reviews - bio 5068

The plasma membrane of eukaryotic cells forms a delicate boundary between the potentially harsh extracellular milieu and the cellular constituents. It allows ion gradients to be established that are necessary for cell excitability and function, and contains an expansive array of transmembrane proteins for recognition, adhesion, nutrient uptake and signalling. Yet, the protein composition of the plasma membrane is never invariant — rather, it is continuously remodelled in response to both extracellular and intracellular cues. Precisely what gets cleared off the cell surface and when and how this occurs are vital for myriad cellular processes, ranging from basic nutrition and cell division to coordinated cell movements or fate changes that underpi n complex embryonic patterning events.

The clathrincoated vesicles that form on the cytosolic leaflet of the cell surface are the archetype for coatassisted sorting events because, in general, the fundamental molecular mechanisms of vesiclemediated transport between organelle compartments seem similar. During biogenesis, vesicular carriers progress through a common series of steps: restricted assembly of coat components at a desig nated site on the donor membrane, linked membrane deformation and cargo concentration to generate a coated bud, membrane scission, uncoating (BOX 1) and, finally, tethering and fusion of the released vesicular intermediate with an acceptor compartment. The variety of transmembrane cargoes gathered into clathrincoated carriers at the plasma membrane necessitates the use of several unrelated sorting signals to prevent competition for entry and to allow flexibility in the temporal regulation of uptake. Sorting signals range from intrinsic

linear peptide sequences to whole folded proteins that are reversibly appended to cargoes1–4 (FIG. 1). Also essential is to ensure that components (such as SNAREs) that are necessary for successful docking and fusion of the incoming vesicle with endosomal elements are collected in the nascent bud. In this Review, recent information on how diverse cargo sorting events occur at clathrincoated structures formed on the surface of eukaryotic cells is discussed. The exceptional variation within and between different classes of internalization signals and the machinery that recognizes them is emphasized in relation to the trafficking requirements of animal cells and the modulation of the abundance of surface cons tituents. Other current reviews deal more thoroughly with the participation of phosphoinositides in coat assembly5, the mechanics of clathrin coat assembly and budding6, and the role of the cytoskeleton7, as well as with various clathrinindependent internalization pathways8.

The core sorting adaptor: AP‑2The term ‘adaptor’ was coined to connote a functional entity that connects transmembrane cargoes to the overlying clathrin coat9. Because AP2 is the principal nonclathrin constituent of purified endocytic clathrincoated vesicles, it is the prototypical, and still the best understood, adaptor. AP2 is a stable complex comprised of four nonidentical polypeptide chains: the ~100 kDa αsubunit, the ~100 kDa β2subunit, the 50 kDa μ2subunit and the 17 kDa σ2subunit10,11. Targeted disruption of the genes that encode AP2, or AP2 RNA interference (RNAi), is lethal in several

Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.e-mail: [email protected]:10.1038/nrm2751

Tickets to ride: selecting cargo for clathrin-regulated internalizationLinton M. Traub

Abstract | Clathrin-mediated endocytosis oversees the constitutive packaging of selected membrane cargoes into transport vesicles that fuse with early endosomes. The process is responsive to activation of signalling receptors and ion channels, promptly clearing post-translationally tagged forms of cargo off the plasma membrane. To accommodate the diverse array of transmembrane proteins that are variably gathered into forming vesicles, a dedicated sorting machinery cooperates to ensure that non-competitive uptake from the cell surface occurs within minutes. Recent structural and functional data reveal remarkable plasticity in how disparate sorting signals are recognized by cargo-selective clathrin adaptors, such as AP-2. Cargo loading also seems to govern whether coats ultimately bud or dismantle abortively at the cell surface.

ClathrinA triskelion-shaped protomer, composed of three heavy and three light chains, that polymerizes into a characteristic polyhedrally structured coat that is found on several intracellular membrane surfaces.

SNARE(Soluble N-ethylmaleimide-sensitive factor attachment protein receptor). A member of a family of transmembrane proteins that assemble into heteromeric four-helix-bundle complexes to fuse membrane bilayers together.

REVIEWS

NATuRe RevIewS | MoleculAr cell Biology vOlume 10 | SePTemBeR 2009 | 583

f o c u s o n e n d o c y to s i s

© 2009 Macmillan Publishers Limited. All rights reserved

mailto:[email protected]

Nature Reviews | Molecular Cell Biology

Nucleation Growth StabilizationFailureDissolution Invagination Scission

Uncoating

Plasma membrane

Transmembrane cargo receptors

CLASPsAP-2 adaptorClathrin trimer

DynaminActin

DynaminA large regulatory GTPase that drives membrane scission when it is polymerized into a spiral at the base of deeply invaginated buds.

AAK1(Adaptor-associated kinase 1). A Ser/Thr protein kinase that regulates endocytosis and cargo recognition.

Type I transmembrane proteinA single-pass transmembrane protein oriented with an extracellular N terminus and a cytosolic C terminus.

metazoan models, including Caenorhabditis elegans12,13. A pivotal role for AP2 is also evidenced by selective caspasedependent proteo lysis of the large subunits in apoptotic cells14. However, in Saccharomyces cerevisiae, deletion of the AP2 sub units is not lethal and has minimal effect on clathrin function15,16.

YXXØ sorting signals. Preferential entry into clathrincoated buds is a regulated step that requires a positive sorting signal to be presented on the cargo protein (TABLE 1). The μ2subunit was the first dedicated cargorecognizing subunit of AP2 to be identified17. evidence shows that YXXØtype sorting signals (in which X is any amino acid and Ø a bulky hydrophobic amino acid) present in cargo molecules (TABLE 1) are decoded through the carboxyterminal extended βsandwich subdomain of the μ2subunit18. In the context of the heterotetrameric AP2 core complex, this βsandwich subdomain of the μ2subunit must be mobile, and its movement depends on the phosphorylation status of residue Thr156 (REF. 19,20). AAK1mediated phosphorylation of this residue shifts the equilibrium of the μ2subunit conformation to the open, YXXØbinding state. The open state is further stabilized by an adjacent phosphatidylinositol4,5bisphosphate (PtdIns(4,5)P2) binding surface on the μ2subunit, which allows the

μ2subunit to simultaneously engage a YXXØ signal and the bilayer21. Reciprocally, release of AP2 from budded vesicles is facilitated by concurrent dephosphorylation of the μ2subunit and PtdIns(4,5)P2 (REF. 22).

Structurally, a YXXØcontaining peptide is bound as a temporary antiparallel βstrand, hydrogen bonded to strand β16 of the μ2subunit and with the Y and Ø side chains accommodated in chemically compatible pockets located on either side of β16 (REF. 18) (FIG. 1b). Directed mutagenesis of residue pairs in the μ2subunit (D176A and w412A23 or F174A and D176A24) disrupts the integrity of the YXXØaccepting surface and interferes selectively with the uptake of cargoes using this signal. The basic engagement pattern can be augmented by additional peptide contacts. For example, Pselectin, an endothelial type I transmembrane protein that functions as a leukocyte receptor, inserts its leu777 residue at position Y–3 (three residues upstream of the Tyr at position 0 of the sorting signal) (TABLE 1) into a depression adjacent to the Yaccommodating pocket in the μ2subunit25. A different sequence, 365YGYeCl from the GABAA (γaminobutyric acid type A) receptor γ2subunit, binds the μ2subunit in a virtually identical manner26 (FIG. 2a). These additional anchor residues in cargoes can make the variant YXXØ signals less dependent on the defining

Box 1 | Clathrin coat assembly

Because the AP‑2 adaptor has low affinity for biological membranes21, assembly zones are probably nucleated de novo when collisions of suitably oriented AP‑2 with the phosphatidylinositol‑4,5‑bisphosphate (PtdIns(4,5)P

2)‑containing plasma

membrane are stabilized by binary associations between AP‑2 appendages and accessory factors6 (see the figure). Avidity effects temporarily retain the complex at the membrane, enabling further lateral extension. Clathrin coat protein is recruited by AP‑2 and accessory proteins and translocates onto the nascent substructures. Lateral associations between the membrane‑tethered triskelia lead to assembly of a polymerized coat. AAK1, the kinase for the μ2‑subunit of AP‑2, enters by interacting with AP‑2 appendages128, and its catalytic activity is stimulated by the overlying clathrin coat129,130.

Phosphorylation of the Thr156 residue of the μ2‑subunit triggers YXXØ‑containing (in which X is any amino acid and Ø a bulky hydrophobic amino acid) cargo recognition19,21. Concurrently, co‑assembled clathrin‑associated sorting proteins (CLASPs), such as disabled 2, autosomal recessive hypercholesterolemia protein, stonin 2, epsin 1 and epidermal growth factor receptor substrate 15, can also cluster cargo molecules within the forming sorting scaffold. Likewise, β‑arrestins can enter the coat to downregulate activated G protein‑coupled receptors97,98. The polyhedral lattice might induce or stabilize thermally driven membrane deformation as the bud makes the transition from a shallow to a deeply invaginated contour131.

Successful cargo capture is monitored, in part, by dynamin41 to prevent abortive dissolution. Late recruitment of dynamin positions an oligomeric dynamin spiral to enwrap the base beneath the bulbous coated bud. Branched actin polymerization occurs at the membrane‑proximal surface of the vesicle, with microfilaments coupled to the assembled coat by accessory proteins. Longitudinal tension, generated by actin linkage, operates together with the radial twisting or constriction of the dynamin spiral on GTP hydrolysis to allow the GTPase to effectively sever the membrane that connects the bud to the cell surface132. On vesicle release, PtdIns(4,5)P

2 is dephosphorylated by synaptojanin 1 to terminate phosphoinositide‑based

membrane associations133, and hydrolysis might commence before scission is complete. The resulting free, coated vesicle quickly moves away from the plasma membrane and is rapidly uncoated; re‑entry of coat components into the soluble pool allows additional rounds of coat construction.

R e V i e W s

584 | SePTemBeR 2009 | vOlume 10 www.nature.com/reviews/molcellbio

R e V i e W s



Ub Lys63

Ub Gly76

Ub Lys48

β16

Tyr350

Leu353

Arg352

Gln351 5

Y0 pocket in µ2

Tyr757

Tyr762

Pro760 Asn759

[DE]XXXL[LIM]

AP-2

α–σ2-subunithemicomplex

Sorting signal:

Modulardomain

CLASP:

pSer/pThr

β-arrestin 1 andβ-arrestin 2

N-terminus ofarrestin

Ubiquitin

Epsins and EPS15

UIM

YXXØ

AP-2

C terminus ofµ2-subunit

[FY]XNPX[YF]

DAB2,ARH and NUMB

PTB

a

b c d

Ø pocket in µ2

Y0 pocket inPTB of DAB2

Tyr residue, can result in higher affinity and are differentially regulated. Phosphorylation of Tyr365, Tyr367 or both impedes the GABAA receptor γ2subunit from binding to AP2 (REF. 26). There is also flexibility in recognition at the distal end of YXXØtype sequences; for example, the ionotropic P2X4 ATP receptor uses a YXXGØ signal27. Because of the altered spacing, in this instance the Ø residue (leu381) projects into the

reciprocal μ2subunit pocket at a different angle, which is facilitated by the flexibility conferred by the preceding Gly380 (REF. 27). Therefore, despite the extra residue, the major features of μ2subunit engagement are conserved. The apparent plasticity in the modes of engagement may reflect merely the limits of convergent evolution on peptide motifs that are able to fit into allied adaptor contact sites with the appropriate affinity.

Figure 1 | Modular sorting codes. a | The major plasma membrane sorting signals for entry into clathrin-coated vesicles (in which X is any amino acid and Ø a bulky hydrophobic amino acid) and the complementary folded modular detection domains (shown as ribbon diagrams) within the cargo-selective clathrin adaptors AP-2 and clathrin-associated sorting proteins (CLASPs). There is little primary-sequence or tertiary-structure similarity between the different folded modules that are involved in cargo selectivity. Acidic cluster and di-Ile sorting signals (TABLE 1) are not included because the precise molecular basis for the recognition of these signals has not yet been established. In addition, although there are available structural data for HIV-1 Rev-binding protein (also known as AGFG1)100 (FIG. 2c) and a computational model for stonin 2 (REF. 102), these CLASPs sort highly restricted cargo molecules by recognizing folded determinants that are not widely represented in otherwise unrelated proteins, unlike the YXXØ, [DE]XXXL[LIM] and [FY]XNPX[YF] peptide sequences. b | Close up of the μ2-subunit of AP-2 (magenta) bound to the YQRL sorting signal in trans-Golgi network integral membrane protein 2 (also known as TGN38) (stick representation: yellow, carbon backbone; red, oxygen; blue, nitrogen; orange, sulphur) (PDB code 1BXX). c | Close up of the phosphoTyr-binding (PTB) domain of disabled 2 (DAB2) (green) engaging the YXNPXY sorting signal in β-amyloid precursor protein (PBD 1M7E). d | Interaction between a ubiquitin (Ub)-interacting motif (UIM) in 26S proteasome regulatory subunit S5A (also known as Rpn10 and PSMD4) (purple) and ubiquitin (yellow) (PBD code 1YX6). The helically structured UIM binds to one face of ubiquitin and the location of important acceptor Lys residues and the Gly residue on the carboxy-terminal of ubiquitin are shown. Y0 represents the Tyr at position 0 in the sorting signal. ARH, autosomal recessive hypercholesterolemia (also known as LDLRAP1).

R e V i e W s




http://www.rcsb.org/pdb/explore/explore.do?structureId=1BXX

http://www.rcsb.org/pdb/explore/explore.do?structureId=1M7E

http://www.rcsb.org/pdb/explore/explore.do?structureId=1YX6

Type II transmembrane proteinA single-pass transmembrane protein oriented with an extracellular C terminus and a cytosolic N terminus.

CD317 (also known as bone marrow stromal antigen 2, tetherin and Hm1.24) is a clinically relevant protein because it counteracts the release of HIv1 particles from the cell surface28,29. It is an unusual type II transmembrane protein that cycles between the cell surface, endosomes and the transGolgi network (TGN)30,31, and it contains a YDYXXv sequence in the cytosolic portion. But, unlike Pselectin and the closely related YGYeCl endocytic signal in the GABAA receptor26, CD317 might use the AP2 αsubunit appendage, which is positioned outside of the heterotetrameric core (BOX 2), for incorporation into clathrincoated buds31. These data reveal the impressive malleability of the AP2 adaptor in recognizing various Tyrbased sorting signals.

Dileucine recognition. Another AP2requiring, constitutively active sorting signal is the [De]XXXl[lIm] type acidic dileu sequence, which is structurally distinct from YXXØ and does not compete with it for entry into

clathrincoated vesicles32. Although data initially suggested that this dileu sequence binds to either the β2 or the μ2subunit, the interaction interface is primarily positioned on the σ2subunit in the AP2 core, adjacent to the PtdIns(4,5)P2binding site on the αsubunit33. The [De]XXXl[lIm] sequence binds in an extended conformation but, like the YXXØ site on the μ2subunit, the contact surface on the σ2subunit is not freely accessible in the basal AP2 conformation. The amino terminus of the associated β2subunit, paticularly residues Tyr6 and Phe7, packs against the binding site, which occludes the two leubinding pockets in the σ2subunit33 (FIG. 2b). This segment of the β2subunit must therefore move to allow dileu engagement and may be regulated by phosphorylation of Tyr6 (REF. 34). Intriguingly, although binding of the dileu signal is associated with a conform ational change in the arrangement of the AP2 core, the μ2subunit can remain in the closed conformation while a [De]XXXl[lIm] peptide is bound33.

Table 1 | Clathrin-dependent endocytic sorting signals

Signal type* example sequence* corresponding cargo protein

recognition protein or domain

refs

YXXØ YTRF YRGV YKKV YQRL YQTI YATL YHEL LGTYGVF YGYECL YEQGL

Transferrin receptor CD-M6PR PAR1 TGOLN2 (TGN38) LAMP1 LRP1 Dishevelled 2 P-selectin GABA

A receptor γ2

P2X4 receptor

µ2-subunit of AP-2

1,55,134

YDYCRV CD317 (BST2; tetherin; HM1.24) α-subunit of AP-2?

[DE]XXXL[LI] pSQIKRLL ERAPLI DKQTLL EKQPLL DQRDLI ENTSLLHPVSLHGMDD

CD4 LIMP2 CD3γ Tyrosinase Ii (CD74) Nef

α–σ2 hemicomplex of AP-2

1,135

II EEAGII K+ channel Kir2.3 AP-2 136

[FY]XNPX[YF] FDNPVY FTNPVY FENPMY YTNPAF FTNAAF GENPIY VVNPKY

LDL receptor LRP1 LRP2 (megalin) Sanpodo P-selectin β1A integrin (1) ‡

β1A integrin (2)‡

PTB domain of ARH, DAB2 and NUMB

1,55

Phosphate group S/ T GPCRs β-arrestin 1 and β-arrestin 2

55,99

Ubiquitin K/C EGFR MHC class I Delta ENaC

UIM of epsins and EPS15

64,137

Acidic cluster QEECPpSDpSEEDE DDQLGEEpSEERDD

Furin CD-M6PR

AP-2? 1,103

*Sequences indicated in single-letter amino acid notation using PROSITE syntax. Ø indicates a bulky hydrophobic amino acid (Leu, Met, Ile, Phe or Val) and X is any amino acid. ‡(1) connotes the first repeat and (2) the second repeat within the cytosolic domain. ARH, autosomal recessive hypercholesterolemia; BST2, bone marrow stromal antigen 2; CD-M6PR, cation-dependent mannose 6-phosphate receptor; DAB2, disabled 2; EGFR, epidermal growth factor receptor; ENaC, epithelial sodium channel; EPS15, EGFR substrate 15; GABA

A, γ-aminobutyric acid type A; GPCR, G protein-coupled receptor; LAMP1, lysosome-associated

membrane protein 1; LDL, low-density lipoprotein; LIMP2, lysosomal integral membrane protein 2; LRP, LDL receptor-related protein; MHC, major histocompatibility complex; Nef, negative factor; PAR1, proteinase-activated receptor 1; pS, phosphoSer; PTB, phosphoTyr-binding; TGOLN2, trans-Golgi network integral membrane protein 2; UIM, ubiquitin-interacting motif.

R e V i e W s


R e V i e W s


http://www.uniprot.org/uniprot/Q10589


Tyr6

Phe7

Lys298

Arg341

Lys298

Arg341

b

c

Leu0Leu+1

Y0 pocket in µ2

Y–2 or Y–3pocket in µ2

Ø pocket in µ2

β16

Tyr367

Leu370

Tyr365

a

Leu173

Leu171

Val157

Leu160

Leu164

Leu163

β2-subunit

α-subunit

σ2-subunit

µ2-subunit

Closed AP-2 core DiLeu-bound AP-2 core

The β2subunit thus shields both AP2 cargobinding sites in the soluble, inactive conformation. There is biochemical evidence for positive linkage between the YXXØ and dileubinding sites35, but the molecular details remain elusive.

The cytosolic portion of the major histocompatibility complex class II coreceptor on T cells, CD4, contains an atypical dileu signal in the SQIKRll sequence, the endocytic activity of which depends on phosphorylation of a Ser residue in the –5 position36. Surprisingly though, crystals of a phosphorylated CD4 peptide bound to AP2 do not directly explain the activating effect of Ser modification33; phosphorylation probably allows CD4 to enter endocytic vesicles by disrupting the inter action of CD4 with p56lCK, a Srcfamily nonreceptor Tyr kinase expressed in lymphoid cells37. CD4 is also a coreceptor for HIv1 on immune cells and, shortly after infection, the HIv1 early viral protein negative factor (Nef) binds to CD4 to promote clearance from the host cell surface. Downregulation of CD4 involves the assembly of a transient CD4–Nef–AP2 complex and requires the canonical eXXXll dileu signal in Nef 38, which binds to AP2. A distal DD residue pair also makes an important contribution to the interaction of Nef with AP2 (REF. 39), because binding of Nef to the α–σ2 hemicomplex of AP2 is inhibited equally by mutation of either the leuleu or the AspAsp doublets to Ala39. So, the diacidic motif in Nef engages AP2 through electrostatic interactions, but it is not an autonomous sorting signal39. The reciprocal contact site on AP2 is located on the αsubunit and comprises a basic patch formed by lys297 and Arg340 (REF. 40). Notably, this patch is adjacent to the [De]XXXl[lIm]binding site on the σ2subunit (FIG. 2b), and lys297 and Arg340 are required in vivo for Nefmediated CD4 internalization and for the formation of the CD4–Nef–AP2 ternary complex40.

There is currently no evidence that cellular [De]XXXl[lIm] sequences also engage the basic patch on the αsubunit. Nef therefore uses a unique and expanded contact site on the α–σ2 hemicomplex. Perhaps the diacidic motif evolved to reinforce the association with AP2, because the eXXXll signal in Nef seems to bind AP2 more weakly than other dileu residues40. Alternatively, tandem engagement of the αsubunit and the σ2subunit by Nef–CD4 might effectively outcompete cellular cargo proteins, and the site is therefore fortuitously exploited as a second contact point on the core when the dileu signal of Nef drives CD4 internalization. However, the pronounced phylogenetic conservation of the basic patch on the αsubunit, but not the analogous γsubunit or δsubunit in AP-1 or AP-3, hints that this region may actually be a dedicated cargobinding interface, perhaps for acidic cluster sorting signals40 (TABLE 1).

Cargoes drive successful coat assembly and budding. The available structural data indicate, against current textbook diagrams, that cargo recognition is unlikely to be the initial step for deposition of AP2 on the plasma membrane. Indeed, AP2 does not assemble

Figure 2 | Structural basis for cargo recognition. a | Close up of the μ2-subunit of AP-2 (magenta) associated with the YGYECL sorting signal (stick representation: yellow, carbon backbone; red, oxygen; blue, nitrogen; orange, sulphur) from the γ-subunit of the GABA

A (γ-aminobutyric acid type A) receptor (Protein Data Bank (PDB) code 2PR9).

This interaction uses the Y–3 (three residues upstream of the Tyr at position 0 of the sorting signal) or Y–2 binding site of the μ2-subunit, which is not typically occupied by most YXXØ signals (in which X is any amino acid and Ø a bulky hydrophobic amino acid). b | The AP-2 adaptor core in the basal, closed conformation (PDB code 2VGL) and in the CD4 (a T-cell surface glycoprotein) diLeu signal-bound conformation (PDB code 2JKR). In the closed state, two amino-terminal aromatic residues in the β2-subunit (green) obstruct the [DE]XXXL[LIM] binding site on the σ2-subunit (gold). Engagement of the pSQIKRLL peptide (in which pS is phosphoSer) in CD4 (stick representation) causes an obvious outward movement of the β2-subunit relative to the σ2-subunit and the α-subunit (blue), and the N-terminus of the β2-subunit is expelled. However, the μ2-subunit (violet) remains closely associated with the β2-subunit and is unable to bind YXXØ signals. The location of the two basic side chains on the αsubunit that contact negative factor (Nef) are shown; note that the Lys298 and Arg341 residues of the murine α-subunit are equivalent to the Lys297 and Arg340 residues in humans40. c | Close up of the HIV-1 Rev-binding protein (also known as AGFG1) (stick representation: cyan, carbon backbone; red, oxygen; blue, nitrogen) interaction with the longin domain of vesicle-associated membrane protein 7 (brown) (PDB code 2VX8). In contrast to part a, the CLASP polypeptide here is in the extended conformation and the cargo presents the folded interaction surface.

R e V i e W s




http://www.uniprot.org/uniprot/P01730

http://www.rcsb.org/pdb/explore/explore.do?structureId=2PR9

http://www.rcsb.org/pdb/explore/explore.do?structureId=2VGL

http://www.rcsb.org/pdb/explore/explore.do?structureId=2JKR

http://www.rcsb.org/pdb/explore/explore.do?structureId=2VX8


Plat

form

sub

dom

ain

Sand

wic

h su

bdom

ain

FXn[FW]n [DE]nX1-2FXX[FL]XXXR WXX[FW]X[DE]n FXDXF or DP[FW]Interactionmotif:

β2-subunit appendage α-subunit appendage

AP-2 coreβ2-subunit

α-subunit

µ2-subunit σ2-subunit

AP-1An AP-2-related heterotetrameric sorting adaptor that couples cargo selection with clathrin assembly at the TGN and/or in endosomes.

AP-3An AP-2-related heterotetrameric sorting adaptor that is involved in sorting transmembrane cargoes through the endosomal system.

on endosomes, where the bulk of cycling receptors are often located at steady state. Strong overexpression of receptors does not increase clathrin coat density at the surface41,42, and AP2 with incapacitated cargo selectively still assembles into clathrincoated vesicles19,23,24. It seems that cargo capture occurs concomitantly with clathrin coat polymerization and invagination, and that the cargo load is dynamically monitored to prevent assembly and budding of empty vesicles43. Recent evidence for this comes from quantitative timeresolved analysis of vast numbers of clathrincoated structures in BSC1 cells overexpressing the transferrin receptor41.

In control cells, only ~40% of clathrin (and AP2)positive structures bud productively into the cell. Approximately 20% fail to bud with a halflife (t1/2) of ~15 s, representing late abortive events41,43. If the surface transferrin receptor level is increased >30fold, these late abortive events are no longer apparent. Cargo capture therefore biases coat formation towards productive budding events41. An early abortive population (~40% of events, with a t1/2 of ~5 s) is insensitive to transferrin receptor levels and is likely to represent transient tethering of AP2 to the membrane, possibly by engaging PtdIns(4,5)P2 (REF. 41).

Box 2 | Appendage interaction hubs

The functionally analogous α‑subunit and β2‑subunit appendages of AP‑2 each consist of an amino‑terminal immuno‑globulin‑like sandwich subdomain that is rigidly affixed to a carboxy‑terminal platform subdomain6,55. An independent interaction surface is present on each subdomain, resulting in four physically separate contact sites per AP‑2 heterotetramer. The matching short interaction motifs are all characterized by aromatic side chains, but the arrangement and selectivity of these anchor residues is different for engagement of each appendage binding site. The sandwich subdomain of the α‑subunit binds to WXX[FW]X[DE]

n (in which X is any amino acid) motifs (see the figure; shown in yellow)

as found in stonin 2, clathrin coat assembly protein AP180 and synaptojanin 1 (Protein Data Bank (PDB) code 1W80). The platform subdomain of the α‑subunit engages either DP[FW] or FXDXF motifs, which are present in clathrin‑associated sorting proteins (CLASPs), such as epsins, epidermal growth factor receptor substrate 15 (EPS15), disabled 2 and HIV‑1 Rev‑binding protein (also known as AGFG1), and also in AAK1 (PDB code 1KY7). Similarly, the sandwich subdomain of the β2‑subunit binds to a Phe‑rich motif of ill‑defined consensus that occurs in EPS15 and AP180 (PDB code 2IV9). This β2‑sandwich site also binds to the clathrin heavy chain in conjunction with a clathrin box that is positioned in the hinge that links the core to the appendage. The platform subdomain of the β2‑subunit binds to a [DE]

nX

1‑2FXX[FL]XXXR sequence

presented in the context of an α‑helix (PDB code 2G30). This restricted β2‑subunit platform motif is present in β‑arrestin, autosomal recessive hypercholesterolemia protein and epsins95,124. The affinity of these different interaction motifs for the appendages ranges from 1– 100 μM and so the motifs have interaction half‑lives of several seconds. During assembly, AP‑2 must be subjected to large numbers of alternative encounters and become momentarily complexed by highly transient interactions. Because CLASPs can interact with each other as well, it is clear that a tremendous degree of intricate protein rearrangement occurs within minutes to drive efficient clathrin coat assembly and release. The complexity of the interaction network poses significant biological challenges and many unanswered questions are raised: for example, are protein–protein interactions prioritized? It is currently unclear how ‘noise’, in the form of promiscuous interactions, is handled and how forward progression is assured.

R e V i e W s


R e V i e W s


http://www.rcsb.org/pdb/explore/explore.do?structureId=1W80

http://www.rcsb.org/pdb/explore/explore.do?structureId=1KY7

http://www.rcsb.org/pdb/explore/explore.do?structureId=1IV9

http://www.rcsb.org/pdb/explore/explore.do?structureId=2G30


AP-2Transferrin

0 s 20 s 60 s

AP-2 Transferrin

20 sec 20 sec

60 sec 60 sec

a

b

Transferrin

AP-2Transferrin

AP-2Transferrin

AP-2

c

10 µm

The uniformity in both size and dynamics of clathrin coated structures in BSC1 cells is remarkable41,43. In most other cultured cells, heterogeneous clathrincoated structures on the surface range from dynamic, diffractionlimited objects to large, apparently sessile patches that are often >500 nm in diameter. The large, longlived patches do have cargo fluxing through44 (FIG. 3): intensity fluctuations45 and coat components or cargoes emanating from the periphery of these regions can be observed by livecell imaging44,46,47. These patches probably correspond to small buds adjacent to the flat clathrin arrays that can be seen by freezeetch electron microscopy (FIG. 3c). Given the positive modulatory role that cargoes play in the overall budding success, enduring clathrincoated patches might occur as a means to stabilize clathrinmediated endocytosis in the face of variable endocytic activity and fluctuating signalling requirements.

Additional sorting adaptorsNot only do different classes of sorting signals not compete with certain others for internalization32,48, but the cellular AP2 concentration can be diminished by ≥90% with the uptake of certain ligands (such as lowdensity lipoprotein (lDl)) into clathrincoated vesicles being barely affected49–53. However, the same is not true for clathrin49,50,54. This indicates that adaptors besides AP2 operate in clathrinmediated endocytosis, and a myriad of monomeric adaptors, designated clathrinassociated sorting proteins (ClASPs) to distinguish them from AP2, have now been characterized. ClASPs almost invariably bind to both AP2 and clathrin through short peptide interaction motifs located in the disordered Cterminal region55. This creates a highly populated interaction web, in which the AP2 appendages and the clathrin terminal domain operate as organizational hubs6,55 (FIG. 4a). The numerous simultaneous contacts

Figure 3 | clathrin‑coated structures at the cell surface. a | Concentration of transferrin receptors at coat assembly zones in HeLa cells stably expressing a yellow fluorescent protein-tagged β2-subunit of AP-2 observed with time-resolved total internal reflection fluorescence microscopy. Fluorescent transferrin is initially diffusely soluble but it quickly concentrates on clustered transferrin receptors in AP-2-containing clathrin-coated structures on the ventral plasma membrane. b | Enlarged images, corresponding to the dashed boxes in part a, reveal two fundamentally important aspects of clathrin-mediated endocytosis. First, clathrin puncta occur at discrete sites on the plasma membrane that can range from diffraction-limited spots (arrows) to large assemblies (arrowheads). Second, selection and rapid concentration of cargoes, due to recognition of sorting signals in transmembrane proteins, occurs at these internalization zones as a prelude to uptake and delivery to the early endosome compartment. c | A freeze-etch micrograph of a representative region of the cytosolic face of the adherent plasma membrane from a HeLa cell. The image illustrates the extent of polymerized polyhedral clathrin lattices (purple) in these cells. It also shows flat, largely hexagonal sheets and the progression from pentagon-containing, shallow curved lattices to a deeply invaginated state just before membrane scission. The large, stable AP-2-positive structures seen by the live-cell imaging probably correspond to regions analogous to these extended arrays.

R e V i e W s





AP-2 ClathrinPtdIns(4,5)P2

AP180

Stonin 2

DAB2

ARH

β-arrestin

EPS15

EpsinsNUMB

HRB

[FY]XNPX[YF]

pSer or pThrSNAREs andfusion factors

Ubiquitin

a

b

[DE]XXXL[LIM]YXXØ

PtdIns(4,5)P2β-arrestin

LDLreceptor

EGFreceptor

GPCRTransferrinreceptor

CD4

DAB2 Epsin ARH

Clathrin Ubiquitin

β2-subunitα-subunit

AP-2

IntegrinA heterodimeric surface receptor that binds the extracellular matrix and couples to the actin cytoskeleton at the internal membrane surface.

that AP2 can establish with ClASPs and other accessory proteins further explain how AP2 can associate with the plasma membrane before binding to cargoes. In addition, because of the considerable connectivity between ClASPs, these alternative adaptors are still able to nucleate the assembly of fewer diminutive but

operational clathrincoated structures when AP2 levels are diminished by RNAi47,49,51,52,56. So, ClASPs can populate incipient bud sites in the apparent absence of AP2.

[FY]XNPX[YF]-selective CLASPs. The phosphoTyrbinding (PTB) domaincontaining subfamily of ClASPs decodes the [FY]XNPX[YF] class of signals, which, despite having an anchoring Tyr side chain, are not normally recognized by the μ2subunit of AP2 (TABLE 1). The PTB domain fold engages the [FY]XNPX[YF] signal in a βaugmentation — a temporary βstrand, with the NP residues stabilizing a βtight turn that re orients the main chain to present the [YF] side chain to an accepting pocket57 (FIG. 1c). Simultaneously, the PTB domain binds PtdIns(4,5)P2 through an adjacent basic contact site57. Showing clear cargo selectivity, ectopic expression of disabled 2 (DAB2) selectively enhances the uptake of [FY]XNPX[YF]containing, but not YXXØcontaining, cargoes47. Reciprocally, trans lational silencing of both DAB2 and autosomal recessive hypercholesterolemia (ARH) protein (also known as lDlRAP1) suppresses lowdensity lipo protein (lDl) uptake without similarly affecting transferrin51–53. DAB2 and ARH are therefore functionally redundant ClASPs that internalize lDl receptor family members in several tissues51–53.

The βchains of integrins contain an activating NPX[YF] sequence in the cytosolic portion that binds to the FERM domain of the focal adhesion protein talin58. The same sequence, or a distal flanking [Fv]XN[PIv]X[YF] sequence in some βintegrins, can bind to PTB domains without insideout integrin activation (that is, stimulated extracellular adhesion due to talin binding)58 and thereby operate as a sorting signal. Several recent studies document the involvement of PTB domaincontaining ClASPs in the endocytosis of integrins47,59–61. Perturbed integrin trafficking leads to defects in cell adhesion, spreading and locomotion. DAB2, NUMB, AP2 and clathrin coregionalize with focal adhesions before disassembly59,60, and RNAi of these coat components perturbs the turnover of focal adhesions60. Accordingly, overexpression of DAB2 accelerates cell spreading on a fibronectin matrix47. Here, the role of DAB2 and NumB is to drive endocytic remobilization of dissociated integrins to the leading edge of motile cells, allowing polarized assembly of new adhesions. Despite some discrepancy over where precisely on the cell surface DAB2 and NumB sort βintegrins59–62, it is clear that the NPX[FY] signal can alternate between binding talin to induce focal adhesions and binding ClASPs to mobilize integrins in a polarized manner. unlike lDl receptor uptake, integrin internalization is slow and depends heavily on AP2 (REFS 59–62), indicating that recognition of additional sorting signals, perhaps in the αsubunit, might be necessary. Several pathogens, including rheovirus62, adenovirus, Yersinia pseudotuberculosis, Staphylococcus aureus and uropathogenic Escherichia coli 63, use β1 integrin as an entry receptor, making PTB domaincontaining ClASPs clinically relevant components.

Figure 4 | The endocytic clathrin sorting interactome. a | The connectivity between the core coat components, clathrin and AP-2, phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P

2) of the phospholipid bilayer and characterized clathrin-associated sorting

proteins (CLASPs), along with the sorting signal recognized. These interactions, typically mediated by short peptide motifs that are often tandemly arrayed in intrinsically unstructured regions6,55 (BOX 2), are a hallmark of CLASPs and underpin their operation as cargo-selective coat constituents. b | A model for coincident packaging of an expanded repertoire of transmembrane cargoes in individual clathrin-coated regions that are populated by various CLASPs. For clarity, the assembling bud site is depicted before membrane deformation and invagination begins and only portions of the clathrin trimer are shown. The multiplicity of AP-2 appendage and clathrin interaction surfaces (BOX 2) presented allows different CLASPs to cluster in the nascent assemblage with reduced overall competition. Importantly, the β2-subunit of AP-2 binds to the clathrin heavy chain using two independent contact sites56,124 and, because of avidity effects, will expel any proteins that were previously bound to the β2-sandwich site at regions of extensively assembled lattices. This might provide some directionality to the assembly process6. However, the privileged β2-platform site, which engages the helical [DE]

nX

1-2FXX[FL]XXXR sequence (in which X is any amino acid), remains accessible.

Ø represents a bulky hydrophobic amino acid. ARH, autosomal recessive hypercholester-olemia (also known as LDLRAP1); DAB2, disabled 2; EGF, epithelial growth factor; EPS15, epidermal growth factor receptor substrate 15; GPCR, G protein-coupled receptor; HRB, HIV-1 Rev-binding protein (also known as AGFG1); LDL, low-density lipoprotein.

R e V i e W s


R e V i e W s



http://www.uniprot.org/uniprot/Q5SW96


FERM domainA tripartite domain found in talin and ERM proteins that binds to integrins, thereby activating their extracellular domain through conformational rearrangements.

Focal adhesionA large localized contact between a cell and the extracellular matrix. It is established through integrins and serves as an intracellular signalling station to arrange actin into stress fibres.

NUMBA PTB domain-containing CLASP, first identified in D. melanogaster, that is involved in asymmetric positioning of cell surface receptors.

Fibronectin A secreted extracellular matrix component that assembles into polymers on binding to integrins.

UBA domain(Ubiquitin-associated domain). A small three-helix fold that binds to ubiquitin monomers or ubiquitin chains.

ENTH domain (Epsin N-terminal homology domain). An all-α-helical module that binds to PtdIns(4,5)P2 and might change the properties of the underlying bilayer.

NPF motifAn Asn-Pro-Phe tripeptide sequence that binds to EH domains and is often tandemly arrayed in disordered polypeptide regions.

EH domain(EPS15 homology domain). An ~100-residue folded domain found in proteins that are involved in endocytosis and vesicular transport.

E3 ubiquitin ligaseAn enzyme that catalyses the transfer of a thioester-linked reactive ubiquitin to an acceptor Lys residue on the substrate protein and determines substrate specificity.

Ubiquitin-selective CLASPsNot all clathrindependent cargoes are taken up constitutively. Numerous signalling receptors, ion channels and transporters persist at the cell surface and are only selectively internalized, in a temporally defined manner. Typically, this requires posttranslational modification of the transmembrane cargo to tag it for uptake. In unicellular S. cerevisiae, the predominant internalization signal is the reversible conjugation of ubiquitin. A subfamily of ClASPs, including epsins and epidermal growth factor receptor substrate 15 (ePS15), which display overall architectural similarity and connectivity to PTB domaincontaining ClASPs, oversee the packaging of ubiquitylated cargoes into endocytic clathrin coats64,65 (FIG. 4a). Cargo selectivity comes from tandemly arrayed ubiquitininteracting motifs (uIms) (FIG. 1d). The yeast proteins ent1, ent2 and ede1 are functional orthologues of metazoan epsin 1, epsin 2 and ePS15, albeit in ede1 a Cterminal UBA domain replaces the two uIms. evidence that these proteins redundantly sort ubiquitylated cargoes comes from gene deletion experiments in S. cerevisiae. endocytosis of Ste2 is blocked in a strain that is deficient in ent1, ent2 and ede1 but can be reconstituted by reexpression of ent1 (REF. 66). To operate as an adaptor ent1 must be able to bind PtdIns(4,5)P2, ubiquitin and clathrin67. ent1, ent2 and epsins have a folded ENTH domain that promotes association with the plasma membrane. Both ePS15 and epsins bind strongly to AP2 appendages, epsins associate with ePS15 through the binding of NPF motifs to EH domains, and epsins also engage clathrin (FIG. 4a,b). These properties of epsins, as with DAB2, allow assembly of clathrin coats in the absence of AP2.

The ubiquitin signal. when it initially seemed that a single ubiquitin molecule could efficiently direct surface proteins to cortical actin patches, which represent the site of clathrinmediated uptake in S. cerevisiae, a beguiling dichotomy was formulated: polyubiquitin chains signal proteasomal degradation, whereas monoubiquitin directs endosomal trafficking68. mounting evidence questions whether this appealing idea is actually correct. In yeast, synthetically monoubiquitylated Ste2 is internalized, but the wildtype protein is normally multiply ubiquitylated69. In cultured cells, a single ubiquitin fused to the cytosolic domain of various transmembrane reporter proteins is a poor internalization signal compared with linearly linked tetraubiquitin70,71.

The epidermal growth factor receptor (eGFR) is often used as an example of a protein that undergoes ubiquitindependent endocytosis, but considerable controversy and confusion surround the mode of eGFR internalization. eGFR contains both YXXØ and dileu signals in the cytosolic domain, and its internalization is sensitive to knock down of AP2 by RNAi72,73. However, it is also clear that ubiquitin is sufficient for uptake of a kinasedefective eGFR mutant74. within 5 min of activation eGFR is normally polyubiquitylated, with the bulk of the additions being lys63linked chains75. The uIms of epsin 1 bind to ubiquitylated eGFR76 and to lys63 (and lys48)linked polyubiquitin, but not to monoubiquitin70,71. This indicates that epsin 1 and ePS15 preferentially recognize

lys63linked ubiquitin chains as a sorting signal. Indeed, a pair of Kaposi’s sarcomaassociated herpesvirusencoded transmembrane E3 ubiquitin ligases (K3 (also known as mIR1) and K5 (also known as mIR2)), which drive the clearance of several transmembrane immunoreceptors off the surface of infected cells to avoid detection by cytotoxic T or natural killer cells, synthesize lys63linked polyubiquitin77. lys63dependent uptake of cargo in yeast78–80 and mammalian81 systems is firmly established.

The basis of epsin 1 and ePS15 selectivity for lys63linked chains seems to be the length of the intervening linker between the tandemly arrayed uIms82,83. uIms engage ubiquitin as an αhelix (FIG. 1d), and short linkers with high helical propensity allow tandem uIms to be configured as a single uninterrupted helix in which the residues that are important for ubiquitin binding are aligned on one face82. In this orientation, uIms with a ~7residue linker have conformational selectivity for lys63linked ubiquitin chains82. Furthermore, although it has long been argued that monoubiquitylation is the main internalization signal in yeast, Rsp5 (the HeCT family e3 ligase that is involved in the trafficking of most transmembrane proteins84) is in fact tailored for synthesizing lys63 linkages of >12 ubiquitin molecules irrespective of the E2 enzyme partner85. The bias of endocytic e3 ligases towards lys63 linkage and the definition of the molecular basis for ‘linkagespecific avidity’82 argue that lys63linked polyubiquitin is generally a preferred sorting signal, not an exception. moreover, several e3 ligases involved in internalization occur in macromolecular complexes with deubiquitylating enzymes86,87, enabling dynamic synthesis and trimming of polyubiquitin chains to occur locally88. Notably, the major ubiquitin receptor on the 26S proteasome, regulatory subunit S5a (also known as Rpn10 and PSmD4), recognizes lys63linked89 as well as lys48linked ubiquitin. Because of the large number of cellular reactions that depend on ubiquitin, tightly regulated and localized cycles of ubiquitylation and disassembly are likely to prevent promiscuous or inappropriate events. Once the designated cargo has been sorted and clustered into a deeply invaginated bud, the membrane geo metry at the constricting base makes diffusional escape of transmembrane proteins improbable. Therefore, rev ersible sorting signals may be removed at this point, making bulk biochemical detection of ubiquitylated cargo challenging.

Intermediate E3 adaptors in S. cerevisiae. Although Rsp5 has three WW domains and some endogenous substrates do use PPXYtype motifs to bind these domains, no transmembrane cargo seems to engage Rsp5 directly through these for internalization90,91. Instead, another posttranslational modification, phosphorylation, is required for the internalization of numerous receptors and transporters80,84,92. when phosphorylated, the manganese transporter Smf1 is bound by two functionally redundant proteins (extracellular mutant protein 21 (ecm21) and Chs5 Spa2 rescue protein 2 (Csr2))90. Remarkably, these proteins are members of the arrestin superfamily, which are only distantly related in sequence to the βarrestins (see below) but are likely to have a conserved fold90,91,93.

R e V i e W s










E2 enzymeThe second intermediate in the three-step process of ubiquitylation. It delivers activated ubiquitin to an E3 ligase.

Deubiquitylating enzymeAn isopeptidase that removes ubiquitin monomers from acceptor proteins or assembled ubiquitin chains, thereby replenishing the pool of ubiquitin and allowing further ubiquitylation.

26S proteasomeA large proteolytic complex that generally degrades polyubiquitylated proteins in the cytosol.

WW domainA small modular domain containing two crucial Trp residues that engage short Pro-rich interaction peptides with micromolar affinity to mediate protein–protein interactions.

G protein-coupled receptor(GPCR). A member of the largest class of signalling receptors, which are defined by seven transmembrane α-helices. GPCRs couple a wide range of stimuli, including light, odorants, hormones and small bioactive molecules, to various intracellular signal transduction pathways.

Heterotrimeric G proteinA member of a family of membrane-apposed effectors of GPCRs that are composed of α, β and γ subunits and stimulate adenylyl cyclase, phospholipase C, ion channels or several other signalling molecules.

Longin domainA modular domain found in several functionally discrete proteins that are involved in protein trafficking.

RetromerA non-clathrin-based sorting coat that is assembled on endosomal tubules.

Nine structurally related proteins, termed arrestinrelated trafficking (ART) adaptors91 (also known as αarrestins93), are encoded in the S. cerevisiae genome, each with one or more PPXY interaction motifs at the C terminus. The PPXY motifs are necessary for αarrestins to link cargo to Rsp5 (REFS 90,91); fusion of the PPXY sequence to the Arg transporter Can1 bypasses the requirement of ART for uptake91. ARTs therefore operate as intermediary e3 ligase adaptors to recruit Rsp5, which can then ubiquitylate both the transmembrane cargo molecule and the ART protein within a ternary complex90,91. Synchronous and extensive ubiquitylation of αarrestin90 might bolster the partitioning of the cargo–ART complex into cortical actin structures to optimize internalization.

β-arrestins in multicellular organismsIn vertebrates, ClASPs of the arrestin superfamily have a wellestablished role in the internalization of many, but not all, G protein-coupled receptors (GPCRs)94. ligand stimu lation leads to hyperphosphorylation of GPCRs, often in the Cterminal cytosolic domain. The high density of receptorattached phosphate groups recruits soluble βarrestins, thereby uncoupling the activated GPCR from the relevant heterotrimeric G protein or proteins. On engaging the cytosolic aspect of activated GPCRs, the AP2binding motif in βarrestin, which otherwise stabilizes the basal, inactive state, becomes accessible95,96. By undergoing a strandtohelix transition, this motif interacts with the β2subunit appendage of AP2, thereby guiding the GPCR–βarrestin complex to preexisting clathrincoated structures97,98. A clathrinbinding sequence present in the Cterminal stretch of βarrestin that restructures when GPCR binds also contributes to coat associations56,96. This mode of engaging the clathrin machinery to effectively internalize stimulated receptors from the surface reveals another variation in cargo selection: temporal activation of a dormant cytosolic ClASP drives prompt and selective uptake. In this case, receptors are clearly the primary contact for the initial deposition of βarrestin on the plasma membrane, although the βarrestin core does have a dedicated PtdIns(4,5)P2binding site99.

Interestingly, there is limited but strong sequence conservation (the arrestin motif 91) between βarrestins and ARTs. This region encompasses residues of the phosphosensing polar core that maintain βarrestin in the closed, basal conformation, suggesting that ARTs might analogously reorganize on engaging a phosphorylated receptor, perhaps properly orienting the distal PPXY motifs for Rsp5 capture. why the array of apparently cargoselective ARTs91 in S. cerevisiae has been augmented in vertebrates with a battery of ClASPs that all directly engage the principal coat machinery probably relates to the complex requirements of multicellular life.

Recognition of membrane fusion factorslittle is currently known about the packaging of SNARes, which generally lack known sorting signals and do not seem to be ubiquitylated, into surface coats. A structural counterpoint to the typical recognition of short poly peptide signals by modular recognition domains in adaptors is the sorting of a vesicleassociated membrane

protein 7 (vAmP7) SNARe complex into clathrincoated buds. vAmP7 is a longin domaincontaining SNARe that is sorted by HIv1 Revbinding protein (HRB; also known as AGFG1) at the plasma membrane100. The architecture of HRB is reminiscent of DAB2 and epsins: a folded Nterminal ArfGAP domain is followed by an essentially unstructured tract that contains AP2, clathrin and ePS15binding motifs (FIG. 4a). HRB localizes to surface clathrin puncta, and RNAi of HRB results in stagnation of vAmP7 at the plasma membrane100,101. For sorting, a stretch of ~20 residues of HRB orders around the globular longin domain of vAmP7, projecting 5 leu residues into an extended hydrophobic furrow100 (FIG. 2c). The recognition process is strikingly opposite to the canonical process, in which a folded domain recognizes an extended sorting signal. Here, a disordered segment of the ClASP contacts a folded cargo sorting determinant. most importantly, in uncomplexed vAmP7 the longin–HRB binding surface is occupied intramolecularly by a SNARe helix. This means that the uncomplexed vAmP7 will not be recognized and so HRB will retrieve only thermodynamically stable cisSNARepaired complexes from the surface100.

SNARes have a crucial role at nerve terminals, working in conjunction with synaptotagmin 1, which imposes Ca2+ sensitivity on SNARemediated synaptic vesicle fusion. Following neurotransmitter exocytosis, synaptotagmin 1 is promptly reinternalized by clathrin coats to allow synaptic vesicle recycling102. A dedicated neuronal ClASP, stonin 2, sorts synaptotagmin 1; a μhomology domain in stonin 2 binds to the edge of the folded C2A domain of synaptotagmin 1 (REF. 102). The structure of the μhomology domain is highly related to the μ2subunit of AP2, but despite cargo engagement using a surface βstrand, recognition is molecularly distinct and occurs on a different subdomain of the μfold102. enrichment of stonin 2 in clathrin puncta depends on binding to AP2. As an expanded cargo repertoire depends on ClASPs that contact AP2, how is competition at the AP2 hub prevented? The answer seems to lie in the multiplicity of interaction surfaces that are mapped on the two appendage domains (BOX 2).

Multiplexed sorting signalsProteins that repeatedly cycle between the TGN, endosomes and the plasma membrane, such as the mannose 6phosphate receptors, have several discrete trafficking signals in the cytosolic domain103. each signal orchestrates a sorting event at a different membrane compartment, directing the receptor into AP1, AP2 or retromer sorting coats. Other receptors have apparent redundancy in entry signals. Alascanning mutagenesis of Pselectin shows only a minor role for Tyr777 in the lXXYXXF motif, whereas distal F780A and N782A mutations have a strong effect25. It seems that a variant [FY]XNPX[YF]type sorting signal, decoded by NumB, predominates in Pselectin104. lDl receptorrelated protein 1 (lRP1) is a large, multifunctional receptor that participates in lipoprotein metabolism and other signalling processes. lRP1 has YXXØ, [FY]XNPX[YF] and dileu signals in the cytosolic domain, with YXXØ being the main internalization determinant105. Similarly, the cytosolic domain

R e V i e W s


R e V i e W s





COPII(Coat protein II). A complex that is involved in packaging cargoes into transport vesicles leaving the ER from discrete exit sites.

DeltaA transmembrane ligand for the Notch receptor that internalizes, along with the extracellular domain of Notch, after proteolysis triggered by Delta engagement.

Notch signallingA conserved pathway in which proteolysis of the ligand-bound Notch receptor releases the intracytoplasmic domain, which enters the nucleus and regulates gene expression.

Imaginal discsSheets of epithelial cells in D. melanogaster larvae that, during pupation, develop into different adult body parts and structures.

of the hyaluronan receptor, which is involved in hepatic glycosaminoglycan clearance, has several redundant signals106. multiple signals can make uptake more rapid105, more robust and less dependent on a single ClASP.

Other receptors use different signals for uptake depending on whether a recycling or degradative trajectory will be followed. Proteinaseactivated receptor 1 (PAR1) is an atypical GPCR that is stimulated by thrombin, which catalyses irreversible PAR1 activation by proteolysis. In the absence of ligand, PAR1 undergoes slow constitutive internalization to generate a reserve pool that can repopulate the surface after clearance of activated, cleaved PAR1. This constitutive uptake requires the de ubiquitylation of lys421 and lys422, which are interlaced in a YKKl sorting signal107. However, internalization of PAR1, which necessarily traffics to the lysosome because it cannot be returned to the inactive state, is AP2 and βarrestin independent107. A different permutation occurs with the type 1 interferonα/β receptor. Here, phosphorylationinduced ubiquitylation of several lys residues in the cytosolic domain is necessary to expose a YveF signal for productive internalization through AP2 (REF. 108).

Dedicated sorting stations?Because of the clear diversification of the sorting machinery at surface clathrincoated structures, a logical extension is the possibility of differentially cargoselective sorting stations on the plasma membrane. Possible reasons to presort cargo at the plasma membrane are to avoid competition, to kinetically segregate entering cargo types or to generate specialized endosomes with distinct functions at different intracellular locations109,110. There are precedents for this: at eR exit sites in S. cerevisiae, three different populations of COPII vesicles are apparently formed that package different cargo subsets111. The TGN is a complex sorting station that directs various cargoes into different transport intermediates destined for spatially discrete acceptor membranes, particularly in polarized epithelia. Several groups have documented that different constitutively internalized receptors112, constitutive and signalling receptors113,114 or different types of signalling receptors115,116 tend to segregate into spatially resolved sorting patches at the plasma membrane. However, the idea of compositionally distinct clathrin coats is currently a hotly debated topic, principally because of decades of accumulated data showing that a wide range of different cargoes copopulate clathrincoated vesicles and that incoming vesicle components are rapidly homogenized by repeated rounds of homotypic earlyendosome fusion117,118.

endogenous βarrestin 2 occupies only a subset of clathrincoated structures following GPCR activation, but overexpressed βarrestin 2 can occupy most clathrincoated structures116. Given that βarrestin 2 can access all clathrin spots at the plasma membrane, irrespective of ongoing constitutive endocytic activity56,96, βarrestin must have access to a fairly privileged surface on AP2 (BOX 2). Activated eGFR also moves into preformed clathrin patches73. widespread colocalization of eGFR with AP2 suggests that epsins and ePS15 must be general constituents of clathrin coats at the surface, which has now been confirmed. Also, because a Delta–lDl

receptor chimaera in Drosophila melanogaster can bypass the requirement for ubiquitin and the epsin homologue liquid facets (lqf) in Notch signalling in imaginal discs119, PTB domaincontaining adaptors must be regularly positioned in coats that normally sort Delta.

In fact, the way that clathrincoated structures form imposes constraints on what cargoes can be packaged and when. AP2, clathrin and ClASPs all seem to enter and exit the assembly zone repeatedly in the course of coatedvesicle assembly. Diffusional exchange is not entirely unexpected as the system is built around interaction motifs embedded in intrinsically disordered, flexible linkers that dock with low individual affinities onto structured domains that act as hubs6. Because of the multi valent and stochastic nature of the assembly process6,67, and the presence of ClASPs and AP2 in most surface puncta47,51,59,96,100,120,121, uniform sorting capability could be anticipated for all patches. How, then, could specialized subsets of coats assemble?

Regulation of CLASPs. In addition to the μ2subunit of AP2, AAK1 phosphorylates the Thr102 residue in NumB, which is positioned in the PTB domain122. Overexpression of AAK1 leads to a dramatic relocalization of NumB from the plasma membrane to perinuclear endosomes, whereas a T102A mutant is found in surface puncta122. NumB is also phosphorylated by calcium/calmodulindependent protein kinase (CAmK) or atypical protein kinase C (aPKC). CAmKmediated phosphorylation of the Ser264 or Ser283 residues in NumB recruits 1433 (a regu lator of phosphoSer/Thrbinding proteins), thereby inactivating AP2 binding123. likewise, aPKC phosphorylates Ser264 and Ser283, as well as Ser7, and overexpressing aPKC leads to the disappearance of NumB from surface clathrin spots59. This suggests that there are several signalling pathways that regulate the presence of NumB in coated buds; for Pselectin, inactivation of NumB would allow accumulation on the surface and enhanced leukocyte adhesion. Differential phosphoryl ation by kinases that are positioned at restricted intracellular locations could thereby lead to the asymmetric incorporation of ClASPs into surface clathrin coats59. NumB is probably more amenable to this type of regulation because it has very limited intrinsic AP2binding ability (FIG. 4a).

Alternatively, preferential entry of receptors into subsets of surface clathrin coats could be due to regional immobilization or asymmetrical partitioning into biophysically distinct microdomains, with release confined to only some surface puncta on activation, or could be due to positive cooperativity115,116. Also, because there are multiple ClASP occupancy options, receptors that display multiplexed sorting signals might directly influence the packaging of other molecules locally and so skew the density or representation of some transmembrane cargoes. Segregation might therefore reflect a secondary consequence of alternative mechanisms of clustering macro molecules in emergent transport vesicles and not an intrinsic difference in the properties of the clathrin coat.

Another possibility is that selective deactivation of ClASPs in nascent buds locally biases cargo loading. The Tyr888 residue in the β2subunit appendage of AP2

R e V i e W s





1. Bonifacino, J. S. & Traub, L. M. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72, 395–447 (2003).

2. Robinson, M. S. Adaptable adaptors for coated vesicles. Trends Cell Biol. 14, 167–174 (2004).

3. Maldonado-Baez, L. & Wendland, B. Endocytic adaptors: recruiters, coordinators and regulators. Trends Cell Biol. 16, 505–513 (2006).

4. Leon, S. & Haguenauer-Tsapis, R. Ubiquitin ligase adaptors: regulators of ubiquitylation and

endocytosis of plasma membrane proteins. Exp. Cell Res. 315, 1574–1583 (2009).

5. Vicinanza, M., D’Angelo, G., Di Campli, A. & De Matteis, M. A. Function and dysfunction of the PI system in membrane trafficking. EMBO J. 27, 2457–2470 (2008).

6. Schmid, E. M. & McMahon, H. T. Integrating molecular and network biology to decode endocytosis. Nature 448, 883–888 (2007).

7. Robertson, A. S., Smythe, E. & Ayscough, K. R. Functions of actin in endocytosis. Cell. Mol. Life Sci.

66, 2049–2065 (2009).8. Mayor, S. & Pagano, R. E. Pathways of clathrin-

independent endocytosis. Nature Rev. Mol. Cell Biol. 8, 603–612 (2007).

9. Pearse, B. M. Receptors compete for adaptors found in plasma membrane coated pits. EMBO J. 7, 3331–3336 (1988).

10. Matsui, W. & Kirchhausen, T. Stabilization of clathrin coats by the core of the clathrin-associated protein complex AP-2. Biochemistry 29, 10791–10798 (1990).

is phosphorylated, and this residue forms a vital part of the privileged platform region that binds βarrestin and ARH95,124. Receptordependent phosphorylation of Tyr888 could therefore prevent the efficient local packaging of GPCRs or lDl receptors. likewise, regional phosphorylation might modulate the propensity of the epsin and ePS15 tandem uIms to adopt a helical conformation and therefore recognize ubiquitin chains. A distinct mechanism for negatively regulating the loading capacity of uIms is through ubiquilin 2 (also known as PlIC2), which has an Nterminal ubiquitinlike domain and a Cterminal uBA domain125. The ubiquitinlike domain, which binds to uIms with higher affinity than ubiquitin, can compete for access to ePS15 or epsin ClASPs and retard clustering in clathrin coats125.

Separation precedes congregation? Despite plausible possible mechanisms for the assembly of differentially selective coats at the surface, there are theoretical objections to their use. It is widely accepted that incoming vesicle components quickly converge in the early endosome compartment117,118. why do endosomes have specialized domains and the characteristic tubulo vesicular morphology that is inextricably linked to their operation as centralized sorting stations if different endosome populations can be generated by presorting at the plasma membrane? Oddly, two ADPresponsive GPCRs, the P2Y1 and P2Y12 purinergic receptors, are initially located in distinct coated vesicles but rapidly merge in peripheral early endosomes115. moreover, if presorting occurs at the surface for delivery to partic ular endosomes, how are these different endosomal acceptors recognized and how is this coupled with the packaging of the necessary recognition tethers and SNARes at nascent buds? Gurken, a membranebound transforming growth factorα that is secreted to different surfaces of the developing D. melanogaster oocyte to establish the anteroposterior and dorsoventral axes, is exported from only a subset of eR exit sites, but this crucially depends on polarized mRNA localization126. The COPII machinery at gurkenexporting zones is not different to other COPII exit sites on the continuous eR, and perturbing gurken mRNA localization results in unrestricted export of the protein in all eR vesicles126. Obviously, what needs to be established next is whether the apparent specialization of clathrin puncta reflects differential segregation and carry ing capacity of surfacederived transporters, or rather reflects the optimization of signalling platforms spatially and temporally, or is merely an indication of different mechanisms for concentrating and packaging various cargoes into clathrincoated structures.

ConclusionsCargo selection in S. cerevisiae apparently uses a limited set of molecular connection elements. metazoans have replaced or diversified the family of ARTs, which do not engage core endocytic components and function as intermediate adaptors, with a broad array of additional sorting signals and cognate ClASPs that physically associate with clathrin and AP2. This variety of adaptors far outstrips anything known for other sorting coats. Current molecular descriptions of sorting mechanisms at clathrincoated buds are beginning to reveal the logic behind the multitude of internalization signals in endocytosis. An important goal is to obtain a complete inventory of sorting signals and cognate ClASPs. Other signals almost certainly remain to be characterized. For example, Niemann–Pick C1like protein 1 (NPC1l1) is a polytopic transmembrane protein that regulates dietary cholesterol uptake by enterocytes. when cellular cholesterol levels are low, NPC1l1 trans locates from endosomes to the apical surface. As membrane cholesterol levels normalize, NPC1l1 returns to its reserve endosomal location127. The nature of the cholesterolsensitive sorting signal is completely unknown: are mammalian ART family proteins involved?

Beside the obvious potential to avoid competition, in numerous cases it is still not clear why one signal would be favoured over another. Perhaps the signal specifies a defined sorting itinerary or dictates clustering into a dedicated subpopulation of coats at the cell surface? Clathrinmediated endocytosis subserves different downstream cellular responses such as migration, trans criptional activation and proliferation. whether particular sorting signals are tailored to specific receptor concentrations at the cell surface and whether this relates to the kinetic parameters that determine the efficiency of uptake are not known. It will also be important to establish whether the cargo checkpoint that assures productive assembly is restricted to AP2, or whether the occupancy of other ClASPs is also monitored while the coat polymerizes. Because SNARe loading is crucial for subsequent endosome fusion, it seems likely that successful incorporation of other cargoes must be checked. The fact that coats still bud when AP2 is almost completely absent is highly suggestive of additional cargo monitoring events. How this proofreading operates at the molecular level and whether selective deactivation of individual ClASPs favours cargoselective buds need to be investigated. The rapid pace of investigation will undoubtedly provide answers, and continued analysis of clathrinmediated endo cytosis in various model systems such as D. melanogaster and zebrafish is certain to provide greater insight into development and organ physiology.

R e V i e W s


R e V i e W s


11. Collins, B. M., McCoy, A. J., Kent, H. M., Evans, P. R. & Owen, D. J. Molecular architecture and functional model of the endocytic AP2 complex. Cell 109, 523–535 (2002).A landmark structural study revealing the molecular basis of AP-2 heterotetrameric core assembly and arrangement and the resulting constraints on cargo recognition.

12. Mitsunari, T. et al. Clathrin adaptor AP-2 is essential for early embryonal development. Mol. Cell. Biol. 25, 9318–9323 (2005).

13. Shim, J. & Lee, J. Molecular genetic analysis of apm-2 and aps-2, genes encoding the medium and small chains of the AP-2 clathrin-associated protein complex in the nematode Caenorhabditis elegans. Mol. Cells 10, 309–316 (2000).

14. Austin, C. D. et al. Death-receptor activation halts clathrin-dependent endocytosis. Proc. Natl Acad. Sci. USA 103, 10283–10288 (2006).

15. Huang, K. M., D’Hondt, K., Riezman, H. & Lemmon, S. K. Clathrin functions in the absence of heterotetrameric adaptors and AP180-related proteins in yeast. EMBO J. 18, 3897–3908 (1999).

16. Yeung, B. G., Phan, H. L. & Payne, G. S. Adaptor complex-independent clathrin function in yeast. Mol. Biol. Cell 10, 3643–3659 (1999).

17. Ohno, H. et al. Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science 269, 1872–1875 (1995).

18. Owen, D. J. & Evans, P. R. A structural explanation for the recognition of tyrosine-based endocytotic signals. Science 282, 1327–1332 (1998).References 17 and 18 identify the μ2-subunit as the AP-2 adaptor subunit that physically recognizes YXXØ sorting signals and highlight the structural basis of the recognition process.

19. Olusanya, O., Andrews, P. D., Swedlow, J. R. & Smythe, E. Phosphorylation of threonine 156 of the μ2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo. Curr. Biol. 11, 896–900 (2001).Demontrates that YXXØ signal recognition by AP-2 is positively regulated by the phosphorylation of the Thr156 residue of the μ2-subunit.

20. Ricotta, D., Conner, S. D., Schmid, S. L., von Figura, K. & Honing, S. Phosphorylation of the AP2 μ subunit by AAK1 mediates high affinity binding to membrane protein sorting signals. J. Cell Biol. 156, 791–795 (2002).

21. Höning, S. et al. Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2. Mol. Cell 18, 519–531 (2005).

22. Semerdjieva, S. et al. Coordinated regulation of AP2 uncoating from clathrin-coated vesicles by rab5 and hRME-6. J. Cell Biol. 183, 499–511 (2008).Reports that the timely exit of AP-2 from budded vesicles is supported by RAB5-dependent dephosphorylation of Thr156 and PtdIns(4,5)P2.

23. Nesterov, A., Carter, R. E., Sorkina, T., Gill, G. N. & Sorkin, A. Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant μ2 subunit and its effects on endocytosis. EMBO J. 18, 2489–2499 (1999).

24. Motley, A. M. et al. Functional analysis of AP-2 α and μ2 subunits. Mol. Biol. Cell 17, 5298–5308 (2006).

25. Owen, D. J., Setiadi, H., Evans, P. R., McEver, R. P. & Green, S. A. A third specificity-determining site in μ2 adaptin for sequences upstream of YxxΦ sorting motifs. Traffic 2, 105–110 (2001).

26. Kittler, J. T. et al. Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor γ2 subunit. Proc. Natl Acad. Sci. USA 105, 3616–3621 (2008).

27. Royle, S. J. et al. Non-canonical YXXGΦ endocytic motifs: recognition by AP2 and preferential utilization in P2X4 receptors. J. Cell Sci. 118, 3073–3080 (2005).

28. Neil, S. J., Zang, T. & Bieniasz, P. D. Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature 451, 425–430 (2008).

29. Van Damme, N. & Guatelli, J. HIV-1 Vpu inhibits accumulation of the envelope glycoprotein within clathrin-coated, Gag-containing endosomes. Cell. Microbiol. 10, 1040–1057 (2008).

30. Rollason, R., Korolchuk, V., Hamilton, C., Schu, P. & Banting, G. Clathrin-mediated endocytosis of a lipid-raft-associated protein is mediated through a dual tyrosine motif. J. Cell Sci. 120, 3850–3858 (2007).

31. Masuyama, N. et al. HM1.24 is internalized from lipid rafts by clathrin-mediated endocytosis through

interaction with α-adaptin. J. Biol. Chem. 284, 15927–15941 (2009).

32. Marks, M. S., Woodruff, L., Ohno, H. & Bonifacino, J. S. Protein targeting by tyrosine- and di-leucine-based signals: evidence for distinct saturable components. J. Cell Biol. 135, 341–354 (1996).

33. Kelly, B. T. et al. A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex. Nature 456, 976–979 (2008).Crystals of [DE]XXX[LIM] peptides bound to the heterotetrameric AP-2 core reveal how diLeu signals are accommodated by the adaptor and that the entrance of cargoes is regulated.

34. Huang, F., Jiang, X. & Sorkin, A. Tyrosine phosphorylation of the β2 subunit of clathrin adaptor complex AP-2 reveals the role of a di-leucine motif in the epidermal growth factor receptor trafficking. J. Biol. Chem. 278, 43411–43417 (2003).

35. Lee, I., Doray, B., Govero, J. & Kornfeld, S. Binding of cargo sorting signals to AP-1 enhances its association with ADP ribosylation factor 1–GTP. J. Cell Biol. 180, 467–472 (2008).

36. Pitcher, C., Honing, S., Fingerhut, A., Bowers, K. & Marsh, M. Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation. Mol. Biol. Cell 10, 677–691 (1999).

37. Laguette, N. et al. Nef-induced CD4 endocytosis in human immunodeficiency virus type 1 host cells: role of the p56lck kinase. J. Virol. 83, 7117–7128 (2009).

38. Craig, H. M., Pandori, M. W. & Guatelli, J. C. Interaction of HIV-1 Nef with the cellular dileucine-based sorting pathway is required for CD4 down-regulation and optimal viral infectivity. Proc. Natl Acad. Sci. USA 95, 11229–11234 (1998).

39. Lindwasser, O. W. et al. A diacidic motif in human immunodeficiency virus type 1 Nef is a novel determinant of binding to AP-2. J. Virol. 82, 1166–1174 (2008).

40. Chaudhuri, R., Mattera, R., Lindwasser, O. W., Robinson, M. S. & Bonifacino, J. S. A basic patch on α-adaptin required for binding of HIV-1 Nef and cooperative assembly of a CD4–Nef–AP-2 complex. J. Virol. 83, 2518–2530 (2009).

41. Loerke, D. et al. Cargo and dynamin regulate clathrin-coated pit maturation. PLoS Biol. 7, e57 (2009).Detailed computational analysis of coat dynamics and internalization in BS-C-1 cells provides novel mechanistic insight into substages of assembly and cargo capture.

42. Santini, F., Marks, M. S. & Keen, J. H. Endocytic clathrin-coated pit formation is independent of receptor internalization signal levels. Mol. Biol. Cell 9, 1177–1194 (1998).

43. Ehrlich, M. et al. Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell 118, 591–605 (2004).Time-resolved imaging of surface clathrin coat behaviour in BS-C-1 cells, showing astonishing uniformity of budding structures and correlating bud failure with a paucity of packaged cargo.

44. Merrifield, C. J., Perrais, D. & Zenisek, D. Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell 121, 593–606 (2005).Using an intelligent oscillating pH imaging format, precise entry of cargo molecules into the cell interior is catalogued, repeated budding events from large, long-lived clathrin-coated structures are observed and actin involvement is shown.

45. Bellve, K. D. et al. Plasma membrane domains specialized for clathrin-mediated endocytosis in primary cells. J. Biol. Chem. 281, 16139–16146 (2006).

46. Keyel, P. A., Watkins, S. C. & Traub, L. M. Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy. J. Biol. Chem. 279, 13190–13204 (2004).

47. Chetrit, D., Ziv, N. & Ehrlich, M. Dab2 regulates clathrin assembly and cell spreading. Biochem. J. 418, 701–715 (2008).

48. Warren, R. A., Green, F. A., Stenberg, P. E. & Enns, C. A. Distinct saturable pathways for the endocytosis of different tyrosine motifs. J. Biol. Chem. 273, 17056–17063 (1998).

49. Motley, A., Bright, N. A., Seaman, M. N. & Robinson, M. S. Clathrin-mediated endocytosis in AP-2-depleted cells. J. Cell Biol. 162, 909–918 (2003).Together with reference 50, this report details the somewhat surprising finding that internalization of

subsets of clathrin-dependent cargo continues efficiently in cells that have been depleted of AP-2 by RNAi.

50. Hinrichsen, L., Harborth, J., Andrees, L., Weber, K. & Ungewickell, E. J. Effect of clathrin heavy chain- and α-adaptin specific small interfering RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells. J. Biol. Chem. 278, 45160–45170 (2003).

51. Keyel, P. A. et al. A single common portal for clathrin-mediated endocytosis of distinct cargo governed by cargo-selective adaptors. Mol. Biol. Cell 17, 4300–4317 (2006).

52. Maurer, M. E. & Cooper, J. A. The adaptor protein Dab2 sorts LDL receptors into coated pits independently of AP-2 and ARH. J. Cell Sci. 119, 4235–4246 (2006).

53. Eden, E. R., Sun, X. M., Patel, D. D. & Soutar, A. K. Adaptor protein Disabled-2 modulates low density lipoprotein (LDL) receptor synthesis in fibroblasts from patients with autosomal recessive hypercholesterolemia. Hum. Mol. Genet. 16, 2751–2759 (2007).

54. Moskowitz, H. S., Yokoyama, C. T. & Ryan, T. A. Highly cooperative control of endocytosis by clathrin. Mol. Biol. Cell 16, 1769–1776 (2005).

55. Owen, D. J., Collins, B. M. & Evans, P. R. Adaptors for clathrin coats: structure and function. Annu. Rev. Cell Dev. Biol. 20, 153–191 (2004).

56. Keyel, P. A. et al. The AP-2 adaptor β2 appendage scaffolds alternate cargo endocytosis. Mol. Biol. Cell 19, 5309–5326 (2008).

57. Stolt, P. C. & Bock, H. H. Modulation of lipoprotein receptor functions by intracellular adaptor proteins. Cell. Signal. 18, 1560–1571 (2006).

58. Wegener, K. L. et al. Structural basis of integrin activation by talin. Cell 128, 171–182 (2007).

59. Nishimura, T. & Kaibuchi, K. Numb controls integrin endocytosis for directional cell migration with aPKC and PAR-3. Dev. Cell 13, 15–28 (2007).

60. Chao, W. T. & Kunz, J. Focal adhesion disassembly requires clathrin-dependent endocytosis of integrins. FEBS Lett. 583, 1337–1343 (2009).

61. Teckchandani, A. et al. Quantitative proteomics identifies a Dab2/integrin module regulating cell migration. J. Cell Biol. 86, 99–111 (2009).

62. Maginnis, M. S. et al. NPXY motifs in the β1 integrin cytoplasmic tail are required for functional reovirus entry. J. Virol. 82, 3181–3191 (2008).

63. Eto, D. S., Gordon, H. B., Dhakal, B. K., Jones, T. A. & Mulvey, M. A. Clathrin, AP-2, and the NPXY-binding subset of alternate endocytic adaptors facilitate FimH-mediated bacterial invasion of host cells. Cell. Microbiol. 10, 2553–2567 (2008).

64. Wendland, B. Epsins: adaptors in endocytosis? Nature Rev. Mol. Cell Biol. 3, 971–977 (2002).

65. Sorkina, T. et al. RNA interference screen reveals an essential role of Nedd4–2 in dopamine transporter ubiquitination and endocytosis. J. Neurosci. 26, 8195–8205 (2006).

66. Shih, S. C. et al. Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis. Nature Cell Biol. 4, 389–393 (2002).

67. Aguilar, R. C., Watson, H. A. & Wendland, B. The yeast Epsin Ent1 is recruited to membranes through multiple independent interactions. J. Biol. Chem. 278, 10737–10743 (2003).

68. Hicke, L. Protein regulation by monoubiquitin. Nature Rev. Mol. Cell Biol. 2, 195–201 (2001).

69. Terrell, J., Shih, S., Dunn, R. & Hicke, L. A function for monoubiquitination in the internalization of a G protein-coupled receptor. Mol. Cell 1, 193–202 (1998).

70. Barriere, H. et al. Molecular basis of Ub-dependent internalization of membrane proteins in mammalian cells. Traffic 7, 282–297 (2006).

71. Hawryluk, M. J. et al. Epsin 1 is a polyubiquitin-selective clathrin-associated sorting protein. Traffic 7, 262–281 (2006).

72. Huang, F., Khvorova, A., Marshall, W. & Sorkin, A. Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference. J. Biol. Chem. 279, 16657–16661 (2004).

73. Rappoport, J. Z. & Simon, S. M. Endocytic trafficking of activated EGFR is AP-2 dependent and occurs through preformed clathrin spots. J. Cell Sci. 122, 1301–1305 (2009).

74. Huang, F., Goh, L. K. & Sorkin, A. EGF receptor ubiquitination is not necessary for its internalization. Proc. Natl Acad. Sci. USA 104, 16904–16909 (2007).

75. Huang, F., Kirkpatrick, D., Jiang, X., Gygi, S. & Sorkin, A. Differential regulation of EGF receptor internalization and degradation by multiubiquitination within the kinase domain. Mol. Cell 21, 737–748 (2006).

R e V i e W s




76. Kazazic, M. et al. Epsin 1 is involved in recruitment of ubiquitinated EGF receptors into clathrin-coated pits. Traffic 10, 235–245 (2009).

77. Duncan, L. M. et al. Lysine-63 linked ubiquitination is required for endolysosomal degradation of class I molecules. EMBO J. 25, 1635–1645 (2006).

78. Galan, J. M. & Haguenauer-Tsapis, R. Ubiquitin Lys63 is involved in ubiquitination of a yeast plasma membrane protein. EMBO J. 16, 5847–5854 (1997).

79. Springael, J. Y., Galan, J. M., Haguenauer-Tsapis, R. & Andre, B. NH4+-induced down-regulation of the Saccharomyces cerevisiae Gap1p permease involves its ubiquitination with lysine-63-linked chains. J. Cell Sci. 112 1375–1383 (1999).

80. Paiva, S. et al. Glucose-induced ubiquitylation and endocytosis of the yeast JEN1 transporter: role of K63-linked ubiquitin chains. J. Biol. Chem. 284, 19228–19236 (2009).

81. Geetha, T., Jiang, J. & Wooten, M. W. Lysine 63 polyubiquitination of the nerve growth factor receptor TrkA directs internalization and signaling. Mol. Cell 20, 301–312 (2005).

82. Sims, J. J. & Cohen, R. E. Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference of rap80. Mol. Cell 33, 775–783 (2009).References 82 and 83 nicely explain how suitably positioned tandem UIMs are selective for Lys63-linked ubiquitin chains.

83. Sato, Y. et al. Structural basis for specific recognition of Lys 63-linked polyubiquitin chains by tandem UIMs of RAP80. EMBO J. 18 Jun 2009 (doi:10.1038/emboj.2009.160).

84. Belgareh-Touze, N. et al. Versatile role of the yeast ubiquitin ligase Rsp5p in intracellular trafficking. Biochem. Soc. Trans. 36, 791–796 (2008).

85. Kim, H. C. & Huibregtse, J. M. Polyubiquitination by HECT E3s and the determinants of chain type specificity. Mol. Cell. Biol. 29, 3307–3318 (2009).

86. Kee, Y., Lyon, N. & Huibregtse, J. M. The Rsp5 ubiquitin ligase is coupled to and antagonized by the Ubp2 deubiquitinating enzyme. EMBO J. 24, 2414–2424 (2005).

87. Shenoy, S. K. et al. β-Arrestin-dependent signaling and trafficking of 7-transmembrane receptors is reciprocally regulated by the deubiquitinase USP33 and the E3 ligase Mdm2. Proc. Natl Acad. Sci. USA 106, 6650–6655 (2009).

88. Kee, Y., Munoz, W., Lyon, N. & Huibregtse, J. M. The Ubp2 deubiquitinating enzyme modulates Rsp5-dependent K63-linked polyubiquitin conjugates in Saccharomyces cerevisiae. J. Biol. Chem. 281, 36724–36731 (2006).

89. Saeki, Y. et al. Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome. EMBO J. 28, 359–371 (2009).

90. Nikko, E., Sullivan, J. A. & Pelham, H. R. Arrestin-like proteins mediate ubiquitination and endocytosis of the yeast metal transporter Smf1. EMBO Rep. 9, 1216–1221 (2008).

91. Lin, C. H., MacGurn, J. A., Chu, T., Stefan, C. J. & Emr, S. D. Arrestin-related ubiquitin-ligase adaptors regulate endocytosis and protein turnover at the cell surface. Cell 135, 714–725 (2008).References 90 and 91 define the role of the yeast β-arrestin-related ARTs in guiding the Rsp5 E3 ligase to transmembrane cargoes to facilitate their ubiqui-tin-dependent internalization.

92. Lundh, F. et al. Molecular mechanisms controlling phosphate-induced downregulation of the yeast Pho84 phosphate transporter. Biochemistry 48, 4497–4505 (2009).

93. Alvarez, C. E. On the origins of arrestin and rhodopsin. BMC Evol. Biol. 8, 222 (2008).

94. Marchese, A., Paing, M. M., Temple, B. R. & Trejo, J. G protein-coupled receptor sorting to endosomes and lysosomes. Annu. Rev. Pharmacol. Toxicol. 48, 601–629 (2008).

95. Edeling, M. A. et al. Molecular switches involving the AP-2 β2 appendage regulate endocytic cargo selection and clathrin coat assembly. Dev. Cell 10, 329–342 (2006).

96. Burtey, A. et al. The conserved isoleucine-valine-phenylalanine motif couples activation state and endocytic functions of β-arrestins. Traffic 8, 914–931 (2007).References 95 and 96 reveal that the AP-2-binding sequence in β-arrestins normally contributes to maintaining the basal conformation of the CLASP but becomes active when a phosphorylated GPCR is bound.

97. Santini, F., Gaidarov, I. & Keen, J. H. G protein-coupled receptor/arrestin3 modulation of the endocytic machinery. J. Cell Biol. 156, 665–676 (2002).

98. Scott, M. G., Benmerah, A., Muntaner, O. & Marullo, S. Recruitment of activated G protein-coupled receptors to pre-existing clathrin-coated pits in living cells. J. Biol. Chem. 277, 3552–3559 (2002).

99. Milano, S. K., Pace, H. C., Kim, Y. M., Brenner, C. & Benovic, J. L. Scaffolding functions of arrestin-2 revealed by crystal structure and mutagenesis. Biochemistry 41, 3321–3328 (2002).

100. Pryor, P. R. et al. Molecular basis for the sorting of the SNARE VAMP7 into endocytic clathrin-coated vesicles by the ArfGAP Hrb. Cell 134, 817–827 (2008).

101. Chaineau, M., Danglot, L., Proux-Gillardeaux, V. & Galli, T. Role of Hrb in clathrin dependent endocytosis. J. Biol. Chem. 283, 34365–34373 (2008).

102. Jung, N. et al. Molecular basis of synaptic vesicle cargo recognition by the endocytic sorting adaptor stonin 2. J. Cell Biol. 179, 1497–1510 (2007).

103. Ghosh, P., Dahms, N. M. & Kornfeld, S. Mannose 6-phosphate receptors: new twists in the tale. Nature Rev. Mol. Cell Biol. 4, 202–213 (2003).

104. Schlüter, T., Knauth, P., Wald, S., Boland, S. & Bohnensack, R. Numb3 is an endocytosis adaptor for the inflammatory marker P-selectin. Biochem. Biophys. Res. Commun. 379, 909–913 (2009).

105. Li, Y., Lu, W., Marzolo, M. P. & Bu, G. Differential functions of members of the low density lipoprotein receptor family suggested by their distinct endocytosis rates. J. Biol. Chem. 276, 18000–18006 (2001).A quantitative study showing that different members of the LDL receptor superfamily have significantly different internalization rates depending on the mix of sorting signals present in the cytosolic domain.

106. Pandey, M. S., Harris, E. N., Weigel, J. A. & Weigel, P. H. The cytoplasmic domain of the hyaluronan receptor for endocytosis (HARE) contains multiple endocytic motifs targeting coated pit-mediated internalization. J. Biol. Chem. 283, 21453–21461 (2008).

107. Wolfe, B. L., Marchese, A. & Trejo, J. Ubiquitination differentially regulates clathrin-dependent internalization of protease-activated receptor-1. J. Cell Biol. 177, 905–916 (2007).

108. Kumar, K. G. et al. Site-specific ubiquitination exposes a linear motif to promote interferon-α receptor endocytosis. J. Cell Biol. 179, 935–950 (2007).

109. McCaffery, G., Welker, J., Scott, J., van der Salm, L. & Grimes, M. L. High-resolution fractionation of signaling endosomes containing different receptors. Traffic 10, 938–950 (2009).

110. Gould, G. W. & Lippincott-Schwartz, J. New roles for endosomes: from vesicular carriers to multi-purpose platforms. Nature Rev. Mol. Cell Biol. 10, 287–292 (2009).

111. Castillon, G. A., Watanabe, R., Taylor, M., Schwabe, T. M. & Riezman, H. Concentration of GPI-anchored proteins upon ER exit in yeast. Traffic 10, 186–200 (2009).

112. Lakadamyali, M., Rust, M. J. & Zhuang, X. Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes. Cell 124, 997–1009 (2006).

113. Leonard, D. et al. Sorting of EGF and transferrin at the plasma membrane and by cargo-specific signaling to EEA1-enriched endosomes. J. Cell Sci. 121, 3445–3458 (2008).

114. Tosoni, D. et al. TTP specifically regulates the internalization of the transferrin receptor. Cell 123, 875–888 (2005).

115. Mundell, S. J., Luo, J., Benovic, J. L., Conley, P. B. & Poole, A. W. Distinct clathrin-coated pits sort different G protein-coupled receptor cargo. Traffic 7, 1420–1431 (2006).

116. Puthenveedu, M. A. & von Zastrow, M. Cargo regulates clathrin-coated pit dynamics. Cell 127, 113–124 (2006).

117. Rink, J., Ghigo, E., Kalaidzidis, Y. & Zerial, M. Rab conversion as a mechanism of progression from early to late endosomes. Cell 122, 735–749 (2005).A beautiful live-cell imaging study showing the temporal relationship between Rab proteins and maturing, cargo-laden endosomes.

118. Driskell, O. J., Mironov, A., Allan, V. J. & Woodman, P. G. Dynein is required for receptor sorting and the morphogenesis of early endosomes. Nature Cell Biol. 9, 113–120 (2007).

119. Wang, W. & Struhl, G. Drosophila Epsin mediates a select endocytic pathway that DSL ligands must enter

to activate Notch. Development 131, 5367–5380 (2004).

120. Morris, S. M. & Cooper, J. A. Disabled-2 colocalizes with the LDLR in clathrin-coated pits and interacts with AP-2. Traffic 2, 111–123 (2001).

121. Rappoport, J. Z., Kemal, S., Benmerah, A. & Simon, S. M. Dynamics of clathrin and adaptor proteins during endocytosis. Am. J. Physiol. Cell Physiol. 291, C1072–C1081 (2006).

122. Sorensen, E. B. & Conner, S. D. AAK1 regulates numb function at an early step in clathrin-mediated endocytosis. Traffic 9, 1791–1800 (2008).

123. Tokumitsu, H. et al. Phosphorylation of Numb regulates its interaction with the clathrin-associated adaptor AP-2. FEBS Lett. 580, 5797–5801 (2006).

124. Schmid, E. M. et al. Role of the AP2 β-appendage hub in recruiting partners for clathrin coated vesicle assembly. PLoS Biol. 4, e262 (2006).

125. N’Diaye, E. N. et al. The ubiquitin-like protein PLIC-2 is a negative regulator of G protein-coupled receptor endocytosis. Mol. Biol. Cell 19, 1252–1260 (2008).

126. Herpers, B. & Rabouille, C. mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum-Golgi units involved in Gurken transport in Drosophila oocytes. Mol. Biol. Cell 15, 5306–5317 (2004).

127. Ge, L. et al. The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1. Cell Metab. 7, 508–519 (2008).

128. Conner, S. D. & Schmid, S. L. Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis. J. Cell Biol. 156, 921–929 (2002).

129. Jackson, A. P. et al. Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor μ2 kinase. J. Cell Biol. 163, 231–236 (2003).

130. Conner, S. D., Schroter, T. & Schmid, S. L. AAK1 mediated μ2 phosphorylation is stimulated by assembled clathrin. Traffic 4, 885–890 (2003).

131. Hinrichsen, L., Meyerholz, A., Groos, S. & Ungewickell, E. J. Bending a membrane: how clathrin affects budding. Proc. Natl Acad. Sci. USA 103, 8715–8720 (2006).

132. Roux, A., Uyhazi, K., Frost, A. & De Camilli, P. GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. Nature 441, 528–531 (2006).

133. Cremona, O. et al. Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 99, 179–188 (1999).

134. Yu, A. et al. Association of Dishevelled with the clathrin AP-2 adaptor is required for Frizzled endocytosis and planar cell polarity signaling. Dev. Cell 12, 129–141 (2007).

135. Janvier, K. et al. Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 γ-σ1 and AP-3 δ-σ3 hemicomplexes. J. Cell Biol. 163, 1281–1290 (2003).

136. Mason, A. K., Jacobs, B. E. & Welling, P. A. AP-2-dependent internalization of potassium channel Kir2.3 is driven by a novel di-hydrophobic signal. J. Biol. Chem. 283, 5973–5984 (2008).

137. Hicke, L. & Dunn, R. Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu. Rev. Cell Dev. Biol. 19, 141–172 (2003).

AcknowledgementsSpace constraints have unfortunately precluded discussion of many excellent and important studies in this area. I am grate-ful to N. Johnson, D. Owen and the anonymous referees for critical comments on the manuscript, to J. Heuser, R. Roth, S. Watkins and J. Thieman for beautiful microscopic images and to members of my laboratory for discussions. Support was from the US National Institutes of Health (R01 DK53249) and the American Heart Association (Award 0540007N).

DATABASESProtein dataBank: http://www.rcsb.org/pdb1BXX | 1IV9 | 1KY7 | 1M7E | 1W80 | 1YX6 | 2G30 | 2JKR | 2PR9 | 2VGL | 2VX8uniProtKB: http://www.uniprot.orgCD317 | CD4 | DAB2 | Ede1 | EGFR | Ent1 | Ent2 | EPS15 | HRB | LDLRAP1 | LRP1 | NUMB | PAR1 | Rsp5 | VAMP7

FURTHER INFORMATIONLinton M. traub’s homepage: http://www.cbp.pitt.edu/faculty/traub.html

All linkS Are AcTive in The online Pdf

R e V i e W s


R e V i e W s


http://www.rcsb.org/pdb

http://www.rcsb.org/pdb/explore/explore.do?structureId=1BXX

http://www.rcsb.org/pdb/explore/explore.do?structureId=1IV9

http://www.rcsb.org/pdb/explore/explore.do?structureId=1KY7

http://www.rcsb.org/pdb/explore/explore.do?structureId=1M7E

http://www.rcsb.org/pdb/explore/explore.do?structureId=1W80

http://www.rcsb.org/pdb/explore/explore.do?structureId=1YX6

http://www.rcsb.org/pdb/explore/explore.do?structureId=2G30

http://www.rcsb.org/pdb/explore/explore.do?structureId=2JKR

http://www.rcsb.org/pdb/explore/explore.do?structureId=2PR9

http://www.rcsb.org/pdb/explore/explore.do?structureId=2VGL

http://www.rcsb.org/pdb/explore/explore.do?structureId=2VX8

http://www.uniprot.org










http://www.uniprot.org/uniprot/Q5SW96






http://www.cbp.pitt.edu/faculty/traub.html

http://www.cbp.pitt.edu/faculty/traub.html

reviews - bio 5068

Documents