Novel binding sites on clathrin and adaptors regulate distinct aspects of coat assembly

Clathrin-coated vesicles (CCVs) sort proteins at the plasma membrane, endosomes and trans Golgi network for multiple membrane traffic pathways. Clathrin recruitment to membranes and its self-assembly into a polyhedral coat depends on adaptor molecules, which interact with membrane-associated vesicle cargo. To determine how adaptors induce clathrin recruitment and assembly, we mapped novel interaction sites between these coat components. A site in the ankle domain of the clathrin triskelion leg was identified that binds a common site on the appendages of tetrameric [AP1 and AP2] and monomeric (GGA1) adaptors. Mutagenesis and modeling studies suggested that the clathrin-GGA1 appendage interface is nonlinear, unlike other peptide-appendage interactions, but overlaps with a sandwich domain binding site for accessory protein peptides, allowing for competitive regulation of coated vesicle formation. A novel clathrin box in the GGA1 hinge region was also identified and shown to mediate membrane recruitment of clathrin, while disruption of the clathrin-GGA1 appendage interaction did not affect recruitment. Thus, the distinct sites for clathrin-adaptor interactions perform distinct functions, revealing new aspects to regulation of CCV formation.     

Cloning and expression of a plant homologue of the small subunit of the Golgi-associated clathrin assembly protein AP19 from Camptotheca acuminata

Clathrin-coated vesicles (CCVs) are involved in selective protein transport in eukaryotes. AP-1 and AP-2 are protein complexes found in the CCVs of the Golgi apparatus and the plasma membrane respectively. AP19 is the smallest polypeptide chain components of AP-1. We have identified a cDNA clone (CAP19) encoding a putative homologue for the assembly protein AP19 from the Chinese medicinal tree, Camptotheca acuminata. The deduced polypeptide contains 161 amino acids and has a predicted M _r of 18 820. DNA blot analysis suggests that the AP19s of C. acuminata are encoded by a small gene family. CAP19 was expressed ubiquitously throughout the plant suggesting that it may be involved in general Golgi-mediated secretion.     

Clathrin-protein interactions

There is a complex network of protein–protein and protein–lipid interactions that underlie clathrin-mediated vesicular traffic in all compartmentalized cells from yeast to man. Major progress has been made in the determination of the three-dimensional structures of many of the components. Recently, there has been an explosion in the identification and characterization of clathrin binding partners. This review integrates the structural and biochemical information that is currently available to present a unified view of how many clathrin binding partners interact with clathrin.     

Characterization of yeast clathrin and anticlathrin heavy-chain monoclonal antibodies

Clathrin-coated vesicles (CVs) were isolated from Saccharomyces cerevisiae by using procedures developed by Mueller and Branton [17]. Triskelions were purified from this material by extraction of CVs to release clathrin and by subsequent fractionation on Sepharose CL-4B. Triskelions were composed of approximately 180,000 Mr heavy chains and a single light-chain type of approximately 38,000 Mr and were able to undergo self-assembly into polyhedral cages. Trypsin digestion of such reassembled cages showed a peptide pattern very similar to that obtained for mammalian clathrin with two fragments of 125,000 and 110,000 Mr, which represent the major portion of the heavy-chain arm, and a polypeptide of approximately 43,000 Mr, which is the presumptive terminal domain. Eight monoclonal antibodies reacting with yeast clathrin heavy chains were produced. All eight bind to the major portion of the heavy-chain arm, and none bind to the terminal domain fragment. Peptide digestion experiments also indicated that at least three major regions on the arm are recognized by these antibodies. These will be useful in further structural and functional studies of clathrin from yeast.     

Coated Vesicles From Locust Oocytes: Isolation and Characterization

Summary

This report demonstrates for the first time the isolation of coated vesicles from insect oocytes. Coated vesicles were purified from oocytes of Locusta migratoria by differential centrifugation and sucrose density centrifugation. The coated vesicles were characterized by electron microscopy, SDS-PAGE and scanning densitometry. Like coated vesicles isolated from pig brain and chicken oocytes, the coated vesicles from locust oocytes contained clathrin as the major protein component. Apart from clathrin, another major protein characteristic of coated vesicles had a molecular weight of about 115,000, and in addition, several minor unidentified bands were identified.     

Multi‐modal adaptor‐clathrin contacts drive coated vesicle assembly

Clathrin‐coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated‐pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the β2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo‐EM structure of clathrin cages assembled in the presence of β2 hinge‐appendage (β2HA). We find that the β2‐appendage binds in at least two positions in the cage, demonstrating that multi‐modal binding is a fundamental property of clathrin‐AP2 interactions. In one position, β2‐appendage cross‐links two adjacent terminal domains from different triskelia. Functional analysis of β2HA‐clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin‐box motif on the hinge and the “sandwich site” on the appendage. We propose that β2‐appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.     

New faces of the familiar clathrin lattice

The clathrin triskelion self-assembles into a lattice that coats transport vesicles participating in several key membrane traffic pathways. A new model of a clathrin lattice at approximately 8 angstrom resolution, generated by Fotin et al. (Nature 2004;432:573) confirmed the basic structural features of clathrin that were defined over many years of biochemical and structural analysis. In addition, new structural features of the clathrin trimerization domain were modelled for the first time, and the predictions correlated well with previous biochemical studies. A second model, placing auxilin within the lattice suggested a possible lattice contact targeted during lattice disassembly (Fotin et al. Nature 2004;432:649). This contact predicts interactions of the newly modelled trimerization domain with a newly defined extension of the clathrin triskelion, the ankle domain. These aspects of the new models were emphasized in the published reports describing them and in recent commentary (Brodsky, Nature 2004;432:568). Also emerging from the new models is a better picture of how the clathrin structure is distributed throughout the lattice, allowing the first predictions of interacting molecular interfaces contributing to contacts in the assembled lattice. The focus of this interchange is to emphasize these additional features revealed by the recently published models from Fotin and colleagues.     

Cryo-electron tomography of clathrin-coated vesicles: structural implications for coat assembly

Clathrin-coated vesicles mediate vesicular traffic in cells. Three-dimensional image reconstructions of homogenous populations of in vitro assembled clathrin coats have yielded a molecular model for clathrin and its interactions with some of its partners. The intrinsic averaging required for those calculations has precluded detailed analysis of heterogeneous populations of clathrin-coated vesicles isolated from cells. We have therefore used cryo-electron tomography to study the lattice organization of individual clathrin-coated vesicles and the disposition of the captured vesicle with respect to the surrounding coat. We find a wide range of designs for the clathrin lattice, with different patterns of pentagonal, hexagonal, and occasionally heptagonal facets. Many coats, even smaller ones, enclose membrane vesicles, which are generally offset from the center of the clathrin shell. The electron density distribution between the coat and the underlying vesicle is not uniform, and the number of apparent contacts that anchor the clathrin lattice to the vesicle membrane is significantly less than the number of clathrin heavy chains in the assembly. We suggest that the eccentric position of the vesicle reflects the polarity of assembly, from initiation of coat formation to membrane pinching.     

Using quantum dots to visualize clathrin associations

Our current understanding of clathrin-mediated endocytosis proposes that the process is initiated at a specialized anatomical structure called a coated pit. Electron microscopy has been required for elucidation of the morphology of coated pits and the vesicles produced therein, and the presence of a bristle coat has been taken as suggestive of clathrin surrounding these vesicles. More recently, immunocytochemical methods have confirmed that endocytic vesicles are surrounded by clathrin and its adaptor proteins, but there is a need to identify precisely and to follow the fate of the cellular organelles seen by fluorescence microscopy. We used quantum immune-electron microscopy to localize clathrin in a human adrenal cortical cell line (SW-13). Clathrin was shown to associate with a variety of vesicle types including the classic clathrin-coated vesicles and pits used in receptor internalization, pentilaminar annular gap junction vesicles, and multivesicular bodies. The images obtained with quantum dot technology allow accurate and specific localization of clathrin and the clathrin adaptor protein, AP-2, with cellular organelles and suggest that some of the structures classified as typical coated vesicles by immunocytochemical light microscopic techniques actually may be membrane bound pits.     

Clathrin coated vesicles in Neurospora crassa

Summary Electropherograms of Neurospora crassa homogenates showed a polypeptide with a mobility slightly lower than that of a standard sample of clathrin (from bovine brain). Subcellular fractionation of the homogenate resulted in a 20-fold enrichment of the putative N. crassa clathrin in the microsomal fraction. Further fractionation of the microsomal fraction by glass bead permeation chromatography yielded a fraction enriched about 150-fold relative to the homogenate. Coated vesicles (42.5 ± 2.5 nm diameter) were found in this preparation by electron microscopy of negatively stained specimens. Ribosomes were virtually absent from this sample. N. crassa clathrin remained associated with the coated vesicles after repeated centrifugation and homogenization steps, even in the presence of 0.4 M-NaCl, but was released by treatment with Tris buffer pH 8.5. However the polypeptide was again sedimentable after dialysis against Mes buffer pH 6.5. Under the electron microscope this sediment resembled the empty coats of higher eukaryotes. The results taken together indicate that a clathrin-like protein occurs in wild type cells of N. crassa.     

In vitro, insulin receptor catalyses phosphorylation of clathrin heavy chain and a plasma membrane 180,000 molecular weight protein

Insulin receptor mutation studies that the receptor tyrosine kinase activity is necessary for receptor endocytosis, and several insulin receptor-containing tissues have a plasma membrane-associated protein (M_r 180,000, p180) whose tyrosine phosphorylation is receptor catalysed. Since clathrin heavy chain (M_r 180,000 in dodecyl sulphate gel electrophoresis) is a major component of coated vesicles, the latter functioning in receptor endocytosis, we investigated whether insulin receptors can catalyse clathrin phosphorylation and whether p180 is clathrin. Bovine brain triskelion or coated vesicles and ³²P-ATP were added to prephosphorylated insulin receptor preparations (wheat ferm agglutinin-purified human placenta membrane proteins). Antiphosphotyrosine immunoprecipitated a phosphorylated 180,000 molecular weight protein. Insulin (10⁻⁷M) increased the rate of phosphorylation. Monoclonal anti-clathrin antibody immunoprecipitated the phosphorylated 180,000 molecular weight protein, whereas monoclonal anti-insulin receptor antibodies (-IR1, MA10) immunoprecipitated both insulin receptors and the phosphorylated 180,000 molecular weight protein. In the absence of added clathrin, anticlathrin immunoprecipitated no proteins, and -IR1 imunoprecipitated only the insulin receptor. Density gradient (glycerol 7.5–30%, w/v) centrifugation separated human placenta microsomal membrane proteins into endosomal, plasma membrane, cytoplasmic and coated vesicle fractions. Antiphosphotyrosine immunoprecipitated phosphorylated-microsomal proteins that centrifugated into endosomal and plasma membrane fractions. Addition of glycerol gradient fractions to a prephosphorylated insulin receptor preparation, however, gave a tyrosine-phosphorylated 180,000 molecular weight protein when cytoplasmic and coated vesicle fractions were added. Taken together these results suggest: (1) that, in vitro, human placenta insulin receptors can phosphorylate bovine brain and human placenta clathrin heavy chain; (2) that both assembled and unassembled clathrin can be phosphorylated; and (3) that p180, the plasma membrane-associated insulin receptor substrate, is not clathrin heavy chain.     

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 .     

Location of auxilin within a clathrin cage

The Dna J homologue, auxilin, acts as a co-chaperone for Hsc70 in the uncoating of clathrin-coated vesicles during endocytosis. Biochemical studies have aided understanding of the uncoating mechanism but until now there was no structural information on how auxilin interacts with the clathrin cage. Here we have determined the three-dimensional structure of a complex of auxilin with clathrin cages by cryo-electron microscopy and single particle analysis. We show that auxilin forms a discrete shell of density on the inside of the clathrin cage. Peptide competition assays confirm that a candidate clathrin box motif in auxilin, LLGLE, can bind to a clathrin construct containing the beta-propeller domain and also displace the well-characterised LLNLD clathrin box motif derived from the beta-adaptin hinge region. The means by which auxilin could both aid clathrin coat assembly and displace clathrin from AP2 during uncoating is discussed.     

The generation of curved clathrin coats from flat plaques

Flat clathrin lattices or 'plaques' are commonly believed to be the precursors to clathrin-coated buds and vesicles. The sequence of steps carrying the flat hexagonal lattice into a highly curved polyhedral cage with exactly 12 pentagons remains elusive, however, and the large numbers of disrupted interclathrin connections in previously proposed conversion pathways make these scenarios rather unlikely. The recent notion that clathrin can make controlled small conformational transitions opens new avenues. Simulations with a self-assembling clathrin model suggest that localized conformational changes in a plaque can create sufficiently strong stresses for a dome-like fragment to break apart. The released fragment, which is strongly curved but still hexagonal, may subsequently grow into a cage by recruiting free triskelia from the cytoplasm, thus building all 12 pentagonal faces without recourse to complex topological changes. The critical assembly concentration in a slightly acidic in vitro solution is used to estimate the binding energy of a cage at 25-40 k(B) T/clathrin.     

Gyrating clathrin: highly dynamic clathrin structures involved in rapid receptor recycling

We report here detection of novel intracellular clathrin-coated structures revealed by continuous high-speed imaging of cells expressing green fluorescent protein fusion proteins. These structures, which we operationally term 'gyrating clathrin' (G-clathrin), are characterized by localized but extremely rapid movement, leading to the hypothesis that they are coated buds on waving membrane tubules. G-clathrin structures have structurally and functionally distinct features. They lack detectable adaptor proteins AP-1 and AP-2 but contain GGA1 [Golgi-localized, gamma-ear-containing, Arf (ADP-ribosylation factor)-binding protein] as well as the cation-dependent mannose-6-phosphate receptor. While they accumulate internalized transferrin (Tf), they do not contain detectable levels of cargos targeted for the late endosome/lysosome pathway such as EGF and dextran. Pulse-chase studies indicate that Tf appears in G-clathrin structures in the cell periphery after sorting endosomes (SEs), but before filling of the perinuclear endocytic recycling compartment. Furthermore, the inhibitors LY294002 and wortmannin, which inhibit direct recycling of Tf from SEs to the plasma membrane, also block its appearance in G-clathrin. These observations suggest that peripheral G-clathrin contributes to rapid recycling, a kinetically defined compartment that has largely eluded structural identification. More generally, the rapid continuous live cell imaging reported here reveals new aspects of membrane trafficking.     

Limited proteolytic digestion of coated vesicle assembly polypeptides abolishes reassembly activity

We have previously identified a fraction containing several assembly polypeptides (AP) that promotes reassembly of clathrin into vesicle-free coat structures [Zaremba S, Keen JH: J Cell Biol 97:1339, 1983]. The AP are prepared from purified bovine brain-coated vesicles by extraction with 0.5 M TRIS-HCl followed by Sepharose CL-4B column chromatography. Centrifugation in sucrose gradients under nonassembly conditions supports earlier observations suggesting that four active polypeptides in the AP preparation, of Mr approximately 110,000, 100,000, 50,000, and 16,500 are present in a discrete complex that is incorporated as a unit into reassembled coats. The 16,500-dalton polypeptide does not coelectrophorese with authentic bovine brain calmodulin and does not exhibit calmodulin's Ca2+-induced shift in electrophoretic mobility. When the partially purified AP fraction was digested with elastase, the Mr approximately 110,000 and 100,000 polypeptides were rapidly degraded with little or no effect on the Mr approximately 50,000 and 16,500 bands. This treatment abolished the in vitro coat-forming ability of the AP fraction and the loss of activity closely parallels the loss of the Mr approximately 100,000 band. Disappearance of the Mr approximately 110,000 and 100,000 bands is accompanied by the generation of new bands at Mr approximately 76,000 and 65,000. When the elastase-treated AP is examined by sucrose gradient sedimentation in nonassembly buffers, the new bands continue to cosediment with the Mr approximately 50,000 and 16,500 polypeptides. This indicates that the elastase digestion has cleaved off a fragment of the Mr approximately 110,000 and 100,000 bands, leaving behind a truncated, inactive AP complex. A protein kinase activity has been detected in coated vesicle preparations that utilizes the 50,000-dalton AP as its preferred substrate [Keen JH, Zaremba S: J Cell Biol 97:174a, 1983]. Elastase treatment does not abolish this activity, indicating that the kinase by itself is not sufficient for maintaining reassembly activity.     

Association of adaptor protein TRIP8b with clathrin

TPR-containing Rab8b-interacting protein (TRIP8b) is a brain-specific hydrophilic cytosolic protein that contains tetratricopeptide repeats (TPRs). Previous studies revealed interaction of this protein via its TPR-containing domain with Rab8b small GTPase, hyperpolarization-activated cyclic nucleotide-regulated channel (HCN) channels and G protein-coupled receptor calcium-independent receptor of α-latrotoxin. We identified clathrin as a major component of eluates from the TRIP8b affinity matrix. In the present study, by in vitro-binding analysis we demonstrate a direct interaction between clathrin and TRIP8b. The clathrin-binding site was localized in the N-terminal (non-TPR containing) part of the TRIP8b molecule that contains two short motifs involved in the clathrin binding. In transfected HEK293 cells, co-expression of HCN1 with TRIP8b resulted in translocation of the channels from the cell surface to large intracellular puncta where both TRIP8b and clathrin were concentrated. These puncta co-localized partially with an early endosome marker and strongly overlapped with lysosome staining reagent. When HCN1 was co-expressed with a clathrin-non-binding mutant of TRIP8b, clathrin did not translocate to HCN1 and TRIP8b-containing puncta, suggesting that TRIP8b interacts with HCN and clathrin independently. We found TRIP8b present in the fraction of clathrin-coated vesicles purified from brain tissues. Stripping the clathrin coat proteins from the vesicles with Tris alkaline buffer resulted in concomitant release of TRIP8b. Our data suggest complex regulatory functions of TRIP8b in neuronal endocytosis through independent interaction with membrane proteins and components of the clathrin coat.     

Does turgor prevent endocytosis in plant cells?

Abstract. The energetics of coated vesicle-mediated endocytosis in turgid plant cells are discussed in terms of the known ATP requirement for clathrin cage dissociation and on the basis of the elementary expression for the energy w = P · v, where P corresponds to the turgor and v to the vesicle volume. The authors' calculations indicate that the possibility for formation of coated vesicles by endocytosis is limited to a rather low turgor range (P below about 0.1 MPa). This view is consistent with reports of well-developed clathrin coats on the plasma membranes in those cells having very low turgor such as root hairs and naked flagellates.     

AAK1-mediated micro2 phosphorylation is stimulated by assembled clathrin

AAK1, the adaptor-associated kinase 1, phosphorylates the μ2 subunit of AP2 and regulates the recruitment of AP2 to tyrosine-based internalization motifs found on membrane-bound receptors. AAK1 overexpression specifically inhibits the AP2-dependent internalization of transferrin receptor and LDL-receptor related protein by functionally sequestering AP2 (Conner and Schmid. J Cell Biol 2003; 162: 773). However, while AAK1 stably associates with AP2 and specifically targets the μ2 subunit in vitro , μ2 phosphorylation in vivo was not altered by overexpression of either wild-type or kinase-inactive AAK1. These results suggested that AAK1 might be tightly regulated in the cell. Here, we report that AAK1 is an atypical kinase that is rate limited by its stable association with AP2 and that clathrin stimulates μ2 phosphorylation by AAK1. Efficient stimulation of AAK1 by clathrin involves multiple interactions between several domains on AAK1 and both heavy and light chains on clathrin. Importantly, incubation of AAK1 with clathrin cages resulted in even greater stimulation when compared to that of unassembled clathrin triskelia. Collectively, our observations indicate that clathrin function is not limited to structural and/or mechanical roles in endocytic vesicle formation: the stimulatory effects of clathrin on AAK1 activity argue that it also plays a regulatory role by modulating the activity of AP2 complexes through activation of AAK1. We suggest a model in which AAK1 is specifically activated in coated pits to enhance cargo recruitment and efficient internalization.