ANALYTICALBIOCHEMISTRY 183,220-224 (1989)

Identification of Covalently Bound Fatty Acids on

Acylated Proteins Immobilized on Nitrocellulose Paper’
Franklin E. Callahan,*,” Helen A. Norman,** T. Srinath,t Judith B. St. John,** Ravi Dhar,?
and Autar K. Mattoo*,
*Plant Molecular Biology and **Weed Science Laboratories, USDA-ARS, Beltsuille Agricultural Research Center, West,
Beltsville. Maryland 20705, and fLaboratory of Molecular Virology, National Institutes of Health, NCI.
Bethesda, Ma&land 20892

Received June 12,1989

A common procedure (see for example Ref. (10)) for

A general method for identification of fatty acids co- identifying protein-bound acyl moieties has been to (a)
valently bound to acylated proteins following their tag putative acylated proteins by in vivo labeling with
electrophoretic transfer onto nitrocellulose paper is de- radioactive fatty acids; (b) resolve these proteins on
scribed. As demonstrated for [3H]palmitoylated RASl SDS4-polyacrylamide gels followed by their visualiza-
protein of Saccharomyces cerevisiae and the acylated tion after fluorographic exposures of the dried gels; (c)
acyl carrier protein of Spirodela oligorrhiza, this pro- elute the acylated protein from gel slices; (d) extract the
cedure alleviates the need for elution of proteins from fatty acid from the eluted protein; and (e) identify the
polyacrylamide gel slices. Fatty acid ligands of such fatty acid by TLC or HPLC. Using such a procedure very
proteins are hydrolyzed directly from their immobi- low recoveries are obtained and hydrophobic proteins
lized state on the nitrocellulose paper, then derivatized (e.g., membrane proteins) and even certain soluble poly-
with p-nitrophenacyl bromide, and finally resolved by peptides (e.g., large subunit of ribulose-1,5-bisphosphate
reversed-phase high-performance liquid chromatogra- carboxylase) are not always easily elutable from gel
phy. The amount of acylated protein required for iden-
slices and usually aggregate in solution.
tification of acyl groups is minimized compared to that
In this paper, we describe a general procedure for iden-
required for more conventional approaches by coupling
tifying fatty acids bound to in vivo radiolabeled, acylated
a radioactive flow detector with the HPLC system.
proteins. This method avoids problems associated with
ic 1999 Academic Press, Inc.
elution of proteins from gel slices. Moreover, the use of
a radioactive flow detector coupled to reversed-phase
HPLC allows qualitative analysis of acyl moieties even
Post-translational modification of proteins by cova- under conditions where absolute amounts of the fatty
lent linkage to fatty acids, i.e., protein acylation, has acids are too low for detection by HPLC alone. The gen-
been observed for a variety of proteins of diverse biologi- eral application of this method is demonstrated by iden-
cal origin (l-7). The acyl groups are bound to the protein tification of the fatty acid ligands of the 36-kDa RASl
backbone either indirectly via glycosyl-phosphatidyhno- membrane protein (11) of Saccharomyces cerevisiae and
sitol bridges (8) or directly through amide or ester bonds the lo-kDa acylatedacyl carrier protein (12) of Spirodela
(9). As interest in determining the physiological role(s) oligorrhiza chloroplast stroma.
of protein acylation increases, the need for sensitive yet
simplified methods for identification of the protein-
MATERIALS AND METHODS
bound fatty acids becomes increasingly important.
In vivo radiolabeling procedures. S. cerevisiae strain
’ This work was supported in part by a U.S.A.-Israel BARD grant. S288C (Mat, gal 2) was grown at 30°C in minimal me-
Mention of a trademark or proprietary product does not constitute
a guarantee or warranty of the product by the U.S. Department of
Agriculture and does not imply its approval to the exclusion of other ’ Abbreviations used: ACP, acyl carrier protein; PMSF, phenyl-
products that may also be suitable. methylsulfonyl fluoride; SDS, sodium dodecyl sulfate; PAGE, poly-
’ Present address: USDA-ARS, Crop Science Research Laboratory, acrylamide gel electrophoresis; TLCK, N-tosyl-L-lysine chloromethyl
P.O. Box 5367, Mississippi State, MS 39762-5367. ketone; TPCK, L-1-tosylamide-2-phenyl ethylchloromethyl ketone:
” To whom correspondence should be addressed. Mops, 4-morpholinepropanesulfonic acid; NP-40, Nonidet-P40.

220
0003.2697/89 $3.00
Copyright Q 1989 by Academic Press, Inc.
All rights of reproduction in any form reserved.
 ANALYSIS OF ACYL MOIETIES ON ACYLATED PROTEINS 221

dium [6.7% yeast nitrogen base (Difco) containing am- containing 1.8 X lo7 cpm of [35S]methionine-labeled pro-
monium sulfate] supplemented with 2% dextrose. Sta- teins or 5.2 X lo8 cpm of [3H]palmitate-labeled proteins
tionary phase cells were inoculated into fresh medium to were used. Seventy microliters of Staph A cells (Calbio-
an optical density of 0.3 and then grown to early expo- them) preadsorbed with anti-rat antibodies (Boehring-
nential phase. For radiolabeling with [35S]methionine er-Mannheim) was added to each of the extracts and in-
(Amersham; sp act 1072 Ci/mmol), 40 ml of culture was cubated at 4°C for 2 h with continuous mixing. The
incubated with 20 &i/ml of the radiolabel for 7 min at extracts were then centrifuged at 10,OOOg for 2 min. The
30°C. For labeling with [3H]palmitic acid, 40 ml of cul- supernatants were treated with monoclonal antibodies
ture was gently pelleted at room temperature and resus- (Y13-259) against RAS (provided by A. Papageorge) and
pended in 5 ml of minimal medium containing 2% dex- allowed to react for 4 h at 4°C with constant mixing. An
trose. Cells (107/ml) were then incubated with 1 mCi/ml additional 70 ~1 of Staph A cells precoated with anti-rat
of 9,10-[3H]palmitic acid (New England Nuclear/Du- antibodies was added to each extract and incubation was
Pont; sp act 28.5 Ci/mmol) for 60 min at 30°C. At the continued for another hour with constant mixing at 4°C.
end of labeling, a mixture of protease inhibitors (TLCK, Staph A cells were collected by centrifugation and
TPCK, PMSF) and cycloheximide was added (each to a washed four times with RIPA buffer, once with RIPA
final concentration of 100 pg/ml). Cells were then chilled buffer containing 1 M NaCl, and, finally, two times with
on ice and collected by centrifugation at 6OOOg for 5 min RIPA buffer (14).
at 4°C. The pelleted cells were washed once with phos- SDS-PAGE and electroblotting of radiolabeled pro-
phate-buffered saline containing PMSF (100 pg/ml). teins. RAS protein immunoprecipitates were solubi-
Axenic S. oligorrhiza plants were grown phototrophi- lized without heating in Laemmli buffer (15). Soluble
tally at 25-28°C under cool white fluorescent lamps (20 proteins of Spirodela were solubilized for electrophoresis
pm01 . me2 . s-‘) for lo-15 days on half-strength Hutner’s as previously described (7). Electrophoresis was carried
mineral medium (13) containing 0.5% sucrose. Prior to out according to Laemmli (15) using a 4.5% stacking gel
pulse labeling, the plants (200-400 mg fresh wt) were and a lo-20% gradient resolving gel. Several lanes of 3H-
transferred for 24-48 h to 35 X lo-mm sterile petri fatty-acid-labeled protein fractions were bordered on
dishes (Falcon style) containing mineral medium lack- each side by a lane containing the [35S]methionine-la-
ing sucrose. Radiolabeling in the light for 3 min was ini- beled “marker” protein fractions. In addition, 14C-meth-
tiated with 1 mCi/ml of 9,10-[3H]palmitic acid (sp act ylated molecular weight standards ( Amersham) were
28.5 Ci/mmol) or 9,10-[3H]myristic acid (sp act 30 Ci/ run alongside these samples. Following SDS-PAGE, the
mmol) in mineral medium at room temperature. Alter- resolved proteins were electrophoretically transferred
natively, plants were incubated in the light with 0.05 onto nitrocellulose paper (0.2 pm, Schleicher & Schuell)
mCi/ml of [35S]methionine (sp act 1072 Ci/mmol) for 1 by the standard electroblotting procedure (16) using 25
h. The labeling media were subsequently removed, and mM Tris-Cl, 0.192 M glycine, 20% methanol, and 0.02%
plants were washed three times with fresh mineral me- SDS as the transfer buffer. We found that longer trans-
dium prior to homogenization. fer times (12 h) at lower voltages (40 V) at 15°C gave
For labeling with fatty acids the stock radioactive superior protein transfer efficiency. After protein blot-
fatty acid solutions were dried under a stream of nitro- ting, the nitrocellulose paper was washed in phosphate-
gen gas and then redissolved in dimethyl sulfoxide. The buffered saline for 5 min, air-dried, marked around the
final concentration of dimethyl sulfoxide in the labeling margins with radioactive ink for orientation, and ex-
medium was kept under 1% (v/v). posed to Kodak XAR-5 X-ray film to locate the [35S]ra-
Cell homogenization and isolation of protein frac- dioactive protein and [14C]protein standard bands.
tions. Total proteins were extracted from the radiola- Identification of RASl and acyl-acyl carrier proteins
beled yeast cells by vortexing with 0.4 g of glass beads on nitrocellulose paper. The autoradiographs were pre-
and 1 ml of RIPA buffer (20 mM Mops, 150 mM NaCl, 1 cisely realigned with the blots to mark the positions of
mM EDTA, 1% NP-40, 0.1% SDS, and 1% aprotinin, the [35S]methionine-labeled, immunoprecipitated RASl
pH 7.0) six times with 1-min bursts. Unbroken cells, cell protein and the 14C-labeled protein molecular weight
debris, and glass beads were removed by centrifugation standards. Fluorographs of the SDS-polyacrylamide
of the extracts at 10,OOOg for 5 min. The supernatant was gels had to be exposed to X-ray films for between 10 and
further clarified by centrifugation at 105,OOOg for I5 min 20 days to visualize the 3H-acylated proteins.
at 4°C. The position of acyl-ACP on the nitrocellulose paper
The method used for isolation of soluble chloro- was detected by immunoreaction of the protein, on a 5-
plast proteins from Spirodela has been previously de- mm-wide strip cut out of the blot, to antibodies against
scribed (7). ACP (12) and by its migration as a lo-kDa band on poly-
Immunoprecipitation of RASl protein of Saccharo- acrylamide gels. Following identifications on the blot,
myces. For immunoprecipitation, cell-free extracts nitrocellulose paper strips containing the putative [3H]-
222 CALLAHAN ET AL.

acyl-ACP and [3H]acyl-RAS1 proteins were excised.

Several strips comprising three to five gel lanes contain-
ing the [3H]acyl-proteins were pooled for subsequent
120-A. Standards
fatty acid analysis. Control strips from other regions of
the blots were also carried through the procedure to mea- L

sure the background levels of radioactivity.

Extraction of covalently bound fatty acids fromproteins
on nitrocellulose paper. Nitrocellulose paper contain-
ing the resolved radiolabeled protein bands of interest
were extracted with 5 ml of chloroform-methanol (2:l)
to remove any noncovalently bound lipids. The nitrocel-
lulose paper was then cut into smaller pieces and re- 1300
6. 3H-Acyl ACP
fluxed for 8 h with 3 ml of methanol-3 N sodium hydrox- t
ide (9:l) at 70°C. After cooling to 25”C, the reaction
mixture was acidified to pH l-2 with 6 N HCl and ex-
tracted three times with 5 ml of petroleum ether. The
organic phases were combined and washed three times
with equal volumes of water. After 200 ~1 of ethanol was
added, the solution was dried completely under nitrogen.
The residue was then immediately redissolved in 0.5 ml
of hexane.
Derivatization and analysis of extracted fatty acids.
The fatty acids extracted from acylated proteins were
converted to p-nitrophenacyl derivatives (17,18). The
derivatization reagent consisted of 0.02 mM p-nitro-
phenacyl bromide (Aldrich) and 0.04 mM diisopro-
pylethylamihe (Sigma) in NJ-dimethylformamide
0 2 4 e 8 10 12 14 13 1.3 20
(Fisher). Prior to use, diisopropylethylamine was dried
ElMion Time, min.
over sodium hydroxide, and N,N-dimethylformamide
was stored over oven-activated molecular sieves. FIG. 1. Reversed-phase HPLC separation ofp-nitrophenacyl deriv-
The fatty acid extracts were evaporated under nitro- atives of authentic fatty acid standards (absorbance at 254 nm) (A)
and of [3H]fatty acids (dpm) extracted from nitrocellulose strips con-
gen and 20 ~1 of the derivatization reagent solution was
taining acyl-ACP of Spirodeh (B) or RASl protein of Saccharomyces
added to vials containing the residue. These were then (C). Spirodela were radiolabeled with either [3H]palmitic (0) or [3H]-
heated for 15 min at 65°C. Derivatization was 92-95% myristic (+) acids, while Saccharomyces was radiolabeled with [3H]-
as assessed by TLC of the fatty acid derivatives on silica palmitic acid (0) only. Following SDS-PAGE of the isolated protein
fractions, electroblotting, and identification of the specific proteins on
gel H using petroleum ether&ethyl ether-acetic acid
the nitrocellulose paper, [3H]fatty acids were extracted, derivatized,
(70:30:1) as the solvent system. Thep-nitrophenacyl de- and resolved by reversed-phase HPLC. The eluant was monitored
rivatives (Rf 0.21) were identified by exposure of the continuously for radioactivity by liquid scintillation counting of each
thin-layer plate to iodine vapor. Aliquots of the deriva- fraction. Identification of the fatty acid corresponding to the peak in
tized fatty acids were transferred to scintillation vials the elution profile was made by comparison to coeluting, derivatized
fatty acid standards (A).
containing 10 ml Aquasol- (DuPont) and counted for
radioactivity.
Resolution of p-nitrophenacyl fatty acids was
achieved using a HPLC system (Waters Assoc., Model mixed with the HPLC eluate by means of a pump ad-
6000A) equipped with a Model U6K universal injector justed to give a flow rate of 2 ml/min. Peak identity was
and a 15 cm X 4.5 mm Ultrasphere-ODS (5 pm) reversed- determined by coelution with derivatives of authentic
phase column (Alltech Assoc., Inc.). Aliquots consisting fatty acid standards. Alternatively, fractions eluting ev-
of 20 ~1 of a derivatized sample and 30 ~1 of mobile phase ery 0.5 min were collected, dried, and counted in toluene-
[methanol-acetonitrile-water (82:9:9)] were injected. liquiflour (DuPont) in order to determine overall recov-
The flow rate was 1 ml/min. The samples were eluted eries from the column.
first through a detector monitoring at 254 nm (Waters
Assoc., Model 450 variable wavelength detector) and
RESULTS AND DISCUSSION
then through a Flow-one Beta (Model 1C) HPLC radio-
active flow detector using a scintillant, Flow-Stint III Two proteins whose acylation is well established were
(Radiomatic Instruments and Chemical Co., Inc.), selected for this study. Yeast RASl protein, a product of
 ANALYSIS OF ACYL MOIETIES ON ACYLATED PROTEINS 223

the RAS gene family (11) involved in the regulation of TABLE 1

cellular growth, is palmitoylated following its synthesis Total Recoveries of Radioactive Fatty Acid Moieties
and processing (56). The acylation of RASl is consid- Hydrolyzed from Acylated RASl and Acyl-ACP
ered necessary for targeting of the mature protein to the
plasma membrane (11). On the other hand, ACP is an Total dpm in integrated peak
important cofactor in lipid metabolism onto which acyl
chains are linked through a thio ester bond with the Acyl-RASl” Acyl-ACP*
phosphopantetheine prosthetic group (19). Rapid turn-
Fraction 16:0 14:o 16:0
over and low abundance of acyl-ACP pools in higher
plants limits their detection in uiuo (12). Thus, these two Petroleum ether-
diverse kinds of protein are acylated for different func- soluble fraction
tions, and assessment of the extent of their acylation is after hydrolysis 1041 f 61 3034 + 202 11,700 *ala
Aliquot injected 582 + 32 1269-c 52 3,909 + 180
evidently important.
Total p-nitrophenacyl
Initially, the extent of in uiuo acylation of the yeast sH-14:O recovered 295 6 832 2 54 32’f 7
RASl protein and plant ACP was determined by the ra- Total p-nitrophenacyl
dioactivity that partitioned into petroleum-ether frac- 3H-16:0 recovered 483 + 24 230f 11 3,203 f 216
tions following hydrolysis of the corresponding nitrocel-
a Cells labeled with [3H]palmitic acid (16:O).
lulose strips. In these particular experiments, we
* Plants labeled with [3H]myristic acid (140) or [“Hjpalmitic acid
recovered 3034 and 11,700 dpm after hydrolysis of [3H]- (16:O).
acyl-ACP isolated from samples labeled with [3H]myris- ’ Background counts were 25-35 dpm.
tate and [3H]palmitate, respectively. Similarly, a total of
1041 dpm was recovered from nitrocellulose strips con-
taining [3H]RAS1 protein. Background levels as deter-
mined by similar treatment of an equivalent number of from acylated ACP indicating that there was no further
strips from other regions of the blot were between 25 and elongation.
35 dpm. No more than these background levels of radio- These results show that acyl groups remain covalently
activity were detected when strips containing [35S]me- linked to these proteins following electrophoretic trans-
thionine-labeled proteins were similarly hydrolyzed and fer but can be hydrolyzed directly from the nitrocellu-
extracted. lose-immobilized protein. More important, this tech-
Identification of the radioactive fatty acids extracted nique allowed identification of the acyl ligand on the
by organic solvents was achieved by reversed-phase protein from only a few excised lanes on nitrocellulose
HPLC analysis of p-nitrophenacyl derivatives of the ex- blots even though the amount of myristic acid or pal-
tracts. Figures 1B and 1C show the elution profiles of the mitic acid in the derivatized extracts was too low for
radioactive fatty acid derivatives recovered from strips direct determination by HPLC (i.e., by absorbance at
containing [3H]acyl-ACP and [3H]acyl-RAS1 protein, 254 nm) .
respectively. Separation of authentic fatty acid stan- Both RASl and ACP are acylated via thio ester link-
dards is compared in Fig. 1A. Table 1 summarizes the ages. The method described here, however, is also appli-
total recoveries obtained of fatty acid moieties hydro- cable to acylated proteins that are modified by amide
lyzed from the acyl-RASl and acyl-ACP. Derivatized ex- linkages. For instance, a similar procedure was used to
tract from in uiuo palmitoylated RASl protein equiva- identify the fatty acid ligand of the 32-kDa photosystem
lent to 582 dpm was injected onto the column and 83% II reaction center protein of chloroplast membranes
(483 dpm) of the total radioactivity eluted as palmitic (20), another protein to which palmitic acid is bound via
acid [Fig. lC, closed symbol; Table 11. In a typical run of an amide bond and whose acylated pool is in very low
the derivatized extract of acyl-ACP from in uiuo myris- abundance (7). In cases where palmitate or myristate la-
tate-labeled Spirodela, out of 1269 dpm, 832 dpm (66%) beling is transient in nature and associated with proteins
was recovered as myristic acid and 230 dpm (18%) as of low abundance, long exposures of fluorographed gels
palmitic acid (Fig. 1B; Table 1). In a run of the equiva- to X-ray films are required for visualization of discrete
lent derivatized extract from palmitate-labeled plants, [3H]acyl-proteins fractionated by SDS-PAGE. Once es-
81% was recovered as palmitic acid, with only back- tablished for each case, the methodology described here
ground radioactivity being associated with the myristic circumvents this requirement and enables rapid identi-
acid peak. The detection of both 3H-radiolabeled myris- fication of the fatty acid(s) bound to an acylated protein.
tic and palmitic acids on ACP when Spirodeh plants
were incubated with [3H]myristic acid (Fig. 1B; Table 1) REFERENCES
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