Abstract
Progress in understanding the biology of protein fatty acylation has been impeded by the lack of rapid direct detection and identification methods. We first report that a synthetic ω-alkynyl-palmitate analog can be readily and specifically incorporated into GAPDH or mitochondrial 3-hydroxyl-3- methylglutaryl-CoA synthase in vitro and reacted with an azido-biotin probe or the fl uorogenic probe 3-azido-7-hydroxycoumarin using click chemistry for rapid detection by Western blotting or flat bed fluorescence scanning. The acylated cysteine residues were confirmed by MS. Second, ω-alkynyl-palmitate is preferentially incorporated into transiently expressed H- or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with ω-alkynyl-myristate or ω-alkynyl-stearate, via an alkali sensitive thioester bond. Third, ω-alkynyl-myristate is specifically incorporated into endogenous co- and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of ω-alkynylpalmitate and ω-alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with ω-alkynyl- palmitate does not affect membrane association of N-Ras. Furthermore, the palmitoylation of endogenous proteins including H- and N-Ras could be easily detected using ω-alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The ω-alkynylmyristate and -palmitate analogs used with click chemistry and azido-probes will be invaluable to study protein acylation in vitro, in cells, and in vivo.
Original language | English (US) |
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Pages (from-to) | 1566-1580 |
Number of pages | 15 |
Journal | Journal of lipid research |
Volume | 51 |
Issue number | 6 |
DOIs | |
State | Published - Jun 1 2010 |
Keywords
- Enzymes
- Membranes
- Mitochondria
- Myristoylation
- Palmitoylation
ASJC Scopus subject areas
- Biochemistry
- Endocrinology
- Cell Biology