Structural insights into the clathrin coat

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Seminars in Cell & Developmental Biology 18 (2007) 448–458

Review

Structural insights into the clathrin coat

Anna Young ∗Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, West Midlands, UK

Available online 16 August 2007

bstract

Clathrin is a cytoplasmic protein best known for its role in endocytosis and intracellular trafficking. The diverse nature of clathrin has recentlyecome apparent, with strong evidence available suggesting roles in both chromosome segregation and reassembly of the Golgi apparatus duringitosis. Clathrin functions as a heterohexamer, adopting a three-legged triskelion structure of three clathrin light chains and three heavy chains.uring endocytosis clathrin forms a supportive network about the invaginating membrane, interacting with itself and numerous adapter proteins.dvances in the field of structural biology have led us to a greater understanding of clathrin in its assembled state, the clathrin lattice. Combining

echniques such as X-ray crystallography, NMR, and cryo-electron microscopy has allowed us to piece together the intricate nature of clathrin-oated vesicles and the interactions of clathrin with its many binding partners. In this review I outline the roles of clathrin within the cell and theecent structural advances that have improved our understanding of clathrin–clathrin and clathrin–protein interactions.

2007 Elsevier Ltd. All rights reserved.

eywords: Endocytosis; Clathrin triskelion; Clathrin lattice

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4491.1. Clathrin-mediated endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449

1.1.1. Initiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4501.1.2. Coated pit formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4501.1.3. Maturation and budding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4501.1.4. Uncoating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450

1.2. Involvement of clathrin in other cellular processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4501.2.1. The role of clathrin in mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4501.2.2. The role of clathrin in Golgi reassembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451

1.3. Involvement of clathrin in disease processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4511.3.1. Ageing and neuronal impairment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4511.3.2. Mis-localisation events and neuronal disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4511.3.3. Vesicular transport defects and related disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451

2. The clathrin triskelion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4512.1. The clathrin heavy chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4522.2. The clathrin hub . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4522.3. The clathrin terminal domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4522.4. Clathrin light chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

3. The clathrin lattice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4533.1. The history of the clathrin lattice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453

3.1.1. Cryo-EM of the clathrin lattice begins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4533.1.2. Triskelion interactions within the lattice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453

∗ Tel.: +44 2476 522568; fax: +44 2476 523701.E-mail address: [email protected].

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A. Young / Seminars in Cell & Developmental Biology 18 (2007) 448–458 449

3.2. Recent structural advances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4543.2.1. Polymorphism in the clathrin lattice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4553.2.2. The location of clathrin light chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4553.2.3. The helical tripod . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4553.2.4. The structure of a clathrin-coated vesicle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455

3.3. Binding partners and the clathrin lattice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4553.3.1. The AP2-bound clathrin lattice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4553.3.2. The auxilin-bound clathrin lattice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4553.3.3. Hsc70 associated with the clathrin lattice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456

4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Clathrin-coated vesicles began to interest scientists in thearly 1960s when Roth and Porter [1] noted the formation ofbristle-coated’ pits and vesicles in the uptake of yolk proteinsn thin sections of mosquito oocytes. By 1969, morphologicaltudies of coated vesicles had begun [2] showing polygonal pro-ein baskets surrounding vesicles, whose surfaces were made upf pentagons and hexagons. Material was also observed betweenhe protein basket and the vesicle of 150–200 A in thickness,hich was subsequently shown to be the adaptor protein layer.Clathrin, the major protein component of a major class of

oated vesicles, was first identified by Pearse in 1975 [3]. Peptideapping of clathrin has shown that the sequence is conserved

hroughout tissues, and across species. Clathrin functions as arimer, and these trimers, or triskelions, are comprised of threeegs joined by a central vertex (Fig. 1). Each leg consists of oneeavy chain and one light chain [4]. The heavy chain molecu-ar weight is approximately 192 kDa. In higher eukaryotes therere two light chain isomers, denoted LCa and LCb, which havepproximate molecular weights of 36 and 33 kDa, respectively5,6]. Under slightly acidic conditions, in vitro, clathrin has thebility to spontaneously assemble into cages [6–8] that resem-le the lattices that form around coated vesicles. These purifiedlathrin cages have been used extensively in structural and bio-hemical studies of model systems to further understand the rolef the clathrin coat in clathrin-mediated endocytosis.

.1. Clathrin-mediated endocytosis

Endocytosis is a fundamental process mediating the uptake ofxtracellular macromolecules into the cytoplasm. These materi-ls enter the cell via endocytic vesicles, which are formed by thenvagination and subsequent budding of the plasma membrane.ndocytosis plays roles in the uptake of nutrients, synapticesicle recycling and the regulation of cell surface signallingeceptors. Clathrin-mediated endocytosis is a major mechanismy which various lipids, proteins and macromolecules are sortednd efficiently internalised. Clathrin and coated vesicles are also
mportant for intracellular trafficking at the trans-golgi networknd endosomes.
Many proteins involved in clathrin-mediated endocytosisave a ubiquitous form and a neuronal counterpart, specifi-

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ally involved in the recycling of synaptic vesicles. For instanceynamin 1 participates in synaptic vesicle recycling, whereasynamin 2 is required for receptor-mediated endocytosis. Aux-lin 1 is a brain-specific form whilst auxilin 2, also called cyclin-associated kinase (GAK), is the ubiquitous form. AP180 andALM (clathrin assembly lymphoid myeloid leukaemia pro-

ein) are also homologues, the latter being the ubiquitous protein.The clathrin-coated vesicle itself is a three-layered structure,

onsisting of an outer clathrin layer, an inner membrane layernd an adaptor protein layer sandwiched between the two. Adap-or protein complexes, of which there are four (termed AP1 to), interact directly with the lipid bilayer, and clathrin inter-cts with these adaptors. Coated vesicles have been reported as

ig. 1. The clathrin triskelion. The overall shape of the triskelion is depicted,ith the three-clathrin heavy chain legs radiating outwards from the centralertex. On one heavy chain each segment has been labelled. The clathrin heavyhain repeat motifs (CHCR0-7) have also been highlighted. This figure wasdapted from Fotin et al. [59].

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50 A. Young / Seminars in Cell & Dev

ein coat around the vesicle is thought to provide mechanicaltrength to the invaginating membrane and to help concentratepecific macromolecules for entry into the cell. The sequentialteps involved in endocytosis are briefly described below.

.1.1. InitiationInitiation of endocytosis is thought to begin by random

ucleation of coated pits, consisting of clathrin, AP2 and acces-ory proteins, on the inner surface of the plasma membrane11]. APs localise lattice formation to specific sites at thelasma membrane and mediate the interaction of clathrin withotential cargo [12]. Both synaptotagmin [13], a type I trans-embrane protein, and phosphotidylinositol-(4,5)-bisphosphate

PtdIns(4,5)P2) [14,15] are able to bind AP2 independently.ne scenario, suggested by Takei and Haucke [16], is that

ynaptotagmin and PtdIns(4,5)P2 together tightly bind AP2 athe membrane, creating a nucleation point for clathrin poly-

erisation and coated pit formation. The proteins epsin andP180/CALM also bind PtdIns(4,5)P2, AP2 and clathrin [17],

nd may draw further AP2 and clathrin to the membrane andynchronise their assembly there [16,18]. Once a nascent pit hasormed it may become abortive or capture potential cargo andontinue its internalisation.

.1.2. Coated pit formationIf a nascent-coated pit is to develop, further cargo must

e acquired. AP2 plays an important role in cargo selection,inding directly to cargo moieties or binding cargo via clathrin-ssociated sorting proteins, such as �-arrestin [19]. The bindingf AP2 to cargo-containing plasma membranes is a two-steprocess. Initially the � subunit weakly binds to PtdIns(4,5)P2.nhancement of the binding affinity of AP2 for protein-sortingotifs is achieved by phosphorylation of residue Thr156 of

he �2 subunit [20] of AP2 by adaptor-associated kinase 121,22]. This allows the �2 subunit to bind both the cargootif and PtdIns(4,5)P2, creating a solid foundation for the for-ation of a clathrin-coated vesicle. The � and � appendages

f AP2 both recruit and bind accessory proteins to the form-ng pit. The � appendage can also bind clathrin. Binding ofhe � appendage to clathrin displaces bound accessory pro-eins, allowing the accessory proteins to interact with AP2,here clathrin has yet to polymerise, ensuring the maturationf the coated pit [23]. CALM plays an important role in regu-ating coated pit formation. RNAi depletion of CALM in HeLaells caused the formation of large, irregular coated pits andlongated tubules attached to the plasma membrane [24]. Inonjunction with clathrin polymerisation, a number of other pro-eins are required to control the invagination of the endocytic

embrane during coated pit formation. Membrane curvature ishought to be driven by several proteins, most notably thoseontaining BAR (Bin/amphiphysin/Rvs) domains [25] such asmphiphysin [26] and endophilin [27]. The crystal structure ofhe human Bin1/amphiphysin BAR domain revealed a crescent-
haped dimer with a highly positively charged concave surface,uggesting that curvature is generated by electrostatic inter-ctions between the membrane and this surface [28]. Epsin,PtdIns(4,5)P2 binding protein involved in clathrin and AP2
1

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ental Biology 18 (2007) 448–458

ecruitment, is also able to induce membrane curvature [29].ssembled clathrin itself has an innate curvature, brought abouty the pentagonal faces of the polyhedral lattice, which canelp to stabilise the bending membrane as a coated pit initiallyorms.

.1.3. Maturation and buddingFurther deformation of the membrane and polymerisation

f clathrin leads to a coated vesicle attached to the plasmaembrane by a narrow neck, which pinches off to form a free

oated vesicle. An essential player in this process is dynamin,hose GTPase activity is intrinsically linked to its role in endo-

ytosis. Amphiphysin, already associated with forming coatedesicles, has binding sites for both clathrin and dynamin. Its thought to recruit dynamin to forming coated vesicles ando facilitate dynamin oligomerisation [30]. Dynamin has threeunctional states, dimers, tetramers and rings or spirals, eachf which has a differing effect on GTP binding and hydrolysis.pon recruitment, dynamin binds to PtdIns(4,5)P2 via its pleck-

trin homology domain [15,31]. Dynamin oligomerisation, fromimers to tetramers, stimulates GTP binding, and ultimatelyeads to vesicle constriction [32,33]. Following constriction,hanges in the conformation of dynamin, from tetramers to ringsr spirals, upon GTP hydrolysis may lead to vesicle scission [34].here are several proposed methods for how this occurs whichre reviewed in Sever et al. [34].

.1.4. UncoatingOnce the vesicle is formed, the clathrin coat must be effi-

iently removed. This uncoating allows the vesicle to fuse withts target membrane and deliver its cargo. Proteins that play a

ajor role in dismantling the clathrin lattice include heat shockognate 70 kDa protein (Hsc70), a constitutively expressed heathock protein, and auxilin. Auxilin, an Hsp40 homologue, bindso clathrin and recruits Hsc70 to the cage via its J-domain. Hsc70an then disassemble the clathrin lattice in an ATP-dependentanner [35,36]. As well as removing the outer clathrin lat-

ice, the complex adaptor protein layer must also be removed.hanges in the phosphorylation state of AP complexes haveeen implicated as a further controlling factor in uncoating.hosh and Kornfeld [37] showed that dephosphorylation of

he �1 subunit of AP1 by protein phosphatase 2A resulted inelease of adaptors from coated vesicles. As previously men-ioned, phosphorylation of the �2 subunit of AP2 enhances itsinding affinity for protein sorting motifs [21,22], implying thatephosphorylation of this subunit is an essential step in uncoat-ng. Synaptojanin and endophilin can act to dephosphorylatehosphoinositide lipids, thus decreasing the affinity of adaptorso the vesicle, allowing the adaptors to be released from the

embrane [38].

.2. Involvement of clathrin in other cellular processes

.2.1. The role of clathrin in mitosisIn addition to its involvement in endocytosis, clathrin has

lso been implicated as having a role in mitosis [39,40]. In000 Okamoto et al. [39] showed that clathrin was localising

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A. Young / Seminars in Cell & Dev

ith the mitotic spindle of cultured cells during mitosis. Thelathrin heavy chain has since been shown to bind to the mitoticpindle, and a construct lacking residues 1–330 was unable toind to the spindle, confirming that the globular N-terminalomain is required [40]. Once bound, clathrin is believed toelp regulate the segregation of chromosomes during mitosisnd to stabilise the fibres connecting the chromosomes to theitotic spindle (kinetochore). Royle and Lagnado [41] have

hown that, for clathrin to stabilise the kinetochore, it muste present as a trimer, and, in this way, can act as a bridgenterconnecting fibres in the mitotic spindle. This highlightshe importance of the clathrin triskelion structure in enablinglathrin to function in the cell. The � subunits of AP complexesave been shown to bind to BubR1 and Bub1, both mitoticheckpoint kinases, in vitro [42]. BubR1 and Bub1 are involvedn controlling chromosome segregation during mitosis. Finally,mmunolocalisation studies showed colocalisation of BubR1nd the AP2 � subunit in the cytosol.

.2.2. The role of clathrin in Golgi reassemblyDuring mitosis, the Golgi apparatus must be reversibly dis-

ssembled into mitotic Golgi clusters for division into daughterells. The organelle then needs to be reassembled to form aunctional unit. Radulescu et al. [43] reversibly induced Golgiisassembly in cultured cells by addition of primary alcohol (1-utanol). Upon removal of alcohol, clathrin and clathrin-coatedesicles were recruited to the fragmented Golgi cisternae and theypical Golgi morphology restored. In addition, clathrin deple-ion in cells prevented the efficient reassembly of the Golgipparatus, strongly suggesting a role for clathrin in its reassem-ly post mitosis.

.3. Involvement of clathrin in disease processes

With roles to play in nutrient uptake, intracellular traffickingnd mitosis it is easy to see how an imbalance or obstruc-ion in clathrin function can cause problems within not onlyhe cell, but the whole organism. Even during normal cellularunction viruses and bacteria have adapted to hijack the pro-ess of clathrin-mediated endocytosis, infecting cells. Foot andouth disease virus [44] and hepatitis C virus [45] both gain

ntry to the cell via the clathrin pathway. Toxins, such as shigaoxin [46], secreted by Shigella dysentriae, and cholera toxin47], secreted by Vibrio cholerae, are internalised by clathrin-ediated endocytosis, where they can disrupt cellular function

nd cause disease.

.3.1. Ageing and neuronal impairmentAs neurons age their rate of endocytosis decreases. Clathrin-

oated vesicles have been found to be more persistent in matureeurons. This affects the cellular response to extracellular sig-als, by altering the regulation of proteins and receptors at thelasma membrane. This impaired regulation may contribute to
he ageing processes of cells and neurons [48]. A substantial per-entage of clathrin-mediated endocytosis in neurons is involvedn the uptake of transferrin. With iron deposition occurring inhe brains of sufferers of Parkinson’s disease and Alzheimer’s
1itO

ental Biology 18 (2007) 448–458 451

isease, a link has been proposed between poorly regulatedlathrin-mediated endocytosis in aging cells and diseases of theervous system [48,49].

.3.2. Mis-localisation events and neuronal diseaseThe mis-localisation of clathrin has been implicated in some

eurological diseases. Nakamura et al. [50] showed that clathrinight chains localise to Alzheimer’s plaques and neurofibril-ary tangles in Alzheimer brains. Analysis, by Western blotting,howed no difference in the total amount of clathrin presentn control or Alzheimer brains, implying that the clathrin lighthains are abnormally distributed. The disruption of clathrin-ediated endocytosis can lead to failures in neuron signalling,hich may contribute to the symptoms of Alzheimer’s dis-

ase [51]. Clathrin may also contribute to frontotemporal lobaregeneration (Pick’s disease), a neuronal disorder caused byorticobasal degeneration and protein deposition in the formf Pick’s bodies. Clathrin light chains have been discovered inick’s bodies, suggesting that mis-localisation events contribute

o disease [52].

.3.3. Vesicular transport defects and related disordersClathrin-mediated vesicular transport is also implicated in

untington’s disease, an inherited neurodegenerative disorderharacterised by progressive cognitive impairment, lack of coor-ination and premature death, caused by a mutated form ofhe protein, Huntingtin. Wild-type Huntingtin has been impli-ated in vesicular trafficking and, when GFP-tagged, the proteinas found to localise to clathrin-coated and trans-Golgi vesicles

53]. Mutant Huntingtin disrupts a number of cellular processes,ncluding clathrin-mediated endocytosis [54]. Oculocerebrore-al syndrome (Lowe syndrome) is an X-linked, recessive geneticisorder that may be linked to intracellular vesicle trafficking.he syndrome is characterised by glaucoma, cataracts, mentalisability, rickets and amino acids in the urine, and is thoughto be caused by mutant oculocerebrorenal syndrome of Lowerotein 1 (OCRL1) [55]. OCRL1 is a type II 5-phosphatase,f the same family as synaptojanin. Its normal function maye involved in regulating vesicular transport between the trans-olgi network and endosomes [56]. This protein can bind to the

lathrin heavy chain and has been shown to promote clathrinssembly into lattices in vitro. This would suggest that mutantCRL1 may cause defects in this trafficking system, contribut-

ng to the symptoms of the syndrome.

. The clathrin triskelion

In order to begin to know how clathrin is able to participate inuch diverse cellular functions one must examine its structure.nderstanding the structure of the basic lattice unit, the triske-

ion, can provide insights as to how the lattice is formed, andow and where the interactions with clathrin’s many bindingartners occur. Purified clathrin trimers were first visualised in
981 [4], using rotary shadowing and negative stain EM. Thesemages showed the presence of a three-legged triskelion struc-ure, with the legs exhibiting a preferential direction of curvature.n EM grids clathrin triskelions appear flattened, but, in reality,

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he heavy chain legs are bent and puckered in solution, givinghem their flexibility to form spherical cages [57], and a rightand twist of the heavy chain legs can be seen as they radiateutwards from the vertex [58,59].

.1. The clathrin heavy chain

Heavy chains can be partitioned into a proximal domain, aistal domain and a terminal domain [60]. The proximal domains located at the C-terminus of the heavy chain, closest to theertex, central to the triskelion. The distal domain follows onrom this forming the ‘knee’, with the chain ending in a globu-ar N-terminal domain attached to an ankle by a flexible linker.be et al. [61] solved the structure of part of the clathrin heavy

hain proximal domain (residues 1210–1516) to 2.6 A reso-ution, using X-ray crystallography. They identified a clathrineavy chain repeat (CHCR) motif, of which seven were foundn total per leg, termed CHCR1-7 (Fig. 1). Each CHCR motif is45 residues in length and forms a right-handed super-helix coilf �-helices. This motif forms a rigid structure of antiparallel-helices, oriented perpendicular to the heavy chain leg, with aontinuous hydrophobic core. Looking closer at the linker con-ecting the terminal domain to the rest of the heavy chain, Fotint al. [59] noticed that residues 395–542 within the linker areonsistent with the CHCR motif. This region is now termedHCR0. CHCR7 and 6 make up the proximal domain. CHCR5

orms the knee and, with CHCR4 and 3 the distal domain. FinallyHCR2, 1 and the C-terminal part of CHCR0 constitute thenkle.

.2. The clathrin hub

The clathrin vertex and the proximal domain together formhe clathrin hub. The hub contains regions which are impor-ant for the trimerisation of heavy chains, the binding ofight chains and lattice assembly [62,63]. The trimerisationomain of clathrin heavy chains has been mapped to betweenesidues 1488–1587, possibly mediated by an �-helical regionetween residues 1522 and 1572. The motif CHRC7 (residues423–1566) enters the vertex, overlapping with the trimerisationomain. This may allow interactions between opposing stackedairpins in the CHCR motifs, helping to mediate trimerisation.ne residue shown to be critical for hub structure and activ-

ty is cysteine 1573 [64]. Mutation of this residue resulted innstability of the trimerisation hub and a loss of ability to selfssemble. The hub is an important feature of the clathrin triske-ion, providing stability and allowing assembly into higher ordertructures.

.3. The clathrin terminal domain

In order for clathrin to function normally in the cell it muste able to interact with multiple adapter proteins. Such interac-
ions are mediated by the clathrin terminal domain. The structuredopted by the clathrin terminal domain is that of a seven-bladed-propeller [65], with the �-sheets organised cyclically about aentral axis. This is attached to the main body of the heavy chain
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ental Biology 18 (2007) 448–458

y an �-zigzag linker. Typical �-propellers structures, such ashe influenza virus glycoprotein neuraminidase [66] and regula-or of chromosome condensation (RCC1) [67], have their proteininding sites located at the centre of the propeller, formed byhe loop regions connecting the motifs. The clathrin �-propelleras three potential protein binding sites identified to date. Oneinding site on the terminal domain is located centrally to theropeller, as for archetypal �-propellers. Peptides with a ‘W box’otif (PWXXW, where X is any amino acid), such as those

ound in amphiphysin 1 and 2 [68], have been shown to bindithin the deep pocket in the centre, formed by the upper regionf blades 1, 4, 6 and 7 [69]. The clathrin terminal domain alsoas a binding site between blades 1 and 2 of the propeller [70].he groove between the first and second blades is wide due toisplacement of the second blade from the centre of the pro-eller. Peptides containing a ‘clathrin-box’ motif (L�X�[D/E],here � is a bulky hydrophobic amino acid) bind to the terminalomain, lining the groove between the blades. A proposed thirdrotein binding site may lie in the groove between blades 4 and, as this is the next largest groove in the propeller, with blade 5ndergoing a similar displacement to blade 2 [70].

.4. Clathrin light chains

Clathrin light chains consist of a conserved C-terminalomain, a central coiled region and an acidic N-terminal domain.he two light chain isomers, LCa and LCb have very similar

unctions, binding and regulating clathrin heavy chain trimeri-ation and assembly, but share only 60% sequence identity.etween species, light chains are highly conserved, retaining5–98% sequence identity. Conserved regions include a 22mino acid sequence near the N-terminus, the clathrin heavyhain binding site, a number of cysteine residues close to the-terminus, and a serine-rich phosphorylation site at the N-

erminus, specific only for LCb [71].In 1983, Ungewickell demonstrated that clathrin light chains

ound to the proximal domains of heavy chains [72]. Residues267–1522 provide the core residues required for light chaininding [73]. These residues extend for approximately 100 And interact with residues 93–160 of LCa or 90–157 of LCb. This-helical central region of the clathrin light chains is essential

or heavy chain binding. Binding of light chains to heavy chainsas even been shown to increase the presence of secondary struc-ure in the light chains [73]. A sequence close to the vertex ofhe heavy chain, between residues 1438 and 1481, has beenroteolytically mapped to be required for mediating light chaininding [74]. Conversely 70 amino acids of the highly conserved-terminal domain of the clathrin light chain are critical forlathrin function and binding to the heavy chain [75]. It has beenroposed that heavy and light chain interactions occur betweenhe helical faces of the heavy chain and the hydrophobic facesf the heavy chain binding site helix of light chains, orientingight chains to the clathrin lattice surface [71,73]. We now know
hat the light chains form helical rods, contacting the proximalomain of the clathrin heavy chain, and reside on the outside ofhe lattice [59]. As well as interacting with the proximal domain,here is evidence to suggest that clathrin light chains also interact

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ith the trimerisation domain (residues 1550–1615). In yeast,utation of heavy chain residue 1590 (1584 in the mammalian

lathrin heavy chain) from glutamate to lysine abolishes clathrinight chain binding and prevents heavy chain trimerisation [76].he cysteine-rich C-terminal domain of clathrin light chains

nteracts strongly with the C-terminal domain of the heavy chain.hese cysteines can readily form disulphide bonds, and we know

hat light chain binding to cysteine 1528 of the heavy chain ismportant for preventing aggregation. It has been suggested thathe light chains may ‘tether’ the legs of the clathrin heavy chain athe trimerisation hub, increasing the stability of the trimerisationomain [64], and allowing stable assemblies to form.

. The clathrin lattice

Clathrin’s main function is to form a supportive lattice aroundnvaginating and budding vesicles, forming the outside of therotein coat. It is important to visualise the lattice as a wholeo gain any understanding of lattice formation, adapter pro-ein interactions and disassembly. Developments in the field oftructural biology have brought us closer to understanding thelathrin lattice, giving us high resolution details, and the bind-ng locations of proteins such as AP2, auxilin and clathrin lighthains.

.1. The history of the clathrin lattice

Both in vivo and in vitro clathrin triskelions are able to poly-erise to form polyhedral cages or lattices. Due to the relatively

arge size of clathrin lattices they are not amenable to struc-
ural studies by X-ray crystallography or NMR. An alternative
ethod that can be used is electron microscopy. Clathrin latticesnd coated vesicles have been extensively studied by negativetain electron microscopy, prior to the introduction of cryo-EM.

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ig. 2. (A) Surface representation of a hexagonal barrel clathrin lattice. The lattice wasing the UCSF Chimera package from the Resource for Biocomputing, VisualizatioIH P41 RR-01081) [91]. (B) A cryo-EM image showing a field of clathrin lattices

aken at 40,000× magnification using a 200 kV JEOL JEM 2011 transmission electroevice camera. The scale bar measures 50 nm.

ental Biology 18 (2007) 448–458 453

he hexagonal and pentagonal nature of clathrin-coated vesi-les was initially described by Kanaseki and Kadota in 19692]. Freeze-etch microscopy of the inner surface of the fibrob-asts plasma membrane also showed the presence of heptagonsithin the honeycomb-like clathrin lattice [77]. Crowther et al.

78] were first to note the ‘hexagonal barrel’ clathrin latticeype, consisting of 36 triskelions forming a closed network of

hexagons and 12 pentagons. This hexagonal barrel structureas a hexagon at each pole, each surrounded by six pentagons,nd finally a ring of six hexagons at the equator completes theattice (Fig. 2).

.1.1. Cryo-EM of the clathrin lattice beginsWith the development of cryo-EM, embedding the specimen

n a thin layer of vitreous ice and maintaining a hydrated state,ore biologically relevant and higher resolution structures could

e achieved. The earliest structure of a clathrin lattice in ice wasublished by Vigers et al. in 1986 [7]. Tilt series of individuallathrin lattices were collected and analysed to produce 3D struc-ures revealing the first details of the inside of the lattice. Theverall height of the hexagonal barrel measured approximately10 A, and the length of individual edges approximately 184 A.rotein can be seen projecting towards the centre of the lattices ‘fingers of density’ creating an inner protein shell. This wasorrectly attributed to the clathrin terminal domains, confirminghe model proposed by Kirchhausen and Harrison in 1984 [60].

.1.2. Triskelion interactions within the latticeThe next breakthrough in clathrin lattice structure came in

998. Smith et al. [58] presented a cryo-EM map of the clathrin
exagonal barrel, assembled with the adaptor protein complexP2, to 21 A resolution. This structure gave us our first real
nsights into how individual triskelions are packed into the lat-ice arrangement of a clathrin barrel. Each triskelion vertex is

s created using a modelled pdb, 1XI4, created by Fotin et al. [59] and displayedn, and Informatics at the University of California, San Francisco (supported by. The black arrow indicates a hexagonal barrel clathrin lattice. The image wasn microscope, fitted with a Gatan 2048 × 2048 pixel ultrascan charge-coupled

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entred at a lattice vertex, and the heavy chain legs, with theirccompanying light chains, spiral outwards from the verticesorming the lattice bars. All heavy chains form two adjacentdges of the polyhedral lattice. The legs appear to interact via theroximal and distal domains, each edge consisting of two anti-arallel proximal domains beneath which lie two anti-parallelistal domains. Distal–distal and proximal–distal interactionsre important for lattice assembly, with the latter forming thetrongest contacts in the lattice [79]. Recombinant hubs, con-isting of the trimerisation and proximal domains, are able torimerise and self-assemble, but cannot form closed lattices. Dis-al domains, correctly oriented through adapter proteins bindingo the terminal domain, are required to form a closed clathrin
attice [80]. Greene et al. [80] proposed that an hypotheticalcounter-hub’, composed of a central adapter protein bound tohree terminal domains, with three distal domains radiating out-ards, allows correct orientation of the triskelion for closed
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ig. 3. (A) Cryo-EM reconstruction of a clathrin hexagonal barrel to 8.2 A (blue). Teavy chain proximal domains, stretching along the bars of the cage radiating outwroximal domain is seen in green. Beneath this is the helical tripod structure (blue) tryo-EM reconstruction of a clathrin hexagonal barrel (blue) associated with auxiliomain and an ankle crossing segment. (D) Cryo-EM reconstruction of a clathrin hexensity (purple) located within the faces of the clathrin lattice (orange). The white arattice. (A) and (B) were taken from Fotin et al. [59], (C) is taken from Fotin et al. [8

ental Biology 18 (2007) 448–458

attice formation. Within a lattice, the terminal domains projectnwards towards the centre, beneath a vertex located two verticesway from that legs triskelion hub. Here, the terminal domainsppeared to adopt ‘hook-like’ protrusions that form the pointsf contact for the inner adaptor protein layer [58]. Other notableeatures are the nodes of density located directly beneath theertices, which are expected to include the clathrin heavy chain-termini.

.2. Recent structural advances

By the end of 2004 the structure of a clathrin lattice was avail-ble at sub-nanometre resolution. Fotin et al. [59] assembled
lathrin in the presence of sub-stoichiometric quantities of AP2nd, using cryo-EM and single particle analysis, achieved mod-ls of a clathrin lattice, with and without clathrin light chains, to.2 and 7.9 A resolution, respectively (Fig. 3A). This structure
he clathrin light chains (yellow) can be seen as rods following the path of theards from the vertices. (B) The helical tripod as described by Fotin et al. Thehat contacts the ankles (red) of adjacent triskelions, stabilising the lattice. (C)n (red) to 12 A resolution. Auxilin sits within the cage, contacting a terminalagonal barrel associated with C58J and Hsc70. The Hsc70 is shown as rings ofrow indicates a possible contact point between the Hsc70 ring and the clathrin2], and (D) is taken from Heymann et al. [90].

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eveals such novel features as a helical tripod structure beneathhe vertex and the location of the clathrin light chains, togetherith information about lattice variation.

.2.1. Polymorphism in the clathrin latticePurified coated vesicle preparations, and solutions of recon-

tituted clathrin lattices, show a wide array of shapes and sizes.he variation of both clathrin lattices and coated vesicles has, in

he past, been solely attributed to the flexibility of the clathrinriskelion knee [81]. The flexible clathrin knee is not hamperedy any protein–protein contacts, allowing the lattice to adapto varying vesicle sizes. The high-resolution cryo-EM map byotin et al. [59] shows that triskelions adopt a slightly more com-act structure when within a lattice. By studying the organisationf both hexagonal barrels and tetrahedral coats (a structure 80 Amaller than a hexagonal barrel) Fotin et al. were able to con-lude that the ability of clathrin to vary its curvature lies also inltering the angle at which the proximal domains cross.

.2.2. The location of clathrin light chainsIn 2002, Chen et al. used mutagenesis studies to help model

he interactions between heavy chains and light chains. Theyositioned the light chains on the surface of the clathrin lat-ice, with heavy chain residues K1415 and K1326 interactingith LCb residues W127 and W105 (or LCa residues W130

nd W108), respectively [73]. The clathrin lattice structure pre-ented by Fotin et al. shows the clathrin light chains as rod-liketructures, located on the outside of the lattice, running parallelo the proximal domain of the heavy chain (Fig. 3A), supportinghe model proposed by Chen et al. The length of this light chainod is approximately 100 A, corresponding to an extended 71-esidue �-helix, with the C-terminus of the light chain closest tohe lattice vertex. This 71-residue �-helix is likely to correspondo the critical C-terminal fragment required for clathrin bindingnd function discovered by Wang et al. [75].

.2.3. The helical tripodFotin et al. also showed that each vertex of the clathrin lat-

ice exhibits a region of three-fold symmetry that arises fromhree helical rods (residues 1598–1630), 50 A in length. This isermed the ‘helical tripod’ (Fig. 3B). The helices of the tripodxtend beneath the clathrin vertex towards the centre of the lat-ice and contact ankle segments in this region. Three ankles fromeighbouring triskelions cross beneath the vertex forming a ‘tri-ngular barrel’ that, when bound by the helical tripod, stabiliseshe cage structure of the lattice. The remaining residues of theeavy chain C-terminus (residues 1631–1675) appear to mergeith the density of the ankles, beneath the vertex, forming an

ankle brace’ [59,82]. The discovery of these interactions high-ights the importance of this site for lattice stability. Studyinghe clathrin lattice in such detail has given much insight intoriskelion interactions, but it is important to remember that thelathrin lattice forms part of an intricate protein network.

.2.4. The structure of a clathrin-coated vesicleAt the start of 2007 Cheng et al. [83] published a cryo-

M structure of a clathrin-coated vesicle. This lower resolution

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ental Biology 18 (2007) 448–458 455

tructure was attained by cryo-electron tomography, producingeconstructions of individual coated vesicles. The EM densityhowed an inner and an outer layer of protein surrounding thencapsulated vesicle. The outer layer corresponds to the polyg-nal lattice, whilst the inner layer represents the clathrin linkernd N-terminal domains. In the outer lattice of the coated vesicleeptagons can be seen, acting to increase the radius of curva-ure of the clathrin lattice. The inner N-terminal domain layerppears to make a number of contacts between the clathrin lat-ice and the vesicle, most likely via adapter proteins. The vesicletself is located off-centre within the lattice, and it has been sug-ested that, where the vesicle and lattice are in close proximitydapters may have preferentially bound, marking this point as thenitiation site for coat assembly. This poses the interesting ques-ion of whether the far side of the coat, lacking vesicle contact,

ay be a sensible starting point for clathrin lattice disassembly.hese recent advances may provide us with a better understand-

ng of assembly and disassembly of the clathrin lattice, and thessociation of clathrin with its many binding partners.

.3. Binding partners and the clathrin lattice

.3.1. The AP2-bound clathrin latticeIn 1998 Smith et al. [58] produced a 21 A resolution map of

lathrin associated with the clathrin adapter AP2. As well as giv-ng us insights into the triskelion–triskelion interactions withinlattice this map provided us with the first structural informa-

ion on the interactions of binding partners within a lattice. AP2orms a shell of continuous density within the centre of the lat-ice. This continuity suggests that AP2 is tightly packed withinhe lattice. At this resolution we cannot see individual AP2 com-lexes. The AP2 density is seen to make contact with the networkf clathrin terminal domains inside the lattice, suggesting thatP2 tethers clathrin to the central vesicle membrane via its ter-inal domains. ter Haar et al. [70] have since demonstrated that

he hinge of the � subunit of AP1, AP2 and AP3, each containingclathrin box motif, binds to the groove between blades 1 and 2f the clathrin terminal domain �-propeller. AP2 also enhanceshe formation of the clathrin hexagonal barrel lattice, which haseen used to advantage in later clathrin reconstructions.

.3.2. The auxilin-bound clathrin latticeUsing cryo-electron microscopy and single particle analy-

is Smith et al. [84] were able to reconstruct a cryo-EM map ofull-length auxilin within a clathrin lattice to approximately 20 Aesolution. Auxilin formed a shell of density within the lattice, atoints making contact with the terminal domains. Auxilin is ableo interact with the clathrin terminal domains via the terminalomain �-propeller and a proposed clathrin box motif (LLGLE)ound in auxilin at residues 496–500. The inner shell of auxilins similar to that of AP2 shown by Smith et al. in 1998 [58],lthough the auxilin shell is approximately 25% thinner than theP2 shell. The similarities seen between the two may explain

uxilin’s ability to assist cage assembly in vitro. By the end of004 Fotin et al. published a cryo-EM density map of a clathrinexagonal barrel associated with a functional fragment of aux-lin (547–910) to 12 A resolution [82] (Fig. 3C). This C-terminal

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uxilin fragment contains both the J-domain and the clathrin-inding domain. With the auxilin binding sites saturated, auxilinas found to bind with a stoichiometry of three per triskelion,

lthough it has been shown that only one per triskelion is requiredo activate three Hsc70s [35]. At this resolution the fragmentppeared to contact two ankle segments, at their point of cross-ng, and a further terminal domain. As previously mentioned, theelical tripod contacts the crossover points of the three ankleseneath the cage vertex, appearing to fix the triskelions in placeithin the lattice [59]. It is also at this site that density, presumed

o be the heavy chain C-terminus (residues 1631–1675), mergesith that at the regions where the ankles cross beneath the ver-

ex [59,82]. It is to this region, critical for lattice stability, thatuxilin binds. The positioning of the auxilin J-domain was suchhat the motifs required for recruitment of Hsc70 are exposed,acing towards the outside of the lattice. Binding of auxilin tohe clathrin barrel has caused the terminal domains to move out-ards, due to repositioning of the ankle. The ankle now appears

lmost parallel to the proximal and distal domains. This rear-angement of the terminal domains has caused a global changeo the entire lattice, increasing its diameter. Fotin et al. have pro-osed that Hsc70 is recruited, by auxilin in its current position,o this area of crucial lattice interactions. Once in the vicin-ty, Hsc70 could bind to the proposed Hsc70 binding sequence,LMLT, found in the clathrin C-terminus, protruding beneath

he lattice vertex [58,82]. This sequence shows similarity to anown Hsc70 binding peptide, FYQLALT [85]. The peptideYQLALT is optimised for binding Hsc70, and hydrophobicYQLALT-like sequences are involved in facilitating proteinolding [85]. This conforms well with the proposal by Fotint al. that the sequence QLMLT at the apparently unstructuredlathrin heavy chain C-terminus may be able to bind Hsc70 andosition it at this important interaction site.

.3.3. Hsc70 associated with the clathrin latticeHsc70 is a constitutively expressed chaperone protein

nvolved in many cellular processes. Among these processesre protein folding, degradation and translocation. Anothernteresting function of Hsc70 is its ability to disassemble theomparatively large clathrin lattice. Mixing Hsc70 and ATPith coated vesicles in vitro causes disassembly of the clathrin

attice [86]. The reaction is stoichiometric, requiring 3 mol ofsc70, hydrolysing 3 mol of ATP [87], to dissociate 1 mol of

lathrin triskelia [86,88]. In 1989 Heuser and Steer visualisedrimeric binding of Hsc70 to the vertices of free clathrin triskeliay freeze-etch electron microscopy [89], but until very recentlyt was uncertain if this reflected Hsc70’s true binding positionithin a clathrin lattice. In contrast to suggestions made by Fotin

t al. [82], Heymann et al. [90] have visualised Hsc70 formingings of density in the polygonal faces of the clathrin latticeFig. 3D). This 26 A resolution structure showed a hexagonalarrel clathrin lattice associated with an AP180-auxilin chimera,58J, and Hsc70, in the presence of an ATP regenerating system.
he system was kept at pH 6.0 to prevent lattice disassembly.58J is a construct of the clathrin binding domain of AP180 and
he J domain of auxilin. The density attributed to Hsc70 formsdiffuse ring, and individual chaperones are not observed. The

ental Biology 18 (2007) 448–458

sc70 seen in this reconstruction may contact the bars of theattice, close to the central vertex. Upon Hsc70 binding a globalistortion is seen in the lattice, with the lattice height shorteningy approximately 20 A and the diameter increasing by approx-mately 10 A. Heymann et al. have suggested that, from thisocation, Hsc70 may interfere with the interactions between theroximal and distal domains that constitute the bars of the cage.his could destabilise the lattice and initiate uncoating and the

emoval of individual triskelia.

. Conclusions

By combining different structural techniques we have learntow clathrin interacts with itself to form an intricate lattice, andow have clathrin lattice structures available at sub-nanometreesolution. These structures reveal important features, such ashe helical tripod and the location and appearance of clathrinight chains. We are now also beginning to understand clathrin’snteractions with just a handful of its numerous binding partners.P2 and auxilin both contact the clathrin terminal domains, anday act as mediators between the clathrin lattice and the internal

arrier vesicle during endocytosis. Clathrin has a diverse role:unctioning in endocytosis, intracellular trafficking and in newlyiscovered roles in chromosome segregation and Golgi reassem-ly during mitosis. With multiple roles throughout the cell innumber of fundamental cellular processes, and the potential

nvolvement of clathrin with a number of well-characterised dis-ases, the structure and function of clathrin-coated vesicles andheir coat proteins will remain an important area of research inuture years.

cknowledgements

I thank C.J. Smith for a critical reading of the manuscript,nd the BBSRC for financial support.

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Structural insights into the clathrin coat