©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inositol Hexakisphosphate Binds to Clathrin Assembly Protein 3 (AP-3/AP180) and Inhibits Clathrin Cage Assembly in Vitro(*)

(Received for publication, September 20, 1994)

F. Anderson Norris Ernst Ungewickell Philip W. Majerus

From the Division of Hematology-Oncology, Departments of Internal Medicine Biochemistry and Molecular Biophysics, and the Center for Immunology in the Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
experimental procedures
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We have isolated an inositol hexakisphosphate binding protein from rat brain by affinity elution chromatography from Mono S cation exchange resin using 0.1 mM inositol hexakisphosphate (InsP(6)). The amino acid sequences of six tryptic peptides from the protein were identical to the sequences predicted from the cDNA encoding a previously isolated protein designated as AP-3 or AP180. This protein is localized in nerve endings and promotes assembly of clathrin into coated vesicles. The isolated protein-bound InsP(6) with a dissociation constant of 1.2 µM and a stoichiometry of 0.9 mol of InsP(6) bound/mol of AP-3. Recombinant AP-3 expressed in Escherichia coli also bound InsP(6) with a similar affinity. InsP(6) inhibited clathrin cage assembly mediated by AP-3, in an in vitro assay, but had little effect AP-3 binding to preformed cages. We speculate that InsP(6) and perhaps highly phosphorylated inositol lipids may play a role in coated vesicle formation.


INTRODUCTION

Inositol polyphosphates containing five- and six-phosphate groups (inositol pentakisphosphate and inositol hexakisphosphate) are ubiquitous in eukaryotic cells, yet their functions remain unknown. In an attempt to define proteins that interact with these moieties Theibert et al.(1) and Chadwick et al.(2) isolated inositol polyphosphate binding proteins from rat and bovine brain, respectively. Both groups showed that the protein isolated was identical to clathrin assembly protein 2 (AP-2)(^1)(3, 4) . This conclusion was based on the finding that the amino acid sequence of the inositol polyphosphate binding proteins was identical to that of the alpha subunit of AP-2. AP-2 is thought to function in the formation of coated vesicles at the plasma membrane by recruiting receptors and clathrin into growing coated pits. Beck and Keen (5) have shown that inositol polyphosphates inhibit AP-2-mediated clathrin assembly in an in vitro assay.

We recently devised a scheme for the isolation of inositol polyphosphate 4-phosphatase, an enzyme of the inositol phosphate signaling pathway, that utilized an affinity elution protocol(6) . In the course of this work, we noted a major contaminant protein of approximately 160 kDa that was eluted from a cation-exchange resin (Mono S) by low concentrations of inositol hexakisphosphate (InsP(6)). We now report that this protein is identical to a protein that was previously isolated as clathrin assembly protein 3 (AP-3), another protein involved in assembly of clathrin-coated vesicles(7, 8, 9) . We also report that both native and recombinant AP-3 bind inositol polyphosphates with high affinity and that InsP(6) inhibits AP-3-mediated clathrin assembly.


experimental procedures

Materials

[^3H]-InsP(6)(24 Ci/mmol) was purchased from Dupont NEN. Mono S 5/5 column was purchased from Pharmacia Biotech Inc. Polyethylene glycol 4000 was purchased from Fluka. Unstripped rat brains were purchased from Pelfreez. Trypsin, sequencing grade, (modified) was obtained from Boehringer Mannheim. Inositol 1,3,4,5,6-pentakisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol 1,4,5-trisphosphate were purchased from Calbiochem. Phytic acid (InsP(6)), bovine -globulin, iodoacetamide, and all other chemicals were purchased from Sigma.

Purification of Rat Brain AP-3

The purification of AP-3 was performed as described(6) . Briefly, a partially purified preparation of inositol-polyphosphate 4-phosphatase was loaded unto a Mono S column in 10 mM Tricine, pH 8.5, containing 1 mM EDTA, 1 mM dithiothreitol, and 20% glycerol, (buffer A). The column was then reequilibrated with 20 mM HEPES, pH 7.5, containing 1 mM EDTA, 1 mM dithiothreitol, and 20% glycerol, (buffer B). AP-3 was then eluted with buffer B containing 0.1 mM InsP(6).

Trypsinization and Peptide Sequencing

Purified AP-3 (200 µg) was dried in a vacuum centrifuge and then resuspended in 50 µl of 0.4 M NH(4)HCO(3) containing 8 M urea. 5 µl of 45 mM dithiothreitol was added, and the sample was incubated for 15 min at 50 °C. The sample was cooled to room temperature and 5 µl of 100 mM iodoacetamide was added, and the sample was incubated in the dark for 15 min at room temperature. Deionized water (140 µl) and 10 µg of modified trypsin was added, and the sample was incubated for 24 h at 37 °C. The sample was evaporated to 100 µl in a vacuum centrifuge, and then 5 µl of trifluoroacetic acid was added, and the sample was degassed overnight in a vacuum desiccator. The sample was then loaded onto an Aquapore BU-300 microbore HPLC column, and peptides were eluted with a gradient of 0-100% acetonitrile in 0.1% trifluoroacetic acid. The eluted peptides were sequenced using an Applied Biosystems model 477A sequencer.

Measurement of AP-3

The amount of AP-3 was determined by amino acid analysis or spectrophotometrically using an extinction coefficient of

of 4.6(10) . Recombinant AP3 was produced by inserting the full-length AP-3 cDNA clone Pst36 (10) into the expression vector PQE 31 containing a histidine tag (Qiagen). The protein was expressed in Escherichia coli (m15[pREP4]) upon induction with 3 mM IPTG for 2 h. The protein was purified from the bacterial lysate by affinity chromatography on a Ni-containing resin according to the manufacturer's instructions followed by gel filtration as will be described in detail elsewhere.

Clathrin Assembly Assay

Bovine brain clathrin was purified from brains (Pelfreeze) as described previously (11) and dialyzed against 10 mM Tris-HCl, pH 8.0, containing 0.02% NaN(3).

Clathrin (200 µg/ml) and AP-3 (50 µg/ml) were incubated with InsP(6) for 10 min on ice. Assembly was induced by addition of 10 times concentrated buffer C to give a final concentration of 1times. The final composition of buffer C was 0.1 mM MES, pH 6.5, 0.5 mM MgCl(2), 1 mM EGTA, and 0.02% NaN(3). After 1 h on ice, assembly was analyzed by ultracentrifugation for 20 min at 100,000 times g in a Beckman TL-100 centrifuge followed by SDS-PAGE of supernatant and pellet fractions as described previously(12) . The estimation of cage content was made by densitometry of Coomassie-stained gels using a molecular dynamics laser densitometer(13) .

Cage Binding Assay

Clathrin cages were assembled in the absence of AP-3 by overnight dialysis of bovine brain clathrin against buffer C containing 3 mM CaCl(2). The cages were collected by ultracentrifugation as described above and resuspended at a concentration of 3.3 mg of clathrin/ml in buffer C without calcium ions. Cages (0.033 mg) were incubated with 3 µg AP-3 in 0.1 ml of buffer C on ice for 60 min. The reaction was terminated by ultracentrifugation as described above, and the supernatant and pellet fractions were analyzed by SDS-PAGE. AP-3 was measured by densitometry of Coomassie-stained gels as described above.

Cage Stability Assay

Clathrin cages were assembled in the absence of AP-3 as described above and were incubated (0.05 mg/ml) in buffer C with or without InsP(6) at 37 °C for 15 min followed by 45 min on ice. Dissociated clathrin was separated from cages by ultracentrifugation, and the amount of clathrin in the supernatant and pellet fractions was determined by densitometry as described above.

Inositol Phosphate Binding Assay

Inositol phosphate binding to AP-3 was determined by a modification the polyethylene glycol precipitation assay of Theibert et al.(1) . AP-3 (1.6 or 3.2 µg) was added to 200 µl of 20 mM HEPES, pH 7.5 containing 1 mg/ml bovine -globulin, 100 mM KCl, 0.1 mM EDTA, [^3H]InsP(6) (23,000 cpm), and the indicated amount of unlabeled inositol phosphate. Samples were incubated for 15 min at room temperature, and then 0.5 ml of ice-cold 25% polyethylene glycol 4000 was added, and each sample was vortexed and centrifuged for 10 min in an Eppendorf centrifuge. The supernatant was aspirated, and each tube was centrifuged again for 10 s. The residual supernatant was aspirated, and the pellet was resuspended in 0.5 ml of deionized water by sonicating each sample for 5 s with a Biosonik IV sonicator (Bronwill, Rochester, NY) at the minimum power setting. Samples were then transferred to scintillation vials with 10 ml of Aquassure (DuPont NEN) scintillation mixture and counted in a Beckman scintillation counter. SDS-PAGE of the supernatant and pellet fractions established that all of the AP-3 was precipitated in this assay.


RESULTS AND DISCUSSION

A purification scheme for the rat brain inositol-polyphosphate 4-phosphatase was recently described that employed affinity elution from a Mono S cation exchange column with InsP(6) and inositol hexasulfate(6) . Prior to the elution of the 4-phosphatase, a major contaminating protein of 160 kDa was eluted with 0.1 mM InsP(6). This protein was nearly homogenous as determined by SDS-PAGE as shown in Fig. 1. We carried out tryptic digestion of the protein in order to determine whether it represented a previously identified protein. We separated the tryptic peptides by HPLC as described under ``Experimental Procedures'' and amino acid sequence was obtained from six peptides as shown in Table 1. A search of the GenBank data base using tblastn (14) indicated that the peptide sequences obtained were identical to the predicted amino acid sequence of a protein that has been previously described by several names including clathrin assembly protein 3/phosphoprotein F1-20/AP 180(10, 15, 16) . The only exception to the sequence identity was at position 854, where the cDNA sequence predicts an arginine residue. There was no arginine signal at this cycle in the sequence, rather we saw a peak that co-migrated with a standard of N-monomethylarginine(17) . Thus it is likely that this residue is methylated in the rat brain protein. The significance of this putative modification is unknown.


Figure 1: SDS-PAGE analysis of the AP-3 protein eluted from Mono S with InsP(6). Protein eluted with 0.1 mM InsP(6) was concentrated and run on a 6% polyacrylamide gel and stained with Coomassie Blue. Left lane, molecular weight markers; right lane, AP-3.





We will refer to the protein as AP-3 in this report. It is a monomeric protein of 897 amino acids that is found exclusively in brain(10, 16, 18) . It has also been isolated from synaptosomes where it was designated as Fl-20(16) . In vivo and in vitro studies have indicated that AP-3 is a phosphoprotein (18) that is abundant in both soluble and vesicle fractions. AP-3 migrates anomalously upon SDS-PAGE in that its apparent molecular mass ranges from 155 to 185 kDa depending on the conditions, while its molecular mass predicted from the cDNA is 91.4 kDa. This retarded gel mobility may result from the fact that AP-3 is a phosphoprotein and has a highly acidic domain of 50 kDa.

AP-3 induces the assembly of clathrin cages in vitro and is about 4 times more effective than AP-1 or AP-2 in the in vitro assembly assays(12) . There is slight homology (10% amino acid identified over 100 amino acids) between the alpha chain of AP-2, which contains the inositol polyphosphate binding site and AP-3 (4.5 standard deviations using the NBRF/PIR relate program)(19) .

Since AP-2 and AP-3 are both involved in clathrin assembly and both were serendipitously isolated as inositol polyphosphate binding proteins, we investigated whether AP-3 binds inositol polyphosphates using an assay similar to that used in the study of AP-2(1) . We measured [^3H]InsP(6) binding in the presence of increasing concentrations of unlabeled inositol polyphosphates as shown in Fig. 2. InsP(6) (IC = 1 µM) was the most effective inositol polyphosphate in displacing [^3H]InsP(6) followed by Ins 1,3,4,5,6-P(5) (IC = 3 µM), Ins 1,3,4,5-P(4) (IC = 10 µM), and Ins 1,4,5-P(3), respectively. This order of specificity is the same as that found in the study of AP-2(1) . Scatchard analysis of the [^3H]InsP(6) displacement curve yielded a K(d) of 1.2 µM and an apparent binding capacity of 0.9 mol of InsP(6) bound per mol of AP-3 as shown in the inset of Fig. 2. The K(d) of InsP(6) for AP-2 has been reported as 12 nM(1) and 0.12 µM(2) , which is significantly lower than the K(d) for AP-3. The relationship between these binding parameters and intracellular events is difficult to fathom since the intracellular concentration of InsP(6) has been estimated to be very high at 50 µM in HL60 cells (20) and 0.7 mM in Dictyostelium discoideum(21) .


Figure 2: The displacement of [^3H]InsP(6) from rat brain AP-3 by increasing concentrations of various inositol phosphates. The amount of [^3H]InsP(6) (23,000 cpm added) bound to rat brain AP-3 (1.6 µg) was determined using the polyethylene glycol precipitation method described under ``Experimental Procedures'' in the presence of unlabeled Ins 1,4,5-P(3) (box), Ins 1,3,4,5-P(4) (), Ins 1,3,4,5,6-P(5) (circle), and P(6) (bullet) Values shown are the average of duplicates. Scatchard analysis of InsP(6) binding is also shown (inset, p = protein concentration (M)).



We also measured the binding of recombinant AP-3 to InsP(6) to exclude the possibility that a contaminating protein accounted for the binding shown in Fig. 2. The recombinant protein bound InsP(6) with an affinity similar to that of purified rat brain AP-3 as shown in Fig. 3. The amount of InsP(6) bound to the recombinant protein is only one-fourth of that bound to the isolated rat brain protein. This may result from proteolysis of the protein as isolated from E. coli, and during subsequent storage since we found that recombinant AP-3 was unstable with respect to InsP(6) binding upon repeated freezing and thawing and storage.


Figure 3: The displacement of [^3H] from recombinant AP-3 by increasing concentration of unlabeled InsP(6). The amount of [^3H]InsP(6) bound (23,000 cpm added) to recombinant AP-3 (2.8 µg) was measured by polyethylene glycol precipitation in the presence of the indicated concentrations of unlabeled InsP(6).



We also investigated whether InsP(6) had an affect on clathrin cage assembly mediated by AP-3 as shown in Fig. 4a. In this experiment, cage assembly was inhibited by InsP(6) with 50% inhibition at 0.1 mM. This concentration is about 100 times the concentration of InsP(6) required for binding to AP-3, but the assays were not performed under the same conditions. The assembly assay was done at pH 6.5, and the binding experiments were done at pH 7.5. We could not do binding assays at the lower pH due to binding of InsP(6) to carrier globulin under this condition. It was also not possible to perform the assembly experiments at pH 7.5 because assembly is very inefficient at elevated pH. The study on the effect of InsP(6) on assembly of clathrin cages promoted by AP-2 also required these high concentrations to demonstrate inhibition(5) . In fact the curve shown in Fig. 4a obtained using AP-3 is almost identical to that found using AP-2(5) . Moreover, Keen and co-workers did not use isolated AP-2 in their experiment, and it is therefore possible that they observed an effect of InsP(6) on assembly mediated in part by contaminating AP-3 in their preparation. Recall that AP-3 is more potent in assembly assays than AP-2(12) . We also determined whether InsP(6) had an effect on clathrin cages preformed in the presence of calcium ions without any AP-3. Upon warming of the cages to 37 °C, there is rapid exchange between free and cage-bound clathrin. There was a modest effect of InsP(6) in this experiment as shown in Fig. 4b. This suggests that the effect of InsP(6) on cage assembly depends on AP-3 and not upon an interaction directly with clathrin. We also found that InsP(6) does not bind to clathrin-Sepharose, further supporting this idea. The stoichiometry of InsP(6) binding to AP-3 is 1:1, while we presume that there are two sites for interaction of AP-3 with clathrin. This notion is further supported by the experiment shown in Fig. 4c where the effect of InsP(6) on the binding of AP-3 to preformed clathrin cages was measured. In this case AP-3 bound to the cages despite InsP(6) implying that it is able to bind by a site distinct from that occupied by InsP(6).


Figure 4: The effect of InsP(6) on clathrin cages. a, clathrin and AP-3 were incubated with or without InsP(6) at the indicated concentrations as described under ``Experimental Procedures.'' In this experiment 100% is the concentration of clathrin cages in the absence of InsP(6). b, the stability of clathrin cages at 37 °C was determined in the presence and absence of InsP(6) as described under ``Experimental Procedures.'' c, binding of AP-3 to preformed clathrin cages was carried out in the presence or absence of InsP(6) as described under ``Experimental Procedures.'' 100% binding corresponds to the amount of AP-3 bound in the absence of InsP(6). In this experiment there was 0.6 mol of AP-3 bound/clathrin triskelion, which is about half of the saturating value.



One difficulty in developing a model for a role of InsP(6) in coated vesicle assembly is that the concentration of InsP(6) in cells is at least an order of magnitude higher than would be required to fully saturate both AP-2 and AP-3. Furthermore, InsP(6) levels do not appear to vary rapidly in cells(20, 21) . Despite these considerations it is tempting to speculate that inositol phosphates participate in this process given that both assembly proteins bind inositol polyphosphates with high affinity. AP-3 is present in neuronal cells in concentrations comparable with those of clathrin, and most of the AP-3 is cytosolic. In differentiated PC12 cells, AP-3 levels exceed those of clathrin by 5-10 times(22) . Perhaps AP-3 is sequestered by its association with cellular InsP(6), thereby preventing coated vesicle assembly. According to this idea, something would be required to displace InsP(6) in order to initiate cage assembly. One possibility is the formation of highly phosphorylated 3-phosphate containing phosphatidylinositols in membranes destined for coated vesicle formation ex. phosphatidylinositol (3, 4, 5) tetrakisphosphate. If AP-3 has a higher affinity for the lipid than for InsP(6) then AP-3 could be recruited to the membranes and initiate the process of clathrin-coated vesicle assembly.

It was recently shown that the yeast gene VPS34 required for vesicular transport to the yeast vacuole encodes for a protein homologous to mammalian phosphatidylinositol 3-kinase(23) . This enzyme is required for formation of the highly phosphorylated phosphatidylinositols. Additionally the phosphatidylinositol 3-kinase binding sites on the PDGF receptor have been shown to be required for PDGF-dependent receptor endocytosis(24) . Further experiments will be required to determine the possible role of soluble and lipid inositol phosphates in the regulation of vesicular transport. In particular it would be interesting to determine if the levels of the highly phosphorylated phosphatidylinositols vary during coated vesicle assembly in synaptosomes.


FOOTNOTES

*
This research supported by grants HL14147 (Specialized Center for Research in Thrombosis), HL16634, training grant HL07088 and funds from the Department of Pathology to EU. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: AP, assembly protein; InsP(6), inositol hexakisphosphate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high performance liquid chromatography; MES, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.