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J Biol Chem, Vol. 273, Issue 14, 8344-8350, April 3, 1998

Specific Interaction of Golgi Coatomer Protein alpha -COP with Phosphatidylinositol 3,4,5-Trisphosphate*

Anu ChaudharyDagger §, Qu-Ming Gu§, Oliver Thum§, Adam A. Profit§, Ying Qi, Loice Jeyakumar, Sidney Fleischer, and Glenn D. PrestwichDagger §par

From Dagger  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

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha -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 beta , beta ', delta , and epsilon -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 alpha -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 alpha -COP-PtdIns(3,4,5)P3 interaction, suggesting an important role for this polyphosphoinositide in vesicular trafficking.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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: alpha -COP, 170 kDa; beta -COP, 110 kDa; beta '-COP, 110 kDa; gamma -COP, 98 kDa; delta -COP, 58 kDa; epsilon -COP, 36 kDa; and zeta -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, alpha -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 alpha -COP has been cloned and characterized (22) from Saccharomyces cerevisiae. Disruption of this gene in yeast has been found to be lethal. alpha -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 alpha -COP (24, 25). beta -COP, which is homologous to the beta ' 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 beta -COP with Golgi membranes has been shown to be enhanced by non-hydrolyzable GTP analogs and AlF4-. beta '-COP is homologous to beta  subunits of heterotrimeric G-proteins. gamma -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 epsilon -COP has been shown to result in temperature-sensitive lethal defects in membrane transport (30). zeta -COP has sequence homology to AP-17 and AP-20 subunits (31). The B complex (alpha -COP, beta '-COP, and epsilon -COP) has been found to interact directly and bind to membranes (32), while gamma -COP and zeta -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).

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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-alpha -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 omega -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-alpha -COP antisera and 1 and 2 µl of anti-beta -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.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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-(omega -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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  		Fig. 1.   Structures of probes used for photoaffinity labeling of Golgi coatomer. Structures of [3H]BZDC derivatives of P-1-O-(omega -aminopropyl)-Ins(1,3,4,5)P4, P-2-O-(omega -aminohexyl)-InsP6, and P-1 phosphotriester analogs of PtdIns(4,5)P2, PtdIns(3,4)P2, and PtdIns(3,4,5)P3.

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 alpha -COP. Labeling was competitively displaced by a 1000-fold excess (0.28 mM) of Ins(1,3,4,5)P4 and InsP6. Thus, both unlabeled Ins(1,3,4,5)P4 and InsP6 appeared to compete for the same binding site as [3H]BZDC-Ins(1,3,4,5)P4.


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  		Fig. 2.   Photoaffinity labeling of Golgi coatomer with [3H]BZDC-Ins(1,3,4,5)P4. Fluorogram of the 10% SDS-PAGE gel, with Coomassie Blue staining shown at right. Arrow indicates labeled protein (alpha -COP) at 170 kDa. Photoaffinity labeling with 0.28 µM [3H]BZDC-Ins(1,3,4,5)P4 was conducted in the absence of competitor (lane a) or in the presence of a 1000-fold excess of InsP6 (lane b) or Ins(1,3,4,5)P4 (lane c).

Coatomer has also been shown to have high affinity binding interactions with InsP6. To study the polypeptides involved in this binding, we employed the P-2-tethered [3H]BZDC-InsP6 probe. Fig. 3 shows the labeling obtained with this probe. [3H]BZDC-InsP6 exhibited intense labeling of most proteins in this partially purified preparation. Interestingly, addition of competitors InsP6 and Ins(1,3,4,5)P4 (0.28 mM) competitively displaced the labeling from beta -COP, beta '-COP, delta -COP, and epsilon -COP, indicating specific labeling of these subunits. However, labeling of the alpha -COP and gamma -COP resisted competitive displacement, suggesting that the soluble probe might not be sufficient to displace a combined electrostatic-hydrophobic interaction afforded by the photoaffinity analog.


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  		Fig. 3.   Photoaffinity labeling of Golgi coatomer with [3H]BZDC-InsP6. Fluorogram of 10% SDS-PAGE gel. Arrow indicates subunits. Competitively labeled proteins (beta -COP, beta '-COP, delta -COP, and epsilon -COP) are at 110, 110, 58, and 36 kDa, respectively. Photoaffinity labeling with 0.28 µM [3H]BZDC-InsP6 was conducted in the absence of competitor (lane a) or in the presence of a 1000-fold excess of InsP6 (lane b) or Ins(1,3,4,5)P4 (lane c).

Evidence from our laboratories using photoaffinity labeling (39, 40) has implicated highly selective binding of PtdInsPn derivatives to a number of proteins important in the budding and fusion of lipid bilayers, as well as those known to be recruited to polyphosphoinositide-rich membranes. Thus, coatomer was labeled with a series of [3H]BZDC-triester-PtdInsPn (n = 2 or 3) probes (40) that have diacylglycerol moieties for membrane anchoring, the correct phosphorylation pattern on the D-myo-inositol ring, and a photoactivatable group that can covalently modify proteins recruited to the membrane surface. Fig. 4 shows that each of the PtdInsPn probes, [3H]BZDC-triester-PtdIns(3,4,5)P3, -PtdIns(3,4)P2, and -PtdIns(4,5)P2 (Fig. 1), labeled only alpha -COP with high subunit and PtdInsPn selectivity.


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  		Fig. 4.   Photoaffinity labeling of Golgi coatomer with [3H]BZDC-PtdInsn probes. Panel A, fluorogram of 10% SDS-PAGE gels, with Coomassie Blue-stained lane at the right. Arrow indicates labeled protein (alpha -COP) at 170 kDa. Lanes a-d, [3H]BZDC-triester-PtdIns(3,4,5)P3 in the absence of competitors (lane a) or in the presence of a 1000-fold excess of PtdIns(3,4,5)P3 (lane b), PtdIns(4,5)P2 (lane c), or PtdIns(3,4)P2 (lane d). Lanes e-h, [3H]BZDC-triester-PtdIns(4,5)P2 in the absence of competitors (lane e) or in the presence of a 1000-fold excess of PtdIns(3,4,5)P3 (lane f), PtdIns(4,5)P2 (lane g), or PtdIns(3,4)P2 (lane h). Lanes i-l, [3H]BZDC-triester-PtdIns(3,4)P2 in the absence of competitors (lane i) or in the presence of a 1000-fold excess of PtdIns(3,4,5)P3 (lane j), PtdIns(4,5)P2 (lane k), or PtdIns(3,4)P2 (lane l). Panel B, quantitation of the photolabeling data in panel A using NIH IMAGE 1.59. Intensity of the photolabeled bands is expressed as a percentage of binding relative to [3H]BZDC-triester-PtdIns(3,4,5)P3 binding. Blanks indicate that no labeling was observed.

Four main points should be emphasized in interpreting the results of the PtdInsPn photoaffinity labeling. First, the labeling profile obtained with the [3H]BZDC-triester-linked bis- and trisphosphoinositides was similar to that observed with the [3H]BZDC-Ins(1,3,4,5)P4 probe. Only the alpha -COP subunit was labeled significantly with each of these probes. This is consistent with the previous observation that centaurin-alpha , a PtdIns(3,4,5)P3-binding protein, was also selectively labeled by this soluble Ins(1,3,4,5)P4 photoaffinity probe (47). The three-carbon linker with the appended benzoyldihydrocinnamoyl amide group appears to act as a 2-desoxy-acylglycerol analog of PtdIns(3,4,5)P3 (40).

Second, the labeling intensities indicated that the relative affinities of the three triester polyphosphoinositide probes for alpha -COP are in the order [3H]BZDC-triester-PtdIns(3,4,5)P3 > -PtdIns(3,4)P2 > -PtdIns(4,5)P2. These data further support the hypothesis that this site is selective for binding to the phosphatidylinositol 3,4,5-trisphosphate.

Third, a 1000-fold molar excess of either PtdIns(3,4,5)P3 or PtdIns(3,4)P2 completely displaced labeling of alpha -COP by [3H]BZDC-triester-PtdIns(3,4,5)P3, but the same excess of PtdIns(4,5)P2 showed only limited displacement. The observation that PtdIns(3,4,5)P3 as well as both PtdIns(4,5)P2 and PtdIns(3,4)P2 completely displaced the covalent modification of alpha -COP by either of the [3H]BZDC-triester-PtdIns(3,4)P2 or -PtdIns(4,5)P2 probes suggests a lack of head group discrimination for the labeling by the two bisphosphoinositide triester probes. This further supports the hypothesis that alpha -COP has a PtdIns(3,4,5)P3-specific binding site.

Fourth, a titration of the competitive displacement of the photoaffinity labeling of the 170-kDa alpha -COP band by three D-3 phosphoinositides was performed (Fig. 5), using the pure synthetic di-C16 polyphosphoinositides (40). The monophosphate PtdIns(3)P showed less than 50% displacement at 1000-fold molar excess relative to the [3H]BZDC-triester-PtdIns(3,4,5)P3 probe, while PtdIns(3,4)P2 showed displacement only at the 1000-fold level. In contrast, even a modest 10-fold excess of PtdIns(3,4,5)P3 showed >50% displacement of the labeling. Because the photoaffinity experiment is intrinsically a non-equilibrium process, "all-or-nothing" displacement is often observed for low affinity competitors.


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  		Fig. 5.   D-3 phosphoinositide specificity of photoaffinity labeling of Golgi coatomer subunits by [3H] BZDC-triester-PtdIns(3,4,5)P3. Fluorogram of 10% SDS-PAGE gels. Arrow indicates labeled protein (alpha -COP) at 170 kDa. Minor nonspecific labeling of other proteins is due to overexposure of film. Lane a, no competitor. Lanes b-e, PtdIns(3)P at 10-, 100-, 500-, and 1000-fold excess; lanes f-h, PtdIns(3,4,5)P3 at 10-, 100-, and 500-fold excess; lanes g-l, PtdIns(3,4)P2 at 10-, 100-, 500-, and 1000-fold excess.

To verify that the labeled 170-kDa band was indeed alpha -COP and not an unrelated protein of molecular weight similar to alpha -COP, immunoprecipitation experiments were performed with the anti-alpha -COP and anti-beta -COP subunit specific antibodies. Fig. 6 shows the SDS-10% PAGE gel and corresponding fluorogram of the immunoprecipitation of the photoaffinity-labeled protein. The anti-beta -COP antisera immunoprecipitated the beta -subunit at 110 kDa, but no radiolabel was present. (The rigorous washing conditions used for the immunoprecipitated, protein A-Sepharose bound protein, including an overnight incubation in 1 M NaCl, effectively dissociates all the COPI subunits and would remove any cargo proteins.) In contrast, the anti-alpha -COP antibody immunoprecipitated the 170-kDa alpha -COP subunit (as seen on the SDS-PAGE gel). Moreover, this subunit contained the [3H]BZDC-triester-PtdIns(3,4,5)P3 label, providing conclusive evidence for the interaction of this subunit with PtdIns(3,4,5)P3. Negligible amounts of radioactivity were detected in the wash buffers, in contrast to the label recovered in the high salt washes of the anti-beta -COP-precipitated proteins. Interestingly, the anti-alpha -COP antibody used was developed against the C terminus of the alpha -subunit, strongly suggesting that the PtdIns(3,4,5)P3-binding site on alpha -COP is not contained within the epitope for antibody recognition on this subunit.


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  		Fig. 6.   Immunoprecipitation of Golgi coatomer subunits employing anti-alpha - and anti-beta -COP antisera. Top, 10% SDS-PAGE of the immunoprecipitation reaction. Lanes a and b, with 1 and 2 µl of anti-beta -COP antisera, respectively. Lanes c and d, with 3 and 4 µl of anti-alpha -COP antisera, respectively. Bottom, fluorogram corresponding to the 10% SDS-PAGE shown above. Partially purified protein was photoaffinity labeled with [3H]BZDC-triester-PtdIns(3,4,5)P3 as described earlier. Immunoprecipitation was performed employing the indicated antibodies. Coatomer dissociation (IP buffer with 1 M NaCl) was then performed as a wash step.

To assess the role of other coatomer subunits in alpha -COP-PtdIns(3,4,5)P3 interaction, coatomer subunits were partially dissociated using a high salt buffer (33). After buffer exchange and gel filtration, the complex of alpha -COP, beta '-COP, and epsilon -COP (B complex, about 316 kDa) was photoaffinity labeled with the [3H]BZDC-triester-PtdIns(3,4,5)P3 probe. Surprisingly, no labeling was observed on this heterotrimeric complex (data not shown), which has been reported to bind to membranes (32, 33).

To examine the possibility that PtdIns(3,4,5)P3 binding might be involved in the ARF-mediated recruitment of coatomer, two experiments were performed. Brefeldin A (BFA), which has been shown to decrease beta -COP binding to membranes (48), had no effect on the [3H]BZDC-triester-PtdIns(3,4,5)P3 photolabeling of alpha -COP (Fig. 7A). This fungal metabolite has been shown to prevent the assembly of coatomer onto the membrane by inhibiting the GTP-dependent interaction of ARF with the Golgi membrane (49). Addition of up to 200 µM BFA did not affect the covalent modification of alpha -COP by the PtdIns(3,4,5)P3 photoaffinity probe. Similarly, addition of up to 10 mM GTP was found to have no effect on this binding. These results further support a specific interaction between PtdIns(3,4,5)P3 and alpha -COP.


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  		Fig. 7.   Effects of BFA, GTP, KCl, and CaCl2 on photoaffinity labeling of alpha -COP. Panel A, effect of BFA (lanes c and d), and GTP (lanes e-i) on the photoaffinity labeling of Golgi coatomer with [3H]BZDC-triester-PtdIns(3,4,5)P3. Fluorogram of the 10% SDS-PAGE gel, labeled as in Fig. 4. Lane a, no competitor; lane b, 0.28 mM PtdIns(3,4,5)P3; lanes c and d have 10 and 200 µM BFA, respectively. Lanes e-i have 1 µM, 100 µM, 1 mM, 5 mM, and 10 mM GTP, respectively. Lanes j and k are identical to lane h (5 mM GTP) with 150 and 500 mM KCl, respectively. Panel B, effect of salt concentrations of photoaffinity labeling coatomer with [3H]BZDC-triester-PtdIns(3,4,5)P3. Lanes a-d each contains 140 mM KCl in buffer A, with increasing amounts of CaCl2 (none, 0.10, 150, and 500 mM, respectively). Lanes e-g show the effects of increasing amounts of KCl in buffer A (300, 500, and 1000 mM, respectively).

Finally, the effects of salts on this interaction were investigated (Fig. 7B), since the high affinity InsP6-coatomer interaction had been reported to exhibit salt dependence (34). Addition of up to 500 mM CaCl2 had no effect on the labeling of alpha -COP by [3H]BZDC-triester-PtdIns(3,4,5)P3. However, presence of greater than 300 mM KCl inhibited labeling. Interestingly, addition of both 5 mM GTP and 150 mM KCl was also found to inhibit the labeling.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha -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 beta -COP, beta '-COP, delta -COP, and epsilon -COP showed InsP6- and Ins(1,3,4,5)P4-competable labeling. The failure of these two ligands to displace labeling of the alpha -COP and gamma -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-alpha (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 Cdelta 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, alpha -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 alpha -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 alpha -COP but not by those raised against beta -COP, verifying the identity of the labeled protein as alpha -COP and not a co-migrating protein. Importantly, the rigorous high salt washes employed prior to electrophoresis of the immunoprecipitated protein ensured that only alpha -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 alpha -COP. In addition, the inability of BFA or GTP to interfere with the PtdInsPn-alpha -COP interaction suggests that separate, non-allosterically regulated binding sites are involved. Thus, the PtdIns(3,4,5)P3-alpha -COP interaction appears to be independent of ARF binding and the coatomer recruitment process.

The inability of the chromatographically isolated B complex of alpha -COP, beta '-COP, and epsilon -COP complex to bind PtdIns(3,4,5)P3 suggests that the PtdIns(3,4,5)P3-alpha -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 alpha -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, alpha -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-alpha -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, alpha -COP has not been implicated as an active participant in COPI recruitment and vesicle coating; beta -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.

    ACKNOWLEDGEMENTS

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-alpha -COP and anti-beta -COP antisera.

    FOOTNOTES

* 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.

par 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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Results
Discussion
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