumi Kimoto, Kanako Matsuzaki, Koko Moriya, Aya Kiwado |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
DNA Complementary Acylation Lipoylation Palmitic Acid Biophysics GTP-Binding Protein alpha Subunits Gi-Go Biology Biochemistry Green fluorescent protein Cell-free system 03 medical and health sciences Protein acylation FLAG-tag Complementary DNA Chlorocebus aethiops Protein biosynthesis Animals Humans Molecular Biology Endodeoxyribonucleases Cell-Free System Cell Biology N-Myristoylation Cyclic Nucleotide Phosphodiesterases Type 2 DNA-Binding Proteins 030104 developmental biology COS Cells lipids (amino acids peptides and proteins) Carrier Proteins Cysteine |
| Zdroj: | Analytical Biochemistry. 511:1-9 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2016.07.025 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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