Clathrin Assembly Proteins and the Organization of the Coated Membrane

CLATHRIN ASSEMBLY PROTEINS AND THE ORGANIZATION OF THE COATED MEMBRANE

James H. Keen OUTLINE I.

INTRODUCTION 11. CLATHRIN 111. ASSEMBLY PROTEINS A. Biochemical Characterization: AP- 1 and AP-2 B. Deep-etch Visualization of AP-2 IV. AP-2-MEDIATED COAT ASSEMBLY IN VITRO V. AP FUNCTIONS IN CELLS: RECEPTOR CLUSTERING AND COATED PIT FORMATION? NOTE ADDED IN PROOF ACKNOWLEDGMENTS REFERENCES Advances in Cell Biology, Volume 3, pages 153-176. Copyright 0 1990 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-013-6

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1.

INTRODUCTION

The structural regularity of the coat profile observed by conventional transmission electron microscopy in chemically fixed cells undoubtedly stimulated the interest of the earliest investigators of coated membrane structure and function. This attention was heightened by the remarkable views of coat structures provided by the freeze-etching technique of Heuser (1980) (Figure IA). The coat structure has been at the focus of two investigative objectives. The first involves structural efforts aimed at determining the composition of this complex structure and how it is assembled. The second objective has been to ascertain precisely how the coated membrane functions in receptor-mediated endocytosis and lysosome biogenesis in particular, two processes with which it is most directly implicated, and more generally in membrane movements and dynamics within cells. Progress along the first line has been slow but steady, with contributions by many different research groups. It is now clear that the polygonal latticework that characterizes the coat (Figure 1A) is composed of at least two different components that can be clearly visualized by deep-etch electron microscopy of coat structures that have been induced to disassemble on a polylysine-coated mica surface (Figure 1 B): these are clathrin triskelia (arrows)and assembly proteins or AP (arrowheads). Analysis of our current understanding of the biochemical properties of clathrin and the AP and their role in the organization of the coat structure will be the focus of the first part of this review. Elucidation of how the coat structure functions at the molecular level in the intact cell is a goal yet to be attained. In receptor-mediated endocytosis, several coat-related processes need to be understood. How is the coat structure involved in clustering receptors so that their concentrations within coated pits are manyfold higher than that in the surrounding plasma membrane? Is the coat structure a driving force for the membrane indentation and formation of the detached intracellular vesicles that characterize endocytosis? How does uncoating of the endocytic vesicle occur and what regulates reassembly of the coat structure at new coated pit sites on the plasma membrane? The available information on the structure, domains, and interactions of the major coat components will be used in the latter part of this review as a basis for several proposals concerning how the coat structure is formed, both in solution and on a cell membrane, and how it may function in receptor-ligand clustering in coated pits and in membrane indentation.

II. CLATHRIN “Clathrin” was the name given to the main structural protein of the coat by Pearse (1975) in view of its formation of an organized lattice or clathrate structure. The protein has a rather unusual extended structure, being composed of 3 usually bent legs joined at a central hub region (Figure 2 ) , consequently the

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Figure 1. Upper: Plasma membrane coated pit in a fibroblast visualized by freeze fracture, deep-etching, and rotary replication. (Courtesy of Dr. John Heuser.) Lower: Clathrin and assembly protein containing coats adsorbed and dissociated upon a polylysine-pretreated mica surface, revealing the surface lattice triskelion components (arrows) and the inner content of assembly protein particles (arrowheads). Scale bar = 100 nm. (From Heuser and Keen, 1988.)

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Figure 2. Electron micrograph of clathrin triskelions after rotary shadowing. (Courtesy of Dr. Daniel Branton.)

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term “triskelion” (Ungewickell and Branton, 198 I ) . Each clathrin molecule is composed of three heavy chains (HC) and three light chains (LC), with apparent M,. on SDS gel electrophoresis of 180 kd and 33-36 kd, respectively (Figure. 3, lane I ) . The rat clathrin heavy chain is the product of a single gene coding for a 191,569-Da polypeptide with only a single message observed in several rat tissues. The deduced amino acid sequence is unrelated to any other known protein but is highly conserved between human, rat, and bovine species (- 99%) (Kirchhausen et al., 1987), and is about 50% identical with the yeast clathrin heavy chain (S. Lemmon, personal communication). Some information is available concerning the orientation of the molecule and its domain organization. The blocked amino terminal of the molecule is almost certainly located at the distal end of each leg, within a compactly folded region of the molecule that has been labeled the terminal domain (Kirchhausen and Harrison, 1984) and which can be readily released on mild protease treatment (Schmid et al., 1982). The precise assignment of the remainder of the primary amino acid sequence to discrete locations on the trimer leg is more speculative. Proteolysis of clathrin that has been assembled into cages yields several discrete fragments (Kirchhausen and Harrison, 1984) that indicate cleavages along the distal leg. The binding site for the clathrin light chains has been localized to the proximal arm of the clathrin leg based on antibody localization (Kirchhausen et al., 1983), antibody inhibition (Blank and Brodsky, 1987), and deductions based on amino acid sequence data (Kirchhausen et al., 1987). These data also led to the suggestion that the immediate C-terminal region of the molecule, which in the mammalian species examined is rich in hydrophobic amino acids, is the region specialized for the coupling and orientation of the three clathrin heavy chains into a functional triskelion. However, in yeast a similar region does not seem to exist, and up to 77 amino acids can be deleted from the C-terminal region of the clathrin heavy chain without affecting trimer formation (S. Lemmon, personal communication). In comparison with the clathrin heavy chain, elucidating how the clathrin light chains are organized is aided by their much smaller size, but perhaps more than outweighed by the apparent diversity and complexity of their organization. Within all non-brain tissues there are two clathrin light chains that differ slightly in apparent M,., 30 and 33 kd, as determined on SDS gel electrophoresis (Brodsky and Parham, 1983). These forms are minor in brain tissue, where two major species are again found, but with higher apparent M,. of -33 and 36 kd. Work from several laboratories (Jackson et al., 1987; Kirchhausen et al., 1987) has shown that within non-brain tissues the two clathrin light chains are coded for by two distinct but related genes to yield polypeptides with large amounts of sequence homology (-60%). Sequence analysis indicates that the charged amino acids in the amino terminal 40% of both chains are overwhelmingly acidic residues. This highly acidic region probably results in reduced SDS binding and anomalously slow migration on SDS gel electrophoresis: the

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16Figure 3. Polypeptide components of the coat structure from bovine brain coated vesicles analyzed by SDS gel electrophoresis. Lane I : Clathrin heavy (HC) and light chains (LC). Lane 2: AP-1. Lane 3: AP-2. Lane 4: AP-2 treated with elastase, which cleaves the 100 kd components to 70 kd species without affecting either the 50 kd or 16 kd polypeptides. predicted molecular weights of all the light chains are 30% less than those calculated from gels. In brain tissue the larger brain-specific light chains are produced by transcriptional splicing events that introduce sequences of either 90-bp (LC,) or 54-bp (LC,). These brain-specific inserts are in approximately the same region of the two light chains and, particularly in LC,, introduce regions of substantial hydro-

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phobicity to the sequence. From antibody studies, these regions are also known to be exposed on the surface of intact coated vesicles (Brodsky et a]., 1987), suggesting that they have been selected for interaction of coats with cellular components specific to neuronal tissues. The central parts of all the light chain sequences contain conserved regions that are characteristic of coiled coil domains and, perhaps as a consequence, have been found to be at least partly homologous to intermediate filament sequences. As noted earlier, these regions are also proposed to be the site of clathrin heavy chain-light chain binding. In summary, the genomic, transcriptional, and sequence organization of the clathrin light chains is complex. Highly conserved and variable regions are contained within the same chain, and differential processing serves to generate specific products in brain. The conserved nature of portions of the clathrin light chains and their diversification at several different levels suggests that the roles that the light chains fulfill must be widespread and yet specialized. However, definition of these functions remains elusive. The light chains do not seem to play an obligatory structural role: clathrin heavy chains can self-assemble (Winkler and Stanley, 1983) and light chains are not required for AP binding or APmediated assembly of clathrin trimers, Keen et al., in preparation). Light chains have been reported to stimulate phosphorylation of the 50-kd assembly polypeptide (Pauloin and Jolles, 1984) and to be required for binding of the uncoating ATPase (Schmid et al., 1984), but the implications of these observations for light chain regulation of coated membrane functions remain to be explored.

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ASSEMBLY PROTEINS

Before presenting an account of our current understanding of assembly protein structure and function, a brief historical background to the discovery of the clathrin assembly proteins may be useful to the reader. One of the most striking early observations was that the coat of the clathrin-coated vesicle could be readily disassembled by various chemical treatments (e.g., pH > 8, 0.5 M TrisHCI) and subsequently reassembled into cages, indistinguishable from those present in the initial coated vesicle preparation, by removing the disassembly promoting agent. Furthermore, reassembly did not require a vesicle membrane, demonstrating that the self-assembly process was intrinsic to the proteins of the peripheral coat structure (reviewed in Keen, 1985). Initial dissociation-reassembly experiments involved unfractionated extracts of coated-vesicle preparations. When these extracts were subjected to gel filtration, a major peak containing clathrin and recognizable by the presence of the 180 kd heavy chain polypeptide (Figure 3, lane 1) was separated from a subsequent fraction whose composition was considerably more heterogeneous. Under certain conditions of ionic strength (<25 mM) and pH (6.M.25), this first clathrin-containing peak reassembled into intact cages with polygonal

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facets, morphologically similar to the initially isolated CVs. This observation demonstrated that the clathrin molecule itself possessed the information for formation of the complete closed latticework. Surprisingly, if the composition of the reassembly medium was adjusted to more closely approximate physiological conditions, either by addition of salts to achieve isotonicity or by raising the pH (>6.5), pure clathrin no longer assembled into cages. But when the second heterogenous peak obtained on gel filtration was recombined with clathrin, assembly of cages did occur. Thus, the term “assembly protein” or “AP” was applied to a component present in the second peak that was required for purified clathrin to be able to assemble into complete cage structures under relatively physiological conditions of pH and ionic,strength (Keen et al., 1979; Zaremba and Keen, 1983). This activity was attributed to specific polypeptides that were shown to be incorporated into assembling clathrin cage structures (Zaremba and Keen, 1983), and were also thought to be involved in clathrin binding to uncoated vesicles (Ungewickell et al., 1982). These initial observations pointed to heterogeneous species with components of M , 1 W 1 2 0 kd, 45-50 kd, and 1 6 1 9 kd. More recent studies have confirmed that there is considerable complexity in the structure and distribution of these proteins (see below). While there were initially some discrepancies among labs concerning the requirement for the APs, probably stemming from experimental differences in the preparation of the proteins, there is now general agreement with the original observation that AP is required for clathrin assembly under physiological solution conditions (Pearse and Robinson, 1984; Prasad et al., 1985; Manfredi and Bazari, 1987). By convention, structures assembled from clathrin alone are termed “cages,” while those containing both clathrin and AP are referred to as “coats”; both are considered “empty” if they do not enclose a vesicle bilayer.

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A.

Biochemical Characterization: AP-1 and AP-2

Two major groups of assembly proteins have been isolated in studies of coated-vesicle proteins obtained from bovine preparations. In studies reported by our laboratory (Keen, 1987), partially purified AP was obtained by gel filtration of Tris-HC1 extracts of coated vesicles. These AP preparations were incubated with Sepharose resin containing covalently linked clathrin trimers in a solution containing 0.5M Tris-HC1 (disassembly conditions) and the Tris-HCI was then gradually removed by dialysis. After equilibrium had been obtained the resin was poured into a column and washed with Tris-HCI-free buffer. When challenged with an excess of the partially purified AP in this way, the clathrin-Sepharose resin excluded small amounts of contaminating polypeptides as expected, but also was found to specifically exclude one group of polypeptides that was subsequently found to possess clathrin cage assembly activity. This fraction, designated AP- 1, comprised approximately 20% of the starting protein and had a polypeptide composition that included two distinct polypeptides in the 100 kd

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region (108 kd and 100 kd), 47 kd, and 19 kd polypeptides (Figure 3, lane 2). While the AP-I fraction can at best be considered only partially purified, it is likely that the 108, 100, 47, and 19 kd polypeptides exist in a discrete functional complex because they co-elute on gel filtration with a Stokes radius comparable to that of the AP-2 (Keen, 1987). When the clathrin-Sepharose column was further washed with a solution containing a gradient of increasing Tris-HC1 concentration, a major peak of protein was eluted at a concentration centered at about 120 mM Tris-HCI. This material, designated AP-2 by virtue of its elution from the clathrin-Sepharose column, comprised approximately 60% of the applied protein. The protein composition of this peak (Figure 3, lane 3) again contained species recognizable in the unfractionated material, but most of them were distinguishable from those present in the AP- 1 fraction. In particular, a group of polypeptides of 1061 15 kd was observed of which a 1 12 and 115 kd doublet were unique to the AP-2, while the major 100 kd band, which could be resolved into a doublet of 99 and 102 kd on more lightly loaded gels, was electrophoretically indistinguishable from a band of similar mobility in the AP-I. In addition to these polypeptides, distinct bands of 50 and 16 kd were also present. Inspection of the elution profile of the major AP-2 polypeptides of 100, 50, and 16 kd molecular weight indicated that they co-eluted from the clathrinSepharose column. Furthermore (assuming equal amounts of dye binding per weight of protein), they were present in molar ratios of 0.99: 1.00:0.65, respectively, suggesting that the AP-2 was a discrete molecular species containing the three polypeptides in equal amounts. To determine how many copies of each of the three polypeptides were present in the AP-2 molecule, we sought to detcrmine the molecular weight of the native complex by low-angle laser light scattering. Two factors made molecular weight measurement by light scattering difficult. The first was that calculation of molecular weight from light scattering data depends on the differential refractive index increment of the protein of interest. It was not feasible to directly measure this parameter for the purified AP-2, but the values for most proteins fall within a fairly narrow range of 0.175-0.185 ml/g. Even if we assumed a considerably more generous range of 0.1&0.20 ml/g, the calculated molecular weight would still be subject to a potential error of less than 25%. While this is unpalatable for a primary molecular weight measurement, it was acceptable for distinguishing between the two choices for AP-2 molecular weight of 166 or 332 kd. The second concern was aggregation. The AP tends to aggregate in the absence of the relatively high concentrations of Tris-HC1 that are used to extract it from coated vesicle preparations. This was a particular concern using the lightscattering technique because the method provides a weight-average value for molecular weight. Fortunately, because of the sensitivity of the method, it was possible to make accurate measurements at low protein concentrations of 2 6 1 2 0 pg/ml. From other experiments (Beck and Keen, in preparation), we knew that

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0.2 M is a high enough Tris-HC1 concentration to essentially block all aggregation of AP-2 at protein concentrations greater than 100 pg/ml, higher than the concentrations used in almost all of our measurements. Sedimentation studies provided gross confirmation that the AP was not aggregated under these conditions. Finally, when purified preparations of AP-2 at the same concentrations used in light scattering were examined by deep-etch electron microscopy (see below), essentially monodisperse fields of uniformly sized particles were observed. Therefore, samples to be measured were diluted from 0.5 A4 into 0.2 M Tris-HC1 immediately before use, were then subjected to a quick ultracentrifugation step to remove aggregates, and finally were filtered through a Duropore membrane immediately before entering the scattering cell. Light scattering measurements at several different protein concentrations yielded a line with slight negative slope, confirming the existence of intermolecular interactions, and a slight downward bend at the highest protein concentrations ( 1 10 pg/ml) suggesting that aggregation was becoming more significant. The fit to a straight line allowed a weight-average molecular weight to be reliably calculated from the extrapolated ordinate intercept, that is, at infinitely dilute protein concentrations. The molecular weight obtained for the AP-2 preparations under these conditions was 343 kd (Keen, 1987). Given the approximately equal molar stoichiometry of 100, 50, and 16 kd species determined by densitometry, the data are consistent with the existence of a dimeric structure of composition, (100),(50),( 16)*, whose molecular weight would be 332 kd. Combining this value with the Stokes radius (6.6 nm) obtained by gel filtration (Keen et al., 1979; Virshup and Bennett, 1988) and the partial molar volume (0.74 cc/g) calculated from the amino acid composition, a frictional ratio (f7fJ of approximately 1.4 can be calculated. Assuming a prolate ellipsoidal shape and hydration of 0.2 g waterlg protein, we can deduce that the AP-2 is a moderately asymmetric particle in solution and has an axial ratio of about 5-6. In the next section, these considerations are integrated with deep-etch electron microscopic observations of the AP-2 to yield an overall model for the structure of the molecule. The AP-1 and AP-2 correspond to two fractions, designated HA-I and HA-11, respectively, previously obtained by repeated hydroxylapatite chromatography of extracts of coated vesicles according to the method of Pearse and Robinson (1984). These workers also suggested a composition of two 100 kd and two 50 kd polypeptides for the HA-I1 (AP-2) complex; several of the lower molecular weight polypeptides were not reported. A hydroxylapatite procedure was also used by Manfredi and Bazari (1987), coming to the same conclusion about the composition of the complex. In contrast, Virshup and Bennett (1988) began with crude brain membranes and employed hydroxylapatite followed by Mono-Q chromatography to obtain an AP-2 preparation. Their structural studies suggest a composition of two 100 kd, one 50 kd and one 16 kd polypeptide. The source of this difference, including the interesting possibility that the smaller AP subunits may undergo reversible dissociation from the complex, is unknown at present.

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Finally, Ahle et al. (1988) have recently published another isolation of the two AP complexes, beginning with stripped brain coated vesicles and using several hydroxylapatite and ion-exchange chromatography steps. Using a panel of antibodies to different components of the complexes, their results support and extend the earlier observations of Pearse and Robinson (1984) that HA-I/AP-1 is restricted to the Golgi region while HA-II/AP-2 is present in plasma membrane coated pits. The similarity in polypeptide species present in the AP-1 and AP-2 initially suggested that several of the groups of polypeptides might be related or have common origins. This does not seem to be the case for the lower molecular weight polypeptides in the two complexes since complete tryptic peptide maps of the 50, 47, 16, and 19 kd polypeptides show no similarities (Ahle et al., 1988). In addition, RNA blot analysis of several rat tissues with a complete cDNA probe for rat brain AP50 reveals only a single message appropriate for a 50 kd translation product, failing to indicate the existence of a higher molecular weight precursor (Thurieau et al., 1988). While the lower molecular weight components do not appear to be related, there is thought to be partial homology between some of the 100 kd polypeptides of the two APs (Pearse and Robinson, 1984; Ahle et al., 1988). Furthermore, there appear to be two distinct 100 kd molecular weight components within the AP-2 complex (Ahle et al., 1988). The AP- 1 and AP-2 display significant differences in their chromatographic properties on both conventional and affinity-based media. On affinity chromatography with clathrin-Sepharose in the presence of both APs, only the AP-2 appears to bind tightly to clathrin. Yet the AP- I polypeptides do promote clathrin assembly and are incorporated into the cage structure. Furthermore, when clathrin is polymerized by a mixture of the two APs, the AP-2 is preferentially incorporated (Zaremba and Keen, 1983; Keen, 1987). Similar preferential binding has been observed when decoration of preformed clathrin cages with AP has been performed (Keen et al., in preparation). In this case, in the preserlce of both assembly proteins the AP-2 is bound while the AP-1 is essentially excluded. Thus, the two assembly proteins have distinguishable affinities for clathrin whether the latter is in the dissociated trimeric state or in an assembled cage structure. Possible implications of these differences are noted on page 174.

B. Deep-etch Visualization of AP-2 The deep-etch visualization of the AP-2, performed in collaboration with Dr. John E. Heuser (Washington University, St. Louis), has been important in helping to decipher the domain structure of the molecule. AP-2 preparations were adsorbed to mica, washed to provide solution conditions favorable for deep-etching, and then subjected to the quick-freeze deep-etch procedure (Heuser, 1983). Tripartite structures are observed (Figure 4, top), containing a

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large central bricklike mass measuring approximately 9 x 7 nm (after correcting for the thickness of the platinum replica) with two smaller appendages. Not all molecules display such appendages, presumably because adsorption to the mica surface is random with respect to the orientation of the appendages. The appendages often appear to be separated from the main body of the particle by up to 6 nm, and the angle between them is quite variable. Bilateral symmetry in appendage orientation is observed among the molecules that have a sharply defined central brick structure and display two appendages: representative images are presented in the gallery of pictures in Figure 4, middle. To gain insight into the domain structure of the AP-2, we treated purified AP-2 with elastase. This treatment had previously been shown to specifically clip the 100-1 15 kd polypeptides into several closely spaced bands of approximately 70 and 30 kd, but did not detectably digest either the 50 kd or 16 kd assembly polypeptides (Figure 3, lane 4). Furthermore, the larger 70 kd fragments derived from the APlOOs remained associated with the intact 50 and 16 kd polypeptides (Zaremba and Keen, 1985). When similar elastase-treated purified AP-2 preparations were examined by deep-etch microscopy, only smooth brick structures were seen, while the appendages were found to be missing (Figure 4, bottom)! The schematic model for the organization of the AP-2 presented in Figure 5 combines these observations with our molecular weight and stoichiometry measurements noted earlier. The molecule is depicted as being dimeric and bilaterally symmetric. The 50 kd and 16 kd polypeptides are contained within the central brick structure while the mass of the 100 kd polypeptides is divided between it and the two elastase-sensitive appendages. Because the hydrodynamic properties of the AP-2 indicate that it is relatively asymmetric in solution (page 162), these appendages are suggested to be extended out from the central core of the molecule in the native structure in solution.

IV. AP-2-MEDIATED COAT ASSEMBLY IN VITRO The properties of the AP-2 structure, deduced from biochemical and biophysical measurements and from the deep-etch electron microscopic images described Figure 4 . Deep-etch visualization of AP-2 molecules. Top: Survey view of affinity purified AP-2 molecules. X 250,000. Scale bar = 100 nm. Middle: Gallery of selected AP-2 molecules displaying two relatively symmetric small appendages on either side of a relatively squared-off 10 X 12 nm central mass. Images are arranged with the widest spans in the left columns and the narrowest spans in the right columns, with intermediates in between. All X 250,000. Window width and height = 75 nm. Bottom: Several examples of the same AP-2 preparation as above, but subjected to brief elastase digestion before adsorption to mica. Note that both appendages are removed by such treatment. All x 250,000. Window width and height = 75 nm. (From Heuser and Keen, 1988.)

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Figure 5 . Schematic diagram of an AP-2 molecule, depicted as being dimeric and bilaterally symmetrical. Placement of the 50-kd and 16-kd polypeptides is entirely speculative; no information is currently available concerning their location beyond the observation that they are not removed when the appendages are proteolytically removed and they are bound only to the 100-kd, but not to each other, on treatment with a cross-linking agent (Virshup and Bennett, 1988). The 100-kd polypeptides are shown as being divided between the main part of the molecule and the two protease-sensitive appendages. The latter are shown as extending out from the central core of the molecule, as in some of the images seen in the bottom row of Figure 4B, because physical measurements of the AP-2 indicate that it is a relatively asymmetric molecule in solution (see text for discussion). (From Heuser and Keen, 1988.)

above, have led us to propose a detailed model for the mechanism by which AP-2 may drive clathrin assembly into cages. That the AP-2 is dimeric in its complement of 100 kd polypeptides and possesses bilateral symmetry suggested to us that the AP-2 might be bivalent in its binding of clathrin. This in turn suggested that the assembly process may be facilitated by a cross-linking function of AP. In this model, shown in Figure 6A and described more completely in the legend, two triskelia would be brought into proximity through bivalent AP-2 binding, followed by overlap of substantial portions of the leg regions of the trimers upon correct orientation, i.e., when the triskelion legs were brought into register. This would stabilize an assembling structure and polymerization would be driven by subsequent addition of triskelia to “free ends” of the bound AP-2 molecules. It should be noted that polymerization would be enhanced beyond that apparent in the simplified drawing in Figure 6A, because for clarity only two of the four trimers interacting along each edge of the polygon are shown. The model as drawn proposes that AP-2 binds to the terminal domain region at the end of the clathrin heavy chain leg and evidence for this interaction has been obtained by deep-etch electron microscopy of mixtures of dissociated clathrin and AP (Figure 7). But in the completed coat structure, electron microscopic studies suggest that three clathrin heavy chain terminal domains are likely to be

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clustered beneath each of the vertices (Heuser and Kirchhausen, 1985; Vigers et al., 1986a). These terminal domains are derived from clathrin trimers whose hubs are located two vertices away from the clustered terminal domains. If each AP molecule bridges an edge of the polygon in the completed coat structure, then each of the two clathrin binding domains of an individual AP-2 would be expected to have multiple clathrin-AP-2 interactions, probably by binding to a clustered group of three clathrin terminal domains. If this is true, it is likely that AP would bind more strongly to clathrin in a coat structure than to an individual clathrin molecule. How well does this model for AP-2-mediated clathrin-coat assembly fit the available data? The stoichiometry of AP-2 100 kd polypeptides with respect to clathrin heavy chains is one way to evaluate this question. Each vertex of the assembled coat is thought to be the location of the hub of a clathrin triskelion (Ungewickell and Branton, 1981; Kirchhausen and Harrison, 1981) and, from the model, each vertex is in contact with one half of an AP-2 molecule (Figure 6B). This indicates that there should be one APlOO polypeptide (one half of the complement of a complete AP-2 molecule) per clathrin trimer (or three clathrin heavy chains). This ratio has been observed when reassembled coats have been isolated from reassembly mixtures by gradient ultracentrifugation and directly analyzed by SDS PAGE and densitometry (Zaremba and Keen, 1983; Keen, 1987). When assembled coats are simply pelleted from solution, higher AP:clathrin ratios are obtained (Keen et al., in preparation; Pearse and Robinson, 1984). This difference is explained by the observation that AP molecules can bind weakly and reversibly to the surface of assembled coats: these molecules are readily removed on gradient ultracentrifugation but not by pelleting in an unfractionated solution, resulting in higher apparent AP: clathrin ratios. Our recent data provide evidence for multiple clathrin-AP-2 interactions and for at least two recognition sites for AP on clathrin (Keen et al., in preparation). We found that AP-2 can bind to preassembled clathrin cages with a stoichiometry that is similar to that found on coassembly of clathrin and AP-2, apparently by diffusing in through the polygonal openings of the cage. Binding of AP-2 to cages, which persists in 0.25 M Tris-HCI, is considerably stronger than the binding of AP-2 to (trimer) clathrin-Sepharose. Somewhat surprisingly, AP also bound to clathrin cages whose terminal domains had been removed by proteolysis. But in this case, binding is abolished by Tris-HCI concentrations greater than approximately 0.1 M Tris-HC1. We interpret these data to indicate that there are at least two distinct clathrin binding sites on the AP-2 molecule. One of these sites recognizes the terminal domain region, probably making multiple contacts with terminal domains derived from adjacent trimers in the coat. The second site can be placed somewhere on the proximal leg or hub region of the clathrin triskelion and is much more sensitive to dissociating agents. If there are indeed two dissimilar 100 kd polypeptides within an individual AP-2 complex (see page 163), are they differentiated for recognition of the distinct sites on clathrin (see above)? Alternatively, one of the APlOO polypep-

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Figure 6. A model for AP-2-mediated clathrin coat assembly. A. Role of AP-2 in initiating assembly. Upper left: AP-2 (shaded rectangle) bridges the terminal domains or distal leg regions of two clathrin trimers (posi168

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tides could possess both binding affinities for clathrin, with the second APlOO specialized for other functions, such as receptor binding (see below). To the extent that these recognition sites act to bind two different clathrin triskelia, coat formation would be promoted by the act of bringing the two trimers into closer proximity and orienting them appropriately for assembly.

V.

AP FUNCTIONS IN CELLS: RECEPTOR CLUSTERING AND COATED PIT FORMATION?

The function of the AP in cells must transcend its narrow ability to drive clathrin assembly into empty (membrane-free) coat structures. Since clathrin alone will spontaneously form cages, the evolution of an unrelated, diverse family of assembly proteins whose function is solely to sustain the assembly process under unfavorable salt conditions seems biologically excessive. Furthermore, coat formation in vivo obviously involves more than just clathrin and AP molecules: empty clathrin cages and AP-containing coats are generally not seen in intact cells. Instead, coat assembly appears to take place exclusively on membrane sites. Thus, while studies of the behavior of clathrin and AP in solution only tell part of the story, they have been productive in revealing some of the underlying characteristics of the assembly system and thereby provide a framework for thinking about how coated membranes are formed and function in cells. In this section, I first focus on two properties of clathrin and AP revealed by solution studies: the intrinsic shape of the clathrin triskelion and the tendency of the AP-2 molecule to self-associate. With the observation that the AP-2 binds to a cell surface receptor (Pearse, 1985), these properties suggest that the AP is multi-

tions d’ and e ) while a second AP-2 molecule is bound at position a. Upper right: Movement of one of the trimers (dashed lines) into overlap with the other (along a-b-c-d) facilitates bridging of a second AP-2 molecule, at a-h, and rigidification of the complex. Additional AP-2 molecules are bound near position i and at g. Lower right: A third clathrin trimer (dorted lines) binds to the available AP-2 site at position i. Lower l e f : The third trimer (dotted lines) has now become aligned with the first two, stabilized by the action of a second AP-2 molecule bridging terminal domains at positions f - g . The three trimers have generated a closed pentagon (a-b-c-fh)and there is multiple leg overlap along a-b-c-d. A “free” AP-2 molecule (near a ) is available for propagation of the assembly reaction. B . At completion, the clathrin domains at each vertex of the assembled coat will interact with one end of a bivalent AP-2 molecule, consistent with biochemical measurements of AP-2 molecular weight and AP-2 :clathrin stoichiometry . (From Keen, 1987.)

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Figure 7. Gallery of selected examples of AP-2 binding to the terminal domains of isolated clathrin triskelia. Window width and height = 100 nm. (From Heuser and Keen, 1987.)

functional in mediating receptor clustering, clathrin binding, and coat assembly as concurrent, or intimately connected, events in cells. In vitro experiments show that under all but the most unphysiological conditions (pH <6), clathrin spontaneously forms cages with surface curvature, rather than flat lattices (reviewed in Keen, 1985). This is consistent with electron microscopic evidence which indicates that the unassembled clathrin triskelion in solution is “puckered” (Kirchhausen et al., 1986). That is to say, the legs of the clathrin triskelion are not coplanar with the central hub, but rather are displaced to one side, yielding a three-legged spiderlike molecule with a raised central hub region. A certain amount of triskelion pucker is required, geometrically, to form a closed coat structure with triskelions at each vertex: perfectly planar molecules are capable of forming only flat lattices. In the cell, flat hexagonal lattices have been observed at sites where membrane is rigidly held, as at adhesion sites (Heuser, 1980) and on polylysine coated surfaces (Larkin et al., 1986; Moore et al., 1987), particularly in chilled cells. These data suggest that mechanical force can flatten the triskelion in situ and that such flat lattices may be special configurations of clathrin, as suggested earlier (Harrison and Kirchhausen, 1983). In turn, that planar lattices are seen only or predominantly at these sites in cells suggests that flattening the triskelion may require considerable energy. As noted above, pure clathrin forms spherical cage structures but there is a detectable variation in the size of the cages formed, from about 80 to 115 nm in diameter as estimated by negative staining. These data indicate that some flexibility in the pucker of the triskelion exists, within narrow limits. In contrast to assembly of pure clathrin alone, when clathrin is assembled in the presence of AP-2 the coats formed are much more sharply centered about 80 nm in diameter (Zaremba and Keen, 1983; Keen, 1987). Formation of these uniformly sized structures depends only on the presence of AP-2 and not on the particular buffer

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conditions in which the assembly reaction is performed, nor on whether assembly proceeds slowly by dialysis or rapidly by dilution. We have interpreted these data to indicate that AP-2 binding to clathrin is effective in inducing either a defined pucker in the triskelion or specific overlap of the assembling clathrin molecules, or both (Zaremba and Keen, 1983). In the proposal below I raise the possibility that the pucker and rigidity of the clathrin triskelion, its consequent tendency to form a curved latticework, and the energy derived from AP binding to clathrin, provide the driving force for indentation of the plasma membrane during endocytosis. Another property of coated membrane components revealed by solution studies is the tendency of brain AP-2 to rapidly self-associate when placed in assembly conditions in the absence of clathrin (Beck and Keen, 1987; Beck and Keen, in preparation). This interaction appears to reflect specific AP-2-AP-2 binding since other proteins in solution are excluded from the growing aggregates. Because various agents that inhibit these AP-2-AP-2 intcractions do not affect AP-2-mediated clathrin coat assembly (at least in the same concentration range), we do not believe that AP-2 self-association initiates coat assembly in solution. But if assembly proteins bind to receptors (Pearse, 1985), then the attribute of self-association may be of considerably greater significance when AP-2 is bound to a membrane (and receptor). That is, AP-2 self-association in solution may be the three-dimensional equivalent of the receptor clustering process that occurs on the two-dimensional membrane surface during endocytosis. Collectively, these properties of the protein components of the clathrin coat provide a framework for suggesting how the coat structure is involved in the processes of receptor clustering and coated pit indentation that are characteristic of receptor-mediated endocytosis (Figure 8). It is hypothesized that the AP-2 is first bound to the membrane, either by recognition of a specific membrane-bound AP-2 binding protein (Virshup and Bennett, 1988) or through binding to the cytoplasmic domain of a cell surface receptor that is destined to cluster in coated pits (Pearse, 1985). Each molecule of AP-2 could come equipped with a bound clathrin triskelion (as indicated in Figure 8), or clathrin could add subsequent to AP-membrane binding. In either case, the strong tendency of AP-2 molecules to self-associate would then lead to clustering of the (clathrintAP-2-receptor complexes to yield the local concentrations of receptor characteristically seen on the plasma membrane surface during endocytosis. Aggregation of receptor-AP-2 complexes could also be driven by the dimerization properties of some receptors themselves, e.g., the EGF receptor (Yarden and Schlessinger, 1987). Polymerization of clathrin on the flat AP-studded membrane surface would be expected to occur rapidly as clathrin trimers, either binding to the clusters of AP-2 or coming pretethered on the associating AP-2 molecules, were brought into proximity and clathrin leg overlap was initiated. Given the observation that the clathrin triskelion resists flattening and that it is bound to the AP-2 by

EXTERIOR

A

PLASM

J

AP-2

ASSOCIATION

1

CLATHRIN POLYMERIZATION

C

D

E

1

MEMBRANE CURVATURE

ASSOCIATION POLYMER It AT ION CURVATURE

Figure 8. Scheme depicting a pathway for coated pit assembly involving AP-2 self-association and clathrin lattice assembly, and based on information derived from solution studies of clathrin and AP-2. Note that the stoichiometry and size 172

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multiple interactions, clathrin polymerization on the membrane-bound AP-2 would begin to form a nonplanar latticework, of curvature similar to that generated during solution assembly. If the AP-2 is indeed tightly linked to integral membrane components (whether binding protein or receptor), the membrane would then be forced to accommodate itself to this curvature, rcsulting in the formation of a coated pit. Clusters of protein thought to be patches of AP-2 have indeed been observed on membranes chemically or enzymatically stripped of clathrin, suggesting that they are coated pit assembly sites underlying the lattice of the coat, although definitive proof that these contain AP is awaited (J. Heuser, R. Anderson, personal communication). In this context it is also of interest that AP-2-AP-2 interactions in solution can be blocked, or reversed, by various physiological polyphosphate-containingcompounds such as PIP, and IP, (Beck and Keen, in preparation). Since the levels of these agents are regulated in cells, these results suggest that polyphosphates may regulate AP-2 function in vivo. In double-label immunofluorescence studies, no detectable difference between clathrin and AP localization has been reported (Robinson, 1987; Ahle et al., 1988). This observation is not inconsistent with the model described above, for if the clathrin-free AP-decorated membrane surface is relatively short-lived com-

of the structures are approximately to scale, but that the number and point of their interactions are meant only to be representative, and not literal. A. AP-2 molecules (cross-hatched squares) are attached to the plasma membrane through direct linkage to cell surface receptors (Y-shaped figures) that traverse the membrane. Clathrin trimers (slightly curved arrowhead shaped figures) are shown bound to the AP-2 through the terminal domain regions at the ends of the clathrin heavy chains (wavy line). Alternate possibilities such as AP-2-receptor interactions mediated by distinct binding proteins, or free AP-2 molecules without tethered clathrin trimers, are not shown for visual clarity. B . Clustering of AP-2-receptor complexes on the planar membrane surface is driven by AP-2-AP-2 interactions. The attached clathrin trimers are brought into proximity, facilitating assembly. C . Clathrin-clathrin interactions initiate lattice assembly. The assembly reaction of the clathrin trimers bound to AP-2 molecules strongly favors formation of a curved lattice with a specific radius of curvature. Pressure is exerted (springlike lines) on the relatively planar membrane surface by this growing curvature of the clathrin lattice while the AP-2 molecules are attached to the membrane through linkage to the receptor. D and E. The membrane has been formed into a curve by accommodation to the curvature of the clathrin lattice; the latter is now a fixed distance from the AP-2 and the membrane. Additional AP-2-receptor complexes with tethered clathrin trimers are recruited to the growing coated pit and further polymerization yields increased membrane curvature.

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pared to a coated pit, it would be exceedingly difficult to detect in the steadystate. A related problem is to determine the factors that regulate the completion of the coat structure of a coated pit and lead to the pinching off of an endocytotic coated vesicle. Since coated pits are readily seen by many different electron microscopic techniques, it is clear that a partially spherical coated membrane surface is a relatively stable and long-lived intracellular structure. This stands in contrast to solution studies in which only completed coat structures or dissociated protein are evident: partial coat structures (e.g., intermediates in the assembly process) are not detectable. If AP-2 self-association plays a role in driving coated pit formation at the plasma membrane, is there any basis for proposing that AP- 1 plays a similar role in the Golgi region? Clathrin coats formed with AP-I are smaller than those formed with AP-2 (Keen, 1987); whether this bears any relation to the smaller size of Golgi coated membrane profiles as compared with those of the plasma membrane is unknown. In preliminary experiments, we have not seen any evidence for AP-1-AP- 1 interactions. Also, AP-1 is not significantly incorporated into aggregates formed in the presence of a large excess of AP-2. The lack of interaction between these two APs and their significantly different affinity for clathrin (page 164) could play a role in partitioning the AP-1 complex to the Golgi region and the AP-2 to plasma membrane coated pits. !n principle, it seems plausible that different assembly proteins may fix slightly different amounts of triskelion pucker, within the narrow range of the latter’s flexibility, yielding different degrees of curvature that may correlate with the varied sizes of coated membrane profiles seen in different tissues and intracellular locations (Pearse and Crowther, 1987). Probably more importantly, different receptor classes and different membrane sites may be distinguished by the individual assembly proteins. Consistent with these notions are the observations that the assembly proteins appear to comprise a family of proteins that are functionally and structurally related, both at the protein (Keen, 1987; Ahle et al., 1988) and gene levels (Kirchhausen et al., in preparation). Many of these ideas and hypotheses will be tested, refined, and resolved by coming studies of the structure and function of the assembly protein family and their interactions with cellular components.

NOTE ADDED IN PROOF The amino acid sequences of two 100-1 15 kd polypeptides in the AP-2 complex have recently become available and have been designated a (M. S. Robinson, J. Cell Biol. 108: 833-842 (19891) and p or APlOSb (T. Kirchhausen, K. L. Nathanson, W. Matsui, A. Vaisberg, E. P. Chow, C. Burne, J. H. Keen and A. E. Davis, Proc. Nat. Acad. Sci. 86: 2612-2616 [ 19891; T. Kirchhausen, personal communication). Comparison of the published sequences shows ap-

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proximately 17% sequence identity, with an alignment score more than thirteen standard deviations greater than the mean of 200 randomly permuted sequences. This statistical evidence of a distant but definite relationship between the two polypeptides is supported by their biochemical similarity in domain structure (pp. 164-166; Zaremba and Keen, 1985) and orientation within the AP-2 complex (Kirchhausen et al., 1989, above). The functional attributes of the individual polypeptides are being evaluated.

ACKNOWLEDGMENTS I am grateful to past and present members of this laboratory, in particular Ken Beck and Jo-Ellen Murphy, for their contributions to work from this laboratory, stimulating discussions, and helpful criticisms; and to Mark Black for critically reading parts of the manuscript. Work in the author’s laboratory was supported by awards from the National Institutes of Health (#GM-28526) and the American Cancer Society (BC-567).

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