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• W2000388340 abstract "Article2 March 1998free access Clathrin self-assembly is regulated by three light-chain residues controlling the formation of critical salt bridges Joel A. Ybe Joel A. Ybe The G.W.Hooper Foundation, Department of Microbiology and Immunology, and Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA, 94143-0552 USA Search for more papers by this author Barrie Greene Barrie Greene The G.W.Hooper Foundation, Department of Microbiology and Immunology, and Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA, 94143-0552 USA Search for more papers by this author Shu-Hui Liu Shu-Hui Liu The G.W.Hooper Foundation, Department of Microbiology and Immunology, and Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA, 94143-0552 USA Search for more papers by this author Ursula Pley Ursula Pley Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, 94305 USA Search for more papers by this author Peter Parham Peter Parham Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, 94305 USA Search for more papers by this author Frances M. Brodsky Corresponding Author Frances M. Brodsky The G.W.Hooper Foundation, Department of Microbiology and Immunology, and Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA, 94143-0552 USA Search for more papers by this author Joel A. Ybe Joel A. Ybe The G.W.Hooper Foundation, Department of Microbiology and Immunology, and Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA, 94143-0552 USA Search for more papers by this author Barrie Greene Barrie Greene The G.W.Hooper Foundation, Department of Microbiology and Immunology, and Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA, 94143-0552 USA Search for more papers by this author Shu-Hui Liu Shu-Hui Liu The G.W.Hooper Foundation, Department of Microbiology and Immunology, and Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA, 94143-0552 USA Search for more papers by this author Ursula Pley Ursula Pley Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, 94305 USA Search for more papers by this author Peter Parham Peter Parham Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, 94305 USA Search for more papers by this author Frances M. Brodsky Corresponding Author Frances M. Brodsky The G.W.Hooper Foundation, Department of Microbiology and Immunology, and Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA, 94143-0552 USA Search for more papers by this author Author Information Joel A. Ybe1, Barrie Greene1, Shu-Hui Liu1, Ursula Pley2, Peter Parham2 and Frances M. Brodsky 1 1The G.W.Hooper Foundation, Department of Microbiology and Immunology, and Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA, 94143-0552 USA 2Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, 94305 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:1297-1303https://doi.org/10.1093/emboj/17.5.1297 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Clathrin self-assembly into a polyhedral lattice mediates membrane protein sorting during endocytosis and organelle biogenesis. Lattice formation occurs spontaneously in vitro at low pH and, intracellularly, is triggered by adaptors at physiological pH. To begin to understand the cellular regulation of clathrin polymerization, we analyzed molecular interactions during the spontaneous assembly of recombinant hub fragments of the clathrin heavy chain, which bind clathrin light-chain subunits and mimic the self-assembly of intact clathrin. Reconstitution of hubs using deletion and substitution mutants of the light-chain subunits revealed that the pH dependence of clathrin self-assembly is controlled by only three acidic residues in the clathrin light-chain subunits. Salt inhibition of hub assembly identified two classes of salt bridges which are involved and deletion analysis mapped the clathrin heavy-chain regions participating in their formation. These combined observations indicated that the negatively charged regulatory residues, identified in the light-chain subunits, inhibit the formation of high-affinity salt bridges which would otherwise induce clathrin heavy chains to assemble at physiological pH. In the presence of light chains, clathrin self-assembly depends on salt bridges that form only at low pH, but is exquisitely sensitive to regulation. We propose that cellular clathrin assembly is controlled via the simple biochemical mechanism of reversing the inhibitory effect of the light-chain regulatory sequence, thereby promoting high-affinity salt bridge formation. Introduction Many cellular processes depend on regulating the spontaneous self-assembly of proteins into macromolecular structures. Such self-assembly reactions include the polymerization of cytoskeletal elements (Egelman and Orlova, 1995), the oligomerization of transcription factors (Wu, 1995) and the assembly of membrane vesicle coats (Pearse and Crowther, 1987). A prerequisite for understanding the cellular control of these processes is to define the molecular basis for the spontaneous protein–protein interactions involved. We therefore set out to establish the biochemical mechanism of spontaneous clathrin polymerization in order to understand the cellular regulation of clathrin-coated vesicle formation. These membrane transport vesicles are composed of a polymerized clathrin coat surrounding a layer of adaptor molecules which interact with membrane proteins. They control receptor-mediated endocytosis at the plasma membrane and the biogenesis of lysosomes and secretory granules in the trans-Golgi network (Schmid, 1997). Clathrin is a triskelion-shaped molecule comprising three heavy chains, each with a bound light-chain subunit. Below pH 6.5, purified clathrin triskelions self-assemble in vitro into a polyhedral lattice (basket) (Brodsky, 1988), but will only form baskets at physiological pH in the presence of stoichiometric quantities of purified AP-1 or AP-2 adaptor molecules or the neuron-specific assembly proteins AP-180 and auxilin (Ahle and Ungewickell, 1986; Keen, 1990; Pearse and Robinson, 1990; Lindner and Ungewickell, 1992). AP-1 and AP-2 adaptors localize clathrin lattice formation to their specific membrane-binding sites in the trans-Golgi network or at the plasma membrane and mediate the interaction of the clathrin lattice with the vesicle cargo (Ohno et al., 1995), so that clathrin polymerization causes receptor sorting by concentrating receptors which are recognized by adaptors. The cellular role of the neuron-specific assembly factors is not yet established, but in vitro studies suggest that they influence the efficiency of assembly (Lindner and Ungewickell, 1992) and disassembly (Ungewickell et al., 1995), a specialized level of regulation that may be required for the extensive membrane traffic at synapses (Morris and Schmid, 1995). To begin to understand how clathrin self-assembly can be controlled so tightly in the cell, we analyzed the self-assembly reaction of a bacterially expressed recombinant hub fragment of the triskelion, which has previously been shown to mimic self-assembly of the whole clathrin molecule, both biochemically and morphologically (Liu et al., 1995). Recombinant hubs, formed from residues 1074–1675 of the clathrin heavy chain, are trimeric structures that reproduce the central portion of the three-legged clathrin triskelion, extending from the vertex to the bend in each leg, comprising the binding sites for clathrin light-chain subunits. Without light-chain subunits, recombinant hubs self-assemble reversibly at physiological pH, while hubs with bound light chains only self-assemble below pH 6.5, similar to purified clathrin (Liu et al., 1995). This inhibition of hub assembly by light-chain subunits appeared to be the key to controlling spontaneous clathrin self-assembly at physiological pH, so we decided to map the light-chain residues responsible for inhibition. In this paper we report that: (i) a conserved negatively charged sequence of three residues (23–25) in the clathrin light-chain subunits regulates the pH dependence of hub assembly; and (ii) two classes of salt bridge play a dominant role in driving clathrin assembly. These findings suggest a simple model for how clathrin light chains prevent non-productive clathrin assembly at physiological pH, making the process dependent on adaptors and assembly proteins within the cell. Results Light-chain residues 23–25 regulate hub assembly Recombinant bovine clathrin hub fragments were reconstituted with wild-type or mutant bovine light chains (Figure 1) and their assembly analyzed at pH 6.2 and 6.7 (Figure 2). Reconstitution was confirmed by Ni2+-affinity purification of the assembled histidine-tagged hubs, and analysis for bound light chains (no polyhistidine tag) by immunoblotting following SDS–PAGE (Figure 1B). All the mutant light chains bound to hub molecules and were reactive with anti-light-chain antibodies, indicating that there were no extreme alterations in their folding or binding properties. Mammalian clathrin has two types of light chain, LCa and LCb, each of which has a neuronal form (nLCa and nLCb) including a short hydrophobic sequence near the C-terminus (Brodsky et al., 1991), which is spliced out in non-neuronal tissue (Wong et al., 1990). It was shown previously that full occupancy of hubs by nLCb strongly inhibited self-assembly at physiological pH (Liu et al., 1995). In initial experiments (Figure 2), a deletion mutant of nLCa (Δ26–56) was found to have a similar effect on pH sensitivity, while another nLCa deletion mutant (Δ22–96) was unable to inhibit assembly at physiological pH, although it bound equally well to the hub. Examination of the differences between these two deletion mutants revealed that one patch of difference, residues 22–25, overlapped with a conserved sequence (23–44) that is shared by all mammalian light chains (LCa and LCb). The sequence of the patch for all mammalian clathrin light chains is GEED in LCa and AEED in LCb (Jackson and Parham, 1988). In addition, in yeast and Aplysia, which have only one clathrin light chain each, the corresponding residues are KDDD and GEVD, respectively (Silveira et al., 1990; Hu et al., 1993). We hypothesized that residues 22–25 might comprise the operative pH control, due to their conserved negative charges. Therefore we generated the nLCa deletion mutant Δ22–25 and tested its ability to regulate the pH sensitivity of hub assembly. Reconstitution of hubs with this mutant demonstrated that the GEED sequence is essential for the light-chain inhibitory activity (Figure 2). To confirm that the negative charge of this sequence was important, a mutant (GQQN) was generated with all three charges neutralized. At pH 6.7, this GQQN mutant bound the hub fragments but did not inhibit hub assembly (Figures 1 and 2). These results established that a minimal acidic patch in the light chains controls the hub self-assembly reaction. Figure 1.Clathrin light-chain mutants and their binding to hub fragments. (A) Schematic alignment of sequences of the wild-type neuronal clathrin light chains (nLCa and nLCb) and the nLCa mutants used for reconstitution of hub molecules. The numbers after Δ in the name of each mutant indicate the segment deleted to create the mutant protein [residue numbers are according to the bovine and human light-chain sequences (Jackson and Parham, 1988)]. The GQQN mutant has nLCa residues 23–25 changed from EED to QQN. In wild-type nLCb, the G at position 22 is A and positions 22–25 are EED, as in nLCa. (B) Reconstituted hub molecules from the assembled fraction of the pH 6.2 experiments shown in Figure 2 were analyzed by SDS–PAGE and immunoblotted with antibodies to detect the hub fragments and associated wild-type or mutant light chain: (1) hub alone, (2) hub + nLCb, (3) hub + nLCa Δ224–243, (4) hub + nLCa Δ22–25, (5) hub+nLCa, (6) hub + GQQN. The arrowheads indicate the light chains and the stars indicate the hub fragments. Note that mutants nLCa Δ22–56 and nLCa Δ22–96 also bind hub molecules (data not shown) and that some of the LCa mutants have heterogeneous migration patterns due to the formation of internal disulfide bonds (Parham et al., 1989). The migration of the molecular mass marker proteins (in kDa) is indicated by the bars on the left of each panel. Download figure Download PowerPoint Figure 2.Identification of clathrin light-chain residues regulating assembly at pH 6.7. Hub molecules were reconstituted with the wild-type or mutant light chain indicated and assembly was measured by light scatter at A320. The value of the light scatter signal, 5 min after changing to the indicated pH from pH 7.9, is plotted. The ratios of this light scatter signal at each pH for the reconstituted hubs are tabulated. Download figure Download PowerPoint Two classes of salt bridges mediate hub assembly Identification of the minimal light-chain sequence regulating the pH dependence of clathrin self-assembly raises the question of how this sequence exerts control. To address this, the biochemical nature of the spontaneous interaction between clathrin heavy chains was investigated. Earlier biochemical studies suggested that salt bridges (Keen et al., 1979) and electrostatic interactions dominate clathrin assembly (Van Jaarsveld et al., 1981; Nandi and Edelhoch, 1984). Therefore, this study investigated the effect of NaCl on the assembly of recombinant hubs that lacked light chain subunits (Figure 3). Recombinant hub molecules were suspended in increasing concentrations of NaCl and self-assembly was then initiated at pH 6.2 (Figure 3A) or pH 6.7 (Figure 3B). Salt inhibition of hub assembly at either pH occurred with two cooperative transitions, indicating that there are two major classes of salt-bridge interaction which mediate hub assembly. At pH 6.2, the transition midpoint for the inhibition of the lower affinity salt bridges was at ∼175 mM NaCl, and for the higher affinity salt bridges was at ∼275 mM NaCl. At pH 6.7, less salt was required to inhibit both classes of salt bridge to their midpoint of assembly, but the decrease was more dramatic for inhibition of the lower affinity salt bridges, whose inhibition midpoint shifted from ∼175 to ∼50 mM NaCl. The considerable weakening of the low-affinity salt bridges at pH 6.7 suggests the possible involvement of histidine residues, whose pKa may be close to this pH. Figure 3.Effect of salt on hub assembly at (A) pH 6.2 and (B) pH 6.7 and (C) on Δhub assembly at pH 6.2 (▴) compared with hub assembly at pH 6.2 (□). Recombinant clathrin hubs without light chains were purified (Liu et al., 1995) and suspended in assembly buffer with the indicated salt concentration. Assembly was measured by light scatter and the signal of each assembly reaction, 5 min after reducing the pH to 6.2 or 6.7, is plotted. Note that the higher light scatter signal produced by hub assembly at pH 6.7, as seen in (B), has been observed previously (Liu et al., 1995) and reflects larger particle formation, probably due to enhanced hydrophobic interactions at higher pH. This effect is also observed in Figures 2 and 4. Download figure Download PowerPoint Critical salt bridges map to distinct subdomains To map which subdomains of the hub sequence contain residues that participate in the salt bridges controlling hub self-assembly, a hub deletion mutant was generated that was missing residues 1074–1212 (Δhub, comprising residues 1213–1675). This deletion removes one-third of the residues that contribute to the interaction with another hub during assembly, and the deleted sequence is predicted to span the length of the proximal leg segment of clathrin (Liu et al., 1995). The Δhub fragment was soluble during bacterial expression and purified readily. Comparing the assembly of purified Δhub with that of hub showed that deletion of the 1074–1212 segment removed residues that are critical for self-assembly at either pH 6.2 or 6.7 (Figure 4). Δhub did not assemble at pH 6.7, while a reduced level of assembly was detected at pH 6.2 compared with the full-length hub. Deletion of residues 1074–1212 apparently eliminated the high-affinity salt bridges that can form at either pH but the low-affinity salt bridges, dependent on low pH for formation, were still present in Δhub. To confirm this, the effect of salt on Δhub assembly at pH 6.2 (Figure 3C) was studied. Indeed, Δhub assembly was inhibited to baseline by 150 mM NaCl, which lies in the same range as the titration midpoint of the low-affinity salt bridges controlling assembly of full-length hub. While these low-affinity salt bridges were detected at pH 6.7 in the salt inhibition study (Figure 3B), they are evidently not sufficient to support hub assembly at physiological pH in the absence of high-affinity salt bridges. Figure 4.Assembly of Δhub recombinant protein compared with hub assembly at pH 6.2 and 6.7. Recombinant hub (residues 1074–1675) and Δhub (residues 1213–1675) proteins were purified and induced to assemble at pH 6.2 or 6.7. Assembly was measured by light scatter over time after addition of concentrated MES buffer at the indicated pH. The value plotted is the light scatter signal for each sample 5 min after the induction of assembly. Download figure Download PowerPoint The assembly properties of the Δhub construct indicated that the two classes of salt bridge are located in two distinct subdomains of the hub molecule. The deleted hub protein sequence from 1074 to 1212 was therefore inspected for conserved, charged residues that could potentially form high-affinity salt bridges at physiological pH (Figure 5). Patches of conserved acidic and basic residues were observed in all eight diverse species whose clathrin heavy-chain sequences are known, in positions equivalent to DD (1132 and 1133) and RKKAR (1161–1165), in the bovine heavy-chain sequence (Figure 5A). The involvement of positively charged residues in clathrin self-assembly had also been suggested previously by the demonstration that the succinylation of arginines and lysines inhibits assembly at low pH (Nandi and Edelhoch, 1984). The Δhub sequence retained residues that formed low-affinity salt bridges at pH 6.2 but not pH 6.7, implicating histidine as a possible partner in the low-affinity salt bridges, since its pKa in a protein can be in this pH range (Hendsch and Tidor, 1994). In addition, the salt titration experiments in Figure 3 were suggestive of histidine involvement in these salt bridges. We therefore searched the Δhub sequence for conserved histidines. Three histidines at positions 1279, 1313 and 1335 of the bovine heavy-chain sequence are present in equivalent positions in seven of the eight known clathrin heavy-chain sequences, and the histidines at 1313 and 1335 are present in all the known sequences (Figure 5B). Close to these histidine residues, all eight heavy-chain sequences have conserved negatively charged residues that are potential salt bridge partners for the histidine residues in the opposing Δhub sequence. This sequence analysis thus revealed that within the heavy-chain domains predicted to form the high- or low-affinity salt bridges, there are conserved residues with properties that are consistent with their participation in such salt bridges. Figure 5.Sequence analysis predicting the residues involved in the formation of salt bridges during clathrin assembly. The clathrin heavy-chain sequences listed are derived from Homo sapiens (Nomura et al., 1994), Rattus norvegicus (Kirchhausen et al., 1987), Bos taurus (Liu et al., 1995), Drosophila melanogaster (Bazinet et al., 1993), Caenorhabditis elegans (Wilson et al., 1994), Dictyostelium discoidum (O'Halloran and Anderson, 1992), Glycine max (Blackbourn et al., 1996) and Saccharomyces cerevisiae (Lemmon et al., 1991). Residue numbering is based on the bovine clathrin heavy-chain sequence. The salt bridge partners of predicted high-affinity salt bridges (A) are labeled a and b, while those of the low-affinity salt bridges (B) are labeled c and d. Acidic residues are shaded and conserved predicted partners are boxed. Download figure Download PowerPoint Discussion Light-chain residues 23–25 inhibit the formation of high-affinity salt bridges between hub molecules The deletion mutagenesis and biochemical experiments reported in this paper, combined with sequence analysis, suggest a remarkably simple salt-bridge formation model (Figure 6) for: (i) how clathrin heavy chains self-assemble spontaneously; (ii) how clathrin light chains inhibit spontaneous assembly at physiological pH; and (iii) how adaptors might reverse the light-chain effect in cells. This assembly model is based on our previous demonstration that the light-chain-occupied hub fragments comprise heavy- and light-chain sequences that are the primary determinants of the clathrin self-assembly reaction (Näthke et al., 1992; Liu et al., 1995). First we suggest that spontaneous self-assembly of the hub fragments depends on high- (Figure 6A) and low-affinity (Figure 6B) salt bridges (Figure 3), formed between conserved residues (Figure 5). These residues are oriented appropriately, i.e. opposite each other, if the sequence forming the hub spans the leg three times, according to our current model (Näthke et al., 1992; Liu et al., 1995), and the hubs are aligned anti-parallel as they would be during self-assembly (Pearse and Crowther, 1987). Figure 6.Model of hub alignment during assembly showing the predicted high- and low-affinity salt bridges and the role of light chains in regulating clathrin assembly. The assembled hub fragments are drawn to be compatible with our earlier folding model predicting that the heavy-chain sequences from 1074 to 1550 span the hub segment three times (Näthke et al., 1992; Liu et al., 1995) and with the fact that hubs self-assemble in an anti-parallel orientation (Pearse and Crowther, 1987). (A) The arrows indicate the predicted interaction between the conserved positively and negatively charged partners of the high-affinity salt bridges, which form at low or physiological pH (missing in the Δhub construct). (B) The dashed arrows are located between patches containing conserved histidines and negatively charged residues that are potential partners for the low-affinity salt bridges, which occur only at low pH (present in the Δhub construct). The asterisks represent variable residues. (C) The central portions of the light chains (striped) are shown in their predicted binding orientation (Näthke et al., 1992; Liu et al., 1995), which would locate the regulatory EED residues (of bovine nLCa and nLCb) in a position to neutralize the electrostatic interactions needed to form the high-affinity salt bridges, depicted in (A). In the presence of light chains, hub interactions are mediated only by the low-affinity salt bridges in (B) and assembly can occur only at low pH. The arrowheads at the N- and C-termini of the light chain indicate ambiguity in the placement of the extreme termini of the light chain (Kirchhausen and Toyoda, 1993; Pishvaee et al., 1997), as discussed in the text. T denotes the trimerization domain. The hub sequences shown and the residue numbering correspond to the bovine clathrin heavy chain (Liu et al., 1995). Download figure Download PowerPoint The assembly properties of the Δhub fragment (Figure 4) localize the high-affinity salt bridges to the 1074–1212 hub subdomain deleted from Δhub. The low-affinity salt bridges involving histidine (Figure 6B) are the ones predicted to induce assembly of the Δhub protein at low pH. Since Δhub and hub with bound light chains have a similar pH dependence of assembly, the light chains must interfere with the high-affinity salt bridges that form between the light-chain-free hubs at physiological pH. The predicted localization of the bound light chain (Figure 6C) within the hub sequence (Kirchhausen et al., 1983; Ungewickell, 1983; Näthke et al., 1992) is consistent with the possibility that the negatively charged residues within the 23–25 sequence could neutralize the conserved positively charged patch on the hub sequence, which, in the absence of a light chain, would form a salt bridge to a conserved negatively charged patch on another hub molecule. The folded orientation of the light chains in Figure 6C has been proposed previously, based on antibody inhibition data and structure predictions (Näthke et al., 1992). Studies of yeast clathrin heavy-chain mutants suggest that light-chain interactions are influenced by, and may extend into, the heavy-chain trimerization region (Pishvaee et al., 1997) beyond the minimal domain that independently binds light chains (Liu et al., 1995). There is also some evidence that the extreme N-terminus of the light chains could extend away from the vertex (Kirchhausen and Toyoda, 1993). These additional hypotheses about light-chain disposition which, for simplicity, are left out of Figure 6C are still consistent with our placement of the residue 23–25 regulatory region and in keeping with the idea that light chains prevent the formation of high-affinity salt bridges at physiological pH. This model of light-chain regulation could also explain why calcium binding by residues 85–96 in the clathrin light chains (Näthke et al., 1990) dramatically enhances clathrin assembly in vitro (Ungewickell and Ungewickell, 1991). If the calcium-binding sequence is oriented appropriately, bound calcium cations could potentially neutralize the negative regulatory sequence in the light chains, freeing up the heavy-chain residues that form the high-affinity salt bridges. While this in vitro effect of calcium on clathrin assembly is explained by our model, the affinity of light chains for calcium is low (25–50 μM) and the calcium concentration required to enhance clathrin assembly is much higher (3 mM) (Liu et al., 1995) than that in the cell (0.1–1 μM) (Näthke et al., 1990). Thus, at physiological concentrations, calcium may instead act to signal clathrin uncoating by low-level binding to the E-F hand motif of residues 85–96 to reveal a binding site for hsc70 (DeLuca-Flaherty et al., 1990; Näthke et al., 1990). In addition, our model also explains the observation that myelin basic protein (MBP) can accelerate clathrin basket formation (Prasad et al., 1995) (also probably non-physiological). The 3-fold stoichiometry of MBP/hub binding necessary for the MBP effect correlates with a single regulatory switch region per light chain. The positively charged MBP may tie up the negative charges in the light-chain switch, allowing high-affinity heavy-chain salt bridges to form. Possible mechanisms for the regulation of clathrin assembly by adaptor proteins Identification of the minimal regulatory switch in clathrin light chains that ‘turns off’ spontaneous salt bridge formation between clathrin heavy chains suggests a very sensitive mechanism for how adaptors could control clathrin assembly in the cell. We predict that adaptors introduce sequences that reverse the light-chain effect and suggest two possible mechanisms by which this could be achieved. First, adaptors could themselves have positively charged residues that interact with the light-chain regulatory sequence and thereby free up the heavy chains to form high-affinity salt bridges. Alternatively, adaptors could align the distal or terminal domain of the triskelion legs with the proximal hub segments to provide the competing residues needed to reverse light-chain inhibition. Adaptors do not directly induce the assembly of hubs with light chains at physiological pH (Liu et al., 1995). Thus, without the distal and terminal domains of the clathrin heavy chain, they are incapable of flipping the assembly switch and allowing salt bridge formation. However, the distal and terminal domains of clathrin could be required for adaptor function either because they determine the correct orientation of regulatory sequences in the adaptors or because they contain regulatory sequences that are held in place by the adaptors. Electron microscopy shows that the AP-2 adaptor binds to the terminal domain of free triskelions (Heuser and Keen, 1988) and biochemical evidence suggests a secondary binding site for adaptors in the hub (Murphy and Keen, 1992; Liu et al., 1995). These physical interactions are compatible with both proposed mechanisms for how adaptors could reverse the light-chain regulatory switch to cause cellular clathrin assembly. Materials and methods Clathrin light chain and heavy chain DNA constructs The construction of nLCa light-chain deletion mutants Δ26–56, Δ22–96 and Δ224–243 has been described previously (Näthke et al., 1990; Pley et al., 1995). The nLCa mutant Δ22–25 was made by digestion of wild-type nLCa cDNA with NaeI and SacII, re-ligation using a double-stranded linker (5′CCTGCCGC3′) to preserve the reading frame and insertion into pET-15b (Novagen)." @default.
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• W2000388340 title "Clathrin self-assembly is regulated by three light-chain residues controlling the formation of critical salt bridges" @default.
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