Receptor-mediated endocytosis by clathrin-coated vesicles

By Dr Tony Jackson *

A review of how research into the components of the clathrin coat has provided insights into the operation of these molecular machines

Abstract

The clathrin-mediated endocytic pathway plays an important role in the selective uptake of proteins at the plasma membrane of eukaryotic cells. Recently, many of the component molecules that contribute to forming the clathrin-coat have been identified and their str

Abstract

The clathrin-mediated endocytic pathway plays an important role in the selective uptake of proteins at the plasma membrane of eukaryotic cells. Recently, many of the component molecules that contribute to forming the clathrin-coat have been identified and their structures determined. This has provided an unprecedented insight into the operation of these molecular machines.

Contents:

Introduction   

Adaptors and accessory proteins 

Clathrin assembly 

Membrane fission and uncoating 

Future prospects

References


Introduction

A typical eukaryotic cell contains a well developed secretory and endocytic pathway composed of discrete intracellular compartments. Most membrane proteins that are transported through the secretory pathway begin their life in the endoplasmic reticulum (ER) where they undergo folding and assembly. Those proteins destined for other compartments (eg. the plasma membrane or lysosomes) move sequentially out of the ER, through the stacks of the Golgi and trans-Golgi network (TGN). Understanding how the right proteins are targeted to the right intracellular location is a major research area in contemporary cell biology [1]. A large part of the answer is that membrane proteins are selectively transported between intracellular compartments by incorporation into distinct classes of coated transport vesicles (Figure 1). Clathrin-coated vesicles have been implicated in several distinct intracellular transport steps. In particular, the internalisation of plasma membrane proteins by receptor-mediated endocytosis (RME) and the transport of lysosomal enzymes from the TGN [2-4]. In this brief overview, I will focus on the role of clathrin in RME since this is the best studied of all the clathrin-mediated mediated transport events.   

Fig1-clathrin-golgi

Figure 1. The distribution of different classes of coated vesicle in the eukaryotic cell. The diagram is highly simplified. For example, only one endosome compartment is shown and the multiple pathways to and from each compartment are omitted for clarity

A wide variety of membrane proteins enter cells by RME. For example, receptors carrying ligands required for growth (eg transferrin, low-density lipoprotein etc) are constantly and efficiently internalised by this process. In addition, RME plays an important role in hormone receptor signalling either by removing hormone-receptor complexes from the surface and attenuating the signal, or in other cases by delivering bound receptors to intracellular compartments for optimum signalling [5]. In neurones, RME plays a special role in rapidly recapturing synaptic vesicle proteins inserted into the synaptic membrane following regulated exocytosis [6]. Possibly for this reason, clathrin is up-regulated in the chick brain following visual imprinting, a model for recognition memory.[7].

The process begins at the under-surface of the plasma membrane with the sequential assembly of coat components to form a clathrin-coated pit. Internalised receptors interact with a class of molecules called adaptors and thus become clustered into the growing coated pit. The adaptors link the membrane proteins with the clathrin that forms the outer layer of the coat. Together with accessory and regulatory molecules, cargo proteins, adaptors and clathrin co-assemble, and the growing coated pit invaginates. In the final stage, the membrane neck is severed to form a closed coated vesicle (Figures 2 and 3).

Adaptors and accessory proteins

Adaptors represent a diverse group of proteins recognising different classes of cargo receptor. The best characterised are a family of closely related proteins called the adaptor proteins (APs) comprising AP1, AP2, AP3 and AP4. Each of these four classes are localised to different intracellular compartments (Figure 1) and vary in their receptor specificity [8-9]. All APs are constructed on a fundamentally similar plan: two 110-130 kDa large subunits (a beta-class adaptin, and one of either an alpha, gamma, delta or epsilon adaptin), a 50 kDa medium chain mu adaptin and a 15-20 kDa small chain sigma adaptin. The two large subunits can be subdivided into a C-terminal appendage domain connected to the N-terminal core domain via an unstructured flexible linker sequence (Figure 2a).

Clathrin-fig2a-gif

Clathrin-fig2b-gif

Figures 2a & b. Structures of AP2 adaptor and clathrin triskelion.

Clathrin-fig2c-gifClathrin-fig2d-gifClathrin-fig2e-gif

Figure 2c-e. Structures of accessory proteins AP180/CALM, epsin and amphiphysin. All structures from http://pdb.ccdc.cam.ac.uk/pdb/. Note the modular construction of many of the components.

The AP2 adaptor is associated with receptor-mediated endocytosis and its structural determination has provided important functional insights [10]. Initially, AP2 interacts with the plasma membrane via binding sites for phosphatidylinositol (4,5)-bisphosphate (PIP2), a lipid particularly concentrated in the plasma membrane. In this respect, it is different from other APs which require the GTP-bound form of the small G-protein Arf1 for membrane binding [9]. Internalised receptors contain short sequence motifs in their cytoplasmic domains that are recognised by APs [11]. One class of signal is of the form YXX-phi, in which phi is a bulky hydrophobic amino-acid, but X is variable. This sequence motif is recognised by the mu subunit [12]. In the atomic resolution structure of the AP2 core domain, the binding site of the mu-2 subunit is occluded, implying a conformational change is needed to allow the mu-2 subunit to bind receptors [10]. This conformational change is driven by the phosphorylation of threonine156 of the mu-2 subunit [13-14]. A good candidate for the enzyme that carries out the phosphorylation is adaptor-associated kinase 1 (AAK1) [15] An inactive form of this enzyme associates at low stoichiometry with cytosolic AP2 and is brought into the growing coated pit as AP2 assembles. Significantly, AAK1 is activated by clathrin, suggesting a simple mechanism by which the functional activation of AP2 is closely coupled to coated pit assembly (Figure 3) [16-17].

A variety of accessory proteins help co-ordinate the coat assembly in receptor-mediated endocytosis (Figures 2 and 3). Proteins such as CALM and its neurone-specific isoform AP180 posses an AP180 N-terminal homology (ANTH) domain that binds PIP2. The rest of the molecule is unstructured and contains multiple short binding motifs for clathrin and the AP2 alpha subunit-appendage domain. AP180 and CALM promote clathrin and AP2-binding as flat lattices and may also modulate coat size [18-19]. Epsin1 was originally identified as an adaptor for ubiquinated receptors [20]. More recently, it has been implicated in membrane deformation that occurs with pit formation. This is mediated by the epsin N-terminal homology (ENTH) domain that also binds PIP2 (Figure 2).  Unlike AP180/CALM, the ENTH domain insets an alpha-helix into the outer leaflet of the membrane and thus induces membrane curvature [21]. The protein Eps15 can bind both epsin and clathrin/AP2 and is localised predominantly to the growing edges of the coated pit [22-23]. As such it may organise the spatial location of epsins and similar molecules. Endophilin I has lysophosphatidic acid acyl transferase activity and could further facilitate invagination by altering the local lipid composition of the membrane [24].

A striking feature of many of the adaptor and accessory proteins is their modular construction. Key interactions between coat components are typically mediated by domains that recognise short sequence motifs, generally only a few amino-acids long. These domains are separated from each other by extended flexible linkers, thus allowing efficient searches for binding partners in the crowded environment of the growing coated pit. Individually, the interactions often have relatively low affinity, but cumulatively these networks of multi-point attachments rapidly build a structure maintained with high avidity. Similarly, when these networks are disrupted, during for example coat dissociation, the network will be rapidly destabilised.

Clathrin assembly

Clathrin assembles onto the adaptors to form the outer layer of the coat. It acts to stabilise the curvature introduced into the growing pit whilst increasing its deformation until the entire region invaginates to form a closed vesicle. The unassembled cytosolic form of clathrin is comprised of three molecules of heavy-chain and three molecules of light chain and is called a triskelion, after its striking resemblance to the heraldic symbol of the same name (read more). The heavy-chain legs are made from a repeated sequence of short alpha-helical hairpins (an alpha-zig-zag) that extends along most of the triskelion length and provides rigidity [25], but the triskelion vertex, formed from three C-terminal heavy-chain domains is puckered and the angle between proximal and distal parts of the leg are flexible. Hence, as triskelions assemble, they tend to form closed cages with striking pentagonal and hexagonal faces (Figures 2 and 3) (See how this occurs). The associated light-chains regulate assembly competence [26]. Under each vertex of the assembled coat lie the heavy-chain N-terminal domains from three separate triskelions which all point inwards [27]. The N-terminal domain adopts a seven-bladed beta-propeller structure with the clefts formed from adjacent blades of the propeller acting as binding sites for short motifs on the adaptors such as the hinge region of the AP beta subunit [28] (Figure 2).

Clathrin-fig3-gif

Figure 3. Outline of the main events in receptor-mediated endocytosis. The AP2 adaptor attaches to the under-surface of the plasma membrane via its PIP2 binding site. As clathrin stabilises this initial interaction it activates mu-2 subunit phosphorylation. This allows the mu-2 subunit (green) to swing out from its normal position in the AP2 core and bind receptors (see text for details). The black outline on the coated vesicle shows the location of an individual triskelion on the assembled coat.

Membrane fission and uncoating

Finally, the membrane connecting the CCV to the plasma membrane is severed. As part of this process, the protein amphiphysin dimerises onto the neck via its N-terminal BAR domain (Figures 2 and 3). The concave face of the dimerised BAR domains is positively charged and will interact with the phospholipid head groups of the membrane, thus stabilising the neck [29]. The GTPase enzyme dynamin binds to the SH3 domain of amphiphysin. Although dynamin drives membrane cleavage, the mechanism is still unclear. A plausible model suggests that dynamin is recruited onto amphiphysin in its GDP-bound form. GTP/GDP exchange facilitates the formation of a polymerised dynamin collar around the neck and GTP hydrolysis drives a conformational change that either directly cleaves the neck or acts indirectly by recruiting other factors [30]. Following detachment from the plasma membrane, the clathrin is quickly uncoated by the combined action of the ATPase Hsp70 and the coat component auxilin (which provides the DnaJ domain for the Hsp70) [31-32]. The APs and accessory proteins are probably uncoated separately. Here the protein synaptojanin may play a role. It is recruited onto the pit by its interaction with amphiphysin and endophilin. Synaptojanin possesses a lipid phosphatase activity which converts PIP2 to PtdIns, thus weakening the attachment of coat components with the vesicle membrane [33-34].

Future prospects

Many of the core structural questions concerning clathrin-coated vesicles in RME are now on the verge of being elucidated. But this still gives a rather static view, whereas real clathrin-coated pits and vesicles are highly dynamic. Recently, live-cell imaging techniques using GFP-tagged coat proteins have been used to follow the assembly of individual pits and vesicles in real time and this is providing new and surprising insights [35]. The increased ability selectively to remove individual coat proteins in vivo should also allow novel experimental questions to be addressed [36-37]. A major unresolved issue is how coat assembly is regulated both temporally and spatially. Cycles of phosphorylation and dephosphorylation of key coat proteins are likely to play an important role here [39-40]. The demonstration that Rab5 controls specific steps in receptor-mediated endocytosis suggests an important regulatory role for small GTPases [41]. Compared to receptor-mediated endocytosis, clathrin-coated vesicles at the TGN rely on different adaptor classes including AP1 and a distinct family of monomeric adaptors, the GGAs [8-9]. What functional aspects are shared with plasma-membrane coats and what aspects are distinct? Transport events between the ER and Golgi are catalysed by COPI and COPII coated vesicles (Figure 1). In both cases, these coats possess two subcomplexes that act in a clathrin-like and an adaptor-like manner. Homologies between these COP components and the clathrin/AP complexes underlie these functional connections, and suggest a deep evolutionary association between all transport vesicles [42]. Thirty years after its first isolation [43], the clathrin-coated vesicle continues to generate new questions.

References

1. Mellman I, Warren G: The road taken: past and future foundations of membrane traffic. Cell 2000, 100:99-112.

2. Kirchhausen T: Clathrin. Annu Rev Biochem 2000, 69:699-727.

3. Brodsky FM, Chen CY, Knuehl C, Towler MC, Wakeham DE: Biological basket weaving: formation and function of clathrin-coated vesicles. Annu Rev Cell Dev Biol 2001, 17:517-568.

4. Smythe E: Clathrin-coated vesicle formation: a paradigm for coated-vesicle formation. Biochem Soc Trans 2003, 31:736-739.

5. Sorkin A, Von Zastrow M: Signal transduction and endocytosis: close encounters of many kinds. Nat Rev Mol Cell Biol 2002, 3:600-614.

6. Slepnev VI, De Camilli P: Accessory factors in clathrin-dependent synaptic vesicle endocytosis. Nat Rev Neurosci 2000, 1:161-172.

7. Solomonia RO, McCabe BJ, Jackson AP, Horn G: Clathrin proteins and recognition memory. Neuroscience 1997, 80:59-67.

8. Owen DJ, Collins BM, Evans PR: Adaptors for clathrin coats: structure and function. Annu Rev Cell Dev Biol 2004, 20:153-191.

9. Robinson MS: Adaptable adaptors for coated vesicles. Trends Cell Biol 2004, 14:167-174.

10. Collins BM, McCoy AJ, Kent HM, Evans PR, Owen DJ: Molecular architecture and functional model of the endocytic AP2 complex. Cell 2002, 109:523-535.

11. Sorkin A: Cargo recognition during clathrin-mediated endocytosis: a team effort. Curr Opin Cell Biol 2004, 16:392-399.

12. Ohno H, Stewart J, Fournier MC, Bosshart H, Rhee I, Miyatake S, Saito T, Gallusser A, Kirchhausen T, Bonifacino JS: Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science 1995, 269:1872-1875.

13. Olusanya O, Andrews PD, Swedlow JR, Smythe E: Phosphorylation of threonine 156 of the mu2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo. Curr Biol 2001, 11:896-900.

14. Ricotta D, Conner SD, Schmid SL, von Figura K, Honing S: Phosphorylation of the AP2 mu subunit by AAK1 mediates high affinity binding to membrane protein sorting signals. J Cell Biol 2002, 156:791-795.

15. Conner SD, Schmid SL: Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis. J Cell Biol 2002, 156:921-929.

16. Jackson AP, Flett A, Smythe C, Hufton L, Wettey FR, Smythe E: Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor micro2 kinase. J Cell Biol 2003, 163:231-236.

17. Conner SD, Schroter T, Schmid SL: AAK1-mediated micro2 phosphorylation is stimulated by assembled clathrin. Traffic 2003, 4:885-890.

18. Morgan JR, Zhao X, Womack M, Prasad K, Augustine GJ, Lafer EM: A role for the clathrin assembly domain of AP180 in synaptic vesicle endocytosis. J Neurosci 1999, 19:10201-10212.

19. Zhang B, Koh YH, Beckstead RB, Budnik V, Ganetzky B, Bellen HJ: Synaptic vesicle size and number are regulated by a clathrin adaptor protein required for endocytosis. Neuron 1998, 21:1465-1475.

20. Legendre-Guillemin V, Wasiak S, Hussain NK, Angers A, McPherson PS: ENTH/ANTH proteins and clathrin-mediated membrane budding. J Cell Sci 2004, 117:9-18.

21. Ford MG, Mills IG, Peter BJ, Vallis Y, Praefcke GJ, Evans PR, McMahon HT: Curvature of clathrin-coated pits driven by epsin. Nature 2002, 419:361-366.

22. Chen H, Fre S, Slepnev VI, Capua MR, Takei K, Butler MH, Di Fiore PP, De Camilli P: Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis. Nature 1998, 394:793-797.

23. Tebar F, Sorkina T, Sorkin A, Ericsson M, Kirchhausen T: Eps15 is a component of clathrin-coated pits and vesicles and is located at the rim of coated pits. J Biol Chem 1996, 271:28727-28730.

24. Schmidt A, Wolde M, Thiele C, Fest W, Kratzin H, Podtelejnikov AV, Witke W, Huttner WB, Soling HD: Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature 1999, 401:133-141.

25. Ybe JA, Brodsky FM, Hofmann K, Lin K, Liu SH, Chen L, Earnest TN, Fletterick RJ, Hwang PK: Clathrin self-assembly is mediated by a tandemly repeated superhelix. Nature 1999, 399:371-375.

26. Brodsky FM, Hill BL, Acton SL, Nathke I, Wong DH, Ponnambalam S, Parham P: Clathrin light chains: arrays of protein motifs that regulate coated-vesicle dynamics. Trends Biochem Sci 1991, 16:208-213.

27. Fotin A, Cheng Y, Sliz P, Grigorieff N, Harrison SC, Kirchhausen T, Walz T: Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 2004, 432:573-579.

28. ter Haar E, Harrison SC, Kirchhausen T: Peptide-in-groove interactions link target proteins to the beta-propeller of clathrin. Proc Natl Acad Sci U S A 2000, 97:1096-1100.

29. Peter BJ, Kent HM, Mills IG, Vallis Y, Butler PJ, Evans PR, McMahon HT: BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 2004, 303:495-499.

30. Sever S: Dynamin and endocytosis. Curr Opin Cell Biol 2002, 14:463-467.

31. Newmyer SL, Schmid SL: Dominant-interfering Hsc70 mutants disrupt multiple stages of the clathrin-coated vesicle cycle in vivo. J Cell Biol 2001, 152:607-620.

32. Lemmon SK: Clathrin uncoating: Auxilin comes to life. Curr Biol 2001, 11:R49-52.

33. Cremona O, Di Paolo G, Wenk MR, Luthi A, Kim WT, Takei K, Daniell L, Nemoto Y, Shears SB, Flavell RA, et al.: Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 1999, 99:179-188.

34. Verstreken P, Koh TW, Schulze KL, Zhai RG, Hiesinger PR, Zhou Y, Mehta SQ, Cao Y, Roos J, Bellen HJ: Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating. Neuron 2003, 40:733-748.

35. Rappoport JZ, Simon SM, Benmerah A: Understanding living clathrin-coated pits. Traffic 2004, 5:327-337.

36. Wettey FR, Hawkins SF, Stewart A, Luzio JP, Howard JC, Jackson AP: Controlled elimination of clathrin heavy-chain expression in DT40 lymphocytes. Science 2002, 297:1521-1525.

37. Motley A, Bright NA, Seaman MN, Robinson MS: Clathrin-mediated endocytosis in AP-2-depleted cells. J Cell Biol 2003, 162:909-918.

38. Hinrichsen L, Harborth J, Andrees L, Weber K, Ungewickell EJ: Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells. J Biol Chem 2003, 278:45160-45170.

39. Hill E, Olusanya O, van der Kaay J, Downes CP, Andrews PD, Swedlow JR, Smythe E: Regulation of clathrin-coated vesicle formation. Biochem Soc Trans 2001, 29:375-377.

40. Korolchuk V, Banting G: Kinases in clathrin-mediated endocytosis. Biochem Soc Trans 2003, 31:857-860.

41. McLauchlan H, Newell J, Morrice N, Osborne A, West M, Smythe E: A novel role for Rab5-GDI in ligand sequestration into clathrin-coated pits. Curr Biol 1998, 8:34-45.

42. McMahon HT, Mills IG: COP and clathrin-coated vesicle budding: different pathways, common approaches. Curr Opin Cell Biol 2004, 16:379-391.

43. Pearse BM: Coated vesicles from pig brain: purification and biochemical characterization. J Mol Biol 1975, 97:93-98.

uctures determined. This has provided an unprecedented insight into the operation of these molecular machines.

Contents:

Introduction   

Adaptors and accessory proteins 

Clathrin assembly 

Membrane fission and uncoating 

Future prospects

References


Introduction

A typical eukaryotic cell contains a well developed secretory and endocytic pathway composed of discrete intracellular compartments. Most membrane proteins that are transported through the secretory pathway begin their life in the endoplasmic reticulum (ER) where they undergo folding and assembly. Those proteins destined for other compartments (eg. the plasma membrane or lysosomes) move sequentially out of the ER, through the stacks of the Golgi and trans-Golgi network (TGN). Understanding how the right proteins are targeted to the right intracellular location is a major research area in contemporary cell biology [1]. A large part of the answer is that membrane proteins are selectively transported between intracellular compartments by incorporation into distinct classes of coated transport vesicles (Figure 1). Clathrin-coated vesicles have been implicated in several distinct intracellular transport steps. In particular, the internalisation of plasma membrane proteins by receptor-mediated endocytosis (RME) and the transport of lysosomal enzymes from the TGN [2-4]. In this brief overview, I will focus on the role of clathrin in RME since this is the best studied of all the clathrin-mediated mediated transport events.   

Fig1-clathrin-golgi

Figure 1. The distribution of different classes of coated vesicle in the eukaryotic cell. The diagram is highly simplified. For example, only one endosome compartment is shown and the multiple pathways to and from each compartment are omitted for clarity

A wide variety of membrane proteins enter cells by RME. For example, receptors carrying ligands required for growth (eg transferrin, low-density lipoprotein etc) are constantly and efficiently internalised by this process. In addition, RME plays an important role in hormone receptor signalling either by removing hormone-receptor complexes from the surface and attenuating the signal, or in other cases by delivering bound receptors to intracellular compartments for optimum signalling [5]. In neurones, RME plays a special role in rapidly recapturing synaptic vesicle proteins inserted into the synaptic membrane following regulated exocytosis [6]. Possibly for this reason, clathrin is up-regulated in the chick brain following visual imprinting, a model for recognition memory.[7].

The process begins at the under-surface of the plasma membrane with the sequential assembly of coat components to form a clathrin-coated pit. Internalised receptors interact with a class of molecules called adaptors and thus become clustered into the growing coated pit. The adaptors link the membrane proteins with the clathrin that forms the outer layer of the coat. Together with accessory and regulatory molecules, cargo proteins, adaptors and clathrin co-assemble, and the growing coated pit invaginates. In the final stage, the membrane neck is severed to form a closed coated vesicle (Figures 2 and 3).

Adaptors and accessory proteins

Adaptors represent a diverse group of proteins recognising different classes of cargo receptor. The best characterised are a family of closely related proteins called the adaptor proteins (APs) comprising AP1, AP2, AP3 and AP4. Each of these four classes are localised to different intracellular compartments (Figure 1) and vary in their receptor specificity [8-9]. All APs are constructed on a fundamentally similar plan: two 110-130 kDa large subunits (a beta-class adaptin, and one of either an alpha, gamma, delta or epsilon adaptin), a 50 kDa medium chain mu adaptin and a 15-20 kDa small chain sigma adaptin. The two large subunits can be subdivided into a C-terminal appendage domain connected to the N-terminal core domain via an unstructured flexible linker sequence (Figure 2a).

Clathrin-fig2a-gif

Clathrin-fig2b-gif

Figures 2a & b. Structures of AP2 adaptor and clathrin triskelion.

Clathrin-fig2c-gifClathrin-fig2d-gifClathrin-fig2e-gif

Figure 2c-e. Structures of accessory proteins AP180/CALM, epsin and amphiphysin. All structures from http://pdb.ccdc.cam.ac.uk/pdb/. Note the modular construction of many of the components.

The AP2 adaptor is associated with receptor-mediated endocytosis and its structural determination has provided important functional insights [10]. Initially, AP2 interacts with the plasma membrane via binding sites for phosphatidylinositol (4,5)-bisphosphate (PIP2), a lipid particularly concentrated in the plasma membrane. In this respect, it is different from other APs which require the GTP-bound form of the small G-protein Arf1 for membrane binding [9]. Internalised receptors contain short sequence motifs in their cytoplasmic domains that are recognised by APs [11]. One class of signal is of the form YXX-phi, in which phi is a bulky hydrophobic amino-acid, but X is variable. This sequence motif is recognised by the mu subunit [12]. In the atomic resolution structure of the AP2 core domain, the binding site of the mu-2 subunit is occluded, implying a conformational change is needed to allow the mu-2 subunit to bind receptors [10]. This conformational change is driven by the phosphorylation of threonine156 of the mu-2 subunit [13-14]. A good candidate for the enzyme that carries out the phosphorylation is adaptor-associated kinase 1 (AAK1) [15] An inactive form of this enzyme associates at low stoichiometry with cytosolic AP2 and is brought into the growing coated pit as AP2 assembles. Significantly, AAK1 is activated by clathrin, suggesting a simple mechanism by which the functional activation of AP2 is closely coupled to coated pit assembly (Figure 3) [16-17].

A variety of accessory proteins help co-ordinate the coat assembly in receptor-mediated endocytosis (Figures 2 and 3). Proteins such as CALM and its neurone-specific isoform AP180 posses an AP180 N-terminal homology (ANTH) domain that binds PIP2. The rest of the molecule is unstructured and contains multiple short binding motifs for clathrin and the AP2 alpha subunit-appendage domain. AP180 and CALM promote clathrin and AP2-binding as flat lattices and may also modulate coat size [18-19]. Epsin1 was originally identified as an adaptor for ubiquinated receptors [20]. More recently, it has been implicated in membrane deformation that occurs with pit formation. This is mediated by the epsin N-terminal homology (ENTH) domain that also binds PIP2 (Figure 2).  Unlike AP180/CALM, the ENTH domain insets an alpha-helix into the outer leaflet of the membrane and thus induces membrane curvature [21]. The protein Eps15 can bind both epsin and clathrin/AP2 and is localised predominantly to the growing edges of the coated pit [22-23]. As such it may organise the spatial location of epsins and similar molecules. Endophilin I has lysophosphatidic acid acyl transferase activity and could further facilitate invagination by altering the local lipid composition of the membrane [24].

A striking feature of many of the adaptor and accessory proteins is their modular construction. Key interactions between coat components are typically mediated by domains that recognise short sequence motifs, generally only a few amino-acids long. These domains are separated from each other by extended flexible linkers, thus allowing efficient searches for binding partners in the crowded environment of the growing coated pit. Individually, the interactions often have relatively low affinity, but cumulatively these networks of multi-point attachments rapidly build a structure maintained with high avidity. Similarly, when these networks are disrupted, during for example coat dissociation, the network will be rapidly destabilised.

Clathrin assembly

Clathrin assembles onto the adaptors to form the outer layer of the coat. It acts to stabilise the curvature introduced into the growing pit whilst increasing its deformation until the entire region invaginates to form a closed vesicle. The unassembled cytosolic form of clathrin is comprised of three molecules of heavy-chain and three molecules of light chain and is called a triskelion, after its striking resemblance to the heraldic symbol of the same name (read more). The heavy-chain legs are made from a repeated sequence of short alpha-helical hairpins (an alpha-zig-zag) that extends along most of the triskelion length and provides rigidity [25], but the triskelion vertex, formed from three C-terminal heavy-chain domains is puckered and the angle between proximal and distal parts of the leg are flexible. Hence, as triskelions assemble, they tend to form closed cages with striking pentagonal and hexagonal faces (Figures 2 and 3) (See how this occurs). The associated light-chains regulate assembly competence [26]. Under each vertex of the assembled coat lie the heavy-chain N-terminal domains from three separate triskelions which all point inwards [27]. The N-terminal domain adopts a seven-bladed beta-propeller structure with the clefts formed from adjacent blades of the propeller acting as binding sites for short motifs on the adaptors such as the hinge region of the AP beta subunit [28] (Figure 2).

Clathrin-fig3-gif

Figure 3. Outline of the main events in receptor-mediated endocytosis. The AP2 adaptor attaches to the under-surface of the plasma membrane via its PIP2 binding site. As clathrin stabilises this initial interaction it activates mu-2 subunit phosphorylation. This allows the mu-2 subunit (green) to swing out from its normal position in the AP2 core and bind receptors (see text for details). The black outline on the coated vesicle shows the location of an individual triskelion on the assembled coat.

Membrane fission and uncoating

Finally, the membrane connecting the CCV to the plasma membrane is severed. As part of this process, the protein amphiphysin dimerises onto the neck via its N-terminal BAR domain (Figures 2 and 3). The concave face of the dimerised BAR domains is positively charged and will interact with the phospholipid head groups of the membrane, thus stabilising the neck [29]. The GTPase enzyme dynamin binds to the SH3 domain of amphiphysin. Although dynamin drives membrane cleavage, the mechanism is still unclear. A plausible model suggests that dynamin is recruited onto amphiphysin in its GDP-bound form. GTP/GDP exchange facilitates the formation of a polymerised dynamin collar around the neck and GTP hydrolysis drives a conformational change that either directly cleaves the neck or acts indirectly by recruiting other factors [30]. Following detachment from the plasma membrane, the clathrin is quickly uncoated by the combined action of the ATPase Hsp70 and the coat component auxilin (which provides the DnaJ domain for the Hsp70) [31-32]. The APs and accessory proteins are probably uncoated separately. Here the protein synaptojanin may play a role. It is recruited onto the pit by its interaction with amphiphysin and endophilin. Synaptojanin possesses a lipid phosphatase activity which converts PIP2 to PtdIns, thus weakening the attachment of coat components with the vesicle membrane [33-34].

Future prospects

Many of the core structural questions concerning clathrin-coated vesicles in RME are now on the verge of being elucidated. But this still gives a rather static view, whereas real clathrin-coated pits and vesicles are highly dynamic. Recently, live-cell imaging techniques using GFP-tagged coat proteins have been used to follow the assembly of individual pits and vesicles in real time and this is providing new and surprising insights [35]. The increased ability selectively to remove individual coat proteins in vivo should also allow novel experimental questions to be addressed [36-37]. A major unresolved issue is how coat assembly is regulated both temporally and spatially. Cycles of phosphorylation and dephosphorylation of key coat proteins are likely to play an important role here [39-40]. The demonstration that Rab5 controls specific steps in receptor-mediated endocytosis suggests an important regulatory role for small GTPases [41]. Compared to receptor-mediated endocytosis, clathrin-coated vesicles at the TGN rely on different adaptor classes including AP1 and a distinct family of monomeric adaptors, the GGAs [8-9]. What functional aspects are shared with plasma-membrane coats and what aspects are distinct? Transport events between the ER and Golgi are catalysed by COPI and COPII coated vesicles (Figure 1). In both cases, these coats possess two subcomplexes that act in a clathrin-like and an adaptor-like manner. Homologies between these COP components and the clathrin/AP complexes underlie these functional connections, and suggest a deep evolutionary association between all transport vesicles [42]. Thirty years after its first isolation [43], the clathrin-coated vesicle continues to generate new questions.

References

1. Mellman I, Warren G: The road taken: past and future foundations of membrane traffic. Cell 2000, 100:99-112.

2. Kirchhausen T: Clathrin. Annu Rev Biochem 2000, 69:699-727.

3. Brodsky FM, Chen CY, Knuehl C, Towler MC, Wakeham DE: Biological basket weaving: formation and function of clathrin-coated vesicles. Annu Rev Cell Dev Biol 2001, 17:517-568.

4. Smythe E: Clathrin-coated vesicle formation: a paradigm for coated-vesicle formation. Biochem Soc Trans 2003, 31:736-739.

5. Sorkin A, Von Zastrow M: Signal transduction and endocytosis: close encounters of many kinds. Nat Rev Mol Cell Biol 2002, 3:600-614.

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Dr Tony Jackson
Department of Biochemistry, University of Cambridge
Email: a.p.jackson@bioc.cam.ac.uk

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