Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

PLoS One. 2015 Aug 26;10(8):e0136360. doi: 10.1371/journal.pone.0136360. eCollection 2015.

Abstract

To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Cell-Free System
  • Chlorocebus aethiops
  • DNA, Complementary / genetics*
  • HEK293 Cells
  • Humans
  • Membrane Proteins / metabolism*
  • Microscopy, Fluorescence
  • Mitochondria / metabolism*
  • Molecular Sequence Data
  • Myristic Acid / chemistry*
  • Protein Biosynthesis
  • Protein Processing, Post-Translational*
  • Sequence Homology, Amino Acid
  • Transfection

Substances

  • DNA, Complementary
  • Membrane Proteins
  • Myristic Acid

Grant support

This work was supported by a Grant-in-Aid for Scientific Research (No. 23580136 and No. 26450125, https://www.jsps.go.jp/j-grantsinaid/index.html) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to TU). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.