©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Clathrin Binding and Assembly Activities of Expressed Domains of the Synapse-specific Clathrin Assembly Protein AP-3 (*)

Weilan Ye (2), Eileen M. Lafer (1)(§)

From the (1) Center for Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center, San Antonio, Texas 78245 and the (2) Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We separately expressed the 58-kDa C-terminal, 42-kDa middle, 16-kDa C-terminal, and 33-kDa N-terminal regions of AP-3 (also called F1-20, AP180, NP185, and pp155), and determined their clathrin binding and assembly properties. The 58-kDa C-terminal region of AP-3 is able to bind to clathrin triskelia and assemble them into a homogeneous population of clathrin cages and will also bind to preassembled clathrin cages. The 42-kDa central region of AP-3 can bind to both clathrin triskelia and to clathrin cages, but cannot assemble clathrin triskelia into clathrin cages. The 16-kDa C-terminal region of AP-3 can bind to clathrin cages, but cannot bind to clathrin triskelia or assemble clathrin triskelia into clathrin cages. The clathrin binding activities of the 42-kDa central region and 16-kDa C-terminal region are weaker than the corresponding activity of either the 58-kDa C-terminal region or full-length AP-3. Previous efforts had mapped a clathrin binding site within the N-terminal 33 kDa of AP-3 (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4401-4408; Morris, S. A., Schroder, S., Plessmann, U., Weber, K., and Ungewickell, E. (1993) EMBO J. 12, 667-675). However, although the N-terminal 33 kDa of AP-3 is able to bind to clathrin triskelia (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4401-4408; Ye, W., and Lafer, E. M. (1995) [Abstract] J. Neurosci. Res. 41, 15-26), it does not promote their assembly into clathrin cages (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4401-4408; Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26) or bind to preassembled clathrin cages (Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26). It appears that the smallest functional unit that carries out all of the reported clathrin binding and assembly properties of AP-3, essentially as well as the full-length protein, is the 58-kDa C-terminal region.


INTRODUCTION

Clathrin-coated vesicles are involved in pathways of receptor-mediated intracellular transport (4) . These include the movement of proteins from the trans-Golgi network to secretory vesicles for regulated secretion, the transfer of lysosomal hydrolases from the trans-Golgi network to lysosomes, receptor-mediated endocytosis, and the biogenesis and recycling of synaptic vesicles. The protein coats of clathrin-coated vesicles have been well characterized (5) . The major coat protein is clathrin (6) . The solution form of clathrin is the triskelion, which consists of three identical clathrin heavy chains (192 kDa) and three clathrin light chains (22-28 kDa) (7, 8, 9, 10, 11) . Triskelions can associate with one another to form icosahedral cages. When these structures form at the surface of a cell membrane, the membrane invaginates into a coated pit, followed by its separation from the parent membrane as a coated vesicle. Coated vesicles also contain one or more of the assembly proteins: AP-1, AP-2, AP-3, or auxilin (12, 13, 14, 15) . The assembly proteins all share the property that they promote the assembly of clathrin triskelia into a homogeneous population of clathrin cages in solution (12, 14, 16, 17) . In vivo, the assembly proteins are believed to sit between the cell membrane and the clathrin cage (18) . The assembly proteins are thought to be involved in directing clathrin cage assembly to a particular cell membrane through an interaction with a membrane receptor (19) .

Much remains to be elucidated concerning the mechanism by which clathrin assembly is promoted by a clathrin assembly protein and how this assembly might be regulated. The assembly proteins AP-1 and AP-2 are tetramers, whereas the assembly proteins AP-3 and auxilin are monomers. AP-1, AP-2, and AP-3 have been cloned and sequenced (20, 21, 22, 23) . As a monomer, AP-3 represents a simple system in which to investigate how a clathrin assembly protein promotes clathrin assembly. AP-3 is the only clathrin assembly protein shown to be synapse-specific (24, 25, 26) , suggesting that it plays a specialized role in the life cycle of the synaptic vesicle (27) . Therefore, understanding how AP-3 functions will also make a contribution toward elucidating the molecular mechanism underlying neurotransmission.

AP-3 was independently discovered in a number of laboratories, and has been known as pp155 (28) , AP180 (16) , NP185 (29) , and F1-20 (23, 30) . pp155, AP180, and NP185 were shown to be the same protein, and renamed AP-3 (1) . F1-20 and AP-3 were then shown to be identical (2, 27) . AP-3 is a neuronal-specific (2, 26, 29) phosphoprotein (23, 28, 31, 32) and glycoprotein (32) , which is unusually acidic (16, 23, 28) , and migrates anomalously on SDS-PAGE()(1, 16, 17, 23) . Two primary functional activities have been found for AP-3. AP-3 is a clathrin assembly protein (16) , as well as a high affinity receptor for specific inositol polyphosphates (33) . These two functions are intimately related, since binding of specific inositol polyphosphates has been shown to inhibit clathrin assembly (33) .

In order to begin to understand the structure-function relationships of AP-3, clathrin binding properties of proteolytic fragments of AP-3 were studied (1, 2) . It was found that the 33-kDa N-terminal region could bind to clathrin triskelia, leading to the conclusion that the 33-kDa N-terminal region was a clathrin binding domain (1) . In this same study it was found that the 33-kDa N-terminal region was insufficient for clathrin assembly. We confirmed these findings utilizing the bacterially expressed N-terminal 33-kDa region of AP-3 and extended them by showing that the 33-kDa N-terminal region could bind to clathrin triskelia, but not to preassembled clathrin cages (3) . This suggested that clathrin triskelion binding and clathrin cage binding are not functionally equivalent. It was also shown that the 33-kDa N-terminal region contained a high affinity binding site for specific inositol polyphosphates (33) . In the current study, we undertook an analysis of the clathrin binding and assembly properties of additional individually expressed regions of AP-3. We found that the 58-kDa C-terminal region of AP-3 had all of the clathrin binding and assembly properties of full-length AP-3. Previous proteolysis studies (1) and sequence analyses (2) led to the suggestion (2) that the 58-kDa C-terminal region could be further divided into a 42-kDa central domain and a 16-kDa C-terminal domain. We found that the 42-kDa central region could bind to clathrin cages and clathrin triskelia and that the 16-kDa C-terminal region could bind to clathrin cages. However, neither of the latter two regions alone could assemble clathrin triskelia into clathrin cages.


EXPERIMENTAL PROCEDURES

Materials

Proteins

All buffers used in the protein work, even if not explicitly indicated in the citations, contained 0.1 mM PMSF. The proteins GST-AP-3 (AS15AS108) (27) , GST-N33 kDa (3) and GST were all expressed and purified exactly as described previously (3) . Bovine brain clathrin was purified from bovine brain clathrin-coated vesicles as described previously (3) , based on subtle modifications of (16) . SDS-PAGE analysis of the purified clathrin revealed three silver-stained bands, corresponding in apparent molecular weight to clathrin heavy chain and to the two clathrin light chains. Clathrin was determined to be free of detectable contaminating AP-3 by Western blot analysis with the F1-20 monoclonal antibody utilizing the ECL detection system (27) . Three cycles of assembly-disassembly was carried out (3) . Protein concentrations were determined spectrophotometrically using extinction coefficients which were calculated from the reported amino acid sequences of clathrin (8, 9, 10) and AP-3 (23) , according to the relation = number of tryptophan residues (5690) + number of tyrosine residues (1280) (34) , and found to be as follows: GST-AP-3 (80, 650) , GST-C58 kDa (63, 440) , GST-M42 kDa (46, 370) , GST-C16 kDa (57, 750) , GST-N33 kDa (57, 890) , GST (40, 680) , and clathrin triskelion (676, 770) . Monoclonal antibody F21-5 was generated by fusion of spleens from mice immunized with clathrin which was free of AP-3 as determined by Western blot analysis with monoclonal antibody F1-20. F21-5 was found to be specific for clathrin heavy chain and did not display cross-reactivity with bacterially expressed or bovine AP-3, clathrin light chains, AP-1 or AP-2.() Monoclonal antibody F1-20 has been shown to be specific for AP-3 (23, 26, 27, 30) .

A construct expressing GST-C58 kDa was constructed as described previously (33) . This construct (pGEX3X-F1-20-COOH58 kDa) was introduced into Escherichia coli BL21 for protein expression. GST-C58 kDa was expressed and purified as described previously (33) . A construct expressing GST-C16 kDa was constructed as follows. Plasmid pGEX3X-F1-20-COOH58 kDa was digested with restriction endonucleases BamHI and StuI, followed by mung bean nuclease treatment and agarose gel electrophoresis. The largest fragment was purified and ligated to form closed circular DNA. The new construct (pGEX3X-F1-20-COOH16 kDa) was introduced into E. coli DH5 for protein expression. GST-C16 kDa was expressed and purified under the same conditions as GST-AP-3 (3) , except that the cells were grown for 2 h following isopropyl-1-thio--D-galactopyranoside induction. A construct expressing GST-M42 kDa was constructed as follows. Plasmid pGEX3X-F1-20-COOH58 kDa was digested with restriction endonucleases StuI and EcoR I, followed by mung bean nuclease treatment and agarose gel electrophoresis. The largest fragment was purified and ligated to form closed circular DNA. The new construct (pGEX3X-F1-20-middle 42 kDa) was introduced into E. coli DH5 for protein expression. GST-M42 kDa was expressed and purified under the same conditions as GST-AP-3 (3) , except that the cells were grown for 4 h following isopropyl-1-thio--D-galactopyranoside induction. Methods

Clathrin Cage Binding Assay

Clathrin cage binding assays were carried out as described (3, 35) , except that all the GST-fusion proteins were used at 20 µM, clathrin was used at 1.8 µM (corresponding to molarity of triskelia; 1.2 mg/ml), and all of the binding reactions were carried out in isolation buffer (0.1 M MES, 1 mM EGTA, 0.5 mM MgCl, 0.1 mM PMSF, pH 6.7). The samples were analyzed by SDS-PAGE followed by Coomassie Blue staining and Western blot analysis. The Western blots were stained with a monoclonal antibody against GST (``GST (12) '' from Santa Cruz Biotechnology, Inc.) and visualized utilizing the ECL detection method, as described (27) . The distribution of the GST-fusion proteins between the pellet and supernatant fractions was quantitated using the Millipore BioImage system with 3cx scanner. Values given in the text for the percent fusion protein bound represent: the percent fusion protein in the pellet in the sample containing clathrin - the percent fusion protein in the pellet in the parallel reaction not containing clathrin.

Clathrin Triskelia Binding Assay

GST-AP-3, GST-C58 kDa, GST-M42 kDa, GST-C16 kDa, and GST were each bound to glutathione-Sepharose (Pharmacia Biotech Inc.) (3) . Rather then eluting the bound proteins with glutathione, each resin was then equilibrated with ice-cold isolation buffer (0.1 M MES, 1 mM EGTA, 0.5 mM MgCl, pH 6.7) containing 0.1 mM PMSF and 0.01% gelatin. 0.4 mg/ml clathrin triskelia in isolation buffer were incubated with an equal volume of each resin at 4 °C for 2 h with rocking. The resins were pelleted by a 5-s microcentrifugation, and the supernatants were removed. The supernatants were labeled F for flow-through in Fig. 2 and correspond to the unbound material. The resins were washed six times with an equal volume of ice-cold isolation buffer containing 0.1 mM PMSF and 0.01% gelatin. The last wash was found to be free of clathrin by Western blot analysis using the clathrin heavy chain-specific monoclonal antibody F21-5, indicating that all nonspecifically bound material was removed. The beads were then eluted by boiling for 5 min in an equal volume of 1 SDS sample buffer, followed by a 5-s microcentrifugation, and the supernatants were removed. These supernatants were labeled B for bound material in Fig. 2. All of the samples were analyzed by SDS-PAGE, followed by Coomassie Blue staining and Western blot analysis using the clathrin heavy chain-specific monoclonal antibody F21-5 with the ECL detection system. A parallel set of experiments was carried out using 0.4 mg/ml BSA instead of clathrin triskelia in the incubation mix to ensure that the resins did not nonspecifically bind to proteins in the solution. These blots were blocked with 0.10% gelatin instead of BSA and stained with a monoclonal anti-BSA antibody (Sigma B2901).


Figure 2: The 58-kDa C-terminal and 42-kDa central region of AP-3 bind to clathrin triskelia. Clathrin triskelia binding assays were carried out as described under ``Experimental Procedures.'' Glutathione-Sepharose coupled to either GST-AP-3 ( lanes 1, 2, 11, and 12), GST-C58 kDa ( lanes 3, 4, 13, and 14), GST-M42 kDa ( lanes 5, 6, 15, and 16), GST-C16 kDa ( lanes 7, 8, 17, and 18), or GST ( lanes 9, 10, 19, and 20) was incubated with an equal volume of either 0.4 mg/ml clathrin triskelia ( lanes 1-10) or 0.4 mg/ml BSA ( lanes 11-20). Following a 2-h incubation at 4 °C with agitation, the beads were pelleted by a 5-s microcentrifugation, and the supernatant was removed (this is the flow-through, corresponding to the unbound material, labeled as F in all odd lanes). The beads were washed six times with an equal volume of ice-cold isolation buffer. The beads were then eluted by boiling for 5 min in an equal volume of 1 SDS sample buffer (this is the eluate, corresponding to the bound material, labeled as B in all even lanes). All of the samples were analyzed by SDS-PAGE followed by Coomassie Blue staining ( upper panel) and by Western blot ( lower panel) using either a clathrin heavy chain-specific monoclonal antibody ( lanes 1-10) or a BSA-specific monoclonal antibody ( lanes 11-20), visualized by the ECL method. Positions of all proteins are indicated using the abbreviations defined in the legend to Fig. 1. Essentially identical results were obtained in three independent experiments.



Clathrin Assembly Assays

3 µM clathrin triskelia were dialyzed overnight at 4 °C against isolation buffer with the addition of 20 µM of either GST, GST-AP-3, GST-C58 kDa, GST-M42 kDa, GST-C16 kDa, GST-N33 kDa, or GST, as indicated in the figure legend to Fig. 3. Following a 3-min centrifugation at 13,600 g to remove nonspecific aggregates, newly assembled clathrin cages were pelleted by ultracentrifugation for 20 min at 100,000 g. The pellet (P) and supernatant (S) fractions were analyzed by SDS-PAGE, followed by Coomassie Blue staining and Western blot analysis using the anti-clathrin heavy chain-specific monoclonal antibody F21-5. The distribution of clathrin heavy chain between the pellet and supernatant fractions was quantitated using the Millipore BioImage system with 3cx scanner. For the quantitative assembly plot (Fig. 4), assays were carried out as described above, except that the concentration of test assembly protein was varied, as indicated on the plot. Each point on the plot represents the average of triplicate determinations. The morphology of the products of the assembly assays using either 20 µM GST-AP-3, 20 µM GST-C58 kDa, or 20 µM GST were evaluated by negative staining electron microscopy, as described (3) .


Figure 3: The 58-kDa C-terminal region of AP-3 assembles clathrin. Clathrin assembly assays were performed as described under ``Experimental Procedures.'' 3 µM clathrin triskelia were dialyzed overnight at 4 °C against isolation buffer with the addition of 20 µM concentrations of either GST-AP-3 ( lanes 1 and 2), GST-C58 kDa ( lanes 3 and 4), GST-M42 kDa ( lanes 5 and 6), GST-C16 kDa ( lanes 7 and 8), GST-N33 kDa ( lanes 9 and 10), or GST ( lanes 11 and 12). Following a low speed spin to remove nonspecific aggregates, newly assembled clathrin cages were pelleted by ultracentrifugation at 100,000 g. The pellet ( P) and supernatant ( S) fractions were analyzed by SDS-PAGE, followed by Coomassie Blue staining ( upper panel) and Western blotting ( lower panel) using an anti-clathrin heavy chain-specific monoclonal antibody. Positions of all the proteins are indicated in the same way as in Fig. 1. Essentially identical results were obtained in three independent experiments.




Figure 4: The 58-kDa C-terminal region of AP-3 assembles clathrin as efficiently as full-length AP-3. Quantitative assembly assays were carried out as described under ``Experimental Procedures.'' 3 µM clathrin triskelia were dialyzed overnight at 4 °C against isolation buffer with the addition of one of the following test assembly proteins at the concentrations indicated in the figure: GST-AP-3 ( closed circle), GST-C58 kDa ( open circle), GST-C16 kDa ( closed square), GST-M42 kDa ( closed triangle), or GST (). Each data point represents the mean from three experiments with the error bars representing the standard deviations derived from three experiments.




RESULTS

The 58-kDa C-terminal Region, the 16-kDa C-terminal Region, and the 42-kDa Central Region of AP-3 Bind to Clathrin Cages

Previous studies had demonstrated that bacterially expressed AP-3 could bind to clathrin cages, whereas the 33-kDa N-terminal region of AP-3 (amino acid residues 1-304) could not bind to clathrin cages (3) . Consequently, we examined the ability of the 58 kDa C-terminal region (amino acid residues 305-901), the 42 kDa central region (amino acid residues 305-744), and the 16 kDa C-terminal region (amino acid residues 745-901) of AP-3 to bind to preassembled clathrin cages (Fig. 1). In this assay, the ability of the indicated GST fusion proteins to co-sediment with the pre-assembled clathrin cages was monitored by Western blot analysis with an anti-GST monoclonal antibody, and subjected to a quantitative analysis, as described under ``Experimental Procedures.'' Consistent with previous observations (3) we found 88% binding of GST-AP-3 ( lanes 1-4) (positive control) and 0.2% binding of GST-N33 kDa ( lanes 5-8) (negative control) to the clathrin cages. In experiments utilizing the newly expressed domains, we found 86% GST-C58 kDa ( lanes 9-12), 42% GST-M42 kDa ( lanes 13-16), and 51% GST-C16 kDa ( lanes 17-20) bound to the clathrin cages. The GST negative control showed 0% binding to the clathrin cages ( lanes 21-24). We conclude that the 58-kDa C-terminal region, the 42-kDa central region, and 16-kDa C-terminal region of AP-3 all bind to clathrin cages.


Figure 1: The 58-kDa C-terminal, 16-kDa C-terminal, and 42-kDa central region of AP-3 all bind to clathrin cages. Clathrin cage binding assays were carried out as described under ``Experimental Procedures.'' 20 µM concentrations of either GST-AP-3 ( lanes 1-4), GST-N33 kDa ( lanes 5-8), GST-C58 kDa ( lanes 9-12), GST-M42 kDa ( lanes 13-16), GST-C16 kDa ( lanes 17-20), or GST ( lanes 21-24) was incubated in the absence (-) or presence (+) of 1.8 µM (corresponding to molarity of triskelia, 1.2 mg/ml) preassembled clathrin cages. Following a low speed spin to remove nonspecific aggregates, all samples were pelleted by ultracentrifugation at 100,000 g. The pellet ( P) and supernatant ( S) fractions were analyzed by SDS-PAGE, followed by Coomassie Blue staining ( upper panel) and Western blotting ( lower panel) using an anti-GST-specific monoclonal antibody visualized by the ECL detection method. Positions of clathrin heavy chain ( Cla HC), GST-AP-3, GST-C58 kDa, GST-M42 kDa, GST-N33 kDa, GST-C16 kDa, clathrin light chains ( Cla LCs), GST, and molecular mass markers ( MW) are indicated. Essentially identical results were obtained in three independent experiments.



The 58-kDa C-terminal Region and the 42-kDa Central Region of AP-3 Bind to Clathrin Triskelia

Previously, we showed that the bacterially expressed 33-kDa N-terminal region of AP-3 could bind to clathrin triskelia, but could not assemble clathrin triskelia into cages or bind to preassembled clathrin cages (3) . This suggested that binding to clathrin triskelia and clathrin cages were not functionally equivalent. In the present study, we examined the ability of the 58-kDa C-terminal region, the 42-kDa central region, and the 16-kDa C-terminal region of AP-3 to bind to clathrin triskelia. For these experiments, we coupled proteins GST-AP-3, GST-C58 kDa, GST-M42 kDa, GST-C16 kDa, and GST to glutathione-Sepharose. A solution of clathrin triskelia, or as a control a solution of BSA, was incubated with each resin. The resins were washed to remove nonspecifically bound protein, followed by solubilization of the bound material by boiling each resin in SDS-sample buffer. The flow-through (labeled F) and bound (labeled B) fractions were analyzed by SDS-PAGE (Fig. 2). The gels in the top panel were stained for total protein with Coomassie Blue, and the gels in the bottom panel were transferred to nitrocellulose and stained with either an anti-clathrin heavy chain-specific monoclonal antibody ( lanes 1-10) or anti-BSA monoclonal antibody ( lanes 11-20). Note that the GST fusion proteins also eluted from the resins by boiling in SDS-sample buffer and showed up in the Coomassie Blue-stained gels. None of the resins retained the BSA ( lanes 11-20), indicating that the resins did not nonspecifically bind to proteins. In experiments utilizing GST-AP-3 and GST-C58 kDa resins, all the clathrin triskelia were found in the bound fractions ( lanes 1-4). This confirmed that GST-AP-3 can bind to clathrin triskelia and demonstrated that GST-C58 kDa can bind to clathrin triskelia. In experiments utilizing GST-M42 kDa resin, clathrin triskelia were found both in the flow-through fraction ( lane 5) and bound fraction ( lane 6). This indicated binding between clathrin triskelia and GST-M42 kDa. In experiments utilizing either GST-C16 kDa, or GST (negative control), essentially all of the clathrin triskelia were found in the flow-through fractions ( lanes 7-10), indicating that clathrin triskelia did not bind to either of these proteins under these assay conditions. We also increased the concentration of clathrin in the assay by a factor of 3 (1.2 mg/ml) and were still unable to detect any binding of clathrin triskelia to GST-C16 kDa.

The 58-kDa C-terminal Region of AP-3 Is Sufficient for Clathrin Assembly

It had been shown previously that bacterially expressed GST-AP-3 could assemble clathrin triskelia into cages, whereas the bacterially expressed 33-kDa N-terminal region of AP-3 was insufficient for clathrin assembly (3) . In order to further localize the regions of AP-3 required for clathrin assembly, we compared the ability of GST-AP-3, GST-C58 kDa, GST-M42 kDa, GST-C16 kDa, GST-N33 kDa, and GST to assemble clathrin triskelia into clathrin cages. In this assay, we monitored clathrin assembly as the movement of clathrin from a 100,000 g soluble form (triskelia) to a 100,000 g insoluble form (cages) following dialysis with a test assembly protein (Fig. 3). In the gel displayed, it is clear that clathrin moves from the supernatant to the pellet in the presence of GST-AP-3 ( lanes 1 and 2) or GST-C58 kDa ( lanes 3 and 4), indicative of clathrin assembly. However, no movement of clathrin is seen from the supernatant to the pellet in the presence of GST-M42 kDa ( lanes 5 and 6), GST-C16 kDa ( lanes 7 and 8), or GST-N33 kDa ( lanes 9 and 10), compared with the negative control GST ( lanes 11 and 12).

When these experiments were carried out over a wider range of protein concentrations, and subjected to a quantitative analysis (Fig. 4), it was found that the 58-kDa C-terminal region of AP-3 assembles clathrin as well as the full-length protein. No significant clathrin assembly was found by GST-M42 kDa, GST-C16 kDa, GST-N33 kDa, or GST. A hallmark of factor-dependent clathrin assembly is the homogeneity of the cages formed. Analysis of the morphology of the cages assembled in the experiment shown in Fig. 3is presented in Fig. 5 . It is apparent that no cages were formed with GST (Fig. 5 A) and that both GST-AP-3 (Fig. 5 B) and GST-C58 kDa (Fig. 5 C) assembled clathrin triskelia into a homogeneous population of clathrin cages. We conclude that the 58-kDa C-terminal region of AP-3 is sufficient to assemble clathrin triskelia into clathrin cages.


Figure 5: Ultrastructural analyses of the cages assembled by the 58-kDa C-terminal region of AP-3 reveals a homogeneous population of clathrin cages. The morphology of the products of the clathrin assembly assays displayed in Fig. 3 were evaluated by negative staining electron microscopy. Electron micrographs are shown of clathrin assembly reactions promoted by either GST (negative control) ( A), GST-AP-3 (positive control) ( B), or GST-C58 kDa (test assembly protein) ( C). The distribution of cage diameters assembled by GST-AP-3 and GST-C58 kDa was indistinguishable.




DISCUSSION

The results of the study presented here were integrated with the previously published studies, to prepare a summary of structure-function relationships for AP-3 (Fig. 6). These relationships are diagrammed against the distribution of acidic and basic residues in the primary structure of AP-3. Previously, it was reported that the 33-kDa N-terminal region of AP-3 contained a high affinity binding site for specific inositol polyphosphates, with similar binding parameters as full-length AP-3 (33) . We show that the 58-kDa C-terminal region of AP-3 is able to bind to clathrin triskelia and assemble them into a homogeneous population of clathrin cages and will also bind to preassembled clathrin cages. We also show that the clathrin triskelia binding, clathrin cage binding, and clathrin assembly activities of the 58-kDa C-terminal region of AP-3 are essentially indistinguishable from that of the full-length protein. We found that the 42-kDa central region of AP-3 can bind to both clathrin triskelia and to clathrin cages, but cannot assemble clathrin triskelia into clathrin cages. We found that the 16-kDa C-terminal region of AP-3 can bind to clathrin cages, but cannot bind to clathrin triskelia or assemble clathrin triskelia into clathrin cages. We noted that the clathrin binding activities of the 42-kDa central region and 16-kDa C-terminal region are weaker than the corresponding activity of either the 58-kDa C-terminal region or full-length AP-3. Previous efforts had mapped a clathrin binding site within the N-terminal 33 kDa of AP-3 (1, 2) . However, although the N-terminal 33 kDa of AP-3 is able to bind to clathrin triskelia (1, 3) , it does not promote their assembly into clathrin cages (1, 3) or bind to preassembled clathrin cages (3) . It appears that the smallest functional unit that carries out all of the reported clathrin binding and assembly properties of AP-3, essentially as well as the full-length protein, is the 58-kDa C-terminal region.


Figure 6: Summary of the structure-function studies of AP-3. A clathrin triskelia binding site had been found in the 33-kDa N-terminal region (1, 3). A high affinity inositol polyphosphate binding site had also been found in the 33-kDa N-terminal region (33). The 33-kDa N-terminal region had been found to not bind to clathrin cages (3) or assemble clathrin triskelia into cages (1, 3). All data concerning the 58-kDa C-terminal region, the 42-kDa central region, and the 16-kDa C-terminal region are presented in this paper. For reference, all of the binding data are shown along with the distribution of acidic ( A) and basic ( B) residues across the mouse AP-3 sequence (23, 27).



The data presented in this study allow us to consider alternative interpretations to previously published studies. In clathrin cage binding assays utilizing a partial trypsin digestion of AP-3, bands with apparent molecular masses of 148, 107, and 33 kDa were found to co-sediment with preassembled clathrin cages (2) . Sequencing of the digestion products revealed that the 33-kDa fragment corresponded to the 33-kDa N-terminal region of AP-3 and that the 107-kDa apparent molecular mass fragment began at amino acid 304. The interpretation presented was that the 33-kDa N-terminal region bound specifically to the cages and that the 107-kDa apparent molecular mass fragment was only retained by the cages due to a noncovalent association with the 33-kDa N-terminal fragment. Our demonstration of clathrin cage binding by the 58-kDa C-terminal region, the 42-kDa central region, and the 16-kDa C-terminal region, coupled with the lack of clathrin cage binding by the 33-kDa N-terminal region (3) , suggest an alternative interpretation of these proteolysis studies. Possibly it was the 107-kDa apparent molecular mass fragment that was bound specifically to the cages, and the 33-kDa N-terminal fragment was only retained by the cages due to a noncovalent association with the 107-kDa apparent molecular mass fragment. Similarly, in the clathrin triskelia binding assays in which it was shown that the 33-kDa N-terminal region could bind to clathrin triskelia, some retention by the clathrin-Sepharose of a 135-kDa apparent molecular mass fragment and a 100-kDa apparent molecular mass fragment were also observed (1) . It was suggested that the 100-kDa apparent molecular mass fragment was not bound directly to the clathrin triskelia, but was retained by a noncovalent association with the 33-kDa N-terminal fragment. Our data allow one to consider the possibility that both the 33-kDa N-terminal fragment and 100-kDa apparent molecular mass fragment were specifically retained by the clathrin-Sepharose.

Analysis (27, 36) of the amino acid sequence of AP-3 (23) revealed clear divisions between the characteristics of the 30-kDa N-terminal region and the 60-kDa C-terminal region. The 60-kDa C-terminal region has an unusual self-repeating primary structure, a preponderance of acidic residues, an unusually high proline, serine, threonine, and alanine content, and is predicted to have a high content of irregular secondary structure. The 30-kDa N-terminal region of AP-3 has an amino acid composition typical of a globular protein, with a roughly equal balance of acidic and basic residues, and is predicted to have a high alpha helix content. It was suggested that AP-3 has an essentially neutral 30-kDa N-terminal domain with an amino acid composition typical of a globular structure and an acidic 60-kDa C-terminal domain with a highly repetitive and redundant amino acid composition (23, 27) . These data were consistent with published proteolysis studies (1) , which had shown that limited digestion with a number of different proteases generated a stable 30-kDa fragment, which was later shown (2) to correspond to the 33-kDa N-terminal region. Other investigators (2) proposed a further division within the 58-kDa C-terminal region into a 42-kDa central acidic domain and a 16-kDa C-terminal domain. These assignments were based on the identification of these regions as products of partial proteolysis reactions (1, 2) and the finding that the 16-kDa C-terminal region was more basic, and had a higher proline content, than any other part of the protein.

The results of this study lend additional support to the assignment of a 33-kDa N-terminal domain and a 58-kDa C-terminal domain. The 33-kDa N-terminal domain is a high affinity receptor for inositol polyphosphates (33) and also binds to clathrin triskelia (1, 3) . The 58-kDa C-terminal domain is a fully functional clathrin binding and assembly domain. Evidence supporting the further subdivision of the 58-kDa C-terminal domain into a 42-kDa central domain, and a 16-kDa C-terminal domain, is more tenuous at this time. We found that each of these proteins is less stable than the 33-kDa N-terminal or 58-kDa C-terminal regions. For example, we were able to identify conditions under which we could stably prepare both the 33-kDa N-terminal region and the 58-kDa C-terminal region free of GST. However, we were not able to identify conditions, even with extensive testing, under which we could prepare stable 42-kDa central region and 16-kDa C-terminal region free of GST (data not shown). The reported binding properties of these fragments are interesting, but their meaning is subject to several possible interpretations. For example, it is possible that the 42-kDa central region, and the 16-kDa C-terminal region, each contain a distinct binding site for clathrin cages. Alternatively, it is possible that in the 58-kDa C-terminal region, residues from both the 42-kDa central region and 16-kDa C-terminal region contribute to a single clathrin binding and assembly site. When expressed and studied separately, the residues can make sufficient contacts to account for the clathrin binding observed in this study, but not to carry out clathrin assembly. Distinguishing between these various possibilities may require the elucidation of the three-dimensional structure of AP-3.

This study raises two intriguing and important questions. Why is there is a binding site for clathrin triskelia in the 33-kDa N-terminal domain, when the 58-kDa C-terminal domain binds and assembles clathrin as well as the full-length protein? How does an inositol polyphosphate binding site in the 33-kDa N-terminal domain modulate the clathrin assembly activity which we show here is present in the 58-kDa C-terminal domain? While at this point in time we can only speculate, it is interesting to consider that the 33-kDa N-terminal domain may function as a regulatory domain, and the 58-kDa C-terminal domain may function as a clathrin assembly domain. In this light, it is provocative that the 33-kDa N-terminal domain binds to a site on clathrin triskelia that either becomes inaccessible or changes conformation upon assembly of the triskelia into cages. Answers to these interesting questions await future studies.


FOOTNOTES

*
This work was supported by NINDS Grant NS29051 (to E. M. L.). 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.

§
To whom correspondence should be addressed: Institute of Biotechnology, University of Texas Health Science Center, 15355 Lambda Dr., San Antonio, TX 78245. Tel.: 210-567-7220; Fax: 210-567-7277; E-mail: lafer@thorin.uthscsa.edu.

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; BSA, bovine serum albumin; GST-AP-3, GST fused with full-length AP-3 (isoform AS15AS108); GST-C58 kDa, GST, fused with amino acids 305-744 of AP-3 (isoform AS15AS108); GST-N33 kDa, GST, fused with amino acids 1-304 of AP-3 (isoform AS15AS108); GST-C16 kDa, GST, fused with amino acids 745-901 of AP-3 (isoform AS15AS108); GST-M42 kDa, GST, fused with amino acids 305-744 of AP-3 (isoform AS15AS108); PMSF, phenymethylsulfonyl fluoride; MES, 4-morpholineethanesulfonic acid.

E. M. Lafer, W. Ye, and N. H. Tannery, manuscript in preparation.


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