Clathrin light chain: importance of the conserved carboxy terminal domain to function in living cells

Clathrin triskelions assemble into coats capable of packaging membrane and receptors for transport to intracellular destinations. A triskelion is formed from three heavy chains bound to three light chains. All clathrin light chains (clc) contain an acidic amino terminal domain, a central coiled segment, and a carboxy terminal domain conserved in amino acid sequence. To assess their functional contribution in vivo, we expressed tagged segments of the Dictyostelium clcA in clc-minus Dictyostelium (clc null) cells. We examined the ability of these clcA fragments to rescue clathrin phenotypic deficiencies, to cluster into punctae on membranes, and to bind to the heavy chain. When expressed in clc null cells, a clcA fragment containing the amino terminal domain and the central coiled domain bound heavy chain but was dispensable for clathrin function. Instead, the carboxy terminal domain of clcA was a critical determinant for association with punctae, for clathrin function and for robust binding to the heavy chain. A 70 amino acid carboxy terminal fragment was necessary and sufficient for full function, and for localization into punctae on intracellular membranes. A shorter 49 amino acid carboxy terminal fragment could distribute into punctae but failed to rescue developmental deficiencies. These results reveal the importance of the carboxy terminal domain of the light chain in vivo.     

Multiple interactions of auxilin 1 with clathrin and the AP-2 adaptor complex

The removal of the clathrin coat is essential for vesicle fusion with acceptor membranes. Disassembly of the coat involves hsc70, which is specifically recruited by members of the auxilin protein family to clathrin lattices. In vitro, this function of auxilin does not require the globular amino-terminal domain of the clathrin heavy chain, which is known to play a prominent role in the interaction of clathrin with adaptors and numerous endocytic accessory proteins. Here we report the unexpected finding that the neuron-specific form of auxilin (auxilin 1) can also associate with the clathrin amino-terminal domain. This interaction is mediated through tandemly arranged sites within the auxilin 1 carboxyl-terminal segment 547-910. The overlapping auxilin 1 fragments 547-714 and 619-738 bind the clathrin terminal domain with high affinity, whereas auxilin 1-(715-901) interacts only poorly with it. All three fragments also associate with the clathrin distal domain and the alpha-appendage domain of AP-2. Moreover, they support efficient assembly of clathrin triskelia into regular cages. A novel uncoating assay was developed to demonstrate that auxilin 1-(715-901) functions efficiently as a cofactor for hsc70 in the uncoating of clathrin-coated vesicles. The multiple protein-protein interactions of auxilin 1 suggest that its function in endocytic trafficking may be more complex than previously anticipated.     

Structural domains of clathrin heavy chains

We used a combination of electron microscopy and proteolytic dissection to study the substructure of the clathrin trimer. The fragments of a heavy chain generated by limited proteolysis of cages were examined by rotary shadowing after disassembly. Correlation of lengths and molecular weights allowed us to map certain cleavage points along an arm and to assign them to positions in a model for a cage. We found that a particularly stable fragment of 52,000-59,000 Mr (depending on the enzyme) corresponded to the knob-like terminal domain at the tip of each arm.     

Recognition sites for clathrin-associated proteins AP-2 and AP-3 on clathrin triskelia.

AP-2 and AP-3 are cellular proteins that drive the in vitro polymerization of clathrin triskelia into cage structures. The interaction of these two types of assembly proteins (APs) with preassembled clathrin cages has been studied in order to identify the sites on the triskelia required for binding. Comparing binding of the APs to intact or to proteolytically clipped cages, we attempted to distinguish between binding to the terminal domain, the globular end of the heavy chain, and binding to the hub of the clathrin triskelia, the portion that remains assembled after trypsin treatment. AP-3 binds to intact clathrin cages but not to those that were treated with trypsin. AP-3 also bound to cages consisting solely of clathrin heavy chains; proteolysis of these cages also eliminated AP-3 binding. In addition, AP-3 did not bind to either isolated hubs or terminal domains that had been immobilized on Sepharose. These data indicate that clathrin light chains are not required for binding of AP-3, and that neither terminal domain nor hubs alone will suffice. However, an intact heavy chain is both necessary and sufficient for the binding of AP-3. Previous work has demonstrated one binding site for AP-2 on proteolyzed cages containing only clathrin hubs; the existence of a second binding site associated with the terminal domain was hypothesized. Here we provide direct evidence for recognition by AP-2 of isolated terminal domains immobilized on Sepharose and show that the core of the AP-2 molecule is responsible for this interaction. These results provide the first demonstration of a functional role for the conserved terminal domain of the clathrin heavy chain.     

Assembly of clathrin molecules on liposome membranes: a possible event necessary for induction of membrane fusion

Below pH6, clathrin induces fusion of liposomes containing phosphatidylserine (PS) [Maezawa et al. (1989) Biochemistry 28, 1422-1428]. Under similar conditions clathrin forms self-aggregates, suggesting that the associated form of clathrin may be involved in the fusion process. For examination of this possibility, the extent of fluorescence energy transfer from N-(p-(2-benzimidazolyl)phenyl)maleimide (BIPM)-labeled clathrin to N-(7-dimethyl-amino-4-methyl-3-coumarinyl)maleimide (DACM)-labeled clathrin in the presence of liposomes and the number of binding sites for clathrin in one liposome were examined in the pH region inducing membrane fusion. A high degree of transfer was observed, and the area on the membrane surface occupied by a clathrin molecule was estimated to be much less than that expected from its size, indicating that clathrin binds to the liposome membrane as an associated form, which may be essential for induction of membrane fusion.     

Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure-function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP-CALM was targeted to the plasma membrane-coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP-CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP-CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin-CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.     

Mechanism of protein-induced membrane fusion: fusion of phospholipid vesicles by clathrin associated with its membrane binding and conformational change

The clathrin-induced fusion of liposome membranes, the membrane binding of clathrin, and the conformational states of clathrin were investigated over a wide pH range using large unilamellar and multilamellar vesicles composed of phosphatidylserine (PS), phosphatidylcholine (PC), PS/PC (2:1), PS/PC (1:1), or PS/PC (1:2). The pH profiles of clathrin-induced fusion of all types of liposomes containing PS showed biphasic patterns. Their pH thresholds were found in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH profiles of clathrin binding to these vesicles, but the pH profiles of binding were different from the biphasic fusion patterns. With PC vesicles, only small degrees of fusion and clathrin binding were observed at pH 2-4. The pH dependences of the conformation and hydrophobicity of clathrin were determined by measuring the extent of the blue shift of the fluorescence maximum of 1-anilinonaphthalene-8-sulfonate in the presence of the protein, the fluorescence intensity of N-(1-anilinonaphthyl-4)maleimide bound to the clathrin molecule, the resonance energy transfer from its tryptophan to anilinonaphthyl residues, the partitioning of the protein in Triton X-114 solution, and the hydrophobicity index of clathrin using cis-parinaric acid. These measurements indicated that conformational change and exposure of hydrophobic regions occur below pH 6 and suggested that clathrin may adopt different conformational states in the pH region where it induced membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transformation by Rous sarcoma virus induces clathrin heavy chain phosphorylation

We have shown that the heavy chain of clathrin is phosphorylated in chicken embryo fibroblast cells transformed by Rous sarcoma virus, but not in normal cells. Approximately 1 mol of phosphate is bound for every 5 mol of heavy chain in the maximally phosphorylated transformed cells. Two-thirds of the phosphate is on serine and one-third on tyrosine residues. Clathrin heavy chain is a substrate for pp60v-src in vitro. Cleveland analysis of the in vivo and in vitro clathrin heavy chain phosphopeptides, generated by protease V8 digestion, show labeled proteolytic fragments of similar molecular weight, suggesting that pp60v-src could be directly responsible for the in vivo phosphorylation of clathrin. Phosphate is equally incorporated into clathrin in both the unassembled and the assembled clathrin pools, whereas [35S]methionine is preferentially incorporated into the assembled pool. In normal cells, clathrin visualized by immunofluorescent staining appears in a punctate pattern along the membrane surface and concentrated around the nucleus; in transformed cells the perinuclear staining is completely absent. The phosphorylation of clathrin heavy chain in transformed cells may be linked to previously observed transformation-dependent alterations in receptor-mediated endocytosis of ligands such as EGF and thrombin.     

The association of clathrin fragments with coated vesicle membranes

The association between clathrin triskelions and the clathrin-stripped membranes of coated vesicles has been investigated using a filter assay to separate bound from unbound clathrin. The filter assay is more sensitive and less cumbersome than a sedimentation assay used previously (1). While confirming the high affinity interaction between clathrin and the vesicle membrane, our results yield Scatchard plots that are curvilinear and consistent with a positively cooperative interaction between clathrin and the vesicle membranes. Controlled digestion with trypsin removes the distal portions of the triskelion legs leaving the proximal 31 nm portions that form the hub of the triskelions. These hubs are trimers of large 112,000- and 124,000-dalton fragments of clathrin heavy chains. They competitively inhibit the binding of 125I-labeled intact triskelions to stripped vesicles with a KI identical to the KD for the association of 125I-labeled intact triskelions to stripped vesicles. Furthermore, these large fragment trimers bind to stripped vesicles with approximately the same high affinity as do intact triskelions and also show evidence of a positively cooperative interaction. It is concluded that clathrin binds to coated vesicles by an interaction that is mediated by the proximal 112,000-dalton fragment of the clathrin heavy chains.     

Structure of the anion-transport protein of the human erythrocyte membrane. Further studies on the fragments produced by proteolytic digestion.

The topology of the human erythrocyte membrane anion-transport protein (band 3) has been investigated by isolation and peptide 'mapping' of the major and minor fragments derived from proteolytic cleavage of the lactoperoxidase 125I-labelled protein in erythrocytes and erythrocyte membranes. The content, in each fragment, of lactoperoxidase 125I-labelled sites (which have a known location in the extracellular or cytoplasmic domain of the protein), together with the location of the sites of proteolytic cleavage yielding the fragments, has allowed us to determine the alignment of the fragments on the linear amino acid sequence and to infer the topology of the polypeptide in the membrane. The results suggest that a region in the C-terminal portion of the polypeptide forms part of the cytoplasmic domain of the protein in addition to a large N-terminal segment. The membrane-bound regions of the protein are located in the C-terminal two-thirds of the molecule. In this region the polypeptide chain traverses the membrane at least four times and an additional loop of polypeptide is either embedded in the membrane or also penetrates through it to the other surface. The location of the lectin receptors on the protein and the site of binding of an anion-transport inhibitor have also been studied.     

Getting in Touch with the Clathrin Terminal Domain

The N‐terminal domain (TD) of the clathrin heavy chain is folded into a seven‐bladed β‐propeller that projects inward from the polyhedral outer clathrin coat. As the most membrane‐proximal portion of assembled clathrin, the TD is a major protein–protein interaction node. Contact with the TD β‐propeller occurs through short peptide sequences typically located within intrinsically disordered segments of coat components that usually are elements of the membrane‐apposed, inner ‘adaptor’ coat layer. A huge variation in TD‐binding motifs is known and now four spatially discrete interaction surfaces upon the β‐propeller have been delineated. An important operational feature of the TD interaction sites in vivo is functional redundancy. The recent discovery that ‘pitstop’ chemical inhibitors apparently occupy only one of the four TD interaction surfaces, but potently block clathrin‐mediated endocytosis, warrants careful consideration of the underlying molecular basis for this inhibition.     

Intramolecular organization of Class I H-2 MHC antigens; localization of the alloantigenic determinants and the beta 2 m binding site to different regions of the H-2 Kb glycoprotein

The intramolecular organization of the membrane integrated Class I major histocompatibility complex (MHC) molecule H-2Kb (Kb) was analyzed. After the removal of the two carbohydrate moieties by glycosidase enzymes, proteolytic digestion of the Kb molecule yielded: 1) several fragments with the beta 2 microglobulin (beta 2 m) subunit still bound and 2) one fragment carrying alloantigenic activity but lacking the beta 2 m. Isolation of the beta 2 m binding fragments showed them to be derived from the C-2 domain by partial N-terminal sequence analysis. One fragment extended to the C-terminus and the other fragment had lost the transmembrane region. Such studies conclusively show that the beta 2 m subunit is bound in the third domain, i.e., C-2, of the Kb 44,000 m.w. heavy chain. The alloantigenic fragment also isolated from the proteolytic digest consists of the first 180 residues of the 44,000 m.w. heavy chain, i.e., domains N and C-1, and carried alloantigenic determinants detected by several monoclonal antibodies as well as alloantisera. The present studies indicate that the external region of the Class I molecules has two functional regions. The first 180 residues bear the recognition elements for the immune system, and the next 90 residues (180-270) are involved in binding to beta 2 m.     

Clathrin in gastric acid secretory (parietal) cells: biochemical characterization and subcellular localization

Clathrin fromH-K-ATPase-rich membranes derived from the tubulovesicular compartmentof rabbit and hog gastric acid secretory (parietal) cells wascharacterized biochemically, and the subcellular localization ofmembrane-associated clathrin in parietal cells was characterizedby immunofluorescence, electron microscopy, and immunoelectronmicroscopy. Clathrin from H-K- ATPase-rich membranes was determinedto be comprised of conventional clathrin heavy chain and a predominanceof clathrin light chain A. Clathrin and adaptors could be induced topolymerize quantitatively in vitro, forming 120-nm-diameter basketlikestructures. In digitonin-permeabilized resting parietal cells, theintracellular distribution of immunofluorescently labeled clathrin wassuggestive of labeling of the tubulovesicular compartment. Clathrin wasalso unexpectedly localized to canalicular (apical) membranes, as were-adaptin and dynamin, suggesting that this membrane domain ofresting parietal cells is endocytotically active. At theultrastructural level, clathrin was immunolocalized to canalicularand tubulovesicular membranes. H-K-ATPase was immunolocalized tothe same membrane domains as clathrin but did not appear to be enrichedat the specific subdomains that were enriched in clathrin. Finally, inimmunofluorescently labeled primary cultures of parietal cells, incontrast to the H-K-ATPase, intracellular clathrin was found not totranslocate to the apical membrane on secretagogue stimulation. Takentogether, these biochemical and morphological data provide a frameworkfor characterizing the role of clathrin in the regulation of membranetrafficking from tubulovesicles and at the canalicular membrane inparietal cells.

     

Alpha-actinin and calmodulin interact with distinct sites on the arms of the clathrin trimer

In the present study, the interaction of alpha-actinin and calmodulin with clathrin heavy chain are demonstrated using Western blot analysis and rotary shadowing electron microscopy. The results show that alpha-actinin and calmodulin bind the clathrin heavy chain. The interaction is specific and affected by calcium. However, the interaction of both proteins with the clathrin heavy chain is distinct; the proteins do not block each other's ability to bind, and they interact with different protein fragments of the clathrin heavy chain. Furthermore, using rotary shadowing the results show that alpha-actinin differentially affected the terminal region of the clathrin trimer. Whereas, the effects of calmodulin were most noticeably detected along the length of trimer arms. The possible existence of distinct binding sites on the arms of the clathrin trimer for these cytosolic proteins supports the contention that these cytosolic proteins play an important role in cellular trafficking.     

Characterization of a membrane-specific tubulin isoform by peptide mapping

A membrane-specific tubulin-like protein, found in preparations of synaptic plasma membranes and brain mitochondria, was analyzed by chemical and proteolytic peptide mapping to determine which part of the molecule was different from cytoplasmic tubulin. The membrane polypeptide was identical to alpha tubulin in the first two-thirds of the molecule containing the amino terminal, as found by peptide mapping. However, some differences were observed in the peptide maps of the carboxy terminal one third of the molecule which includes a domain that is important in the regulation of tubulin self-assembly.     

Hrs recruits clathrin to early endosomes

The hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, has been implicated in intracellular trafficking and signal transduction. Hrs contains a phosphatidylinositol 3-phosphate-binding FYVE domain that contributes to its endosomal targeting. Here we show that Hrs and EEA1, a FYVE domain protein involved in endocytic membrane fusion, are localized to different regions of early endosomes. We demonstrate that Hrs co-localizes with clathrin, and that the C-terminus of Hrs contains a functional clathrin box motif that interacts directly with the terminal beta-propeller domain of clathrin heavy chain. A massive recruitment of clathrin to early endosomes was observed in cells transfected with Hrs, but not with Hrs lacking the C-terminus. Furthermore, the phosphatidylinositol 3-kinase inhibitor wortmannin caused the dissociation of both Hrs and clathrin from endosomes. While overexpression of Hrs did not affect endocytosis and recycling of transferrin, endocytosed epidermal growth factor and dextran were retained in early endosomes. These results provide a molecular mechanism for the recruitment of clathrin onto early endosomes and suggest a function for Hrs in trafficking from early to late endosomes.     

Clathrin domains involved in recognition by assembly protein AP-2

The domains on clathrin responsible for interaction with the plasma membrane-associated assembly protein AP-2 have been studied using a novel cage binding assay. AP-2 bound to pure clathrin cages but not to coat structures already containing AP that had been prepared by coassembly. Binding to preassembled cages also occurred in the presence of elevated Tris-HCl concentrations (greater than or equal to 200 mM) which block AP-2 interactions with free clathrin. AP-2 interactions with assembled cages could also be distinguished from AP-2 binding to clathrin trimers by sodium tripolyphosphate (NaPPPi), which binds to the alpha subunit of AP-2 (Beck, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4442-4447). At concentrations of 1-5 mM, NaPPPi blocked clathrin-triskelion binding; in contrast, interactions with cages persisted in the presence of 25 mM NaPPPi. To begin to identify the region(s) of the clathrin molecule important in recognition by AP-2, clathrin cages were proteolyzed to remove heavy chain terminal domains and portions of the distal leg as well as all of the light chains. AP-2 bound to these "clipped cages"; however, unlike the interaction with native cages, binding of AP-2 to clipped cages was sensitive to the lower concentrations of both Tris-HCl and NaPPPi which disrupt interactions of AP-2 with clathrin trimers. Reconstitution of the clipped cages with clathrin light chains did not restore resistance of AP-2 binding to Tris-HCl. We conclude that one binding site for AP-2 resides on the hub and/or proximal part of the clathrin triskelion whereas a second site is likely to involve the terminal domain and/or distal leg; the second site is manifested only in the assembled lattice structure. We suggest that these two distinct binding interactions may be mediated by the two unique large subunits within the AP-2 complex, acting sequentially during assembly.     

A PH domain within OCRL bridges clathrin‐mediated membrane trafficking to phosphoinositide metabolism

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5‐phosphatase domain followed by an ASH and a RhoGAP‐like domain. Their divergent NH2‐terminal portions remain uncharacterized. We show that the NH2‐terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin‐coated pits, was earlier shown to contain another binding site for clathrin in its COOH‐terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2‐terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin‐binding site in OCRL maps to an unusual clathrin‐box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin‐dependent membrane trafficking.     

NH2-terminal localization of that part of the staphylococcal enterotoxins polypeptide chain responsible for binding with membrane receptor and mitogenic effect

It is established that the part of the SEA and SEC2 polypeptide chain responsible for the binding of these toxin proteins with the membrane receptor on the surface of rabbit thymocytes and mitogenic effect is localised in the NH2-terminal region of the molecule. The SEC2 splits in two fragments T1 (17 kdalton) and T2 (12.5 kdalton) under limited proteolysis by trypsin in the presence of 2-ME. The amino acid terminal residues of SEA, SEC2 and their proteolytic fragments are also studied.     

Sequence of critical events involved in fusion of phospholipid vesicles induced by clathrin.

Membrane fusion induced by clathrin is accompanied by several events such as conformational change, membrane binding and association of clathrin, and membrane aggregation (Maezawa et al. (1989) Biochemistry 28, 1422-1428; Maezawa and Yoshimura (1990) Biochem. Biophys. Res. Commun. 173, 134-140). To clarify the sequence of these events, we examined their time-courses by reducing the pH of the medium from 7.4 to a given pH in the range of 3.5-5.0 at 25 degrees C or 10 degrees C. Large unilamellar vesicles composed of phosphatidylserine and phosphatidylcholine were used in most experiments. The half-time for conformational change of clathrin was less than those for membrane binding and association of clathrin. The half-times and the initial rates of membrane binding and association of clathrin were similar order of magnitude, although the pH-profiles of the initial rates of the two events were somewhat different. Membrane aggregation started after membrane binding of clathrin. A lag phase was observed in the time-course of membrane fusion, whereas there was no lag phase in membrane binding and association of clathrin and membrane aggregation. Moreover, the lag time before fusion was independent of the clathrin concentration, although the initial rates of these three events were dependent on it, suggesting that the three reactions are not responsible for the lag phase before fusion, and that there is some other event(s) in the lag time. On the other hand, there was a threshould-pH in the pH profile of the lag-time and the threshold-pH coincided with the critical pH at which the final associated state of clathrin was apparently reversed in the presence and absence of liposomes, suggesting that the event(s) in the lag phase may be related to this final associated state of clathrin molecules on the liposome membranes. These results indicate that clathrin-induced fusion of liposomes is initiated through the following sequential events: conformational change of clathrin, membrane binding and association of clathrin, which occur simultaneously but independently, membrane aggregation, an event(s) in the lag phase, and actual fusion.