Protein Protocols Protein Protocols

Protein Palmitoylation 633
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Analysis of Protein Palmitoylation
Morag A. Grassie and Graeme Milligan
1. Introduction
The incorporation of many membrane proteins into the lipid environment is based
on sequences of largely hydrophobic amino acids that can form membrane-spanning
domains. However, a number of other proteins are membrane-associated, but do not
display such hydrophobic elements within their primary sequence. Membrane association
in these cases is often provided by covalent attachment, either cotranslationally or
posttranslationally, of lipid groups to the polypeptide chain. Acylation of proteins by
either addition of C14:0 myristic acid to an N-terminal glycine residue or addition of
C16:0 palmitic acid by thioester linkage to cysteine residues, in a variety of positions
within the primary sequence, has been recorded for a wide range of proteins.
Palmitoylation of proteins is not restricted to thioester linkage and may occur also
through oxyester linkages to serine and threonine residues. Furthermore, thioester linkage
of fatty acyl groups to proteins is not restricted to palmitate. Longer chain fatty
acids, such as stearic acid (C18:0) and arachidonic acid (C20:4), have also been
detected. Artificial peptide studies have provided evidence to support the concept that
attachment of palmitate to a protein can provide sufficient binding energy to anchor a
protein to a lipid bilayer, but that attachment of myristate is insufficient, in isolation, to
achieve this.
Mammalian proteins that have been demonstrated to be palmitoylated include a
range of G protein-coupled receptors and G protein α subunits, members of the Src
family of nonreceptor tyrosine kinases, growth cone-associated protein GAP 43,
endothelial nitric oxide synthase, spectrin, and glutamic acid decarboxylase. Since
many of these proteins play central roles in information transfer across the plasma
membrane of cells, there has been considerable interest in examining both the steadystate
palmitoylation status of these proteins and, because the thioester linkage is labile,
the possibility that it may be a dynamic, regulated process (1–7). Palmitoylation thus
provides a means to provide membrane anchorage for many proteins and, as such, can
allow effective concentration of an enzyme or other regulatory protein at the twodimensional
surface of the membrane. Turnover of the protein-associated palmitate
may regulate membrane association of polypeptides and, thus, their functions.
There has been considerable pharmaceutical interest in the development of smallmol-wt
inhibitors of the enzyme farnesyl transferase, since attachment of the farnesyl
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
633