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PubMed1997

A giant protein that stimulates guanine nucleotide exchange on ARF1 and Rab proteins forms a cytosolic ternary complex with clathrin and Hsp70.

We have recently identified an unusually large human protein that stimulates guanine nucleotide exchange on ARF1 and Rab proteins (EMBO J 15: 4262-4273, 1996). This protein, designated p532 based on its predicted molecular weight (EMBO J 15: 5738, 1996), contains multiple structural domains including two regions of seven internal repeats highly related to the cell cycle regulator RCC1, a guanine nucleotide exchange factor for the small GTP-binding protein Ran, seven beta-repeat domains characteristic of the beta subunit of heterotrimeric G proteins, three putative SH3 binding sites, a putative leucine-zipper and a carboxy-terminal HECT domain characteristic of E3 ubiquitin-protein ligases. Some of these domains are known to be involved in protein-protein interactions, suggesting the existence of p532-interacting proteins. To identify some of these proteins, we used the carboxy-terminal RCC1-like domain (RLD) of p532 in the yeast two-hybrid system. We report here the isolation of a clone that encodes the last 654 amino acid residues of the clathrin heavy chain. This interaction involves amino acid residues 1315-1557 of the clathrin heavy chain and the carboxy, but not the amino-terminal RLD of p532. p532 has been located in the cytosolic fraction as well as in the Golgi apparatus. The interaction between p532 and clathrin only occurs in the cytosol and is mediated by the formation of an ATP-dependent ternary complex with the heat shock protein, Hsp70. These observations suggest that p532 is involved in membrane transport processes.

ADP-Ribosylation Factor 1↗

PubMed1988

Molecular characterization of the AP180 coated vesicle assembly protein.

Recently, a new clathrin assembly protein (AP180) has been purified from coated vesicles of bovine brain (Ahle & Ungewickell, 1986). This protein has been shown to promote polymerization of clathrin into a homogeneous population of baskets under conditions where pure clathrin does not polymerize by itself. We have purified this protein from coated vesicles by a simpler method than has been reported. The method involves a gel filtration step on a Sephacryl S-300 column, in 0.5 M Tris-HCl, pH 8.0, and a hydroxylapatite column eluted with 10 mM sodium phosphate/0.5 M Tris-HCl, pH 7.0. By running SDS gels over an extended period of time (5-15% gradient gel, 10 mA for the first 12 h followed by 20 mA for the next 3-4 h) after the marker dye entered the electrode buffer, we have been able to separate AP180 from clathrin heavy chain on the gels. This enabled us to determine its stoichiometry to clathrin heavy chains in isolated coated vesicles and assembled baskets, and was helpful in the purification procedure. The apparent molecular weight of the pure protein on SDS gels was about 180,000, yet gel filtration yielded values of about 120,000. Thus, we undertook the molecular weight determination by another independent method, sedimentation equilibrium analysis, and found a molecular weight of 115,000 and a sedimentation coefficient of 3.50 +/- 0.05 S. Circular dichroism data revealed that it has 30% helical structure, 14% beta-structure, 27% beta-sheet, and the rest random peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2001

Human Vam6p promotes lysosome clustering and fusion in vivo.

Regulated fusion of mammalian lysosomes is critical to their ability to acquire both internalized and biosynthetic materials. Here, we report the identification of a novel human protein, hVam6p, that promotes lysosome clustering and fusion in vivo. Although hVam6p exhibits homology to the Saccharomyces cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p, the presence of a citron homology (CNH) domain at the NH(2) terminus is unique to the human protein. Overexpression of hVam6p results in massive clustering and fusion of lysosomes and late endosomes into large (2-3 microm) juxtanuclear structures. This effect is reminiscent of that caused by expression of a constitutively activated Rab7. However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7. Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein. Both the CNH and clathrin heavy chain repeat domains are required for induction of lysosome clustering and fusion. This study implicates hVam6p as a mammalian tethering/docking factor characterized with intrinsic ability to promote lysosome fusion in vivo.

Adaptor Proteins, Vesicular Transport↗

PubMed1993

Clathrin facilitates the internalization of seven transmembrane segment receptors for mating pheromones in yeast.

The role of clathrin in endocytosis of the yeast phermone receptors was examined using strains expressing a temperature-sensitive clathrin heavy chain. The yeast phermone receptors belong to the family of seven transmembrane segment, G-protein-coupled receptors. A rapid and reversible defect in uptake of radiolabeled alpha-factor pheromone occurred when the cells were transferred to the nonpermissive temperature. Constitutive, pheromone-independent internalization of newly synthesized a-factor phermone receptor was also rapidly inhibited in mutant strains at the nonpermissive temperature. In both cases residual endocytosis, 30-50% of wild-type levels, was detected in the absence of functional clathrin heavy chain. Once internalized, the a-factor receptor was delivered to the vacuole at comparable rates in chc1-ts and wild-type cells at the nonpermissive temperature. Clathrin heavy chain was also required for maximal uptake of a mutant a-factor receptor which is dependent on pheromone for internalization. In the presence of a-factor, the internalization rate of the mutant receptor in chc1-ts cells at the nonpermissive temperature was 2.5 times slower than the rate observed for endocytosis of the mutant receptor in wild-type cells. These experiments provide in vivo evidence that clathrin plays an important role in the endocytosis of the seven trans-membrane segment pheromone receptors in yeast.

Biological Transport↗

PubMed1983

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.

Animals↗

PubMed2005

Clathrin heavy and light chain isoforms originated by independent mechanisms of gene duplication during chordate evolution.

In humans, there are two isoforms each of clathrin heavy chain (CHC17 and CHC22) and light chain (LCa and LCb) subunits, all encoded by separate genes. CHC17 forms the ubiquitous clathrin-coated vesicles that mediate membrane traffic. CHC22 is implicated in specialized membrane organization in skeletal muscle. CHC17 is bound and regulated by LCa and LCb, whereas CHC22 does not functionally interact with either light chain. The imbalanced interactions between clathrin subunit isoforms suggest a distinct evolutionary history for each isoform pair. Phylogenetic and sequence analysis placed both heavy and light chain gene duplications during chordate evolution, 510-600 million years ago. Genes encoding CHC22 orthologues were found in several vertebrate species, with only a pseudogene present in mice. Multiple paralogons surrounding the CHC genes (CLTC and CLTD) were identified, evidence that genomic or large-scale gene duplication produced the two CHC isoforms. In contrast, clathrin light chain genes (CLTA and CLTB) apparently arose by localized duplication, within 1-11 million years of CHC gene duplication. Analysis of sequence divergence patterns suggested that structural features of the CHCs were maintained after gene duplication, but new interactions with regulatory proteins evolved for the CHC22 isoform. Thus, independent mechanisms of gene duplication expanded clathrin functions, concomitant with development of neuromuscular sophistication in chordates.

Animals↗

PubMed2004

Leishmania major: clathrin and adaptin complexes of an intra-cellular parasite.

To investigate the role of clathrin-mediated trafficking during the Leishmania lifecycle, open reading frames encoding clathrin heavy chain and the beta-adaptins, major components of the adaptor complexes, have been analysed both in silico and experimentally. The Leishmania genome encodes three beta-adaptins, which arose at a time predating speciation of these divergent trypanosomatids. Unlike Trypanosoma brucei, both clathrin heavy chain and beta-adaptin1 are constitutively expressed throughout the Leishmania life cycle. Clathrin relocalises in amastigotes relative to promastigotes, consistent with developmental alterations to the morphology of the endo-membrane system.

Adaptor Protein Complex beta Subunits↗

PubMed2005

Shiga toxin regulates its entry in a Syk-dependent manner.

Shiga toxin (Stx) is composed of an A-moiety that inhibits protein synthesis after translocation into the cytosol, and a B-moiety that binds to Gb3 at the cell surface and mediates endocytosis of the toxin. After endocytosis, Stx is transported retrogradely to the endoplasmic reticulum, and then the A-fragment enters the cytosol. In this study, we have investigated whether toxin-induced signaling is involved in its entry. Stx was found to activate Syk and induce rapid tyrosine phosphorylation of several proteins, one protein being clathrin heavy chain. Toxin-induced clathrin phosphorylation required Syk activity, and in cells overexpressing Syk, a complex containing clathrin and Syk could be demonstrated. Depletion of Syk by small interfering RNA, expression of a dominant negative Syk mutant (Syk KD), or treatment with the Syk inhibitor piceatannol inhibited not only Stx-induced clathrin phosphorylation but also endocytosis of the toxin. Also, Golgi transport of Stx was inhibited under all these conditions. In conclusion, our data suggest that Stx regulates its entry into target cells.

Clathrin Heavy Chains↗

PubMed2001

Internalization of cholera toxin by different endocytic mechanisms.

The mechanism of cholera toxin (CT) internalization has been investigated using Caco-2 cells transfected with caveolin to induce formation of caveolae, HeLa cells with inducible synthesis of mutant dynamin (K44A) and BHK cells in which antisense mRNA to clathrin heavy chain can be induced. Here we show that endocytosis and the ability of CT to increase the level of cAMP were unaltered in caveolin-transfected cells grown either in a non-polarized or polarized manner. Treatment of Caco-2 cells with filipin reduced CT-uptake by less than 20%, suggesting that caveolae do not play a major role in the uptake. Extraction of cholesterol by methyl-beta-cyclodextrin, which removes caveolae and inhibits uptake from clathrin-coated pits, gave 30-40% reduction of CT-endocytosis. Also, CT-uptake in HeLa K44A cells was reduced by 50-70% after induction of mutant dynamin, which inhibits both caveolae- and clathrin-dependent endocytosis. These cells contain few caveolae, and nystatin and filipin had no effect on CT-uptake, indicating major involvement of clathrin-coated pits in CT-internalization. Similarly, in BHK cells, where clathrin-dependent endocytosis is blocked by induction of antisense clathrin heavy chain, the CT-uptake was reduced by 50% in induced cells. In conclusion, a large fraction of CT can be endocytosed by clathrin-dependent as well as by caveolae- and clathrin-independent endocytosis in different cell types.

Animals↗

PubMed2001

Clathrin box in G protein-coupled receptor kinase 2.

beta(1)-Adrenergic receptor (beta(1)AR) shows the resistance to agonist-induced internalization. However, beta(1)AR can internalize as G protein-coupled receptor kinase 2 (GRK2) is fused to its carboxyl terminus. Internalization of the beta(1)AR and GRK2 fusion protein (beta(1)AR/GRK2) is dependent on dynamin but independent of beta-arrestin and phosphorylation. The beta(1)AR/GRK2 fusion protein internalizes via clathrin-coated pits and is found to co-localize with the endosome that contains transferrin. The fusion proteins consisting of beta(1)AR and various portions of GRK2 reveal that the residues 498-502 in the carboxyl-terminal domain of GRK2 are critical to promote internalization of the fusion proteins. This domain contains a consensus sequence of a clathrin-binding motif defined as a clathrin box. In vitro binding assays show that the residues 498-502 of GRK2 bind the amino-terminal domain of clathrin heavy chain to almost the same extent as beta-arrestin1. The mutation of the clathrin box in the carboxyl-terminal domain of GRK2 results in the loss of the ability to promote internalization of the fusion protein. GRK2 activity increases and then decreases as the concentration of clathrin heavy chain increases. Taken together, these results imply that GRK2 contains a functional clathrin box and directly interacts with clathrin to modulate its function.

Amino Acid Sequence↗

PubMed2005

Golgi vesiculation induced by cholesterol occurs by a dynamin- and cPLA2-dependent mechanism.

It was recently demonstrated that an increase in the cellular cholesterol level leads to vesiculation of the Golgi apparatus. This vesiculation affects the entire Golgi apparatus and is a reversible process. We have now started to elucidate the mechanism behind this cholesterol-induced vesiculation of the Golgi apparatus. Transient transfection of cells with dominant negative mutant constructs of dynamin 1 and 2 inhibited the vesiculation; expression of dynK44A in HeLa cells stably transfected with this construct had the same effect. However, the vesiculation seems to be independent of clathrin, as cholesterol-induced vesiculation still occurred following knock down of clathrin heavy chain in HeLa cells using RNA interference as well as in BHK cells where expression of antisense to clathrin heavy chain had been induced. Importantly, the cPLA2 inhibitor MAFP and the chelator BAPTA-AM that binds cytosolic Ca2+ inhibited the cholesterol-induced vesiculation, suggesting involvement of a cPLA2 that requires cytosolic Ca2+ for translocation to membranes. Furthermore, in response to an increased cellular cholesterol level, an EGFP-cPLA2 fusion protein translocated to the Golgi apparatus. Thus, our results demonstrate that the cholesterol-induced vesiculation of the Golgi apparatus is mediated by a cPLA2- and dynamin-dependent mechanism.

Animals↗

PubMed1997

Purification of clathrin heavy and light chain from Dictyostelium 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 approximately 2 mg of clathrin triskelions from 150-200 g of Dictyostelium cells. One additional step purified the 30-kDa clathrin light chain to homogeneity. Glycerol gradient centrifugation was used to determine an S value of 7.9 for purified clathrin. Rotary shadowed images of Dictyostelium clathrin 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 in Dictyostelium clathrin demonstrates the potential of this model organism for the study of clathrin structure and function.

Animals↗

PubMed2002

Mixed lineage kinase 2 interacts with clathrin and influences clathrin-coated vesicle trafficking.

Mixed lineage kinase 2 (MLK2) is a protein kinase that signals in the stress-activated Jun N-terminal kinase signal transduction pathway. We used immunoprecipitation and mass spectrometric analysis to identify MLK2-binding proteins in cell lines with inducible expression of green fluorescent protein-tagged MLK2. Here we report the identification of clathrin as a binding partner for MLK2 in both cultured cells and mammalian brain. We demonstrate that clathrin binding requires a motif (LLDMD) located near the MLK2 C terminus, which is similar to "clathrin box" motifs important for binding of clathrin coat assembly and accessory proteins to the clathrin heavy chain. A C-terminal fragment of MLK2 containing this motif binds strongly to clathrin, and mutation of the LLDMD sequence to LAAAD completely abrogates clathrin binding. We isolated clathrin-coated vesicles from green fluorescent protein-MLK2-expressing cells and from mouse brain lysates and found that MLK2 is enriched along with clathrin in these vesicles. In addition, we demonstrated that endogenous MLK2 co-immunoprecipitates with clathrin heavy chain from the vesicle-enriched fraction of mouse brain lysate. Furthermore, overexpression of MLK2 in cultured cells inhibits accumulation of labeled transferrin in recycling endosomes during receptor-mediated endocytosis. These findings suggest a role for MLK2 and the stress-signaling pathway at sites of clathrin activity in vesicle formation or trafficking.

Amino Acid Motifs↗

PubMed2006

Identification of putative androgen receptor interaction protein modules: cytoskeleton and endosomes modulate androgen receptor signaling in prostate cancer cells.

We have developed a novel androgen receptor (AR) expression system in the 293 human embryonic kidney cell line that recapitulates AR biochemical activity as a steroid hormone receptor in prostate cancer cells. We used this system to identify putative AR-binding proteins in the cytosolic and nuclear compartments of mammalian cells using a large scale co-immunoprecipitation strategy coupled to quantitative mass spectrometry. For example, the heat shock 70 and 90 chaperones, which are known regulators of steroid hormone receptor, were identified as AR-binding proteins. AR purification enriched for proteins involved in RNA processing, protein transport, and cytoskeletal organization, suggesting a functional link between AR and these protein modules in mammalian cells. For example, AR purification in the nuclear compartment led to the specific enrichment of alpha-actinin-4, clathrin heavy chain, and serine-threonine protein kinase C delta. Short interfering RNA knockdown studies and co-transcriptional reporter assays revealed that clathrin heavy chain possessed co-activator activity during AR-mediated transcription, whereas alpha-actinin-4 and protein kinase C delta displayed both co-activator and co-repressor activity during AR-mediated transcription that was dependent upon their relative expression levels. Lastly immunohistochemical staining of prostate tissue showed that alpha-actinin-4 levels decreased in the nucleus of high grade cancerous prostate samples, suggesting its possible deregulation in advanced prostate cancers as previously observed in late stage metastatic breast cancers. Taken together, these findings suggest AR binds to specific protein modules in mammalian cells and that these protein modules may provide a molecular framework for interrogating AR function in normal and cancerous prostate epithelial cells.

Androgen-Binding Protein↗

PubMed1990

Localization of anti-clathrin antibody in the sarcomere and sensitivity of myofibril structure to chloroquine suggest a role for clathrin in myofibril assembly.

Immunofluorescence microscopy has been used to demonstrate that X22, a monoclonal antibody specific for clathrin heavy chain, localizes in repetitive bands that appear soon after the fusion of skeletal myoblasts into multinucleate fibers. This organization has been found in cultures containing myotubes that develop in vitro from explants of newborn rat hindlimb cells and in myotubes derived from the L8E63 myogenic line. Bands were also prominent in skinned fibers prepared from adult rat soleus muscle and in cardiac myocytes grown in vitro from 4-day heart ventricles. Immunofluorescence banding was localized in the sarcomere as a doublet, with one element on either side of the Z line. Evidence that supports the conclusion that the reaction with X22 antibody is specific and indicative of the localization of clathrin in the sarcomere includes: (1) Identical titration of X22 antibody reactivity with the determinant in coated vesicles and in the sarcomere. (2) Conditions (eg., pH and Tris) that disrupt clathrin baskets or prevent its assembly likewise disrupt the localization of X22 in bands. (3) Chloroquine inhibits both the normal trafficking of clathrin in the cell and X22 banding in the sarcomere. (4) Immunoblot analysis of myotube lysates reveals a single band with an electrophoretic mobility identical to the 180,000-Da clathrin heavy chain. (5) The assembly of clathrin into sarcomeric bands occurs early in the development of the myofibrillar apparatus. Quantitation of the appearance of X22 banding in primary cultures of myotubes indicates that it precedes that of other myofibrillar proteins and that assembly takes place in the following order: X22, titin, myosin heavy chain, actin, and desmin. The assembly of myosin, titin, and actin into sarcomeric bands, as well as X22, is inhibited by chloroquine. Upon prolonged exposure to chloroquine previously assembled proteins are drastically reduced or no longer evident in the sarcomere. On the basis of these results and considering the role of clathrin in intracellular transport and its capacity to interact with actin and alpha-actinin, we suggest that clathrin may have diverse roles in the assembly, integrity, and functioning of the sarcomere and its integration with the sarcolemma. The early organization of X22 into bands further suggests that clathrin may also function early in the assembly of the contractile system.

Actins↗

PubMed2001

A novel clathrin homolog that co-distributes with cytoskeletal components functions in the trans-Golgi network.

A clathrin homolog encoded on human chromosome 22 (CHC22) displays distinct biochemistry, distribution and function compared with conventional clathrin heavy chain (CHC17), encoded on chromosome 17. CHC22 protein is upregulated during myoblast differentiation into myotubes and is expressed at high levels in muscle and at low levels in non-muscle cells, relative to CHC17. The trimeric CHC22 protein does not interact with clathrin heavy chain subunits nor bind significantly to clathrin light chains. CHC22 associates with the AP1 and AP3 adaptor complexes but not with AP2. In non-muscle cells, CHC22 localizes to perinuclear vesicular structures, the majority of which are not clathrin coated. Treatments that disrupt the actin-myosin cytoskeleton or affect sorting in the trans-Golgi network (TGN) cause CHC22 redistribution. Overexpression of a subdomain of CHC22 induces altered distribution of TGN markers. Together these results implicate CHC22 in TGN membrane traffic involving the cytoskeleton.

Cells, Cultured↗

PubMed2002

Enthoprotin: a novel clathrin-associated protein identified through subcellular proteomics.

Despite numerous advances in the identification of the molecular machinery for clathrin-mediated budding at the plasma membrane, the mechanistic details of this process remain incomplete. Moreover, relatively little is known regarding the regulation of clathrin-mediated budding at other membrane systems. To address these issues, we have utilized the powerful new approach of subcellular proteomics to identify novel proteins present on highly enriched clathrin-coated vesicles (CCVs). Among the ten novel proteins identified is the rat homologue of a predicted gene product from human, mouse, and Drosophila genomics projects, which we named enthoprotin. Enthoprotin is highly enriched on CCVs isolated from rat brain and liver extracts. In cells, enthoprotin demonstrates a punctate staining pattern that is concentrated in a perinuclear compartment where it colocalizes with clathrin and the clathrin adaptor protein (AP)1. Enthoprotin interacts with the clathrin adaptors AP1 and with Golgi-localized, gamma-ear-containing, Arf-binding protein 2. Through its COOH-terminal domain, enthoprotin binds to the terminal domain of the clathrin heavy chain and stimulates clathrin assembly. These data suggest a role for enthoprotin in clathrin-mediated budding on internal membranes. Our study reveals the utility of proteomics in the identification of novel vesicle trafficking proteins.

Adaptor Protein Complex 1↗

PubMed2005

Lowe syndrome protein OCRL1 interacts with clathrin and regulates protein trafficking between endosomes and the trans-Golgi network.

Oculocerebrorenal syndrome of Lowe is caused by mutation of OCRL1, a phosphatidylinositol 4,5-bisphosphate 5-phosphatase localized at the Golgi apparatus. The cellular role of OCRL1 is unknown, and consequently the mechanism by which loss of OCRL1 function leads to disease is ill defined. Here, we show that OCRL1 is associated with clathrin-coated transport intermediates operating between the trans-Golgi network (TGN) and endosomes. OCRL1 interacts directly with clathrin heavy chain and promotes clathrin assembly in vitro. Interaction with clathrin is not, however, required for membrane association of OCRL1. Overexpression of OCRL1 results in redistribution of clathrin and the cation-independent mannose 6-phosphate receptor (CI-MPR) to enlarged endosomal structures that are defective in retrograde trafficking to the TGN. Depletion of cellular OCRL1 also causes partial redistribution of a CI-MPR reporter to early endosomes. These findings suggest a role for OCRL1 in clathrin-mediated trafficking of proteins from endosomes to the TGN and that defects in this pathway might contribute to the Lowe syndrome phenotype.

Animals↗