Current Protocols in Protein Science
UNIT 14.2 (original pub 1996)
Analysis of Protein Acylation
This is the post-peer-reviewed (but not final) version of the following article: Zeidman, R.,
Jackson, C.S., and Magee, A.I. 2009. Analysis of Protein Acylation. Curr. Protoc. Protein Sci.
55:14.2.1-14.2.12. © 2009 by John Wiley & Sons, Inc., which has been published in final form
at http://www.mrw.interscience.wiley.com/emrw/9780471140863/cp/cpps/toc
Original authors: Caroline S. Jackson and Anthony I. Magee
National Institute for Medical Research
London, United Kingdom
List of current authors and affiliations:
Ruth Zeidman, Caroline S. Jackson and Anthony I. Magee
Molecular Medicine
National Heart & Lung Institute
Imperial College London
London SW7 2AZ
UK
Author for correspondence, with full mailing address, tel, fax, email:
Anthony I. Magee
Molecular Medicine
National Heart & Lung Institute
Imperial College London
Sir Alexander Fleming Building
South Kensington campus
London SW7 2AZ
1
UK
Tel. +44 (0)20 7594 3135
FAX +44 (0)20 7594 3015
2 figures
0 tables
0 multi-line equations
3-7 key terms for indexing:
Acylation
Palmitoylation
Myristoylation
Fatty acids
Protein modification
Abstract of up to 150 words:
Proteins can be acylated with variety of fatty acids attached by different covalent bonds,
influencing among other things their function and intracellular localization. This unit describes
methods to analyse protein acylation, both the levels of acylation and also the identification of the
fatty acid and the type bond present in the protein of interest. Protocols are provided for metabolic
labelling of proteins with tritiated fatty acids, for the utilization of the differential sensitivity to
cleavage of different types of bonds in order to distinguish between them, and for the separation
of the fatty acids associated with proteins by thin layer chromatography enabling their
identification.
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INTRODUCTION
Protein acylation is the covalent attachment of fatty acids to a protein; the most commonly added
fatty acids are myristate (14:0) and palmitate (16:0). Incorporation of radiolabeled fatty acids into
the protein of interest is still the “gold standard” for analysis of this modification. First,
radiolabeled fatty acids are used to label eukaryotic cells in vitro (see Basic Protocol 1). The
radiolabeled material produced can then be analyzed by various methods: the type of fatty acid
linkage can be determined (see Basic Protocol 2), the nature of the protein-bound label can be
determined to check for interconversion (see Basic Protocol 3), and the protein-bound fatty acid
can be identified (see Basic Protocol 4).
BASIC PROTOCOL 1: BIOSYNTHETIC LABELING WITH FATTY ACIDS
To identify proteins that are modified with fatty acid groups, cultured cells are incubated first in
medium containing sodium pyruvate, which acts as a source of acetyl-CoA and minimizes
interconversion of the fatty acid to other metabolites, and then with [3H]fatty acids. Fatty acids
tritiated at positions 9 and 10 provide the best combination of high specific activity and
detectability for in vitro labeling, and because the tritium label is distant from the carboxyl end
where -oxidation occurs, reincorporation of label is minimized.
Materials
Cells for culture
Complete tissue culture medium appropriate for cells
Labeling medium: complete tissue culture medium containing the relevant dialyzed serum and 1
mM sodium pyruvate, 37C
5 to 10 µCi/µl [9,10(n)-3H]fatty acid, e.g., [9,10(n)-3H]palmitic acid or [9,10(n)-3H]myristic acid (30
to 60 Ci/mmol; Amersham GE Healthcare, American Radiolabeled Chemicals, or NEN
PerkinElmer) in ethanol
PBS, pH 7.2 (APPENDIX 2E), ice-cold
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1% (w/v) SDS or SDS sample buffer (for SDS-PAGE, when using adherent or nonadherent cells
respectively; UNIT 10.1) or RIPA lysis buffer (for immunoprecipitation; UNIT 13.2)
5 SDS sample buffer (see recipe)
Cell scrapers
Nitrogen gas
Additional reagents and equipment for immunoprecipitation (UNIT 13.2), SDS-PAGE (UNIT
10.1), treating a gel with sodium salicylate (UNIT 14.3) or DMSO/PPO solution (UNIT 10.2), and
fluorography (UNIT 10.2)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper
sterile technique should be used accordingly.
NOTE: All culture incubations are performed in a humidified 37C, 5% CO2 incubator unless
otherwise specified.
1. On the day before the labeling experiment, split the cells into fresh complete tissue culture
medium.
Set up the cells at two split ratios; then choose the culture closest to 70% to 80% confluency for
labeling.
2. The next day, replace the medium with a minimum volume of 37C labeling medium. Incubate
1 hr.
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Cells in suspension should be used at a cell density of 106 to 107 cells/ml. For adherent cells that
are 70% to 80% confluent, the minimum amount of medium necessary to cover the dish e.g.,
1.5 ml for 60-mm dishes and 3 ml for 100-mm dishesshould be used.
3. Add 2 to 10 µCi/µl [9,10(n)-3H]fatty acid to a concentration of 50 to 500 µCi/ml. Incubate up to
24 hr.
Cells vary in the rate and extent of incorporation (see Critical Parameters), so both the amount of
label and the duration of incubation need to be optimized. Labeling cells overnight in the
presence of 200 µCi/ml [3H]fatty acid will maximize the chances of detecting labeled proteins.
The amount of label and/or time of incubation can then be reduced if good incorporation of label
is achieved, or increased if poor incorporation is attained.
Short labeling times (e.g., pulses on the order of minutes up to 2 hr) require amounts of label at
the higher end of the indicated range. In this case, uptake is relatively low and the medium plus
label can be reused one or more times. The level of label in the medium can be monitored by
scintillation counting. For longer incubations the interconversion of fatty acids becomes a greater
problem, and the protein-bound fatty acid label should be analyzed (see Basic Protocols 3 and
4).
If the [3H]fatty acid is not supplied in ethanol or if the concentration is too low, remove the solvent
by blowing nitrogen over the solution in its original container until dry. Be careful to remove all
traces of potentially toxic solvent e.g. toluene. Dissolve the label in ethanol at a concentration of
2 to 10 µCi/µl. Do not transfer into another container or evaporate the solvent in a plastic
container, as this will cause a significant loss of label that will adhere to the side of the container.
For adherent cells
5
4a. Place the dish on ice and aspirate the medium. Wash the cells twice with ice-cold PBS and
lyse the cells by adding 1% SDS for SDS-PAGE (UNIT 10.1) or RIPA lysis buffer for
immunoprecipitation (UNIT 13.2), using 100 µl of 1% SDS for a 60-mm dish or 300 µl for a 100mm dish, or 1 ml RIPA lysis buffer.
CAUTION: Radioactive medium and washes must be disposed of appropriately.
5a. Using a cell scraper, remove the lysed cells from the dish and transfer them to a 1.5-ml
microcentrifuge tube. Add 20 µl lysate to 5 µl of 5 SDS-PAGE sample buffer. Use all of RIPA
lysate for immunoprecipitation. Resuspend immunoprecipitate in 20 µl SDS sample buffer.
For SDS-PAGE, use DTT at a concentration 20 mM, and do not boil the samples, but incubate
them only 3 min at 80C. This is necessary because the thioester linkage of the fatty acid is
susceptible to cleavage by nucleophiles. In this respect DTT is a safer option, but -mercaptoethanol can be used with caution.
For nonadherent cells
4b. Microcentrifuge the cell suspension 1 min at 6000 rpm, 4C, to pellet the cells. Decant the
supernatant and wash the cell pellet once by resuspending it in 1 ml ice-cold PBS and
centrifuging again.
5b. Lyse the cells by resuspending the cell pellet in 100 µl SDS-PAGE sample buffer for
discontinuous SDS-PAGE (UNIT 10.1) or 1 ml RIPA lysis buffer for immunoprecipitation (UNIT
13.2) for 106 to 107 cells. Resuspend immunoprecipitate in 20 µl SDS sample buffer.
CAUTION: Radioactive medium and washes must be disposed of appropriately.
For analysis of total protein-bound fatty acid label, lyse the cells in 100 µl 1% SDS.
6
For SDS-PAGE, use DTT at a concentration 20 mM, and do not boil the samples, but incubate
them only 3 min at 80C. This is necessary because the thioester linkage of the fatty acid is
susceptible to cleavage by nucleophiles. In this respect DTT is a safer option, but -mercaptoethanol can be used with caution.
6. Analyze whole-cell lysate or immunoprecipitate on an SDS-PAGE minigel, using 20 µl lysate
per lane. Store remaining lysate at 20C.
7. Treat the gel with sodium salicylate (UNIT 14.3) or DMSO/PPO solution (UNIT 10.2). Using
preflashed film, fluorograph the gel (UNIT 10.2) at 80C.
Typical exposure times are overnight to 1 month. Usually, a one week test exposure would be
done and subsequent exposure times are adjusted depending on the result.
BASIC PROTOCOL 2: ANALYSIS OF FATTY ACID LINKAGE TO PROTEIN
To determine the type of linkage by which the [3H]fatty acid is attached to the protein (i.e.,
thioester, oxyester, or amide linkage), the fatty acid is selectively cleaved from the protein. The
most convenient method is to run replicate lanes on an SDS-PAGE gel, cut the lanes apart, and
analyze each lane separately.
Materials
Lysate or immunoprecipitate from [3H]fatty acid-labeled cells (see Basic Protocol 1, step 6)
0.2 M potassium hydroxide (KOH) in methanol
Methanol
1 M hydroxylamineHCl, titrated to pH 7.5 with NaOH
1 M TrisCl, pH 7.5 (APPENDIX 2E)
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Additional reagents and equipment for SDS-PAGE (UNIT 10.1), treating a gel with sodium
salicylate (UNIT 14.3) or DMSO/PPO solution (UNIT 10.2), and fluorography (UNIT 10.2)
1. Run an SDS-PAGE gel (UNIT 10.1) using 20 µl lysate or immunoprecipitate from [3H]fatty
acid-labeled cells in each of four lanes.
2. Cut the four lanes apart and transfer each lane to a 15-ml tube containing one of the following
solutions:
0.2 M KOH in methanol
Methanol
1 M hydroxylamineHCl
1 M TrisCl, pH 7.5.
Incubate 1 hr at room temperature with shaking.
The 0.2 M KOH in methanol will cleave thio- and oxyesters, but not amides; 1 M
hydroxylamineHCl will rapidly cleave thioesters but will cleave oxyesters only poorly, and will not
cleave amides. Methanol and 1 M TrisCl serve as controls.
3. Wash each gel strip three times, 5 min each time, with water. Treat the strips with sodium
salicylate (UNIT 14.3) or DMSO/PPO solution (UNIT 10.2), and fluorograph using preflashed film
at 80C.
Typical exposure times are overnight to 1 month. Usually, a one week test exposure would be
done and subsequent exposure times are adjusted depending on the result. Cleavage is
measured as a reduction in the fluorographic signal compared to those for controls, and can be
quantitated by densitometric scanning of the lane or scintillation counting of excised bands.
8
Bands with fatty acids linked to the protein by thioesters will be missing or greatly reduced in
lanes treated with 0.2 M KOH in methanol and 1 M hydroxylamineHCl; oxyesters will be greatly
reduced or missing in the lane treated with 0.2 M KOH in methanol and may be slightly reduced
in the lane treated with 1 M hydoxylamineHCl; and amide linkages will not be affected by any of
these treatments, so that proteins with amide-linked fatty acids will appear in all four lanes.
BASIC PROTOCOL 3: ANALYSIS OF TOTAL PROTEIN-BOUND FATTY ACID LABEL IN
CELL EXTRACT
Due to problems of interconversion of fatty acids by -oxidation and chain elongation and of
reincorporation of label into other metabolic precursors, the protein-bound label derived from
[3H]fatty acids should ideally be analyzed, especially for experiments with long labeling
incubations. This protocol is used to determine how much of the label has been converted into
other fatty acids or metabolites during the incubation; a different procedure must be used to
determine whether the fatty acid on the protein of interest is different from that added during
labeling (see Basic Protocol 4).
Materials
0.1 M HCl/acetone, 20C
Lysate from [3H]fatty acid-labeled cells in 1% SDS (see Basic Protocol 1, step 4a or 5b)
1% (w/v) SDS
2:1 (v/v) chloroform/methanol
Diethyl ether
6 M HCl (concentrated HCl diluted 1:1 with H2O)
Hexane
5 to 10 µCi/µl [9,10(n)-3H]fatty acid standards (30 to 60 Ci/mmol; Amersham GE Healthcare,
American Radiolabeled Chemicals, or NEN PerkinElmer) in ethanol
90:10 (v/v) acetonitrile/acetic acid
EN3HANCE spray (PerkinElmer)
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15-ml polypropylene centrifuge tubes
Mistral 3000i benchtop centrifuge with swing-out four-bucket rotor or equivalent
Nitrogen gas
30-ml thick-walled Teflon container with an air-tight screw top
110C oven
Thin-layer chromatography tank
RP18 thin-layer chromatography plate (e.g., Merck)
Kodak BioMax MS film, preflashed
Precipitate protein
1. Add 5 vol of 0.1 M HCl/acetone to 100 µl lysate from [3H]fatty acid-labeled cells in 1% SDS in
a 15-ml polypropylene tube. Incubate 1 hr at 20C.
This will precipitate the protein.
2. Centrifuge 10 min at 1500  g (1000 rpm in Mistral 3000i swing-out rotor), 4C, to pellet the
precipitate. Remove the supernatant and allow the pellet to air dry gently.
Remove free label
3. Dissolve the pellet in a minimum volume of 1% SDS and transfer to a 1.5-ml microcentrifuge
tube. Add 5 vol of 0.1 M HCl/acetone. Incubate 1 hr at 20C.
4. Repeat steps 2 and 3.
These precipitation steps concentrate the protein and remove much of the SDS and free label.
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5. Add 500 µl of 2:1 chloroform/methanol and vortex. Centrifuge 10 min at 1000 rpm, 4C, and
remove the supernatant. Repeat this step at least three times until no more free label is extracted
into the organic solvent, as determined by scintillation counting of the supernatant.
6. Add 100 µl diethyl ether to the pellet and vortex. Centrifuge 10 min at 1000 rpm, 4C, and
decant the supernatant. Dry the pellet by placing the microcentrifuge tube under a gentle stream
of nitrogen.
7. Place the tube into a 30-ml thick-walled Teflon container with a air-tight screw top containing 1
ml of 6 M HCl. Flush the tube and container with nitrogen. Close the lid tightly and incubate in an
oven 16 hr at 110C.
This hydrolyzes the fatty acids from the protein.
Extract hydrolyzed fatty acids
8. Extract the contents of the tube twice with 0.5 ml hexane and pool the extracts. Dissolve the
residue in 0.5 ml of 1% SDS. Determine the radioactivity in the hexane extracts and in the
residue.
Fatty acids will be extracted into hexane, while label incorporated into sugars and amino acids
will be mainly in the hexane-insoluble residue.
9. Evaporate the hexane extracts just to dryness with a gentle stream of nitrogen. Dissolve in 2 to
5 µl of 2:1 chloroform/methanol.
It is important not to overdry the sample because it may then be difficult to dissolve.
Identify fatty acids
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10. Preequilibrate a thin-layer chromatography tank with 90:10 acetonitrile/acetic acid for 15 min.
11. Spot resuspended hexane extract onto an RP18 thin-layer chromatography plate. Dilute 1 µl
[9,10(n)-3H]fatty acid standards in ethanol to give 1 µCi/µl and spot 0.5 µl in parallel lanes.
Develop the plate in 90:10 acetonitrile/acetic acid. Air dry the plate.
12. Detect the radioactivity by spraying the plate with En3hance spray and exposing it to
preflashed Kodak BioMax MS film overnight or longer at 80C. Identify the fatty acids.
See Figure 14.2.1 for an example of a typical fluorogram.
BASIC PROTOCOL 4: ANALYSIS OF FATTY ACID LABEL IDENTITY
This protocol is used to identify the labeled fatty acid(s) associated with a specific protein band
on an SDS-PAGE gel. Following electrophoresis, the band of interest is located either by
comparison with molecular weight standards or by fluorography of a sodium salicylate-treated gel
(see UNIT 14.3). DMSO/PPO-treated gels cannot be used. The labeled material is analyzed by
thin-layer chromatography.
Materials
SDS-PAGE gel of lysate from [3H]fatty acid-labeled cells
Additional reagents and equipment for analysis of protein-bound label (see Basic Protocol 3)
1. Excise the band(s) of interest from a wet or dried (fluorographed) SDS-PAGE gel. Wash three
times with shaking, 5 min each, with 0.5 ml water.
If the gel is fluorographed it should be treated with sodium salicylate (UNIT 14.3), not
DMSO/PPO solution. The dried gel piece will rehydrate and the salicylate will be washed out
during the washes.
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2. Place the gel piece in a 1.5-ml microcentrifuge tube and lyophilize.
3. Hydrolyze the fatty acids in the band and identify them by thin-layer chromatography (see
Basic Protocol 3, steps 7 to 12).
REAGENTS AND SOLUTIONS
Note
Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock
solutions, see APPENDIX 2E; for suppliers, see SUPPLIERS APPENDIX.
SDS sample buffer (for discontinuous systems), 5
3.125 ml 1 M TrisCl, pH 6.8 (0.313 M final)
1 g SDS (10% final)
5 mg bromphenol blue (0.05% final)
5 ml glycerol (50% final)
H2O to 10 ml
Store at room temperature
Add DTT to appropriate concentration just before use
Warm the solution before use because it tends to solidify.
COMMENTARY
Background Information
The two most common acyl groups that modify proteins are 14-and 16-carbon saturated fatty
acids, myristic and palmitic acid, respectively (Fig. 14.2.2), and they occur both on different and
on overlapping sets of proteins. By increasing the hydrophobicity of the protein, these fatty acid
moieties can play a role in localization of the protein to the membrane and sometimes to specific
13
types of membrane structurese.g. cholesterol- and sphingolipid-rich lipid rafts (Zacharias et al.,
2002). Identifying the type of acylation of a protein and determining whether the level of
modification can be affected by stimuli can provide more information on the mechanisms of
action of proteins involved in signaling pathways.
Fatty acids are used in labeling cells in vitro because they will diffuse across the plasma
membrane and then be converted to acyl-CoA by the action of the enzyme acyl-CoA synthetase.
This activated form of the fatty acid is the substrate for protein-acyl transferases (PATs) that
transfer the acyl group to the protein. Tritiated fatty acids are most commonly used in biosynthetic
labeling of proteins, but fluorescent analogs of fatty acids have been used to study palmitoylation
of rhodopsin (Moench et al., 1994a,b), and [-125I]iodo-fatty acids have been used to study
myristoylation of v-src (Peseckis et al., 1993) and palmitoylation of Sonic hedgehog (Buglino and
Resh, 2008). ω-azido-fatty acids can also be used to metabolically label cells, followed by
labeling with phosphine-biotin and detection in a Western blot with streptavidin-HRP (Hang et al.,
2007). Acylation can also be detected by chemically labeling the fatty acids in lysed cells using
the acyl-biotin exchange (ABE) method. After blocking free sulfhydryl groups palmitoyl-thioester
bonds are cleaved, generating a free sulfhydryl group that is labeled with a sulfhydryl-specific
biotin-conjugated compound (1-biotinamido-4-[4’(maleimidomethyl)cyclohexanecarboxamido]butane ;Btn–BMCC), which subsequently can be
detected by streptavidin-HRP (Drisdel and Green, 2004, Drisdel at al., 2006, Wan et al., 2007).
With methods that rely on a chemical reaction for detection of acylation there is a concern that the
level of acylation might be underestimated due to inefficiency of the chemical reaction.
Furthermore, in the ABE method, palmitoylation may be below the detection threshold if the level
of palmitoylation is low or the turnover rate high, as only steady state levels are measured. The
ABE method can also give false positives, e.g. with other types of this ester. The reader is refered
to the original publications for these methods.Therefore, metabolic labeling with tritiated fatty
acids is still the most reliable and commonly used acylation detection method.
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A wide variety of proteins are myristoylated, including viral structural proteins and many proteins
involved in cell signaling, such as the  subunits of trimeric G proteins, cytoskeletal-bound
anchoring proteins, and the Src family of tyrosine kinases (Resh, 1999). Myristoylation, usually an
irreversible modification, most commonly occurs co-translationally via an amide linkage to an
NH2-terminal glycine residue but it has also been reported to occur post-translationally for several
proteins, including PAK2 (Vilas et al., 2006), gelsolin (Sakurai and Utsumi, 2006), actin (Utsumi et
al., 2003) and BID (Zha et al., 2000) following caspase cleavage. Myristoylation is dependent on
the removal of the initiator methionine and has been shown to occur by the time the nascent
polypeptide is 100 amino acids long. Inhibitors of protein synthesis will therefore block
myristoylation. The enzyme responsible for NH2-terminal myristoylation, N-myristoyl transferase
(NMT), was first isolated from Saccharomyces cerevisiae; both the yeast and human homologs
have been well characterized and show different protein substrate specificities . Substrate
specificity of yeast NMT is determined by recognition of a sterically unhindered NH 2-terminal
glycine followed by an amino acid sequence that conforms to the following criteria: no charged
residue or proline at position 2; any amino acid at positions 3 and 4; serine, alanine, glycine,
cysteine, asparagine or threonine at position 5; and no proline at position 6 (Farazi et al. 2001).
Residues C-terminal to this region appear to be important in recognition of the  subunits of
trimeric G proteins and the Src family of tyrosine kinases (Glover et al., 1988; Gordon et al.,
1991).
The NMT enzymatic reaction proceeds by formation of a myristoyl-CoA-enzyme complex,
subsequent binding of the peptide, transfer of the myristate moiety to the peptide, release of CoA,
and release of the myristoylated peptide (Rudnick et al., 1991). Several assays have been
developed for this enzyme (e.g., King and Sharma, 1991; Rudnick et al., 1992; French et al.,
1994, Pennise et al. 2002; Takamune et al., 2002). A large number of NMT inhibitors have been
identified (reviewed in Selvakumar et al., 2007), including proteins, histidine analogs, myristic acid
analogs and myristoyl-CoA variants which inhibit acyl CoA synthetase and therefore block the
15
conversion of fatty acids to acyl CoA; including 2-hydroxymyristic acid that is converted to 2hydroxymyristoyl-CoA, a potent inhibitor of NMT, and other synthetic organic compounds.
Myristoylation of a protein can be necessary for its activity, e.g., the transforming activity of Src
(Kamps et al., 1986). Myristoylation alone may not be sufficient for a protein to be localized to the
membrane; for this further lipid modification, such as palmitoylation, prenylation or cooperative
interaction with protein sequences, is required (Resh, 2006a).
Palmitoylation (with and without myristoylation) occurs on many signaling molecules, including
rhodopsin, -subunits of G proteins, Ras, G-protein coupled receptors and Src-family tyrosine
kinases. Palmitoylation is a post-translational event occurring via a thioester linkage to a cysteine
residue. Sometimes, the thioester bond can be chemically rearranged to form a stable attachment
of the palmitate through an amide bond to an immediately adjacent N-terminal glycine instead.
This is seen in, for instance, Hh/Shh (Pepinsky et al., 1998) and Gs (Kleuss and Krause, 2003).
Where it occurs with NH2-terminal myristoylation, the myristoylation is usually a prerequisite for
palmitoylation. This may be because the enzyme responsible for palmitoylation recognizes the
myristoylated protein or, more likely, because myristoylation brings the protein to the correct cell
location for palmitoylation to occur. Palmitoylation is also found in conjunction with prenyl
modification at the C-terminus of proteins belonging to the Ras superfamily; it is responsible for
the localization of these proteins to the membrane (Newman and Magee, 1993). This
palmitoylation is dependent on prior modification of the protein by prenylation. G protein subunits
such as the 1 subfamily and members of the Src family of tyrosine kinases have an NH 2terminal amino acid sequence of Met-Gly-Cys, where the initiator methionine is removed and
replaced by myristate and the cysteine is palmitoylated (Resh, 1994). Palmitoylation is a
reversible modification and has been shown to be dynamic in vivo, with the level of palmitoylation
changing in response to various stimuli such as receptor activation, insulin, and growth factors
(James and Olsen, 1989; Jochen et al., 1991; Wedegaertner et al., 1995). This phenomenon is
thought to play a role in switching on or off signaling molecules by altering either the localization
16
of the molecules or their presentation to other signaling molecules with which they interact.
Proteins can be auto-palmitoylated; alternatively the process can be catalyzed by protein-acyl
transferases (PATs) and there has been progress in the identification and characterization of
PATs in recent years. The DHHC family of PATs are transmembrane proteins, all having
cysteine-rich domains containing a conserved aspartate-histidine-histidine-cysteine (DHHC)
motif, which is required for the PAT activity. Originally discovered in yeast (Lobo et al., 2002, Roth
et al., 2002), there are now 23 mammalian DHHC proteins known (Fukata et al. 2004) and work
is emerging describing their function and substrates, which are intracellular proteins, for example
Ras, G protein subunits, eNOS, vacuole proteins, G protein-coupled receptors and the neuronal
PSD-95 protein (Smotrys and Linder, 2004).
Some secreted proteins and peptides, like Hedgehog (Hh)/Sonic hedgehog (Shh), Wnts and
ghrelin, require acylation for their function and are palmitoylated by a different group of PATs
called MBOAT (membrane-bound-O-acyltransferase) family, which do not have any apparent
sequence homology and where only a small subset of members are known to transfer fatty acids
and other lipids to proteins (Miura and Treisman, 2006). Rasp/Skinny hedgehog (Ski)/Hedgehog
acyl transferase (Hhat) is the PAT for Hh/Shh (Buglino and Resh, 2008), Porcupine (Porc) is the
PAT for Wnt (Zhai et al., 2004) and GOAT is the PAT for ghrelin (Yang et al, 2008).
Proteins are depalmitoylated by acyl protein thioesterases (TEs). So far, two have been
described, APT1, which has been shown to depalmitoylate Gi, H-Ras and eNOS (Duncan and
Gilman, 1998, Duncan and Gilman, 2002 and Yeh et al., 1999), and PPT1, which removes
palmitate as a step in the protein degradation process (Verkruyse and Hofmann, 1996).
Inhibitors of palmitoylation include fatty acid analogues, including 2-bromopalmitate, a nonmetabolizable palmitate analogue, and natural antibiotics, like cerulenin, which inhibits fatty acid
synthesis, and tunicamycin, which is structurally similar to palmitoyl-CoA (Resh, 2006b). More
specific small molecule inhibitors of individual PAT subgroups are also emerging (Ducker et al.,
2006).
17
Analysis of the type of fatty acid linkage present should always be performed. Post-translational
myristoylation via a thioester linkage has been found in platelets (Muszbek and Laposata, 1993).
The term "palmitoylation" is not strictly accurate because other long-chain fatty acids, such as
stearate (18:0) and oleate (18:1), can also be thioesterified to proteins; "S-acylation" or
“thioacylation” are becoming more commonly used to describe this modification. The acylating
activities seem to be relatively unspecific for chain length and degree of unsaturation, and utilize
acyl-CoAs partly in proportion to their abundance in the cell hence the predominance of
palmitate. This and the potential for interconversion of labeled fatty acids necessitate analysis of
the chain length of the attached label. Other more complicated methods can be used to identify
the type of fatty acid attached to the protein; these include gas chromatography, reversed-phase
HPLC, and mass spectroscopy (Aitken, 1992). Mass spectrometry can also be used for
identifying acyl chains attached to proteins, by comparison of acylated and deacylated peptides
from the digested protein of interest, giving the stoichiometry of the acylation and the exact mass
of the modifying group. (Liang et al., 2004)
Further information about the cellular localization of acylated proteins can be found by detergent
extraction of the cell lysate. Extraction with Triton X-114 (which has the property of phase
separation at 30C) can distinguish between hydrophobic and hydrophilic proteins (Aitken, 1992).
Extraction of membranes with non-ionic detergents at 4C and 37C can identify proteins that are
associated with detergent resistant membranes (DRMs) and provide preliminary evidence for
association with lipid rafts (LR) or membrane lipid microdomains (Janes et al, 1999). Full
characterization of lipid raft/membrane lipid microdomain association requires several
complementary techniques and is beyond the scope of this review.
Critical Parameters and Troubleshooting
Cells should be subconfluent; it is recommended that the cells be plated at two split ratios so the
culture closest to 70% to 80% confluency can be selected to be used for labeling.
18
The pH of all solutions should be 7.5 in order to avoid hydrolysis of labile thioesters; the high pH
of SDS-PAGE buffers does not seem to be a problem when using minigels, where the running
times are relatively short. Dithiothreitol (DTT) or 2-mercaptoethanol should be used with care, as
these will also cleave thioester bonds; for SDS-PAGE a maximum concentration of 20 mM DTT
should be used and samples should not be boiled but incubated only 3 min at 80C.
The use of short labeling times (especially for palmitoylation) will reduce reincorporation of the
label.
The ability to detect myristoylation and/or palmitoylation of a protein using these methods will
depend on the ability of the cells to take up radiolabeled compounds and incorporate them into
metabolic precursors; the pool sizes of endogenous fatty acids and fatty acyl-CoA esters; the
expression level and activities of the NMT, PATs and thioesterases; the abundance, rate of
synthesis, and turnover of the protein(s) and modification(s) of interest; and the efficiency of
antibodies for immunoprecipitation.
Anticipated Results
Typically, it is possible to detect myristoylated proteins using fluorographic exposure times
ranging from 1 week for high-level expression of protein in transformed cells (e.g., Lck in
LSTRA cells) to 1 to 4 weeks for a well-expressed endogenous protein. For a poorly expressed
protein, exposure times of 1 to 3 months have been used.
Time Considerations
In vitro labeling experiments require 2 to 3 days for growing and labeling the cells. Harvesting the
cells, preparing the lysate for SDS-PAGE (with or without prior immunoprecipitation) and SDSPAGE require 1 to 2 days plus time for fluorography.
19
Linkage analysis of the labeled proteins takes 1 to 2 hr after the gel has been run. Analysis of the
label in cell lysates requires 1 day to precipitate the protein, remove free label, and hydrolyze the
sample. A second day is required to extract the label and perform thin-layer chromatography, and
fluorography requires an overnight exposure. Analysis of the protein-bound label in gel bands
takes ~3 days after fluorography (dried gel) and a similar amount of time from a wet gel, except
the analysis begins after the gel has been run.
Literature Cited
Aitken, A. 1992. Structure determination of acylated proteins. In Lipid Modification of Proteins, A
Practical Approach (N.M. Hooper and A.J. Turner, eds.) pp. 63-88. Oxford University Press,
Oxford.
Buglino, J.A. and Resh, M.D. 2008. Hhat is a palmitoylacyl transferase with specificity for Npalmitoylation of sonic hedgehog. J. Biol. Chem. 283:22076-22088.
Drisdel, R.C. and Green, W.N. 2004. Labeling and quantifying sites of protein palmitoylation.
Biotechniques. 36:276–285
Drisdel R.C., Alexander, J.K., Sayeed, A., and Green, W.N. 2006. Assays of protein
palmitoylation. Methods. 40:127-134.
Ducker, C.E., Griffel, L.K., Smith, R.A., Keller, S.N,, Zhuang, Y., Xia, Z., Diller, J.D., and Smith,
C.D. 2006. Discovery and characterization of inhibitors of human palmitoyl acyltransferases. Mol.
Cancer Ther. 5:1647-1659
Duncan, J.A. and Gilman, A.G. 1998. A cytoplasmic thioesterase that removes palmitate from G
protein  subunits and p21 Ras. J. Biol. Chem. 273:15830-15837.
20
Duncan, J.A. and Gilman, A.G. 2002. Characterization of Saccaromyces cervisiae acyl-protein
thioesterase 1, the enzyme responsible for G protein  subunit deacylation in vivo. J. Biol. Chem.
277:31740-31752.
Farazi, T.A., Waksman, G., and Gordon, J.I. 2001. The biology and enzymology of protein Nmyristoylation. J. Biol. Chem. 276:39501-39504.
French, S.A., Christakis, H., O'Neill, R.R., and Miller, S.P.F. 1994. An assay for myristoylCoA:protein N-myristoyltransferase activity based on ion-exchange exclusion of [3H]myristoyl
peptide. Anal. Biochem. 220:115-121.
Fukata, M., Fukata, Y., Adesnik, H., Nicoll, R.A., and Bredt, D.S. Identification of PSD-95
palmitoylating enzymes. 2004. Neuron. 44:987-996.
Glover, C.J., Goddard, C., and Felsted, R.L. 1988. N-Myristoylation of p60src. Biochem. J.
250:485-491.
Gordon, J.I., Duronio, R.J., Rudnick, D.A., Adams, S.P., and Gokel, G.W. 1991. Protein Nmyristoylation. J. Biol. Chem. 266:8647-8650.
Hang, H.C., Geutjes, E.J., Grotenbreg, G., Pollington, A.M., Bijlmakers, M.J., and Ploegh, H.L.
2007. Chemical probes for the rapid detection of fatty-acylated proteins in mammalian cells. J.
Am. Chem. Soc. 129:2744-2475.
James, G. and Olson, E.N. 1989. Identification of a novel fatty acylated protein that partitions
between the plasma membrane and cytosol and is deacylated in response to serum and growth
factor stimulation. J. Biol. Chem. 264:20988-21006.
21
Janes, P.W., Ley, S.C., and Magee A.I. 1999. Aggregation of lipid rafts accompanies signaling via
the T cell antigen receptor. J. Cell Biol. 147:447-461.
Jochen, A., Hays, J., Lianos, E., and Hager, S. 1991. Insulin stimulates fatty acid acylation of
adipocyte proteins. Biochem. Biophys. Res. Commun. 177:797-801.
Kamps, M.P., Buss, J.E., and Sefton, B.M. 1986. Rous sarcoma virus transforming protein
lacking myristic acid phosphorylates known polypeptide substrates without inducing
transformation. Cell 45:105-112.
King, M.J. and Sharma, R.K. 1991. N-Myristoyl transferase assay using phosphocellulose paper
binding. Anal. Biochem. 199:149-153.
Kleuss, C. and Krause, E. 2003. Gs is palmitoylated at the N-terminal glycine. EMBO J. 22:826–
832.
Liang, X., Lu, Y., Wilkes, M., Neubert, T.A., and Resh, M.D. 2004. The N-terminal SH4 region of
the Src family kinase Fyn is modified by methylation and heterogeneous fatty acylation: role in
membrane targeting, cell adhesion, and spreading. J. Biol. Chem. 279:8133-8139.
Lobo S., Greentree W.K., Linder M.E., and Deschenes R.J. 2002. Identification of a Ras
palmitoyltransferase in Saccharomyces cerevisiae. J. Biol. Chem. 277:41268-41273.
Miura, G.I. and Treisman, J.E. 2006. Lipid modifications of secreted proteins. Cell Cycle. 5:11841189.
Moench, S.J., Terry, C.E., and Dewey, T.G. 1994a. Fluorescence labeling of the palmitoylation
sites of rhodopsin. Biochemistry 33:5783-5790.
22
Moench, S.J., Moreland, J., Stewart, D.H., and Dewey, T.G. 1994b. Fluorescence studies of the
location and membrane accessibility of the palmitoylation sites of rhodopsin. Biochemistry
33:5791-5796.
Muszbek, L. and Laposata, M. 1993. Myristoylation of proteins in platelets occurs predominantly
through thioester linkages. J. Biol. Chem. 268:8251-8255.
Newman, C.M.H. and Magee, A.I. 1993. Post-translational processing of the ras superfamily of
small GTP-binding proteins. Biochim. Biophys. Acta 1155:79-96.
Pennise, C.R., Georgopapadakou, N.H., Collins, R.D., Graciani, N.R., and Pompliano, D.L. 2002.
A continuous fluorometric assay of myristoyl-coenzyme A:protein N-myristoyltransferase. Anal.
Biochem. 300:275–277.
Pepinsky, R.B., Zeng, C., Wen, D., Rayhorn, P., Baker, D.P., Williams, K.P., Bixler, S.A.,
Ambrose, C.M., Garber, E.A., Miatkowski, K., Taylor, F.R., Wang, E.A., and Galdes, A. 1998.
Identification of a palmitic acid-modified form of human Sonic hedgehog. J. Biol. Chem.
273:14037-14045.
Peseckis, S.M., Deichaite, I., and Resh, M.D. 1993. Iodinated fatty acids as probes for myristate
processing and function. J. Biol. Chem. 268:5107-5114.
Resh, M.D. 1994. Myristoylation and palmitoylation of Src family members: The fats of the matter.
Cell 76:411-413.
Resh, M.D. 1999. Fatty acylation of proteins: new insights into membrane targeting of
myristoylated and palmitoylated proteins. Biochimica et Biophysica Acta 1451:1-16
Resh, M.D. 2006a.Trafficking and signalling by fatty-acylated and prenylated proteins. Nature
Chem. Biol. 2:584-590.
23
Resh, M.D. 2006b. Use of analogs and inhibitors to study the functional significance of protein
palmitoylation . Methods. 40:191-197.
Roth, A., Feng, Y., Chen, L., and Davis, N. 2002. The yeast DHHC protein Ark1p is a palmitoyl
transferase. J. Cell. Biol.159: 23-28.
Rudnick, D.A., McWherter, C.A., Rocque, W.J., Lennon, P.J., Getman, D.P., and Gordon, J.I.
1991. Kinetic and structural evidence for a sequential ordered bi bi mechanism of catalysis by
Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase. J. Biol. Chem.
266:9732-9739.
Rudnick, D.A., Duronio, R.J., and Gordon, J.I. 1992. Methods for studying myristoyl-CoA:protein
N-myristoyltransferase. In Lipid Modification of Proteins, A Practical Approach (N.M. Hooper and
A.J. Turner, eds.) pp. 37-61. Oxford University Press, Oxford.
Sakurai, N. and Utsumi, T. 2006. Posttranslational N-myristoylation is required for the antiapoptotic activity of human tGelsolin, the C-terminal caspase cleavage product of human gelsolin.
J. Biol. Chem. 281:14288-14295.
Selvakumar, P., Lakshmikuttyamma, A., Shrivastav, A., Das, S. B., Dimmock, J. R., and Sharma,
R. K. 2007. Potential role of N-myristoyltransferase in cancer. Progress in Lipid Res. 46:1-36.
Smotrys, J.E., and Linder, M.E. 2004. Palmitoylation of intracellular signalling proteins:
Regulation and function. Annu. Rev. Biochem. 73:559-587.
Takamune, N., Hamada, H., Sugawara, H., Misumi, S., and Shoji, S. 2002. Development of an
enzyme-linked immunosorbent assay for measurement of activity of myristoyl-coenzyme
A:protein N-myristoyltransferase. Anal. Biochem. 309:137–142.
24
Utsumi, T., Sakurai, N., Nakano, K., and Ishisaka, R. 2003. C-terminal 15 kDa fragment of
cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage and
targeted to mitochondria. FEBS Lett. 539:37-44.
Vilas, G.L., Corvi, M.M., Plummer, G.J., Seime, A.M., Lambkin, G.R., and Berthiaume, L.G. 2006.
Posttranslational myristoylation of caspase-activated p21-activated protein kinase 2 (PAK2)
potentiates late apoptotic events. Proc. Natl. Acad. Sci. U. S. A. 103:6542-6547.
Verkruyse, L.A. and Hofmann, S.L. 1996. Lysosomal targeting of palmitoyl-protein thioesterase.
J. Biol. Chem. 271:15831-15836.
Wan, J., Roth, A.F., Bailey, A.O., and Davis, N.G. 2007. Palmitoylated proteins: purification and
identification. Nat. Protoc. 2:1573-1584.
Wedegaertner, P.B., Wilson, P.T., and Bourne, H.B. 1995. Lipid modifications of trimeric G
proteins. J. Biol. Chem. 270:503-506.
Yang, J., Brown, M.S., Liang, G., Grishin, N.V., and Goldstein, J.L. 2008. Identification of the
acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell. 132:387396.
Yeh, D.C., Duncan, J.A., Yamashita, S., and Michel, T. 1999. Depalmitoylation of endothelial
nitric-oxide synthase by acyl-protein thioesterase 1 is potentiated by Ca2+-calmodulin. J. Biol.
Chem. 274: 33148-33154.
Zacharias, D.A., Violin, J. D., Newton, A. C., and Tsien, R. Y. 2002. Partitioning of Lipid-Modified
Monomeric GFPs into Membrane Microdomains of Live Cells. Science. 296:913-916.
25
Zha, J., Weiler, S., Oh, K.J., Wei, M.C., and Korsmeyer, S.J. 2000. Posttranslational Nmyristoylation of BID as a molecular switch for targeting mitochondria and apoptosis. Science.
290:1761-1765.
Zhai, L., Chaturvedi, D., and Cumberledge, S. 2004. Drosophila Wnt-1 undergoes a hydrophobic
modification and is targeted to lipid rafts, a process that requires porcupine. J. Biol. Chem.
279:33220–33227.
Key Reference
Casey, P.J. and Buss, J.E. 1995. Lipid modification of proteins. Methods Enzymol., Vol. 250.
A compilation of methods used in studying lipid modification of proteins.
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Figure 14.2.1 Fluorogram of thin-layer chromatography plate showing analysis of acylated nerve
growth factor (NGF) receptor. Outside lanes, migration of 0.5 µCi [3H]palmitate and [3H]myristate
standards. Lane 1, NGF receptor immunoprecipitated from cells labeled with [ 3H]palmitic acid.
Lane 2, NGF receptor immunoprecipitated from cells labeled with [ 3H]myristic acid. Although the
cells were labeled with different fatty acids, the protein was labeled with palmitic acid due to
chain elongation of [3H]myristic acid to [3H]palmitic acid by the cells. Exposure for standards, 1
week; exposure for lanes 1 and 2, 1 month.
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Figure 14.2.2 Structures of myristic and palmitic acids.
28