From the Signalling Programme, Babraham Institute,
Cambridge CB2 4AT, United Kingdom, the § Department of
Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom,
and the ¶ Department of Biochemistry, University of Cambridge,
Cambridge CB2 1GA, United Kingdom
Received for publication, November 13, 2000, and in revised form, December 18, 2000
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ABSTRACT |
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Phosphatidic acid (PA) is an important bioactive
lipid, but its molecular targets remain unknown. To identify such
targets, we have synthesized and coupled PA to an agarose-based matrix, Affi-Gel 10. Using this matrix as an affinity reagent, we have identified a substantial number of potential PA-binding proteins from
brain cytosol. One class of such proteins is known to be involved in
intracellular traffic and it included coatomer, ADP-ribosylation factor
(Arf), N-ethylmaleimide-sensitive factor (NSF), and
kinesin. Binding of these proteins to PA beads was suppressed by
soluble PA, and it occurred preferentially over binding to beads
coupled to phosphatidylinositol (4,5)-bisphosphate. For
coatomer, Arf, and NSF, we verified direct binding to PA beads using
purified proteins. For recombinant Arf1 and Arf6, binding to PA
required myristoylation. In addition, for NSF and Arf6, an ATPase and a GTPase, respectively, binding to PA beads was extremely sensitive to
the nucleotide state of the protein. Binding to PA may be a property
linking together distinct participants in one complete round of
membrane transport from a donor to an acceptor compartment.
The lipid membranes of eukaryotic cells are dynamic in composition
and appear to be ideal nucleation sites for selective recruiting in a
time-dependent manner a variety of cytosolic proteins to specific microdomains. Diacylglycerol was among the first lipids to
emerge as a specific binding partner for protein kinase C, and more
recently the phosphoinositides are attracting considerable attention in
their roles of recruiting important signaling proteins to defined
membrane compartments (1-5).
PA1 synthesized via the
glycerol-3-phosphate or the hydroxyacetone phosphate pathway is an
important intermediate in the biosynthesis of glycerophospholipids and
triacylglycerols (6). The contribution of PA to lipid-based signaling
is less well understood. Apart from the biosynthetic routes mentioned
above, PA can be rapidly elevated in cells through hydrolysis of
phosphatidylcholine by phospholipase D (PLD) (7, 8). Because activation
of PLD has been implicated in the regulation of cellular processes
ranging from growth control to traffic, it is generally assumed that
PA-interacting proteins downstream of PLD activation exist to mediate
those functions (9, 10). However, because PA elevated through PLD
activation is unstable and rapidly converted to diacylglycerol via
hydrolysis by PA hydrolases, in pathways where PLD activation is
measurable it is not always clear whether PA or diacylglycerol is the
relevant signaling lipid (11).
One protein that has been shown to interact directly with PA is the
serine/threonine kinase Raf-1, a component of the mitogen-activated protein kinase cascade (12, 13). Translocation of Raf-1 from the
cytosol to intracellular membranes (primarily endosomes) depends on the
presence of PA and can be blocked by point mutations that abolish this
interaction. The discovery of at least one protein that appears to
interact directly with PA raises the possibility that additional such
proteins exist, and their identification should be of considerable interest.
In addition to a function in signaling, PA has been proposed to have a
role in intracellular traffic. It was originally shown that artificial
vesicles made with a mixture of lipids including PA were better able to
bind coatomer (a coat protein complex involved in transport between the
endoplasmic reticulum and the Golgi complex) than their counterparts
prepared without PA (14). In addition, assays that recapitulate vesicle
formation in vitro with pure lipid and protein components
revealed that acidic phospholipids such as PA enhanced vesicle
formation (15-17). In other experiments, PA supplied exogenously to
cells was able to rescue a block in endoplasmic reticulum to Golgi
transport of viral proteins imposed by primary alcohols (18). In
addition, altering PA levels was shown to disrupt the structure of the
Golgi complex and affect traffic through this organelle (19). Recent
reports have also indicated that PA synthesized via acylation of lysoPA
may be involved in vesicle fission ("pinching off") in the
Golgi complex or at the plasma membrane (20, 21). The inherent
instability of PA as discussed above complicates any attempt to assign
significance to PA as opposed to diacylglycerol in some of these
experimental settings. More importantly perhaps, because PA can
stimulate formation of PI (4,5)P2 by
phosphatidylinositol 4-phosphate 5-kinase directly (22, 23), the
search for the relevant lipid(s) in those pathways becomes even more
complex and must include PI (4,5)P2. PI (4,5)P2 has been shown to influence on its own the activation cycle of several
small GTPases such as Arf that are crucially involved in membrane
transport pathways (24). PI (4,5)P2 also interacts with
other proteins involved in trafficking such as dynamin and clathrin-associated protein complex 2 (25).
One way to identify PA-binding proteins is to use this lipid in an
immobilized form and to isolate proteins that bind to it. A similar
reagent containing immobilized PI (4,5)P2 can be used in
parallel to determine whether candidate proteins show differential affinity for those two lipids. In this work, we have synthesized and
used such reagents to identify a set of traffic-related proteins that
showed strong binding to immobilized PA but not to immobilized PI
(4,5)P2.
Synthesis of Affi-Gel 10 PA and Affi-Gel 10 PI
(4,5)P2 Matrices--
The amino functionalized PA
(Fig. 1A, 2) was prepared in seven steps from the
commercially available
(S)-(+)-1,2-O-isopropylideneglycerol (Fig.
1A, 3) (Aldrich). The coupling of 2 with the N-hydroxysuccinimide activated ester-agarose resin,
Affi-Gel 10 (Bio-Rad), required the solvent combination
chloroform-methanol-water (0.8:1.0:0.2) to afford the PA-affinity
reagent (1). Excess resin (~5 equivalents of
N-hydroxysuccinimide groups) was used. The resulting loading
capacity was estimated to be 2.6 µmol/ml. The estimation was done by
1H NMR analysis of the concentration of residual
2 in the reaction mixture in the presence of the internal
standard 1,3,5-myo-inositol orthoformate. In addition to the
2-palmitoyl derivative shown here, a 2-lauroyl derivative was prepared
that showed identical binding characteristics. As a control of binding
specificity we have also prepared and employed a
3-( Preparation of Membrane and Cytosolic Fractions from Sheep
Brain--
All steps including centrifugation were carried out at
4 °C. 180 g of deep-frozen sheep brain was broken into small
pieces and resuspended in ice-cold buffer (30 mM Tris-HCl,
pH 7.6, 80 mM NaCl, 2 mM EGTA, 0.5 mM EDTA, 2 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of
antipain, pepstatin A, aprotinin, and leupeptin) to a volume of 500 ml
for 10-15 min until thawed. The tissue was then blended in a polytron
until it reached a fine consistency and was centrifuged at 10,000 rpm
for 30 min. The supernatant from this spin contained the membrane and
cytosolic fractions. For preparing cytosol, the supernatant from above
was centrifuged again for 1 h at 100,000 × g. The
new supernatant was dialyzed for 24 h against buffer containing 50 mM Hepes, pH 7.2, and 90 mM KCl. The dialyzed
material was centrifuged again for 1 h at 100,000 × g, and the supernatant (cytosol) was frozen in liquid
nitrogen and stored at Binding Reactions and Regeneration of Beads--
Cytosol at 6 mg/ml was mixed 1:1 with lysis buffer (50 mM Tris-HCl, pH
8.0, 50 mM KCl, 10 mM EDTA, 1% Nonidet P-40,
0.6 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin,
and 1 µg/ml trypsin inhibitor) on ice and centrifuged at maximum
speed in a microcentrifuge to remove any aggregates. The PA beads,
which were stored in water plus 0.02% sodium azide, were equilibrated with three washes in ipp buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% Tween 20, and
0.02% sodium azide) and were resuspended in this buffer to give a 10%
(v/v) suspension. In a typical binding reaction, 450 µl of diluted
cytosol were mixed with 35 µl of bead solution in a 500-µl
microcentrifuge tube on ice. A few air bubbles were created to effect
better mixing. The tubes were put in a rotator at 4 °C for 1.5-2 h.
Following binding, the beads were washed quickly three times with ipp
buffer and resuspended in Laemmli sample buffer, and bound proteins
were analyzed by SDS-PAGE. In some experiments, soluble PA was included in the binding reaction as follows: L-
The beads could be reused up to five times. At the end of an
experiment, beads were collected using ipp buffer and centrifuged. To
the pellet, sample buffer (50 mM Tris-HCl, pH 7.4, 2% SDS, 100 mM dithiothreitol) was added to make a 25% bead
suspension for 10 min. The beads were then washed six times with ipp
buffer and were stored at 4 °C as a 10% suspension in ipp buffer.
Identification of Polypeptide Bands by Mass
Spectroscopy--
Coomassie-stained gel bands were excised and
fragmented. The gel pieces were subjected to the following steps, 30 min each at room temperature on a tube rotator, and then the washes
were discarded: 1) wash with 250 µl 200 mM ammonium
bicarbonate/50%MeCN; 2) reduce with 5 mM
tris(2-carboxyethyl)phosphine in 200 µl of 200 mM
ammonium bicarbonate, 50% MeCN; 3) alkylate in the dark by adding 20 µl of 0.25 M iodoacetamide (final concentration, 23 mM); 4) wash as in step 1; and 5) dry under vacuum. Gel
pieces were reswollen at 0 °C in 40 µl of 100 mM
ammonium bicarbonate containing 10 µg/ml modified trypsin (Promega).
After 45 min, excess solution was aspirated (54), and digestion was
allowed to proceed at 37 °C, for 22 h. The digest supernatant
was acidified with 2 µl of 10% formic acid, and peptides were
recovered by immobilization and subsequent desalting on a Vydac C8
guard cartridge (µPurifierTM; LC Packings, Amsterdam, The
Netherlands). Washing was with 1% formic acid, and peptides were
eluted to a nanospray needle (Protana, Odense, Denmark) using
1-2 µl of 0.1% formic acid, 70% methanol. Peptide masses were
examined using a Finnigan LCQ ion trap mass spectrometer fitted with a
Protana nanospray interface. All significant peaks were examined for
charge state and monoisotopic mass value in zoomscan mode and subjected
to collision induced dissociation. Mass/fragmentation data sets were
submitted to Mascot for data base searching (55). Results were
scored by Mascot, and only those scoring significantly higher
(p < 0.05) than a chance match were regarded as
positive identifications. Only peptides giving matching fragmentation
data were scored; matching by peptide mass only was not included. The
number of peptides matched per protein discussed in this work were 12 for kinesin, 17 for puromycin-sensitive aminopeptidase, 12 for
neurochondrin, and 5 for NSF.
Antibodies and Purified Proteins--
The following antibodies
were used in immunoblots: mouse monoclonal 1D9 to Arf, a kind gift of
Dr. Richard Kahn; mouse monoclonal M3A5 to Arf6 and Arf1 Transient Expression--
Plasmids expressing wild
type and mutant Arf1 and Arf6 genes were a kind gift of Dr. Julie
Donaldson (45). Transfection into COS-7 cells was using DEAE-dextran as
described before (57). Detection of Arf1 and Arf6 proteins by
immunoblotting or immunofluorescence was done using antibodies to the
hemagglutinin epitope.
Synthesis of PA and PI (4,5)P2 Matrices--
To
identify potential PA-binding proteins we synthesized a solid phase
PA-based matrix as shown in Fig.
1A. In this reagent, the head
group of the phospholipid is expected to be exposed to the solvent (and
therefore available for binding), whereas the fatty acid at the 3 position anchors the PA to the Affi-Gel 10 resin. Although the
2-palmitoyl derivative is shown here, we have also synthesized and used
a 2-lauroyl derivative, which showed identical binding characteristics.
In addition, we have used a 2-palmitoyl PI (4,5)P2
immobilized Affi-Gel 10 reagent (shown in Fig. 1B) to
directly compare affinities of candidate proteins to the two
lipids.
Binding of Cytosolic Proteins to PA Beads--
We initially used
brain cytosol as a source of material. In preliminary experiments we
found that cytosol without detergent gave unacceptably high binding to
the beads (data not shown). We used nonionic detergent in the cytosol
solution to overcome this problem, because other types of detergent
(ionic or zwitterionic) appeared to reduce binding to very low levels.
When cytosol supplemented with Nonidet P-40 was incubated with the PA
beads and unbound material was removed with several washes, a
reasonable number of polypeptides were seen to be in the PA-bound
fraction (Fig. 2). This pattern of
staining is very reproducible. If those polypeptides were binding
specifically to immobilized PA, then soluble PA should reduce their
binding to the beads. Initial attempts at such inhibition of binding
using dipalmitoyl PA (the same lipid as that coupled to the beads) were
unsuccessful, perhaps because of the poor solubility of this lipid.
Inhibition of binding was obtained, however, with shorter chain analogs
of PA (dilauroyl, C:12 or dioctanoyl, C:8), which also showed superior
solubility and sonication properties. When cytosol was preincubated
with soluble dilauroyl PA, a substantial number of PA-bound bands were
reduced in intensity (Fig. 2, asterisks). Interestingly, the
presence of soluble PA also seemed to enhance the binding of some
proteins to the PA beads, whereas binding for other polypeptides was
unaffected. Here we deal with polypeptides whose binding to the PA
beads is reduced in the presence of soluble PA.
Mass spectrometry was used to establish the identity of the PA-binding
polypeptides. In addition, we determined directly the identity of some
polypeptides using immunoblotting. From both protocols, we have
identified 15 known and 5 novel proteins in the PA-bound fraction from
brain cytosol. An additional 8-10 polypeptides have been obtained from
brain membranes extracted with detergent, and their identification by
mass spectrometry is in progress. In this work, we will discuss
proteins involved in intracellular traffic (coatomer, Arf, kinesin, and
NSF) whose binding to PA beads has been characterized further. From the
remaining known proteins, we have cloned, expressed, and verified
binding to PA beads of nordin (or neurochondrin), thought to be
involved in somatodendritic functions in neuronal cells (26, 27), and puromycin-sensitive aminopeptidase, a protease thought to be involved in cell growth (28). The Raf-1 kinase bound to the PA beads at levels
comparable with neurochondrin and puromycin-sensitive aminopeptidase.
Identification of
Additional traffic-related proteins were identified from mass
spectrometric analysis of the PA-bound bands shown in Fig. 2. One such
was NSF, an ATPase involved in numerous transport steps (31, 32), and
its presence in the PA-bound fraction was verified by immunoblotting
(Fig. 3B). Interestingly, little SNAP (soluble NSF attachment protein), which is a
binding partner of NSF (33), was detected in the PA-bound fraction
(Fig. 3B). A second protein identified from mass
spectrometric analysis of the PA-bound bands shown in Fig. 2 was the
heavy chain of kinesin, a microtubule motor protein involved in
numerous transport steps (34, 35). The presence of kinesin in the
PA-bound fraction was verified using two monoclonal antibodies to this
protein (Fig. 3C) (36). There was no NSF or kinesin bound to
uncoupled or to PI (4,5)P2 beads (data not shown but see
Fig. 5).
For all traffic-related proteins discussed above, we verified that
binding to PA beads could be competed with exogenously supplied short
chain PA (Fig. 3D and data not shown for NSF). In all cases,
dilauroyl PA inhibited binding at lower doses than dioctanoyl PA (data
not shown). We speculate that this may be related to the height of the
head group in the mixed micelles used for competition, with the
"taller" dilauroyl lipid having a more accessible head group to
serve as a competitor in the binding reaction. Because we used the
shorter chain lipid in the competition reaction, we also synthesized
the 2-lauroyl-PA resin and were able to show that its binding
characteristics were very similar to the 2-palmitoyl counterpart (data
not shown). We have tried to compete binding to PA beads with a number
of other reagents, in all cases obtaining negative results. Those
included phosphate ions (up to 100 mM), glycerolphosphate
(up to 50 mM), ATP (up to 10 mM), GTP (up to 10 mM), and dipalmitoyl phosphatidylcholine (up to 300 µM) (data not shown).
Binding of Coatomer Complex but Not of Other Coats to PA
Beads--
The observed binding of
Because coatomer and Arf are capable of interacting in vivo
(40, 41) and in vitro (42, 43), we investigated whether the
presence of one would affect the ability of the other to bind to PA
beads under nonactivating conditions for Arf. When purified coatomer
was incubated with PA beads in the presence of increasing amounts of
purified Arf, there was no effect of Arf on the amount of coatomer
bound (Fig. 4C). The converse experiment (increasing coatomer in the presence of constant Arf) showed similar results (data
not shown). Thus, both coatomer and Arf bound to PA independently of
each other and without assistance from other factors. For interaction of activated Arf with coatomer during PA binding, see below.
Arf Binding to PA Beads Requires Myristoylation and Depends on
Nucleotide State--
We adapted the binding reaction to lysates from
tissue culture cells, because this would allow us to address binding of
candidate proteins after transient overexpression or after manipulating cellular function. We found that lysis with nonionic detergent (0.4%
Nonidet P-40) but not with other types of detergent maintained the
specificity of binding (data not shown). Using those conditions, we
examined PA and PI (4,5)P2 binding of transiently expressed Arf1 and Arf6 mutants.
Most members of the Arf family are cytosolic proteins which translocate
to membranes upon binding GTP (44). Binding to PA in this context may
be understood as an additional way to enhance the affinity of the
activated protein to membrane subdomains. A divergent member of the Arf
family is Arf6, which is membrane-bound and cycles between endosomal
and plasma membranes depending on its activation state (45). It was
therefore of interest to determine whether Arf6 also bound to PA beads.
COS cells were transfected with plasmids encoding hemagglutinin-tagged
Arf6 or Arf6 mutants predicted to be in the GDP-bound form (T27N), in
the GTP-bound form (Q67L), or missing a myristoylation site (G2A).
Binding to PA or PI (4,5)P2 beads was assayed from Nonidet
P-40 lysates (Fig. 5A).
Whereas binding to PI (4,5)P2 was very low, the four Arf6 proteins differed significantly in their binding to PA. Wild type Arf6
and the T27N GDP-bound mutant showed significant binding to PA that was
comparable with endogenous
The relative amounts of endogenous The Inactive Form of NSF Binds to PA--
To determine whether NSF
binding to PA is direct, recombinant NSF (46) was used in the binding
assay (Fig. 6A). In this experiment we also asked whether the nucleotide state of NSF is important for PA binding and whether binding occurs to other lipid beads. No binding of recombinant NSF to PI (4,5)P2 beads or
to uncoupled Affi-Gel beads was detected (Fig. 6A,
lanes 7 and 8). Binding to PA beads was very
dependent on the nucleotide state of the protein. Under conditions
where NSF was in the ATP-bound state and unable to hydrolyze the
nucleotide, i.e. in the presence of Mg ions and a
nonhydrolyzable analog of ATP (ATP We have synthesized and started to use a PA-specific affinity
matrix to identify potential protein targets that bind to this lipid.
PA-interacting proteins will be downstream of PLD activation and
perhaps following acylation of lysophosphatidic acid (20, 21). PI
(4,5)P2 has also been implicated, directly or indirectly, in several pathways of intracellular transport (25), frequently in
settings where PA is also a candidate. To differentiate binding partners for these lipids, we have also synthesized and used a PI
(4,5)P2-specific affinity matrix. In this work we
concentrate on traffic-related proteins that bound strongly to PA and
less well to PI (4,5)P2.
In designing this reagent, we have assumed that binding of candidate
proteins to PA should be of sufficient strength to occur in detergent
solution and withstand several washes. We expect that if such proteins
show strong binding in detergent solution, their binding to PA in the
cellular setting may be comparable if not stronger. In addition, by
presenting PA to the candidate proteins in the form of a
two-dimensional patch on the agarose matrix, we have hoped to emulate
the physiological state (and perhaps the local concentration) of this
lipid on cellular membranes.
The PA beads are a novel reagent, and it was important to establish the
specificity of protein binding. Firstly, we have shown throughout this
work that candidate proteins binding to PA do not bind to PI
(4,5)P2 under conditions where a known PI
(4,5)P2 target (PLC Binding of coatomer to PA beads involved the entire complex and did not
require any additional components such as Arf. This observation is
consistent with our earlier report that coatomer bound better to
artificial liposomes containing PA than to those made in its absence
(14). Interestingly, Arf itself (both from cytosolic sources and from
tissue culture lysates) also bound to PA beads. Recent work is
suggesting a complex set of relationships between coatomer, Arf, and
sorting signals on the surface of membranes, leading to the formation
of coated vesicles containing specific cargo (47). An important
requirement of these models is the need to concentrate on membrane
subdomains all of the interacting protein complexes. We suggest that
PA, given its good affinity for both coatomer and Arf, is ideally
suited to serve as a nucleation site for the initiation of a budding reaction.
The differential binding of Arf6 to the PA beads depending on
activation state may be of relevance in explaining the trafficking of
this protein. The GDP-bound form of Arf6 is in endosomal compartments, but activation and binding to GTP result in translocation to the plasma
membrane (48). How can a protein that is always membrane-bound accumulate on two separate membranes depending on activation state? Our
data indicate that the relative PA content of the two membranes may be
of some influence because the activated form of Arf6 has strong
affinity for this lipid. In this context it is interesting that
treatment of cells with phorbol esters (which can activate protein
kinase C that in turn activates PLD to produce PA) results in Arf6
translocation to the plasma membrane (49).
The significance of the NSF-PA interaction is more difficult to
evaluate primarily because this protein is acquiring with time novel
and unexpected functions that do not fit readily within a single
experimental paradigm. Originally thought to mediate vesicle fusion
directly, it was later shown that NSF, through its ATPase activity,
functions primarily as a chaperone to maintain unpaired v-SNARE (SNAP
receptors) and t-SNARE complexes (50). In addition, a function of NSF
in reassembly of Golgi stacks following mitosis was shown not to depend
on its ATPase activity (51). Recently NSF was identified as a binding
partner of two plasma membrane proteins: A single round of protein transport entails cargo concentration and
budding from the donor membrane, vesicle movement, and vesicle
tethering and fusion with the acceptor membrane. It is interesting that
proteins involved in all three of these steps show affinity for PA.
This affinity could be relevant for a specific transport step following
signal-dependent activation of PLD such as the need to
down-regulate by internalization and subsequent degradation an
activated receptor. Affinity for PA could also be relevant for all
transport steps involving the proteins in question, but this would
require that mechanisms of PA formation and consumption underlie those
steps. Thus, because PA can be made by basal or signal-stimulated
cellular pathways, it is possible that it has evolved into a versatile
regulator of membrane transport in housekeeping settings,
signal-dependent settings, or both.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminoacyl)-functionalized PI (4,5)P2 (Fig.
1B, 4) coupled via the amino function to an
Affi-Gel 10 solid support. Details of both syntheses will be reported
separately.2
70 °C. The pellet from the first 100,000 × g spin was resuspended in homogenization
buffer (20 mM Hepes, pH 7.2, 1 mM EDTA, 0.2 M sucrose plus protease inhibitors as above) and placed in
a steel Dounce homogenizer for 20 strokes. The homogenate was
centrifuged at 2500 rpm to remove debris, and the supernatant that
contained membranes was centrifuged at 100,000 × g for
1 h. The pellet after this spin (membranes) was resuspended by
homogenization to a total of 20 ml of homogenization buffer, frozen in
liquid nitrogen, and stored at
70 °C.
-phosphatidic acid
(dioctanoyl, dilauroyl, or dipalmitoyl purchased from Sigma) that was
dissolved in chloroform at 10 mg/ml and kept at
20 °C was warmed
to room temperature and put into a glass tube. The lipid solution was dried for 10 min under a stream of nitrogen. To the dried lipid film,
lysis buffer was added carefully to avoid creating any bubbles. The
tubes were then sonicated for 5 min until a clear solution was
obtained. This solution was stable for a few hours at room temperature
until ready to use. In competition experiments, the cytosol was mixed
1:1 with lysis buffer that had been supplemented with PA solution and
kept on ice for 15 min. Lipid beads were then added, and the binding
reaction was carried out as described above.
-cop, a kind gift
of Dr. Thomas Kreiss; mouse monoclonals H1 and H2 to brain kinesin, a
kind gift of Dr. George Bloom; mouse monoclonal to PLC
, a kind gift
of Drs. Matilda Katan and Matthew Jones; rabbit polyclonal to
protein kinase C, a kind gift of Dr. Null Divecha; mouse monoclonal
100/2 to
-adaptin (AP-2 coat) and 100/3 to
-adaptin (AP-1 coat)
purchased from Sigma; and mouse monoclonal 6B7-3 to
/
-SNAP and
9G7-3 to NSF purchased from Stressgen. The following purified proteins
were used: native coatomer, a kind gift of Dr. Gerry Waters, 50% pure,
prepared and used as described (14, 37); native Arf, 20% pure and
containing primarily (>70%) Arf1, a kind gift of Dr. Alex Brown,
prepared and used as described (56); and recombinant NSF and SNAP, more
than 90% pure (46), a kind gift of Dr. Sidney Whiteheart.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, synthesis of PA-Affi-Gel affinity
matrix. The -amino functionalized phosphatidic acid (2)
synthesized from the optically pure glycerol derivative (3)
was coupled with N-hydroxysuccinimide-activated ester resin,
Affi-Gel 10, to give the matrix (1). The Affi-Gel-10 bead is
shown as a shaded sphere, and it is not drawn to scale.
B, structure of PI (4,5)P2 Affi-Gel 10 beads (4)
used throughout this work as a control of specificity of binding.
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Fig. 2.
Identification of putative PA-binding
proteins in brain cytosol. Cytosol at 3 mg/ml and containing 0.5%
Nonidet P-40 was mixed with PA beads for 1 h followed by washes,
SDS-PAGE, and silver staining. Soluble dilauroyl PA at 50 µM was added to some samples for 15 min before addition
of the PA beads as indicated. Bands marked by
asterisks are candidate PA-binding proteins based on their
reduced intensity in the presence of soluble PA.
-cop Coatomer, Arf, NSF, and Kinesin in the
PA-bound Fraction--
Immunoblotting of PA-bound fractions with
antibodies to the
-cop coatomer subunit or to native Arf revealed
binding of these proteins to the PA beads (Fig.
3A). There was no binding to
uncoupled Affi-Gel beads or aggregation in the absence of any beads
(data not shown). More importantly,
-cop and Arf bound weakly to
2-palmitoyl PI (4,5)P2 beads (Fig. 3A),
indicating that binding was to the head group and not to the acyl chain
of PA. To verify that the PI (4,5)P2 beads bound proteins
known to interact with this lipid, we probed the blots with antibodies
to PLC
1, a protein known to bind PI (4,5)P2 through its
pleckstrin homology domain with a Kd value in the
range of 0.1-10 µM (29, 30). Under conditions where
binding of
-cop and Arf to PI (4,5)P2 was minimal, there was strong
binding of PLC
(Fig. 3A). Conversely, there was
undetectable PLC
bound to the PA beads (Fig. 3A). As an
additional control of the specificity of binding, we determined that
protein kinase C, a protein abundant in the cytosol and known to bind to acidic phospholipids (such as phosphatidylserine) and to
diacylglycerol (a dephosphorylated PA) did not bind to the PA or PI
(4,5)P2 beads (Fig. 3A).
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Fig. 3.
Binding of -cop,
Arf, NSF, and kinesin to PA beads. A, brain cytosol was
mixed with PA beads or three different concentrations of PI
(4,5)P2 beads as shown for 1 h. The concentration of
lipid on the input beads was 150 nmol for PA and 120 nmol for the 2×
PI (4,5)P2. Following washes, the proteins bound to the
beads were analyzed by SDS-PAGE and antibodies to
-cop, PLC
, Arf,
or protein kinase C (PKC). B and C,
brain cytosol was mixed with PA beads for 1 h followed by washes,
SDS-PAGE, and electrotransfer. The blots were probed with antibodies to
NSF or to
/
SNAP (B) or with two anti-kinesin
antibodies (C). D, soluble dilauroyl PA was added
to the binding reaction at the concentrations shown for 15 min before
the addition of PA beads. Following binding, SDS-PAGE, and
electroblotting, the blots were probed with antibodies to
-cop, Arf,
and kinesin as shown.
-cop to PA beads raises the
question of whether the entire coatomer complex (37) is present in the
PA-bound fraction and whether binding is direct. Using native coatomer
that was 50% pure and did not contain any Arf, we found all coatomer
subunits in the PA-bound fraction (Fig.
4A, lanes 6 and
7). Prominent noncoatomer-related bands contaminating the starting preparation were totally excluded from the bound fraction (Fig. 4A, bands marked with
asterisks). We also attempted to disassemble coatomer using
high salt to find which of the subunit(s) contain(s) PA-binding sites.
We were unable to answer this question because conditions of
disassembly also interfere with binding to the PA beads (data not
shown). It was of interest to ask whether coats related to coatomer
also showed binding to PA beads. The PA-bound fraction from cytosol was
resolved in triplicate and probed with antibodies to
-cop,
-adaptin (a subunit of the AP-1 coat involved in Golgi traffic (38))
and
-adaptin (a subunit of the AP-2 coat involved in endosome and
plasma membrane traffic (39)). Only
-cop showed strong binding to
the PA beads (Fig. 4B).
View larger version (40K):
[in a new window]
Fig. 4.
Binding of entire coatomer, but not of other
coats to PA beads. A, native coatomer ~50% pure
(shown in lanes 1 and 2 at two different
concentrations) was mixed with lysis buffer and centrifuged to remove
aggregates. The supernatant (input coatomer; lanes 3 and
4) was mixed with PA beads (lanes 6 and
7) or with uncoupled Affi-Gel beads (lane 5) for
1 h followed by washes and SDS-PAGE. The gel was stained with
silver. Bands marked with asterisks contaminate
the starting coatomer preparation but are eliminated from the sample
bound to the beads. B, cytosol was mixed with PA beads for
1 h followed by washes. The sample was divided into three aliquots
and resolved separately by SDS-PAGE followed by electroblotting. Each
of the blots was probed separately with antibodies to equivalent
subunits (in the 100-110-kDa range) of AP-1, AP-2, or coatomer.
C, purified Arf-free coatomer (lane 1; 10% of
input) diluted to 15 µg/reaction was mixed with the indicated amount
of purified coatomer-free Arf (lane 2; 30% of input) for 15 min before binding to PA beads for 1 h, washes, SDS-PAGE, and
immunoblotting. In this experiment, the gel was transferred overnight,
and the Arf staining required longer exposures (shown for lanes
3-6).
-cop binding. Binding of the Q67L
GTP-bound mutant to PA was more than 15-fold higher, whereas the mutant
lacking myristate showed undetectable binding. These results suggest
that binding of Arf6 to PA requires myristoylation and depends on the
nucleotide state of the protein. In a similar experiment we compared
the affinity of the wild type Arf1 and the corresponding mutants with
PA (Fig. 5B). Again, binding to PA was totally eliminated
for the mutant lacking the myristoylation site (Arf1 G2A).
Interestingly, the differences in PA binding between the GTP-bound
(Arf1 Q71L) and the GDP-bound (Arf1 T31N) forms of Arf1 were much
smaller than the corresponding differences for Arf6. Although
interpretation of this result depends entirely on how well the Arf1
proteins retain their nucleotide in comparison with the Arf6 proteins
after lysis, it is nevertheless important to note that there is a
significant difference between Arf1 and Arf6 with respect to PA
binding.
View larger version (45K):
[in a new window]
Fig. 5.
Characteristics of Arf binding to PA
beads. A, COS cells were transfected with plasmids
expressing wild type Arf6, Arf6 Q67L predicted to be predominantly in
the GTP-bound state, Arf6 T27N predicted to be predominantly in the
GDP-bound state, and Arf6 G2A lacking a myristoylation signal. 40 h post-transfection, cells were lysed in 0.4% Nonidet P-40, and the
lysates were incubated with PA or PI (4,5)P2 beads as
indicated for 1 h. Following washes, SDS-PAGE, and
electrotransfer, the blots were probed with antibodies to -cop,
PLC
, and hemagglutinin epitope (used as an epitope tag for Arf6).
The amount of Arf6 bound to the beads, as a percentage of total input
lysate (representing 3% in this experiment), is shown at the
bottom of the relevant lanes. Quantitation was
from the original film using NIH Image software. B,
identical experiment but using plasmids expressing the corresponding
Arf1 proteins. C, COS cells were transfected with a control
plasmid (0) or with plasmids encoding Arf1Q71L at two different
concentrations as indicated. Notice that the relative binding to PA for
-cop was unchanged in comparison to increasing amounts of
hemagglutinin-Arf1(Q71L) bound (HA-Arf1).
-cop binding to PA beads were
unchanged for the four Arf1 proteins (Fig. 5B), indicating that Arf1 and coatomer interact with PA independently of one another and not as a complex. To explore this further, we transfected COS cells
with two different amounts of plasmid expressing the active form of
Arf1 (Q71L) and assayed recombinant Arf1 and endogenous
-cop binding
to PA for all conditions (Fig. 5C). We saw that
-cop
binding to PA remained constant, although the amount of Arf Q71L
increased as a function of the input plasmid. Because at the highest
plasmid concentration more than 50% of the COS cells express the Arf1
Q71L protein as judged by immunofluorescence (data not shown), we
expected to detect increased
-cop binding as a function of increased
Arf1 Q71L binding if coatomer and activated Arf1 bound to the PA beads
as a complex. Because we did not detect any increase in
-cop
binding, we conclude from this experiment that Arf1 and coatomer do not
bind to PA beads as a complex.
S), binding to PA was undetectable
(Fig. 6A, lane 3). Strongest binding to PA was
obtained with no nucleotide and in the presence of EDTA (Fig.
6A, lane 6), conditions that presumably restrict
NSF to the ADP-bound state. The range of binding affinities when
combinations of ATP, ATP
S, EDTA, and Mg ions were used is also
consistent with the idea that strongest binding to PA occurs for the
ADP conformation of NSF (Fig. 6A, lanes 2,
4, and 5, and data not shown). A hallmark of NSF
function is its sensitivity to alkylation by NEM, which is thought to
inhibit the ATPase activity of the protein. We examined NSF binding to
PA after NEM treatment in the presence of ATP
S, EDTA, or ATP
S
plus Mg ions (Fig. 6B). In general, NEM treatment enhanced
NSF binding to PA (Fig. 6B, compare lanes 2-4
with corresponding lanes 5-7). More importantly, NEM-treated NSF was fully capable of PA binding even in the presence of
ATP
S and Mg (Fig. 6B, compare lane 5 with
lane 8). Thus, PA binding of NSF appears to occur when the
protein is in an inactive form.
View larger version (42K):
[in a new window]
Fig. 6.
Characteristics of NSF binding to PA
beads. A, recombinant Myc-tagged NSF (lane
1; 5% of input in the binding reaction) was diluted in base
buffer (50 mM Hepes, pH 7.6, 90 mM KCl, 0.4%
Nonidet P-40, 0.1% bovine serum albumin) and divided into seven equal
aliquots. The indicated components were added (100 µM
ATP S, 2 mM MgCl2 or 2 mM EDTA as
required), and the samples were incubated at room temperature for 10 min. At the end of this incubation the samples were returned on ice,
and three different types of beads were added: PA (lanes
2-6), PI (4,5)P2 (lane 7), or uncoupled
Affi-Gel-10 (lane 8). Following binding for 1 h,
washes, SDS-PAGE, and electrotransfer, the blots were probed with
anti-Myc antibodies. B, recombinant Myc-tagged NSF was
treated for 30 min on ice with 3 mM NEM (lanes 2 and 6-8) or with 3 mM NEM plus 5 mM
dithiothreitol (lanes 1 and 3-5). Both samples
then received 4 mM dithiothreitol to neutralize the NEM.
The samples were diluted in base buffer as above and centrifuged to
remove any aggregates. The resultant supernatants (shown in lanes
1 and 2 and representing 5% of input in the binding
reaction) were divided into three equal aliquots, and the indicated
components were added (100 µM ATP
S, 2 mM
MgCl2, or 2 mM EDTA as required) for 10 min at
room temperature. At the end of this incubation the samples were
returned on ice, and PA beads were added. Following binding for 1 h, washes, SDS-PAGE, and electrotransfer, the blots were probed with
anti-Myc antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) binds to the PI (4,5)P2
beads. Because the PA and PI (4,5)P2 beads share the same
palmitoyl moiety, binding of candidate proteins to one but not the
other may be independent of the structure of the acyl chain,
i.e. it is probably to the head group or possibly to the
head group because it is restrained by the acyl chain. A second way to
address specificity was to use soluble PA as a competitor of candidate
protein binding to the PA beads. Using shorter chain analogs of PA-such
as dilauroyl or dioctanoyl, we were able to show for all proteins
discussed in this work that soluble PA in the range of 50-100
µM was able to reduce binding to the beads by 75-95%.
Competition of the binding reaction using phosphate, glycerolphosphate,
or ATP was negative, reinforcing the specificity of candidate proteins
to PA. An additional finding that pertains to the specificity of the PA
beads is that related proteins (such as coatomer versus the
AP-1 and AP-2 coats) show very different binding affinities. Thus,
coatomer bound to PA, whereas the other coats did not. Arguing for
specificity was also the observation that the binding of Arf1 and Arf6
to the PA beads was significantly different, especially with respect to
the activation state of the protein. Thus, binding to immobilized PA is
not a general phenomenon for all proteins that cycle between membrane and cytosol but rather is restricted to a subpopulation of this group.
We are currently addressing the possibility that all these proteins
share a common structural determinant for PA binding.
-arrestin, a protein that
mediates internalization of G protein-coupled receptors, and the AMPA
receptor, an ionotropic glutamate receptor involved in synaptic
transmission (52, 53). Our data indicate that the ADP-bound form of
purified NSF has good affinity for PA. In the context of its life
cycle, this affinity can be relevant either for recruiting NSF to a
PA-rich membrane subdomain for a round of activation or for maintaining
GDP-bound NSF on PA-containing membranes following ATP hydrolysis for
subsequent reactivation. In both of these cases, PA may provide
in trans a phosphate group to stabilize ADP-bound NSF.
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ACKNOWLEDGEMENTS |
---|
This work would not have been possible without the generosity of many colleagues who provided us with antibodies, plasmids, and purified proteins (see "Experimental Procedures"). We also thank Phil Hawkins and Len Stephens for crucial advice in setting up these experiments.
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FOOTNOTES |
---|
* This work was supported by the Biotechnology and Biological Sciences Research Council, the Cambridge Commonwealth Trust, and a Committee of Vice-Chancellors and Principals studentship (to Z.-Y. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Signalling
Programme, Babraham Inst., Babraham, Cambridge CB2 4AT, UK.
Tel.: 44-1223-496323; Fax: 44-1223-496043; E-mail:
nicholas.ktistakis@bbsrc.ac.uk.
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M010308200
2 Z. Y. Lim, J. W. Thuring, A. B. Holmes, M. Manifava, and N. T. Ktistakis, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PA, phosphatidic
acid;
PLD, phospholipase D;
PLC, phospholipase C;
PI
(4, 5)P2, phosphatidylinositol (4,5)-bisphosphate;
Arf, ADP-ribosylation factor;
PAGE, polyacrylamide gel electrophoresis;
ATPS, adenosine 5'-O-(thiotriphosphate).
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REFERENCES |
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