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Download fileBifunctional Fatty Acid Chemical Reporter for Analyzing S‑Palmitoylated Membrane Protein–Protein Interactions in Mammalian Cells
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posted on 2015-01-21, 00:00 authored by Tao Peng, Howard C. HangStudying
the functions of S-palmitoylated proteins in cells can
be challenging due to the membrane targeting property and dynamic
nature of protein S-palmitoylation. New strategies are therefore needed
to specifically capture S-palmitoylated protein complexes in cellular
membranes for dissecting their functions in vivo.
Here we present a bifunctional fatty acid chemical reporter, x-alk-16,
which contains an alkyne and a diazirine, for metabolic labeling of
S-palmitoylated proteins and photo-cross-linking of their involved
protein complexes in mammalian cells. We demonstrate that x-alk-16
can be metabolically incorporated into known S-palmitoylated proteins
such as H-Ras and IFITM3, a potent antiviral protein, and induce covalent
cross-linking of IFITM3 oligomerization as well as its specific interactions
with other membrane proteins upon in-cell photoactivation. Moreover,
integration of x-alk-16-induced photo-cross-linking with label-free
quantitative proteomics allows identification of new IFITM3 interacting
proteins.