From The University of Utah, Department of Medicinal
Chemistry, Salt Lake City, Utah 84112-5820, the
§ Departments of Chemistry and Biochemistry and Cell
Biology, The University at Stony Brook, Stony Brook, New York
11794-3400, and the ¶ Department of Molecular Biology, Vanderbilt
University, Nashville, Tennessee 37235
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ABSTRACT |
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The phosphoinositide binding selectivity of Golgi
coatomer COPI polypeptides was examined using photoaffinity analogs of
the soluble inositol polyphosphates Ins(1,4,5)P3,
Ins(1,3,4,5)P4, and InsP6, and of the
polyphosphoinositides PtdIns(3,4,5)P3,
PtdIns(4,5)P2, and PtdIns(3,4)P2. Highly
selective Ins(1,3,4,5)P4-displaceable photocovalent
modification of the -COP subunit was observed with a
p-benzoyldihydrocinnamide (BZDC)-containing
probe, [3H]BZDC-Ins(1,3,4,5)P4. A more highly
phosphorylated probe, [3H]BZDC-InsP6
probe labeled six of the seven subunits, with only
,
',
, and
-COP showing competitive displacement by excess InsP6.
Importantly,
[3H]BZDC-triester-PtdIns(3,4,5)P3,
the lipid with the same phosphorylation pattern as
Ins(1,3,4,5)P4, showed specific,
PtdIns(3,4,5)P3-displaceable labeling of only
-COP.
Labeling by the PtdIns(4,5)P2 and
PtdIns(3,4)P2 photoaffinity probes was less intense and
showed no discrimination based on PtdInsPn ligand. Thus,
both the D-3 and D-5 phosphates are critical for the
-COP-PtdIns(3,4,5)P3 interaction, suggesting an
important role for this polyphosphoinositide in vesicular
trafficking.
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INTRODUCTION |
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Coatomer, a complex of seven polypeptides, is the major component
of the non-clathrin (COPI) membrane coat (1). These coat proteins play
a key role in regulating intracellular membrane traffic (2, 3). The
coatomer complex consists of seven subunits: -COP, 170 kDa;
-COP,
110 kDa;
'-COP, 110 kDa;
-COP, 98 kDa;
-COP, 58 kDa;
-COP,
36 kDa; and
-COP, 20 kDa. An eighth essential component is a small,
N-terminal myristoylated GTP-binding protein ADP-ribosylation factor
(ARF)1 (4), which is required
for coatomer assembly on the donor membrane leading to the formation of
coated vesicles (5, 6). ARF associates with
PtdIns(4,5)P2-rich membranes and initiates budding by
providing a binding site for coatomer (7, 8). The conversion of the
inactive, soluble ARF-GDP to active, membrane-associated ARF-GTP form
requires a GTP exchange factor and exposes the buried N-myristoyl group necessary for membrane localization
(9-11). Both the stimulation of phospholipase D by ARF and the
activation of ARF GTPase activity (12) that is important in uncoating
of the vesicles require PtdIns(4,5,)P2 (13, 14). In
addition to phospholipase D, essential roles for phosphatidylinositol
transfer protein, free diacylglycerol, and protein kinase C have been
recently incorporated into models for the budding and vesicle
scission processes (15, 16).
The detailed roles of COPI polypeptides in the directional transport of membrane proteins require further investigation (17). COPI proteins were first proposed to mediate non-selective transport of proteins from the endoplasmic reticulum (ER) through the Golgi complex to the cell surface (8, 18). Evidence in support of the anterograde role, in which COPI-coated vesicles, phospholipase D, and a novel p24 family protein are involved in moving newly translated proteins from the ER to the Golgi, was recently summarized (19). In an alternative model, COPI may be involved principally in selective retrograde transport of membrane proteins from the Golgi complex to the ER by selective association with a dilysine retrieval motif; a separate set of coat proteins, COPII, was identified that seems to function only in the anterograde vesicular transport of cargo from the ER to the Golgi (20). The recent demonstration that yeast COPII directs the formation of vesicles from the ER, and that these vesicles capture both cargo and necessary components of the molecular secretory apparatus further supports the importance of the COPII in anterograde traffic. The most recent data suggests that COPI may be involved in traffic in both directions, as two distinct populations of Golgi-associated COPI-coated vesicles were found in pancreatic cells, each with a different cargo implicating an intended transport direction for the vesicle population (21).
The polypeptide subunits of COPI have been characterized, and selected
aspects of their individual roles are known. First, -COP is the
clathrin-like subunit. It is localized to coated transport vesicles and
coated buds of Golgi membranes derived from Chinese hamster ovary cells
(22). The gene for
-COP has been cloned and characterized (22) from
Saccharomyces cerevisiae. Disruption of this gene in yeast
has been found to be lethal.
-COP has also been shown to be required
for ER localization of dilysine-tagged proteins (23). A novel human
gene, HEP-COP, has been isolated, the product of which is highly
homologous to yeast
-COP (24, 25).
-COP, which is homologous to
the
' subunit of assembly protein 2 (AP-2) (26), has been reported to be essential for transport of protein from the ER to Golgi in
vitro (27). The binding of
-COP with Golgi membranes has been
shown to be enhanced by non-hydrolyzable GTP analogs and AlF4
.
'-COP is homologous to
subunits of heterotrimeric G-proteins.
-COP binds to dilysine motifs
of membrane proteins (28) and is related to Sec 21, a secretory mutant
of the yeast S. cerevisiae (29). A single point mutation in
-COP has been shown to result in temperature-sensitive lethal
defects in membrane transport (30).
-COP has sequence homology to
AP-17 and AP-20 subunits (31). The B complex (
-COP,
'-COP, and
-COP) has been found to interact directly and bind to membranes
(32), while
-COP and
-COP form a second subcomplex (33).
Direct binding studies using purified coatomer isolated from bovine liver cytosol show that coatomer specifically binds both Ins(1,3,4,5)P4 and InsP6 with affinities of 0.1 and 0.2 nM, respectively (34). The degree of phosphorylation of the inositol polyphosphates (InsPn) has been proposed to dictate the order of binding to coatomer (InsP6 > InsP5 > InsP4) (35). Since dissociation of the COPI polypeptides can only be accomplished under conditions that would not permit measurement of InsPn binding, the affinities of individual COPI subunits for InsPn are unknown. Moreover, binding of coatomer complexes to phosphatidylinositol polyphosphates (PtdInsPns) remains unreported. To address the InsPn and PtdInsPn binding specificities of individual COPI subunits, and to obtain evidence to support the roles of these high affinity interactions in vesicular trafficking, we employed a photoaffinity labeling approach with benzophenone-containing InsPn and PtdInsPn analogs (36). The benzophenone photophore allows handling in ambient light, activation at wavelengths >320 nm, and covalent labeling of active site residues in hydrophobic regions of proteins with high efficiency (37, 38). Photoaffinity analogs of soluble inositol polyphosphates Ins(1,4,5)P3, Ins(1,3,4,5)P4, and InsP6 (39, 40) and the lipid-containing polyphosphoinositides PtdIns(3,4,5)P3 (41), PtdIns(4,5)P2 (41), and PtdIns(3,4)P2 (42) are used herein to determine the polyphosphoinositide selectivity for the COPI subunits in Golgi coatomer (39).
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EXPERIMENTAL PROCEDURES |
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Materials--
The specific activities of the
[3H]BZDC-PtdInsPn and InsPn derivatives
used were 42.5 Ci/mmol. D-myo-InsP4, PtdIns(3,4,5)P3, PtdIns(4,5)P2, and
PtdIns(3,4)P2 were synthesized from
methyl--D-glucopyranoside as described
previously (41-43). PtdIns(3)P was prepared
similarly.2 All
phosphoinositides had sn-1,2-dipalmitoyl acyl chains in the diacylglycerol moiety. InsP6 was obtained from Sigma as the
monopotassium salt. Anti-COP antisera were gifts from Dr. C. Harter
(University of Heidelberg). All other chemicals were commercial
products of reagent grade. Solutions were made in
Nanopure® water.
Synthesis of
p-[2,3-3H2]Benzoyldihydrocinnamoyl (BZDC)
Derivatives of Phosphatidylinositol and Inositol
Polyphosphates--
The [3H]BZDC analogs of
PtdInsPn and InsPn were prepared for
Ins(1,4,5)P3, Ins(1,3,4,5)P4,
Ins(1,2,3,4,5,6)P6, PtdIns(4,5)P2,
PtdIns(3,4)P2 and PtdIns(3,4,5)P3 via synthesis of
an -aminoalkyl ester of one of the phosphates followed by amidation
using [3H]BZDC-NHS ester (40). Each tritiated
photoaffinity ligand was purified by ion exchange, and purified probes
were obtained by elution with 0.4 M triethylammonium
bicarbonate buffer (for [3H]BZDC-InsPns) or
0.8-0.9 M triethylammonium bicarbonate (for
[3H]BZDC-PtdInsPn). Thus,
P-2-O-(6-aminopropyl)-InsP6 and its
[3H]BZDC photoaffinity label were prepared as
described earlier (36, 44). P-1-tethered
[3H]BZDC-Ins(1,3,4,5)P4 was prepared by
coupling the
1-O-(3-aminopropyl)-D-myo-Ins(1,3,4,5)P4 (43) with the [3H]BZDC-NHS ester. The synthesis of
O-(3-aminopropyl)-tethered phosphotriester analogs of
PtdIns(3,4,5)P3, PtdIns(4,5)P2, and PtdIns(3,4)P2 was accomplished using the coupling reagent
2-cyano-ethyl-N,N,N',N'-tetra(isopropyl)phosphordiamidite (37, 38, 41). This introduced a reactive aminopropyl group at the P-1
phosphate to which the [3H]BZDC moiety was then
attached.
Purification and Photoaffinity Labeling of Golgi
Coatomer--
Coatomer was purified from bovine liver cytosol as
described (45) to give material of about 60% purity after Mono Q
chromatography. An aliquot of this partially purified Golgi coatomer (6 µg, 0.3 µM) was incubated with 30 µl of buffer A (150 mM KCl, 10% glycerol, 0.5 mM dithiothreitol,
25 mM HEPES/KOH, pH 8.9) and either
[3H]BZDC-InsPn or
[3H]BZDC-PtdInsPn (0.5 µCi, 0.28 µM). The specificity and affinity of binding were
determined by adding an aliquot of an aqueous solution or suspension of
the corresponding unlabeled PtdInsPn or InsPn (0.28 mM) directly into the incubation mixture. Samples were
equilibrated at 4 °C for 10-15 min in a 96-well plate. The wells in
the plate were then aligned with the axis of a UV light source with
minimum distance maintained between the bottom of the wells and the
bulb. The samples were photolysed for 45 min at 4 °C (360 nm at 1900 µW/cm2) (32). Following irradiation, sample buffer (5 ×)
was added to the samples and the proteins were separated by SDS-PAGE
(10% Laemmli gels), stained with Coomassie Blue; the gel was processed for fluorography as described (32), and exposed to XAR-5 x-ray film for
7-14 days at 80 °C. There was no covalent incorporation of the
photolabel in the absence of UV irradiation. Fluorograms were digitized
on a UMAX-UC 840 scanner using Adobe Photoshop (Macintosh version
2.0.1). The densities of the bands on the fluorogram were determined by
NIH IMAGE 1.59 to calculate relative incorporation.
Disassembly of Golgi Coatomer (33)-- Protein (30 µg, 0.05 µM) was incubated in 1 ml of buffer B (1 M NaCl, 20 mM Tris, pH 7.5, 2 mM EDTA, 1 mM dithiothreitol, 0.5% Triton X-100) and gently shaken and incubated for 1 h at 4 °C. This mixture was applied to a gel filtration high performance liquid chromatography column (TSK-GEL, G3000SW, 7.5 mm inner diameter), flow rate 0.5 ml/min, 30-min run, isocratic elution using buffer C (25 mM Tris, pH 7.46, 0.4 M NaCl). The high performance liquid chromatography system was calibrated using molecular weight protein markers, and 1-min fractions were collected. The 16-min fraction, corresponding to mass 315 kDa, was photolabeled as described above.
Immunoprecipitation of Golgi Coatomer Subunits
(46)--
Coatomer (6 µg, 0.3 µM) was photoaffinity
labeled with [3H]BZDC-PtdIns(3,4,5)P3 (0.5 µCi, 0.28 µM) as described above. The labeled mixture
was then incubated for 2 h at 4 °C with the indicated antibodies (3 and 4 µl of anti--COP antisera and 1 and 2 µl of anti-
-COP antisera, respectively) in 250 µl of immunoprecipitation buffer (IP buffer: 20 mM Tris-HCl, pH 7.5, 2 mM
EDTA, 0.15 M NaCl, 0.5% Triton X-100). Protein A-Sepharose
beads (20 µl) (Pharmacia) were then added to the mixture and
incubated for another 2 h at 4 °C. The beads were washed once
with IP buffer, and then incubated overnight in IP buffer containing 1 M NaCl. Subsequent wash steps were performed in IP buffer
(3 ×) and finally in phosphate-buffered saline, pH 7.4. Sample buffer
(5 ×) was added to the washed pellets and the immunoprecipitate was
separated by SDS-PAGE (10% Laemmli gels). The gel was stained with
Coomassie Blue, and processed for fluorography as described above.
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RESULTS |
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Synthesis of Photoaffinity Labels--
Five photoaffinity labels
(Fig. 1) were used to study the coatomer
subunit specificity and selectivity for different InsPn and
PtdInsPn probes. Each InsPn and PtdInsPn photoaffinity probe was prepared from the corresponding 1- or 2-O-(-aminoalkyl)-InsPn or -PtdInsPn
derivative with the heterobifunctional reagent
[3H]BZDC-NHS ester (38) and had the same nominal specific
activity of 42.5 Ci/mmol. This ensured that levels of radioactivity in each probe corresponded to equivalent concentrations, thereby allowing
direct comparison of relative efficiencies of photoattachment.
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Photoaffinity Labeling of Golgi Coatomer-- Selectivity of the probes for coatomer subunits was determined by photoaffinity labeling experiments employing [3H]BZDC-InsPn or [3H]BZDC-PtdInsPn probes. Specific binding was determined by competitive displacement of photocovalent modification in the presence of a 1000-fold excess of unlabeled (Ptd)InsPn. The specificity could be approximated by the difference between the total binding (no competitor) and binding in the presence of the competing ligand. Since coatomer has been demonstrated to bind Ins(1,3,4,5)P4 and InsP6 with subnanomolar affinities (0.1 and 0.2 nM, respectively (34)), we initially employed the [3H]BZDC-InsP4 and [3H]BZDC-InsP6 probes for photoaffinity labeling of the COPI subunits.
Fig. 2 shows the photolabeling of coatomer polypeptides with the P-1-tethered [3H]BZDC-Ins(1,3,4,5)P4 probe. This probe exhibited specific labeling of the 170-kDa subunit
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DISCUSSION |
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Benzophenone-containing photoaffinity labels (37) have proven to be extremely useful as tools for identification of new PtdInsPn- and InsPn-binding proteins (47), characterization of their ligand-binding sites, and verification of their PtdInsPn and InsPn selectivity (36). The advantages of benzophenone over the classical arylazide photochemistry include improved chemically stability of ligands and adducts, stability in ambient light, low background from nonspecific labeling, and the efficient C-H insertion of the triplet diradicaloid intermediate formed by irradiation at 360 nm (39). Herein we report an application of this photochemical technique to study the subunit specificity of these benzophenone-tethered InsPn and Ptd-InsPn probes with Golgi coatomer COPI polypeptides.
Initially, photoaffinity labeling studies on bovine coatomer employed
soluble Ins(1,3,4,5)P4 and InsP6 photoprobes,
since it had been reported that these ligands bound to coatomer with subnanomolar affinities (34). We found that
[3H]BZDC-Ins(1,3,4,5)P4 exhibited
exquisite selectivity for labeling of the -COP subunit, with
complete displacement by the soluble ligand Ins(1,3,4,5)P4.
Interestingly, [3H]BZDC-InsP6 exhibited a
much more complex labeling profile. Virtually all COP subunits were
photocovalently modified, but only
-COP,
'-COP,
-COP, and
-COP showed InsP6- and
Ins(1,3,4,5)P4-competable labeling. The failure of these
two ligands to displace labeling of the
-COP and
-COP subunits
can be attributed to a dual hydrophobic-electrostatic interaction of
the phosphoinositide-like photoprobe with the protein that could not be
disrupted by the electrostatic component only.
The probes employed in this study (Fig. 1) are more hydrophobic than
the endogenous ligands Ins(1,3,4,5)P4 and InsP6
due to the presence of the photoactivatable BZDC moiety and the
aminopropyl phosphate ester. Indeed, BZDC-Ins(1,3,4,5)P4 is
a reasonable structural surrogate for the inositol phospholipid
PtdIns(3,4,5)P3 (40), as previously observed for the
PtdIns(3,4,5)P3-binding protein centaurin- (47).
Analogously, [3H]BZDC-Ins(1,4,5)P3 has been
used as a probe to study the PtdIns(4,5)P2-binding sites of
the pleckstrin homology domain of recombinant phospholipase C
1 isozyme (50) and of recombinant human profilin
I.3
Coatomer has been shown to be similar to AP-2 and cardiac AP-3 in that all three proteins formed K+ channels when incorporated into planar lipid bilayers (34, 51), and each exhibited high affinity binding to certain InsPns. PtdIns(3,4,5)P3 has been shown to be a high affinity ligand for AP-2 (52) and AP-3 (53). Phosphorylated phosphatidylinositol may cooperate with membrane proteins in the recruitment of cytosolic proteins for certain vesicle coats (50). It has been postulated that the binding of a coat protein to the head group of a phospholipid may orient the coat protein and facilitate side-to-side association through homophilic-heterophilic interaction with other proteins to generate the coat (54). To test this hypothesis, we examined PtdInsPn-coatomer interactions using photoaffinity labeling.
Photoaffinity labeling with the
[3H]BZDC-triester-PtdInsPn
(n = 2 and 3) probes was highly specific, in analogy to that observed with [3H]BZDC-Ins(1,3,4,5)P4.
Thus, -COP was labeled exclusively, and the rank order of labeling
intensities was
[3H]BZDC-triester-PtdIns(3,4,5)P3 > -PtdIns(3,4)P3 > -PtdIns(4,5)P2. Similarly, the concentration dependence of displacement of the labeling
of
-COP by
[3H]BZDC-triester-PtdIns(3,4,5)P3
showed that among the D-3 phosphoinositides, only
PtdIns(3,4,5)P3 showed full competitive displacement below the 1000-fold molar excess, with the monophosphate PtdIns(3)P and
the bisphosphate PtdIns(3,4)P2 showing substantially lower affinity. The photoaffinity-labeled 170-kDa protein was uniquely immunoprecipitated by antibodies against
-COP but not by those raised against
-COP, verifying the identity of the labeled protein as
-COP and not a co-migrating protein. Importantly, the rigorous high salt washes employed prior to electrophoresis of the
immunoprecipitated protein ensured that only
-COP was present in the
170-kDa band.
A human phosphatidylinositol (PI)-specific 3-kinase activity has been implicated in non-clathrin-mediated Golgi membrane traffic (55, 56). This PI 3-kinase complex has been related to the yeast Vps34p-Vps15p protein sorting. Our data thus reflect the potential role of Golgi coatomer as a ligand for PtdInsPns and emphasize the potential role of a PI 3-kinase on its recruitment to membranes. Coatomer bound to the products (PtdIns(3,4,5)P3 and PtdIns(3,4)P2) of a PI 3-kinase with higher affinity than a potential substrate PtdIns(4,5)P2. Also, the substrate PtdIns(4,5)P2 was unable to displace the binding of the product PtdIns(3,4,5)P3.
The phosphoinositide products of PI 3-kinase have pivotal roles in regulation of protein trafficking, cell survival, cell growth, actin rearrangement, and cell adhesion (57). Indeed, the actions of a variety of proteins implicated in membrane trafficking and in exo- and endocytosis are modulated by interactions with PtdInsPns (54). For example, PtdIns(3,4,5)P3 binds specifically and saturably to soluble AP-2, and this binding inhibits the clathrin binding and assembly activities of this heterotetrameric protein (52). Similarly, the brain-derived assembly protein AP-3 (a.k.a. AP-180) also showed preferential binding to and functional regulation by PtdIns(3,4,5)P3 (53). In the synaptic vesicle cycle, synaptotagmin I acts as a bimodal calcium-regulated switch, binding with high affinity to PtdIns(3,4,5)P3-containing liposomes at [Ca2+] below 1 µM, but preferentially to PtdIns(4,5)P2-containing liposomes at calcium concentrations above 10 µM. In addition, phospholipase D is activated by polyphosphoinositides (13) and has been shown to mediate ARF-dependent formation of Golgi-coated vesicles (14). Ktistakis and co-workers (14) have demonstrated that purified coatomer binds selectively to artificial lipid vesicles that contain phosphatidic acid and PtdIns(4,5)P2.
We investigated the effects of salt concentration on the photoaffinity
labeling of the COPI polypeptides, since binding of InsPns to
coatomer was previously reported to be highest at pH 8.9 with 140 mM KCl (34) and decreased with increased salt
concentrations. In corroboration of these results, no photoaffinity labeling was observed at or below pH 7.5 (data not shown). Moreover, no
labeling was observed above 300 mM KCl, while up to 500 mM CaCl2 had no apparent effect on labeling.
High (millimolar) GTP concentrations were reported to block the
K+ channel activity on coatomer (34) but had little effect
on its InsPn binding. The results herein reflect on a similar behavior for the interaction of PtdInsPns with -COP. In
addition, the inability of BFA or GTP to interfere with the PtdInsPn-
-COP interaction suggests that separate,
non-allosterically regulated binding sites are involved. Thus, the
PtdIns(3,4,5)P3-
-COP interaction appears to be
independent of ARF binding and the coatomer recruitment process.
The inability of the chromatographically isolated B complex of -COP,
'-COP, and
-COP complex to bind PtdIns(3,4,5)P3
suggests that the PtdIns(3,4,5)P3-
-COP binding may
involve a more complex set of protein-protein interactions. Thus,
conformational changes due to subunit interactions may be required to
permit PtdInsPn binding to
-COP. Alternatively, the observed
failure of the B complex to undergo photoaffinity labeling could be an
artifact of a non-reversible effect resulting from the buffer
conditions required for subunit dissociation. However, the
physiological significance of this dissociated B complex is not clear,
despite reports of its binding to membranes (33).
In conclusion, the data presented offer the first evidence for a
specific interaction of one, and only one, polypeptide subunit of Golgi
coatomer, -COP, with the polyphosphoinositide
PtdIns(3,4,5)P3. Moreover, these data also demonstrate the
specificity of interactions of the soluble inositol polyphosphates
Ins(1,3,4,5)P4 and InsP6 with individual
coatomer polypeptides. This result offers a new perspective on the
potential role of PI 3-kinase in non-clathrin-mediated Golgi membrane
traffic. Moreover, while the 3-phosphate on the inositol ring plays a
critical role in defining this interaction, the 5-phosphate is also
required for maximal binding activity. In addition, the importance of a
hydrophobic group, either the aminopropyl BZDC group or the
diacylglycerol moiety, suggests that the
Ptd(3,4,5)InsP3-
-COP interaction might be important for
the recruitment (or its inhibition by conformational change) of
coatomer to membranes during the budding and/or coating process. To
date,
-COP has not been implicated as an active participant in COPI
recruitment and vesicle coating;
-COP has been more fully examined
for its importance for coat assembly and in the budding reaction (27).
Our results suggest that a re-examination of protein-polyphosphoinositide, as well as protein-protein interactions, will further illuminate the complex process of vesicular trafficking. Finally, the results reported herein represent an application of a new
class of Ptd(3,4,5)InsPn photoaffinity probes that sample the
interface between the charged phosphoinositide head group and the lipid
bilayer. Additional examples of the uses of these
[3H]BZDC-triester-PtdInsPn probes for
characterization of other protein targets will be presented in due
course.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. D. G. Ahern of
NEN Life Science Products (Boston, MA) for providing the
[3H]BZDC-NHS ester and Dr. J. D. Olszewski for
preparation of an initial sample of
[3H]BZDC-InsP6. We acknowledge the generous
assistance of Drs. J. Ostermann and C. Chee in the preparation of
coatomer. We are also grateful to Drs. F. T. Wieland and C. Harter
for providing the anti--COP and anti-
-COP antisera.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health Grants NS 29632 (to G. D. P.) and HL 32711 (to S. F.).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: The University of
Utah, Dept. of Medicinal Chemistry, 30 South, 2000 East, Rm. 201, Salt
Lake City, UT 84112-5820. Tel.: 801-585-9051; Fax: 801-585-9053; E-mail: gprestwich{at}deans.pharm.utah.edu.
1 The abbreviations used are: ARF, ADP-ribosylation factor; AP, assembly protein; BFA, brefeldin A; COP, Golgi coat protein; ER, endoplasmic reticulum; [3H]BZDC, [3H]-p-benzoyldihydrocinnamoyl; Ins(1,3,4,5)P4, D-myo-inositol 1,3,4,5-tetrakisphosphate; InsPn, D-myo-inositol polyphosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdInsPn, phosphatidylinositol polyphosphate; PI, phosphoinositide; IP, immunoprecipitation; PAGE, polyacrylamide gel electrophoresis.
2 J. Chen, unpublished results.
3 A. Chaudhary, J. Chen, Q.-M. Gu, W. Witke, D. Kwiatkowski, and G. D. Prestwich, submitted for publication.
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REFERENCES |
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