Predominance of clathrin light chain LCb correlates with the presence of a regulated secretory pathway

Two forms of clathrin light chains, LCa and LCb, are expressed in all mammalian and avian tissues that have been examined, whereas only one type is found in yeast. Regions of structural dissimilarity between LCa and LCb indicate possible functional diversity. To determine how LCa and LCb might differentially influence clathrin function, light chain expression patterns and turnover were investigated. Relative expression levels of the two light chains were determined in cells and tissues with and without a regulated secretory pathway. LCa/LCb ratios ranged from 5:1 to 0.33:1. A higher proportion of LCb was observed in cells and tissues that maintain a regulated pathway of secretion, suggesting a specialized role for the LCb light chain in this process. The ratio of light chains in assembled clathrin was found to reflect the levels of total light chains expressed in the cell, indicating no preferential incorporation into triskelions or coated vesicles. The half-lives of LCa, LCb, and clathrin heavy chain were determined to be 24, 45, and 50 h, respectively. Thus, LCa is turned over independently of the other subunits. However, the half-lives of all three subunits are sufficiently long to allow triskelions to undergo many rounds of endocytosis, minimizing the possibility that turnover contributes to regulation of clathrin function. Rather, differential levels of LCa and LCb expression may influence tissue specific clathrin regulation, as suggested by the predominance of LCb in cells maintaining a regulated secretory pathway.     

Identification of the phosphorylation sites of clathrin light chain LCb

Clathrin light chains, LCa and LCb, are products of two closely related genes whose mRNAs undergo differential splicing to result in at least four different light chain isoforms. The physiological significance of clathrin light chain diversity remains unclear. To date, the only evidence for a functional distinction of LCa and LCb is the preferential phosphorylation of LCb, which takes place at serine residues and is mediated by coated vesicle-associated casein kinase II. As a first step toward determining the function of light chain diversity, we have mapped the in vitro phosphorylation sites on LCb. We use [32P]ATP to phosphorylate LCb within coated vesicles, followed by sequencing of 32P-labeled chymotryptic peptides thereof, to identify serine residues at positions 11 and 13 as the phosphorylation sites. We find that phosphorylation of LCb within coated vesicles can be inhibited by four monoclonal antibodies specific for different epitopes of the clathrin light chains.     

Structure of human clathrin light chains. Conservation of light chain polymorphism in three mammalian species

Complementary DNAs (cDNA) encoding the brain and non-brain forms of the human clathrin light chains LCa and LCb have been isolated, sequenced, and compared with their homologues in cow and rat. The significant differences that distinguish LCa from LCb and the brain from non-brain forms show remarkable preservation in all three species. These features include the position and sequence of the brain-specific inserts, a totally conserved region of 22 residues near the amino terminus, the LCb-specific phosphorylation site, the heavy chain binding site, and a distinctive pattern of cysteine residues near the carboxyl terminus. Unorthodox sequences for translation initiation and polyadenylation are found for LCb contrasting with LCa which exhibits orthodox regulatory sequences. Small insertions in human LCa revealed a duplicated sequence of 13 residues that flank the 22-residue conserved region. Only the carboxyl-terminal copy of this sequence is present in LCb. All sequences are consistent with the heavy chain binding site comprising an alpha-helical central region of the light chains. The hydrophobic face of this helix, which is presumed to interact with the heavy chain, is highly conserved between LCa and LCb, whereas the hydrophilic face shows considerable divergence. To help define the carboxyl-terminal limit of the heavy chain binding region, the epitope recognized by the CVC.6 monoclonal antibody was localized to residues 192-208 of LCa with glutamic acid 198 being of most importance. The faithful preservation of clathrin light chain polymorphism in three mammalian species provides evidence supporting a functional diversification of the brain and non-brain forms of LCa and LCb.     

Chromosomal Location and Some Structural Features of Human Clathrin Light-Chain Genes (CLTA and CLTB)

Two human clathrin light-chain genes have been defined. The gene (CLTA) encoding the LCa light chain maps to the long arm of chromosome 12 at 12q23-q24 and that encoding the LCb light chain (CLTB) maps to the long arm of chromosome 4 at 4q2-q3. Isolation and characterization of partial genomic clones encoding human LCa and LCb reveal the neuron-specific insertions of the LCa and LCb proteins to he encoded by discrete exons, thus proving that clathrin light chains undergo alternate mRNA splicing to generate tissue-specific protein isoforms. The insertion sequence of LCb is encoded by a single exon and that of LCa by two exons. The first of the two neuron-specific LCa exons is homologous to the corresponding LCb exon. An intronic sequence of the LCb gene with similarity to the second neuron-specific exon of the LCa gene has been identified.     

Differential control of clathrin subunit dynamics measured with EW-FRAP microscopy

The clathrin triskelion is composed of three light chain (LC) and three heavy chain (HC) subunits. Cellular control of clathrin function is thought to be aimed at the LC subunit, mainly on the basis of structural information. To test this hypothesis in vivo, we used evanescent-wave photobleaching recovery to study clathrin exchange from single pits using LC (LCa and LCb) and HC enhanced green fluorescent protein fusion constructs. The recovery signal was corrected for cytosolic diffusional background, yielding the pure exchange reaction times. For LCa, we measured an unbinding time constant tau(LEa) = 18.9 +/- 1.0 seconds at room temperature, faster than previously published; for LCb, we found tau(LCb) = 10.6 +/- 1.9 seconds and for HC tau(HC) = 15.9 +/- 1.0 seconds. Sucrose treatment, ATP or Ca(2+) depletion blocked exchange of LCa completely, but only partially of HC, lowering its time constant to tau = 10.0 +/- 0.9 seconds, identical to the one for LCb exchange. The latter was also not blocked by Ca(2+) depletion or sucrose. We conclude that HCs bound both to LCa and to LCb contribute side by side to pit formation in vivo, but the affinity of LCa-free HC in pits is reduced, and the Ca(2+)- and ATP-mediated control of clathrin function is lost.     

Tropomyosin-like properties of clathrin light chains allow a rapid, high-yield purification

The light chains (LCa and LCb) of bovine brain clathrin are resistant to heat denaturation by boiling, a property shared by tropomyosin (Bailey, K., 1948, Biochem. J., 43:271-281). Light chains were partially purified by boiling and centrifugation of a Tris-extract of crude membranes prepared from bovine brains (Keen, J. H., M. C. Willingham, and I. H. Pastan, 1979, Cell., 16:303-312). Contaminant polypeptides were then removed by size-exclusion high-pressure liquid chromatography. The purified light chains were separated from each other by using an immunoaffinity column prepared from a monoclonal antibody CVC.7 specific for LCa and not LCb.     

Mechanism of clathrin basket dissociation: separate functions of protein domains of the DnaJ homologue auxilin

Auxilin was recently identified as cofactor for hsc70 in the uncoating of clathrin-coated vesicles (Ungewickell, E., H. Ungewickell, S.E. Holstein, R. Lindner, K. Prasad, W. Barouch, B. Martin, L.E. Greene, and E. Eisenberg. 1995. Nature (Lond.). 378: 632-635). By constructing different glutathione-S-transferase (GST)-auxilin fragments, we show here that cooperation of auxilin's J domain (segment 813-910) with an adjoining clathrin binding domain (segment 547-814) suffices to dissociate clathrin baskets in the presence of hsc70 and ATP. When the two domains are expressed as separate GST fusion proteins, the cofactor activity is lost, even though both retain their respective functions. The clathrin binding domain binds to triskelia like intact auxilin with a maximum stoichiometry of 3 and concomitantly promotes their assembly into regular baskets. A fragment containing auxilin's J domain associates in an ATP-dependent reaction with hsc70 to form a complex with a half-life of 8 min at 25 degrees C. When the clathrin binding domain and the J domain are recombined via dimerization of their GST moieties, cofactor activity is partially recovered. The interaction between auxilin's J domain and hsc70 causes rapid hydrolysis of bound ATP. Release of inorganic phosphate appears to be correlated with the disintegration of the complex between auxilin's J domain and hsc70. We infer that the metastable complex composed of auxilin, hsc70, ADP, and P(i) contains an activated form of hsc70, primed to engage clathrin that is brought into apposition with it by the DnaJ homologue auxilin.     

The occurrence of disulphide bonds in purified clathrin light chains.

Three forms of clathrin light chain contain two cysteine residues. These are the predominant brain-specific forms of LCa and LCb and the non-brain form of LCb. After purification in the absence of thiols they contain intramolecular disulphide bonds. The reduced and the oxidized forms show differences in electrophoretic mobility, explaining the variable and heterogeneous patterns observed on electrophoresis. Accessibility of the thiol groups in the free light chains is greater than when they are associated with the heavy chain. In contrast the cysteine residues of the clathrin heavy chain are completely inaccessible in the absence of denaturants and are not found in disulphide bonds. The antigenic properties of the oxidized and the reduced forms of the clathrin light chains are similar, as is their capacity to bind to the clathrin heavy chain. After isolation in the presence of 10 mM-iodoacetamide, the light-chain cysteine residues are fully alkylated. The results are consistent with the reduced form being the native state and the light-chain disulphide bonds an artifact of isolation.     

Clathrin light and heavy chain interface: alpha-helix binding superhelix loops via critical tryptophans

Clathrin light chain subunits (LCa and LCb) contribute to regulation of coated vesicle formation to sort proteins during receptor-mediated endocytosis and organelle biogenesis. LC binding to clathrin heavy chain (HC) was characterized by genetic and structural approaches. The core interactions were mapped to HC residues 1267-1522 (out of 1675) and LCb residues 90-157 (out of 228), using yeast two-hybrid assays. The C-termini of both subunits also displayed interactions extending beyond the core domains. Mutations to helix breakers within the LCb core disrupted HC association. Further suppressor mutagenesis uncovered compensatory mutations in HC (K1415E or K1326E) capable of rescuing the binding defects of LCb mutations W127R or W105R plus W138R, thereby pinpointing contacts between HC and LCb. Mutant HC K1415E also rescued loss of binding by LCa W130R, indicating that both LCs interact similarly with HC. Based on circular dichroism data, mapping and mutagenesis, LCa and LCb were represented as alpha-helices, aligned along the HC and, using molecular dynamics, a structural model of their interaction was generated with novel implications for LC control of clathrin assembly.     

The calcium-binding site of clathrin light chains

Clathrin light chains are calcium-binding proteins (Mooibroek, M. J., Michiel, D. F., and Wang, J. H. (1987) J. Biol. Chem. 262, 25-28) and clathrin assembly can be modulated by calcium in vitro. Thus, intracellular calcium may play a regulatory role in the function of clathrin-coated vesicles. The structural basis for calcium's influence on clathrin-mediated processes has been defined using recombinant deletion mutants and isolated fragments of the light chains. A single calcium-binding site, formed by residues 85-96, is present in both mammalian light chains (LCa and LCb) and in the single yeast light chain. This sequence has structural similarity to the calcium-binding EF-hand loops of calmodulin and related proteins. In mammalian light chains, the calcium-binding sequence is flanked by domains that regulate clathrin assembly and disassembly.     

Somatic cell mapping of bovine clathrin light chain genes: identification of a new bovine syntenic group

Two bovine DNA probes (LCa and LCb) complementary to the clathrin light chain genes were hybridized to DNAs from bovine hamster hybrid somatic cell lines retaining different combinations of bovine chromosomes. Concordancy of retention of the clathrin genes was compared to existing syntenic data for the domestic cow. LCb identified a single locus. CLTB, concordant with the genes encoding bovine anti-Müllerian hormone (AMH) and bovine osteonectin from bovine syntenic group U22. LCa recognized two loci. CLTAL1 from a previously unidentified bovine syntenic group. U25, and CLTAL2 which is concordant with GGTB2, a gene marker for bovine syntenic group U18.     

Identification of the universal cofactor (auxilin 2) in clathrin coat dissociation

Uncoating of clathrin-coated vesicles in neuronal cells requires hsc70 in concert with the cofactor auxilin which contains a J-domain as well as a domain with homology to dual specific phosphatases and tensin, known as PTEN. The question of whether an analogous factor operates in other cell types has until now remained unanswered. Here we show that it is the recently discovered and widely expressed cyclin G-associated protein kinase which fulfils the function of neuronal auxilin in hsc70-mediated clathrin coat dissociation. GAK possesses a J-domain, which stimulates the hsc70 ATPase, it competes with auxilin for clathrin binding and at sufficiently high concentrations acts as a clathrin assembly protein. Moreover, GAK binds to the gamma- and alpha-appendage domains of the adaptor proteins AP-1 and AP-2 in vitro and phosphorylates their medium chains. Cells that transiently overexpress GAK are impaired in respect of receptor-mediated endocytosis. In transfected cells clathrin is dislodged from coated pits/vesicles and co-localizes with GFP-GAK in the form of large aggregates. The cellular distribution of membrane-associated adaptors was unaffected by overexpression of GAK. Our results point to a hsc70/auxilin-based uncoating system as a ubiquitous feature of eukaryotic cells.     

Dominant-interfering Hsc70 mutants disrupt multiple stages of the clathrin-coated vesicle cycle in vivo

Within the clathrin-coated vesicle (CCV) cycle, coat assembly drives the internalization of receptors from the cell surface and disassembly allows for the processing of internalized ligands. The heat shock cognate protein, hsc70, has been implicated in regulating coat disassembly. We find that in cells overexpressing ATPase-deficient hsc70 mutants, uncoating of CCVs is inhibited in vivo, and the majority of unassembled cytosolic clathrin shifts to an assembled pool that cofractionates with AP1 and AP2. Surprisingly, this assembled pool of coat proteins accumulates in the absence of cargo receptors, suggesting that disruption of hsc70 activity may cause misassembly of empty clathrin cages. The strongest effect of overexpression of hsc70 mutants is a block in transferrin receptor (TfnR) recycling, which cannot be accounted for by the degree of inhibition of uncoating of endocytic CCVs. These results suggest that hsc70 participates in multiple transport and/or sorting events between endosomal compartments. Additionally, the mutant-expressing cells are defective at internalizing transferrin. In the most potent case, the initial rate of uptake is inhibited 10-fold, and TfnR levels double at the cell surface. Our findings demonstrate that hsc70 indeed regulates coat disassembly and also suggest that this chaperone broadly modulates clathrin dynamics throughout the CCV cycle.     

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.     

Molecular and functional characterization of clathrin- and AP-2-binding determinants within a disordered domain of auxilin

Uncoating of clathrin-coated vesicles requires the J-domain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the alpha-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of beta-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.     

Uncoating of clathrin-coated vesicles by uncoating ATPase from developing peas.

A cytosolic ATPase (an enzyme that dissociates clathrin from clathrin-coated vesicles in the presence of ATP) was isolated from developing pea (Pisum sativum L.) cotyledons using chromatography on ATP-agarose. After chromatography on phenyl Sepharose, the fraction with uncoating activity was enriched in a doublet of 70-kD peptides. Using chromatofocusing, it was possible to produce fractions enriched in the upper component of the doublet of 70-kD peptides; these fractions still retained ATP-dependent uncoating activity. In western blot analysis, antibodies against a member of the 70-kD family of heat-shock proteins interacted with the upper component of the doublet of the 70-kD peptides from the phenyl Sepharose-purified fractions. On the basis of these data, it appears that the uncoating ATPase may be a member of the 70-kD family of heat-shock proteins. The uncoating activity removed clathrin from both pea and bovine brain clathrin-coated vesicles. The uncoating ATPase from bovine brain also uncoated coated vesicles from peas. Pea clathrin-coated vesicles that were prepared by three different methods were uncoated to different extents by the plant uncoating ATPase. Different populations of clathrin-coated vesicles from the same preparation showed differential sensitivity to the uncoating ATPase. Limited proteolysis of the clathrin light chains in the protein coat abolished the susceptibility of the clathrin-coated vesicles to the uncoating ATPase. The properties of the uncoating ATPase isolated from developing pea cotyledons are similar to those of uncoating ATPases previously described from mammalian and yeast systems. It appears that despite dissimilarities in composition of the clathrin components of the vesicles from the respective sources, uncoating is achieved by a common mechanism.     

Yeast clathrin has a distinctive light chain that is important for cell growth

The structure and physiologic role of clathrin light chain has been explored by purification of the protein from Saccharomyces cerevisiae, molecular cloning of the gene, and disruption of the chromosomal locus. The single light chain protein from yeast shares many physical properties with the mammalian light chains, in spite of considerable sequence divergence. Within the limited amino acid sequence identity between yeast and mammalian light chains (18% overall), three regions are notable. The carboxy termini of yeast light chain and mammalian light chain LCb are 39% homologous. Yeast light chain contains an amino-terminal region 45% homologous to a domain that is completely conserved among mammalian light chains. Lastly, a possible homolog of the tissue-specific insert of LCb is detected in the yeast gene. Disruption of the yeast gene (CLC1) leads to a slow-growth phenotype similar to that seen in strains that lack clathrin heavy chain. However, light chain gene deletion is not lethal to a strain that cannot sustain a heavy chain gene disruption. Light chain-deficient strains frequently give rise to variants that grow more rapidly but do not express an immunologically related light chain species. These properties suggest that clathrin light chain serves an important role in cell growth that can be compensated in light chain deficient cells.     

Trimeric binding of the 70-kD uncoating ATPase to the vertices of clathrin triskelia: a candidate intermediate in the vesicle uncoating reaction

Clathrin-coated vesicles were uncoated with the 70-kD "uncoating ATPase" from bovine brain, and the molecular products were visualized by freeze-etch electron microscopy. This yielded images of released clathrin triskelia with up to three 70-kD uncoating ATPase molecules bound to their vertices. Likewise, incubation of soluble clathrin triskelia with purified uncoating ATPase also led to trimeric binding of the ATPase to the vertices of clathrin triskelia. However, this occurred only when either EDTA or nonhydrolyzable analogues of ATP were present, in which case the ATPase also appeared to self-associate. When ATP was present instead, no 70-kD ATPases could be found on clathrin triskelia and all ATPases remained monomeric. These observations support the notion that ATP controls an allosteric conversion of the 70- kD uncoating ATPase between two different molecular conformations, an ATP-charged state in which the molecule has relatively low affinity for itself as well as low affinity for clathrin, and an ATP-discharged state in which both of these affinities are high. We presume that in vivo, the latter condition is brought about by ATP hydrolysis and product release, at which point the ATPase will bind tightly to clathrin and/or self-associate. We further propose that these reactions, when occurring in concert within a clathrin lattice, will tend to destabilize it by a mechanism we call "protein polymer competition". We stress the analogies between such a mechanism of uncoating and the ATP-driven events in muscle contraction. Finally, we show that under experimental conditions in which the uncoating ATPase fully removes the coats from brain coated vesicles, identical aliquots of the enzyme do not affect plasmalemmal coated pits in situ. This remarkable selectivity, the mechanism of which remains a complete mystery, is at least consistent with the idea that the 70-kD ATPase indeed plays a role in uncoating coated vesicles after they have formed in vivo.     

Human erythrocyte clathrin and clathrin-uncoating protein

Clathrin, a Mr = 72,000 clathrin-associated protein, and myosin were purified in milligram quantities from the same erythrocyte hemolysate fraction. Erythrocyte clathrin closely resembled brain clathrin in several respects: (a) both are triskelions as visualized by electron microscopy with arms 40 nm in length with globular ends and a flexible hinge region in the middle of each arm, and these triskelions assemble into polyhedral "cages" at appropriate pH and ionic strength; (b) both molecules contain heavy chains of Mr = 170,000 that are indistinguishable by two-dimensional maps of 125I-labeled peptides; and (c) both molecules contain light chains of Mr approximately 40,000 in a 1:1 molar ratio with the heavy chain. Erythrocyte clathrin is not identical to brain clathrin since antibody raised against the erythrocyte protein reacts better with erythrocyte clathrin than with brain clathrin and since brain clathrin contains two light chains resolved on sodium dodecyl sulfate gels while the light chain of erythrocyte clathrin migrates as a single band. The erythrocyte Mr = 72,000 clathrin-associated protein is closely related to a protein in brain that mediates ATP-dependent disassembly of clathrin from coated vesicles and binds tightly to clathrin triskelions (Schlossman, D. M., Schmid, S. L., Braell, W. A., and Rothman, J. E. (1984) J. Cell Biol. 99, 723-733). The erythrocyte and brain proteins have identical Mr on sodium dodecyl sulfate gels and identical maps of 125I-labeled peptides, share antigenic sites, and bind tightly to ATP immobilized on agarose. Clathrin and the uncoating protein are not restricted to reticulocytes since equivalent amounts of these proteins are present in whole erythrocyte populations and reticulocyte-depleted erythrocytes. Clathrin is present at 6,000 triskelions/cells, while the uncoating protein is in substantial excess at 250,000 copies/cell.     

ATP- and Cytosol-dependent Release of Adaptor Proteins from Clathrin-coated Vesicles: A Dual Role for Hsc70

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.