Detection of co- and posttranslational protein N-myristoylation by metabolic labeling in an insect cell-free protein synthesis system.
To establish a simple and sensitive method to detect protein N-myristoylation, the usefulness of a newly developed cell-free protein synthesis system derived from insect cells for detecting protein N-myristoylation by in vitro metabolic labeling was examined. The results showed that in vitro translation of cDNA coding for N-myristoylated protein in the presence of [(3)H]myristic acid followed by SDS-PAGE and fluorography is a useful method for rapid detection of protein N-myristoylation. Differential labeling of N-myristoylated model proteins with [(3)H]leucine, [(3)H]myristic acid, and [(35)S]methionine revealed that the removal of the initiating Met during the N-myristoylation reaction could be detected using this system. Analysis of the N-myristoylation of a series of model proteins with mutated N-myristoylation motifs revealed that the amino acid sequence requirements for the N-myristoylation reaction in this system are quite similar to those observed in the rabbit reticulocyte lysate system. N-myristoylation of tBid (a posttranslationally N-myristoylated cytotoxic protein that could not be expressed in transfected cells) was successfully detected in this assay system. Thus, metabolic labeling in an insect cell-free protein synthesis system is an effective strategy to detect co- and posttranslational protein N-myristoylation irrespective of the cytotoxicity of the protein.
Sakurai
Nagisa
N
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan.
Moriya
Koko
K
Suzuki
Takashi
T
Sofuku
Kozue
K
Mochiki
Hiroyuki
H
Nishimura
Osamu
O
Utsumi
Toshihiko
T
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002474:Cell-Free System / Q000378:metabolism; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D047468:Immunoprecipitation; D007313:Insecta / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D019814:Myristic Acid / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D013460:Sulfur Isotopes / Q000737:chemistry; D014316:Tritium / Q000737:chemistry
D013460:Sulfur Isotopes; D019814:Myristic Acid; D014316:Tritium; D008715:Methionine