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.     

Analysis of clathrin light chain-heavy chain interactions using truncated mutants of rat liver light chain LCB3

Clathrin light chains are extended molecules located along the proximal segment of each of the three heavy chain legs of a clathrin trimer. All mammalian light chains share a central segment with 10 repeated heptad motifs believed to mediate the interaction with clathrin heavy chains. In order to test this model in more detail, we have expressed intact rat liver clathrin light chain LCB3 in Escherichia coli and find that it binds tightly to calf clathrin heavy chains. Using a set of expressed truncated mutants of LCB3, we show that the presence of seven to eight heptads is indeed necessary for a successful interaction. More extensive deletions of the central segment completely abolish the ability to bind to heavy chains. Neither the amino- nor the carboxyl-terminal domain is essential for binding, but competition experiments show that the presence of the carboxyl-terminal domain does enhance the interaction with heavy chains.     

Compromise of clathrin function and membrane association by clathrin light chain deletion

While clathrin heavy chains from different species are highly conserved in amino acid sequence, clathrin light chains are much more divergent. Thus clathrin light chain may have different functions in different organisms. To investigate clathrin light chain function, we cloned the clathrin light chain, clcA, from Dictyostelium and examined clathrin function in clcA– mutants. Phenotypic deficiencies in development, cytokinesis, and osmoregulation showed that light chain was critical for clathrin function in Dictyostelium . In contrast with budding yeast, we found the light chain did not influence steady-state levels of clathrin, triskelion formation, or contribute to clathrin over-assembly on intracellular membranes. Imaging GFP-CHC in clcA– mutants showed that the heavy chain formed dynamic punctate structures that were remarkably similar to those found in wild-type cells. However, clathrin light chain knockouts showed a decreased association of clathrin with intracellular membranes. Unlike wild-type cells, half of the clathrin in clcA– mutants was cytosolic, suggesting that the absence of light chain compromised the assembly of triskelions onto intracellular membranes. Taken together, these results suggest a role for the Dictyostelium clathrin light chain in regulating the self-assembly of triskelions onto intracellular membranes, and demonstrate a crucial contribution of the light chain to clathrin function in vivo .     

Biochemical and immunological studies on clathrin light chains and their binding sites on clathrin triskelions

Clathrin light chains from bovine brain tissue (LC alpha and LC beta) are monomeric proteins with an average mol. wt. of approximately 33,000, as determined by sedimentation equilibrium. Solution studies on purified light chains indicate a large Stokes radius (Re = 3.3 nm) and little defined secondary structure. Both light chains bind specifically and with high affinity (KA approximately 5 x 10(7)/M) to overlapping sites on clathrin heavy chains. These binding sites are contained within a 125,000 dalton heavy chain fragment that forms truncated triskelions with legs, 15 nm shorter than those of intact triskelions. As judged by immuno-electron microscopy, light chain-specific IgG molecules bind mostly to the center of triskelions, but there are also sites that are scattered some 16 nm along the proximal part of triskelion legs. From heterologous binding experiments using human placenta light chains and heavy chain fragments from bovine brain clathrin, it is concluded that the domains of light and heavy chains that are involved in the interaction are conserved across tissue and species boundaries.     

Purification of Clathrin Heavy and Light Chain fromDictyostelium discoideum

Clathrin, a protein important for endocytosis, is a hexamer composed of three heavy chains and three light chains. We report here the purification scheme used to isolate the clathrin protein from the simple eukaryote,Dictyostelium discoideum.Using a combination of differential centrifugation and column chromatography, we isolated ∼2 mg of clathrin triskelions from 150–200 g ofDictyosteliumcells. One additional step purified the 30-kDa clathrin light chain to homogeneity. Glycerol gradient centrifugation was used to determine anSvalue of 7.9 for purified clathrin. Rotary shadowed images ofDictyosteliumclathrin revealed trimeric molecules with extended legs measuring 48 ± 5 nm, similar in length to the legs of mammalian and yeast clathrin triskelions. The single clathrin light chain proved resistant to heat treatment, a property also similar to light chains from other species. The conservation of these physical properties inDictyosteliumclathrin demonstrates the potential of this model organism for the study of clathrin structure and function.     

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.     

Light chain C-terminal region reinforces the stability of clathrin heavy chain trimers

The self-assembly of clathrin into lattices relies on the ability of heavy chain legs to form a three-legged pinwheel structure. We investigated the role of light chains in clathrin trimerization by challenging recombinant hub (plus and minus light chain) with an anionic detergent. The binding of light chain increases the amount of detergent needed to induce detrimerization, suggesting light chains reinforced hub trimers. We also show that light chain C-terminal residues are important for enhancing the in vitro assembly of hub at low pH. We assessed how much the C-terminus of light chain contributed to the stability of the trimerization domain by adding full-length and truncated light chains to trimer-defective hub mutants, C1573S and C1573A. Adding full-length LCb to C1573S caused some retrimerization, but little activity was restored, suggesting the majority of oligomeric C1573S was nonnative. A larger percentage of monomeric C1573A could be retrimerized into an assembly-competent form by adding intact LCb. We also discovered that C-terminally deleted light chains produced a heterogeneous population of hubs that were smaller than native hubs, but were assembly active. We propose a model showing how light chains reinforce the puckered clathrin triskelion. Finally, the ability of light chains to retrimerize C1573A hub suggests that the structural role of light chain may be conserved in yeast and mammals.     

Huntingtin-interacting protein 1 (Hip1) and Hip1-related protein (Hip1R) bind the conserved sequence of clathrin light chains and thereby influence clathrin assembly in vitro and actin distribution in vivo

Clathrin heavy and light chains form triskelia, which assemble into polyhedral coats of membrane vesicles that mediate transport for endocytosis and organelle biogenesis. Light chain subunits regulate clathrin assembly in vitro by suppressing spontaneous self-assembly of the heavy chains. The residues that play this regulatory role are at the N terminus of a conserved 22-amino acid sequence that is shared by all vertebrate light chains. Here we show that these regulatory residues and others in the conserved sequence mediate light chain interaction with Hip1 and Hip1R. These related proteins were previously found to be enriched in clathrin-coated vesicles and to promote clathrin assembly in vitro. We demonstrate Hip1R binding preference for light chains associated with clathrin heavy chain and show that Hip1R stimulation of clathrin assembly in vitro is blocked by mutations in the conserved sequence of light chains that abolish interaction with Hip1 and Hip1R. In vivo overexpression of a fragment of clathrin light chain comprising the Hip1R-binding region affected cellular actin distribution. Together these results suggest that the roles of Hip1 and Hip1R in affecting clathrin assembly and actin distribution are mediated by their interaction with the conserved sequence of 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.     

Clathrin assembly involves a light chain-binding region

Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly.     

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.     

Bovine brain clathrin light chains impede heavy chain assembly in vitro

Intact bovine brain clathrin triskelia, comprising three heavy and three light chains, require either 2 mM calcium or the assistance of protein co-factors for efficient assembly into regular cage structures (Keen, J. H., Willingham, M. C., and Pastan, I. (1979) Cell 16, 303-312). In contrast light chain-free heavy chains assemble readily in the absence of co-factors or calcium. Reconstitution of intact clathrin from heavy and light chains restores the calcium requirement. Our data indicate that light chains impede assembly by creating a kinetic trap rather than by perturbing the affinity of heavy chains for each other. This property suggests a function for light chains as regulatory subunits for clathrin assembly.     

Clathrin heavy chain, light chain interactions

Purified pig brain clathrin can be reversibly dissociated and separated into heavy chain trimers and light chains in the presence of non-denaturing concentrations of the chaotrope thiocyanate. The isolated heavy chain trimers reassemble into regular polygonal cage structures in the absence of light chains. The light chain fraction can be further resolved into its two components L alpha and L beta which give different one-dimensional peptide maps. Radiolabelled light chains bind with high affinity (KD < 10(-10) M) to heavy chain trimers, to heavy chain cages and to a 110,000 mol. wt. tryptic fragment of the heavy chain. Both light chains compete with each other and with light chains from other sources for the same binding sites on heavy chains and c.d. spectroscopy shows that the two pig brain light chains possess very similar structures. We conclude that light chains from different sources, despite some heterogeneity, have a highly conserved, high affinity binding site on the heavy chain but are not essential for the formation of regular cage structures.     

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.     

A 220 kDa polypeptide, immunolocalized to epithelial tight junctions, is associated with brain clathrin preparations

Antibodies were raised in rabbits to highly purified preparations of bovine brain clathrin. The serum stained by immunofluorescence rat liver sections at tight junctions in a pattern that was identical to that previously reported (B. R. Stevenson et al.: J. Cell Biol. 103, 755-766 (1986] in which a monoclonal antibody specific to a 220 kDa (ZO-1) liver tight junction component was used. The serum also stained regions of the cell surface corresponding to the positions of intercellular junctions in confluent MDCK and HepG-2 cell cultures. Analysis of brain clathrin preparations resolved by polyacrylamide gel electrophoresis by immunoblotting with the serum indicated reaction with clathrin heavy and light chains as well as towards a 220 kDa polypeptide that was a minor component. Affinity purification of the serum provided antibodies directed mainly to clathrin light chains and these antibodies, as well as an independent antiserum to clathrin heavy chains, immunofluorescently stained liver tissue and cells in a manner typical of coated membranes/vesicles. These results suggested, by difference, that antibodies to a 220 kDa polypeptide, a minor constituent in brain clathrin preparations, were responsible for staining intercellular tight junctions in epithelia. The 220 kDa polypeptide present in brain clathrin preparations was demonstrated to be immunologically distinct from liver myosin heavy chain as well as erythrocyte and brain ankyrin. Comparison by two-dimensional mapping of the 220 kDa in brain clathrin with the clathrin heavy chain (180 kDa) polypeptide showed they were different proteins, but the 220 kDa polypeptide present in rat liver tight junctions was highly similar to the 220 kDa present in bovine brain clathrin preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Antibodies to brain clathrin recognise plant coated vesicles

Coated vesicles isolated from carrot suspension culture cells were immune-blotted against four antibodies to porcine brain clathrin. Positive cross-reaction was obtained with three antibodies. Two of these cross-reacted with both the heavy clathrin chain and the putative light chains. Three out of five antibodies immunofluorescently stained permeabilised carrot suspension culture cells.     

Possible involvement of clathrin in neuritogenesis induced by a protease inhibitor (benzyloxycarbonyl-Leu-Leu-Leu-aldehyde) in PC12 cells.

Our previous reports showed that benzyloxycarbonyl (Z)-Leu-Leu-Leu-al (ZLLLal) induces neurite outgrowth in PC12 cells, and that 33-, 35-, and 180-kDa proteins from PC12 cells elute specifically from a Leu-Leu-Leu-al (LLLal)-coupled affinity column. Several lines of evidence suggest that the 33-, 35-, and 180-kDa proteins are components of clathrin, well-known for its role in endocytosis. Separation of clathrin into its heavy and light chains showed that the clathrin heavy chains have the ability to bind to a LLLal affinity column directly. Furthermore, ZLLLal enhances the rate of polymerization of clathrin triskelion to the coat structure. ZLLL-COOH does not cause neurite outgrowth in PC12 cells, and has no effect on the rate of clathrin polymerization. On immunocytochemical analysis of PC12 cells with an anti-clathrin heavy chain antibody, enhanced staining of the clathrin heavy chain was observed concomitantly with neurite outgrowth initiated by ZLLLal, but not by NGF. This study provides new insights into both the role of the clathrin molecule and the regulatory mechanism of neurite outgrowth.     

Clathrin self-assembly is regulated by three light-chain residues controlling the formation of critical salt bridges.

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.     

A Comparison of GFP‐Tagged Clathrin Light Chains with Fluorochromated Light Chains In Vivo and In Vitro

Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)‐tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has not been investigated yet thoroughly. As an alternative approach, we labeled purified LCs either with Alexa 488 or Cy3 dye and compared them with GFP‐tagged LC variants. Cy3‐labeled light chains (Cy3‐LCs) were microinjected into HeLa cells either directly or in association with heavy chains. Within 1–2 min the Cy3‐LC heavy chain complexes entered clathrin‐coated structures, whereas uncomplexed Cy3‐LC did not within 2 h. These findings show that no significant exchange of LCs occurs over the time–course of an endocytic cycle. To explore whether GFP‐tagged LCs behave functionally like endogenous LCs, we characterized them biochemically. Unlike wild‐type LCs, recombinant LCs with a GFP attached to either end did not efficiently inhibit clathrin assembly in vitro, whereas Cy3‐ and Alexa 488‐labeled LC behaved similar to wild‐type LCs in vitro and in vivo. Thus, fluorochromated LCs are a valuable tool for investigating the complex behavior of clathrin in living cells.