Localization of clathrin light-chain sequences mediating heavy-chain binding and coated vesicle diversity

At least four distinct forms of clathrin light chains are found in mammalian cells. This molecular variability derives from tissue-specific patterns of expression of LCa and LCb genes. Sequence analysis shows an overall homology of 60% between LCa and LCb and the presence of brain-specific insertion sequences. These findings suggest that the different light chains have both shared and specialized functions. To address this question we have used a panel of monoclonal antibodies to identify two structurally and functionally distinct regions in the clathrin light-chain sequences. One region (residues 158-208) is exposed in native clathrin structures (triskelions and coated vesicles) and includes the brain-specific insertion sequences. The second region (residues 93-157), which is cryptic in native clathrin structures, is involved in binding the clathrin heavy chain and contains the region of strongest homology with intermediate filament proteins.     

Clathrin light chains and secretory vesicle binding proteins are distinct

Recently, several groups have initiated studies on cytosolic proteins that bind to isolated secretory vesicle membranes in the presence of Ca2+ in order to identify proteins that may regulate exocytosis. Two major chromaffin granule binding proteins, of molecular weights 32,000 (32K) and 34,000 (34K), were reported to have the same mobility on one-dimensional SDS gels as clathrin-associated light chains from the adrenal medulla, and the 34K granule binding protein the same one-dimensional peptide map as the 34K clathrin light chain. These observations support the hypothesis that Ca2+-dependent recruitment of soluble light chains to the vesicle membrane may nucleate the assembly of a clathrin coat and initiate endocytosis. Here we report that two-dimensional peptide maps of the clathrin light chains and of all chromaffin granule membrane binding proteins in the 30K range are distinct, and therefore fail to support this hypothesis. It has also been suggested that some or all of the vesicle binding proteins require calmodulin for their interaction with the membrane. However, we find that antagonism of calmodulin by trifluoperazine does not prevent the association of the other cytosolic proteins with the chromaffin granule membrane.     

Internal control of the coated vesicle pp50-specific kinase complex

The polyhedral surface lattice of coated vesicles consists of three-legged hexameric protein complexes called triskelions which constitute the basic assembly unit. The triskelion is a molecular complex of molecular weight 630,000 (Mr 630K) composed of three clathrin heavy chains (subunit 180K) and three light chains (subunits 33K and 36K) (refs 2,3). The presence of additional coated vesicle-specific proteins in the 100-130K and 50-55K range have been reported. We previously described the presence of a cyclic nucleotide- and Ca2+-independent protein kinase activity in coated vesicles which was confirmed by others. This protein kinase specifically phosphorylates the 50K protein (pp50). In this report, we show that the coated vesicle kinase and its 50K protein substrate are part of a stable multimolecular system. In addition we show that the clathrin-light chain complex stimulates the pp50 phosphorylation and only light chains are implicated in this stimulation and that the pp50 phosphorylation does not seem to be affected by the vesicle.     

Clathrin self-assembly is mediated by a tandemly repeated superhelix.

Clathrin is a triskelion-shaped cytoplasmic protein that polymerizes into a polyhedral lattice on intracellular membranes to form protein-coated membrane vesicles. Lattice formation induces the sorting of membrane proteins during endocytosis and organelle biogenesis by interacting with membrane-associated adaptor molecules. The clathrin triskelion is a trimer of heavy-chain subunits (1,675 residues), each binding a single light-chain subunit, in the hub domain (residues 1,074-1,675). Light chains negatively modulate polymerization so that intracellular clathrin assembly is adaptor-dependent. Here we report the atomic structure, to 2.6 A resolution, of hub residues 1,210-1,516 involved in mediating spontaneous clathrin heavy-chain polymerization and light-chain association. The hub fragment folds into an elongated coil of alpha-helices, and alignment analyses reveal a 145-residue motif that is repeated seven times along the filamentous leg and appears in other proteins involved in vacuolar protein sorting. The resulting model provides a three-dimensional framework for understanding clathrin heavy-chain self-assembly, light-chain binding and trimerization.     

Molecular model for a complete clathrin lattice from electron cryomicroscopy

Clathrin-coated vesicles are important vehicles of membrane traffic in cells. We report the structure of a clathrin lattice at subnanometre resolution, obtained from electron cryomicroscopy of coats assembled in vitro. We trace most of the 1,675-residue clathrin heavy chain by fitting known crystal structures of two segments, and homology models of the rest, into the electron microscopy density map. We also define the position of the central helical segment of the light chain. A helical tripod, the carboxy-terminal parts of three heavy chains, projects inward from the vertex of each three-legged clathrin triskelion, linking that vertex to 'ankles' of triskelions centred two vertices away. Analysis of coats with distinct diameters shows an invariant pattern of contacts in the neighbourhood of each vertex, with more variable interactions along the extended parts of the triskelion 'legs'. These invariant local interactions appear to stabilize the lattice, allowing assembly and uncoating to be controlled by events at a few specific sites.     

Recruitment of cytosolic proteins to a secretory granule membrane depends on Ca2+-calmodulin

An increase in free calcium triggers catecholamine secretion from chromaffin cells and calmodulin is strongly implicated as the intracellular Ca2+ receptor. In our recent studies of calmodulin action in the chromaffin cell, micromolar Ca2+ concentrations resulted in calmodulin and cytosolic proteins becoming bound to the chromaffin granule membranes. We now report that calmodulin is bound with high affinity to granule membrane proteins of molecular weights (Mrs) 25,000 and 22,000 (25K and 22K) at low Ca2+ (less than 10(-8) M) and to proteins with Mrs 69K and 50K at high Ca2+ (greater than 1 microM). Other cytosolic components (Mrs 70K, 36K, 34K and 32K) require calmodulin for their interfraction with membrane. These proteins separately bound to calmodulin-Sepharose at high Ca2+ concentrations. Although the functions of these adrenal proteins have not been established, the 34K and 32K Mr components co-migrate with clathrin light chains isolated from medullary coated vesicles and the Mr 34K components from both sources share the same one-dimensional peptide map. These interactions were observed at micromolar Ca2+ levels at 'intracellular' conditions of pH and ionic strength and would be expected to occur during secretion from the chromaffin cell.     

Clathrin is required for the function of the mitotic spindle

Clathrin has an established function in the generation of vesicles that transfer membrane and proteins around the cell. The formation of clathrin-coated vesicles occurs continuously in non-dividing cells, but is shut down during mitosis, when clathrin concentrates at the spindle apparatus. Here, we show that clathrin stabilizes fibres of the mitotic spindle to aid congression of chromosomes. Clathrin bound to the spindle directly by the amino-terminal domain of clathrin heavy chain. Depletion of clathrin heavy chain using RNA interference prolonged mitosis; kinetochore fibres were destabilized, leading to defective congression of chromosomes to the metaphase plate and persistent activation of the spindle checkpoint. Normal mitosis was rescued by clathrin triskelia but not the N-terminal domain of clathrin heavy chain, indicating that stabilization of kinetochore fibres was dependent on the unique structure of clathrin. The importance of clathrin for normal mitosis may be relevant to understanding human cancers that involve gene fusions of clathrin heavy chain.     

The synthesis and in vivo assembly of functional antibodies in yeast

The yeast Saccharomyces cerevisiae can synthesize, process and secrete higher eukaryotic proteins. We have investigated the expression of immunoglobulin chains in yeast and demonstrate here the synthesis, processing and secretion of light and heavy chains, the glycosylation of heavy chain, the intracellular localization of these foreign proteins by immunofluorescence, and the detection of functional antibodies in cells co-expressing both chains. This may provide the basis of a microbial fermentation process for the production of monoclonal antibodies. The co-expression of light and heavy chains in Escherichia coli has been reported but functional antibodies were not assembled in vivo. Furthermore, only low-level assembly of these chains was found in vitro.     

Clathrin is a key regulator of basolateral polarity

Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells.     

Two monoclonal antibodies against different antigens using the same VH germ-line gene

Immunoglobulin diversity seems to arise largely by three mechanisms: (1) the existence of several germ-line genes, which must be rearranged before expression--that is, V and J for the light (L) chains, V, D and J for the heavy (H) chains; (2) somatic events, including mutations and gene conversion; and (3) combinatorial association of heavy and light chains, leading to the proposal that random pairing of p X H and q X L chains might generate p X q antibody molecules expressing discrete specificities. As heavy and light chains derived from the same immunoglobulin molecule would frequently reassociate preferentially, it is likely that only a fraction of potential heavy--light pairs actually provides "valid' antibodies. As a consequence of combinatorial heavy--light chain pairing, antibodies of discrete specificities sharing the same VH region, associated with distinct light chains (or vice versa) should be encountered. We report here that two heavy chains, derived from the same VH germ-line gene, may be present in anti-NP or anti--GAT antibodies, depending on their association with a specific lambda or kappa light chain, respectively.     

Induction of light chain expression in a pre-B cell line by fusion to myeloma cells

Pre-B cells, the first cells in the B-lymphocyte differentiation pathway which express immunoglobulin, have recently been shown to express cytoplasmic mu heavy chain (H) but not light chain (L). If, as is believed, pre-B cells are the precursors of immature B lymphocytes, which express surface IgM, the differentiation of pre-B cells to immature B lymphocytes must be accompanied by the expression of light chains. In this case, it should be possible for the progeny of a single pre-B cell to express a variety of light chains in association with the same heavy chain. We have tested this hypothesis by hybridizing a pre-B cell line 18-81 expressing only cytoplasmic mu chains with variant myeloma cells which do not express light chains. Hybridization of B-lymphoma cells with myeloma cells usually produces a hybrid with the phenotype of the more differentiated parent. In this case, the fusion resulted in the induction of light chain expression from the 18-81 genes and we have been able to demonstrate that independent hybrids express different light chains, in accordance with the hypothesis that a pre-B cell committed to expression of a single mu heavy chain can generate progeny expressing different slight chains.     

Structural basis for recognition of acidic-cluster dileucine sequence by GGA1

GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-interacting proteins) are critical for the transport of soluble proteins from the trans-Golgi network (TGN) to endosomes/lysosomes by means of interactions with TGN-sorting receptors, ADP-ribosylation factor (ARF), and clathrin. The amino-terminal VHS domains of GGAs form complexes with the cytoplasmic domains of sorting receptors by recognizing acidic-cluster dileucine (ACLL) sequences. Here we report the X-ray structure of the GGA1 VHS domain alone, and in complex with the carboxy-terminal peptide of cation-independent mannose 6-phosphate receptor containing an ACLL sequence. The VHS domain forms a super helix with eight alpha-helices, similar to the VHS domains of TOM1 and Hrs. Unidirectional movements of helices alpha6 and alpha8, and some of their side chains, create a set of electrostatic and hydrophobic interactions for correct recognition of the ACLL peptide. This recognition mechanism provides the basis for regulation of protein transport from the TGN to endosomes/lysosomes, which is shared by sortilin and low-density lipoprotein receptor-related protein.     

A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins

Ubiquitination is a post-translation modification in which ubiquitin chains or single ubiquitin molecules are appended to target proteins, giving rise to poly- or monoubiquitination, respectively. Polyubiquitination targets proteins for destruction by the proteasome. The role of monoubiquitination is less understood, although a function in membrane trafficking is emerging, at least in yeast. Here we report that a short amino-acid stretch at the carboxy-termini of the monoubiquitinated endocytic proteins Eps15 and eps15R is indispensable for their monoubiquitination. A similar sequence, also required for this modification, is found in other cytosolic endocytic proteins, such as epsins and Hrs. These sequences comprise a protein motif, UIM (ref. 6), which has been proposed to bind to ubiquitin. We confirm this for the UIMs of eps15, eps15R, epsins and Hrs. Thus, the same motif in several endocytic proteins is responsible for ubiquitin recognition and monoubiquitination. Our results predict the existence of a UIM:ubiquitin-based intracellular network. Eps15/eps15R, epsins and Hrs may function as adaptors between ubiquitinated membrane cargo and either the clathrin coat or other endocytic scaffolds. In addition, through their own ubiquitination, they may further contribute to the amplification of this network in the endocytic pathway.     

Inhibition of phosphorylcholine binding to antibodies using synthetic peptides

The amino-acid sequence Phe-Tyr-Met-Glu is unique to phosphorylcholine (PC)-binding antibodies. It occurs in the first complementarity-determining region (CDR1) of the immunoglobulin heavy chains in 89% of all the anti-PC myeloma and hybridoma proteins but is not present in 490 other immunoglobulin heavy chains, 854 light chains or in 2,260 other unrelated proteins. This unique tetrapeptide therefore seems to be involved in PC binding. Here we compare the effectiveness of Phe-Tyr-Met-Glu and other structurally related peptides in inhibiting the binding of PC to PC-binding proteins McPC603 and HOPC8. We also test a surface-simulation peptide that was constructed to mimic the combining site of McPC603. Our data suggest that all these peptides inhibit the binding of PC to PC-binding proteins non-specifically and we show by computer modelling that the surface-simulation peptide does not duplicate the combining site of McPC603.     

Correlation between immunoglobulin light chain expression and variant translocation in Burkitt's lymphoma

Burkitt's-type lymphomas-leukaemias (BL) are monoclonal proliferations of malignant B lymphocytes. Irrespective of whether they carry the Epstein-Barr virus (EBV) genome, these tumour cells have been shown consistently to have one of the specific reciprocal chromosome translocations, t(8; 14), t(2; 8) or t(8; 22), involving the long arm of chromosome 8 (on 8q24) and chromosome 14, 2 or 22 (on 14q32, 2p12 and 22q11, respectively). The latter chromosomes have been shown recently to carry genes for immunoglobulin (Ig) heavy chains, and kappa and lambda light chains, respectively. Furthermore, the localization of kappa light chains within 2pcen-2p13 encompasses the breakpoint observed in Burkitt's translocation (2p12). It was therefore considered of interest to determine whether the expression of immunoglobulin chains in BL cells is related to the type of chromosomal anomalies observed. We report here that there is a direct relationship between expression of immunoglobulin light chains and specific type of translocation: BL cells with t(8; 22) express lambda chains, whereas those with t(2; 8) express kappa chains.     

A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene

Of the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains. Recent data suggest that pre-B cells express mu chains on the membrane together with the 'surrogate' light chains lambda 5 and V pre B (refs 2-7). This complex could control pre-B-cell differentiation, in particular the rearrangement of the light-chain genes. We have now assessed the importance of the membrane form of the mu chain in B-cell development by generating mice lacking this chain. We disrupted one of the membrane exons of the gene encoding the mu-chain constant region by gene targeting in mouse embryonic stem cells. From these cells we derived mice heterozygous or homozygous for the mutation. B-cell development in the heterozygous mice seemed to be normal, but in homozygous animals B cells were absent, their development already being arrested at the stage of pre-B-cell maturation.     

拟南芥网格蛋白重链突变体的遗传分析

利用PCR方法分离筛选了拟南芥网格蛋白重链T-DNA插入突变体chc1和chc2,并观察了CHC功能缺失后对植株发育的影响.结果表明：拟南芥网格蛋白重链CHC1或CHC2功能缺失引起部分植株发育异常,表现出生长素相关的发育表型,如幼苗子叶出现棒状、喇叭状和扇形等各种异常表型;通过人工杂交未能获得chc1chc2纯合双突变体,表明CHC1和CHC2功能同时缺失可能引起花粉致死或胚胎致死.这些遗传分析结果表明CHC在植物发育过程中具有重要的生物学功能.     

Control of tension development in scallop muscle fibres with foreign regulatory light chains

We have shown previously that chemically skinned fibre bundles from scallop muscle can be desensitized to the action of calcium by the removal of the regulatory light chains of myosin and that sensitivity can be restored by the re-addition of scallop light chains. We have now confirmed these results and extended our observations to fibre bundles from which one or both of the regulatory light chains per myosin have been removed (by treatment with EDTA at 7 and 25 degrees C, respectively) and replaced by the corresponding light chains from other species. In the case of a double substitution--where both light chains were replaced--sensitivity was restored completely by light chains from other molluscs and from gizzard muscle; for a single substitution there was restoration of sensitivity by all the light chains tested.