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Home , Caveolae, Caveolin, Clathrin, Endocytosis, SNARE (protein), Synaptobrevin

CHAPTER 6

Clathrin Adaptor in Cargo Linton M. Traub*

Abstract ukaryotic cells continuously remodel the and composition of the plasma in response to the extracellular milieu. Membrane retrieval typically involves Einward budding of small bilayer-encapsulated vesicles that shutde protein and lipid from the surface to internal endosomal elements. -mediated endocytosis is a domi­ nant pathway for internalization in many types, and a range of dedicated signals are used to ensure selective sorting in this pathway. Evidence suggests that the clathrin coat uti­ lizes a diverse collection of clathrin-associated sorting proteins (CLASPs) to ensure the effi­ cient and noncompetitive concentration of a wide variety of sorting signals within transport vesicles forming at the cell surface. Introduction The limiting membrane of eukaryotic cells represents the primary interface with the exterior environment. It performs a vital barrier function that, by tighdy controlling the entry of molecules into the cell interior, contributes to long-term cell survival. The plasma membrane is also a platform for first detecting and then responding to extracellular signals, ions and nutrients, as well as potential pathogens. This is because a large array of receptors, ion channels, pumps and transporter proteins are positioned at the cell surface of a typical eukary­ otic cell. Some receptors (mosdy handling nutrient uptake or protein delivery) flux constandy through the plasma membrane while others, primarily involved in , only exit the cell surface en masse following ligand stimulation. The process of regulated removal of certain receptors from the surface is perhaps best understood from the point of development,^ where failure to quantitatively clear a particular (s) from the plasma membrane can ultimately result in inappropriate cell fate determination due to erroneous cellular responses to local morphogens or growth factors. Sorting Signals for Selective Internalization In principle, internalization of transmembrane proteins (along with bound ligands) could be either an active or a passive process. There are certainly examples of proteins being retained at the plasma membrane through stabilizing interactions with , lipid rafts, or other proteinaceous components, like scaffolding proteins with PDZ domains for example. Yet, it is generally accepted that endocytic uptake is governed by a positive signal;^ in other words, there

•Correspondence Author: Linton M. Traub—Department of Cell and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST Pittsburgh, Pennsylvania 15261, U.S.A. Email: [email protected] Endosomesy edited by Ivan Dikic. ©2006 Landes Bioscience and Springer Science+Business Media. Clathrin Adaptor Proteins in Cargo Endocytosis 63

is a process of preferential sorting of select transmembrane proteins for enrichment within forming carrier vesicles. This is because many proteins/receptors stagnate at the cell surface, redistributing into a diffuse pattern over the plasma membrane, when sorting signals within the cytosolic domain are inactivated either by inherited defects or directed mutation(s), or if the cytosolic region is simply truncated. Indeed, numerous, structurally discrete sorting signals that specify rapid internalization are now known^ (Table 1). Clathrin-Mediated Endocytosis There are several ways in which preferential entry of transmembrane proteins and bound ligands into the can occur. These include the budding of small (--60-100 nm) membrane bound vesicles in -dependent,^^ clathrin-dependent,^^'^'^ and uncoated caveolin-independent endocytosis, ' as well as through larger forms of membrane in­ ternalization vehicle, as in macropinocytosis and . Of these, probably the best characterized at the mechanistic level is clathrin-mediated endocytosis. Clathrin-coated buds and clathrin-coated vesicles were originally discovered in thin-section electron micrographs on the basis of a highly characteristic brisde-like matrix deposited on the cytosolic aspect of these structures'^ (see Fig. lA). The unusual membrane invaginations also displayed striking concentration of electron dense material within the lumen, located opposite to the bristle coat. These landmark morphological findings accurately mirror the two principal fiinctional activities of clathrin-coated vesicles (and coat-dependent sorting in general); assembly of a polymeric, -oriented coat while simultaneously gathering select cargo molecules into the invaginating coated vesicle.

Table 1, Clathrin •dependent endocytic sorting signals

Examples Recognition Signal Type Sequence Protein Protein/Domain References

YXX0^ YTRF receptor AP-2, \a subunit 7, 19 YSKV CI^-MPR YRGV CD^-MPR YQRL TGN38/46 YQTI LAMP-1 YATL LRP1 [DE]XXXL[LI] ERAPLI LIMP-II AP-1,Y/a1 7,47 DKQTLL CD3-Y hemlcomplex EKQPLL tyrosinase AP-2, (x/a2 DQRDLI li hennicomplex? [FY]XNPXY FDNPVY LDL receptor ARH, Dab2,Numb 7, 19 FTNPVY LRP1 PTB domain FENPMY megalin p-? YTNPAF Sanpodo Phosphorylation - GPCRs P-arrestin 1/2 19,64 EGFR , epsIS 102, 103 Notch UIM^ domain? Delta ENaC^ ^ Consensus sequences indicated in single letter amino acid notation using PROSITE syntax. 0 indicates a bulky hydrophobic amino acid; Leu, Met, lie, Phe, Val, '^CI-MPR is cation-independent mannose 6-phosphate receptor. ^ CD-MPR is cation-dependent mannose 6-phosphate receptor. "ENaC is epithelial sodium channel. ^UIM is ubiquitin interaction motif. 64 Endosi

Figure 1. Clathrin-coat morphology. A) Thin-section electron micrograph of a purified rat brain nerve ending (synaptosome) revealing the characteristic cytosol-oriented 'bristle' matrix (arrows) enveloping several cIathrin-coated vesicles budding from the presynaptic plasma membrane (PM). At the nerve ending, coated vesicles retrieve membrane components to replenish the pool of vesicles (arrowheads) required for sustained . A mito­ chondrion (asterisk) is positioned in close proximity to the synaptic region. B) Freeze-etch electron micrograph of the cytoplasmic face of the ventral plasma membrane of a cultured cell revealing the typical polyhedral clathrin lattice. The progressive invagination of coated mem­ brane is clearly seen in different intermediates of the budding process. This image was graciously provided by John Heuser.

The 200 A bristle-like structures radiating oflF clathrin-coated correspond to the regular coat assemblage. The major constituent is, of course, clathrin, a tri-legged protein complex composed of three 192-kDa heavy chains and three '-25-kDa regulatory light chains, one bound to each heavy chain (Fig. 2). Clathrin trimers can self-assemble onto spherical polyhedral assemblies in vitro, which are highly reminiscent of coated vesicles in vivo. It is the geometric nature of the triskelion that dictates the formation of the hexagon/pentagon-containing lattice, the most recognizable feature of the clathrin coat (Fig. IB). The orientation and rigidity of the fibrous heavy chains imparts an inherent sidedness to the triskelion; when assembled upon biological membranes, the amino-terminal region of the heavy chain, which folds into a 7-bladed P-propeller structure, ^^ is oriented closest to the bilayer. The carboxyl termini, which bundle together at the central vertex in a helical tripod arrangement, are positioned furthest from the membrane surface. However, clathrin does not have the capacity to associate with bilayers direcdy. Instead, adaptor proteins couple the clathrin lattice with the underlying membrane by synchronously binding to the clathrin terminal domain, to , and to cargo sorting signals. The AP-2 Adaptor Complex A second major protein constituent of the endocytic clathrin coat is the AP-2 adaptor, ^^ composed of four distinct subunits; two large --lOO-kDa subunits (a and p2), a 50-kDa medium subunit (|Ll2), and a 17-kDa small subunit (a2).^^ The functional complex has a characteristic architecture of two small appendages projecting oflFa larger globular core through Clathrin Adaptor Proteins in Cargo Endocytosis 65

HIP1 4I~ CALM AP-2 ANTH a subunir appendage AP180 If adaptor wmm MS^W • nSsubunit core epsin [52 subunit J

(i2 subunit appendage Numb

Dab2 heavy chain terminal domain ARH

(3-arrestJn / clathrin Ptdlns(4,5)P2 binding

Figure 2. The clathrin-AP-2-CLASP interaction network. Schematic depiction of the protein- protein interactions between clathrin, AP-2 and selected CLASPs is shown. Images are modeled on the known molecular structures of the coat components, but are not drawn to scale. The clathrin terminal domain and the AP-2 appendages serve as platforms to coordinate the protein- protein interaction networksestablishedduringcoatassembly. The location of interaction motifs used to engage these binding platforms are indicated by color-coded vertical lines. In AP180 and CALM, where the interaction motifs have not been defined precisely, interaction is generally signified by a horizontal line. The location of the three tandem UlMs in epsin 1 (ovals) is shown. pGPCR indicates phosphorylated (ligand-activated) -coupled receptor, while l/LWEQ is a modular F- bindingfold that is also conserved in the S. cerevisiae F4IP1 orthologue Sla2p. protease-sensitive stalks of apparendy variable length.^ Structural studies show the core is a heterotetramer comprised of the amino-terminal trunk regions of the large chains complexed with the smaller subunits (see Fig. 2). The two appendages correspond to the independently folded carboxy-terminal segments of the large a and p2 subunits,^^'^ each attached to the respective trunk through an intrinsically unstructured intervening polypeptide hinge (for a comprehensive review of adaptor structure, see ref 19). A five residue interaction motif (LLNLD; termed the clathrin box) is positioned within the pliable disordered hinge of the P2 subunit^^ and this binds direcdy to the clathrin terminal domain p-propeller. The AP-2 core complex seems to oversee coat operations close to the membrane. A patch of basic amino acids near the amino terminus of the OC subunit binds to phosphoinositides, specifically phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-^^' A second PtdIns(4,5)P2-contact site is located on the |l2 subunit but, in the resting (cytosolic) state, the two chains are not positioned appropriately to allow simultaneous lipid binding. Importandy, the |Ll2 subunit also binds to a common class of sorting signal, the -based YXX0 motif (where X is any amino acid and 0 represents a residue with a bulky hydrophobic side chain), found for example in the transferrin receptor, the mannose 6-phosphate receptors and the low density (LDL) receptor-related protein 1 (LRPl) (Table 1). Classical models for clathrin-based sorting do not accommodate phospholipid support, and posit simply that coat assembly proceeds via the concerted linking of cargo with clathrin through the bifunctional AP-2 adaptor to fabricate a polymeric coat that progressively invaginates before budding into the cytoplasm. The final scission step is a complex process, handled by the large GTPase 66 and several accessory proteins such as , endophilin and 9.^^'^^ Upon release of the transport vesicle, rapid, ATP-dependent uncoating allows both AP-2 and clathrin to reenter a soluble pool to initiate new rounds of coat assembly, while the vesicle is primed for fusion with acceptor endosomal elements. But the structure of the AP-2 core reveals that in the basal state the surface of the \l2 subunit that recognizes the YXX0 sequence is clearly inaccessible.^^ This region of |Ll2 packs up against the adjacent p2 trunk and phosphorylation of |Ll2 at Thr 156 is necessary to unleash the subunit for productive interactions with YXX0 sequences.^^'^^ The repositioning of phosphorylated jLl2 allows simultaneous binding of the YXX0 signal and PtdIns(4,5)P2^^ strengthening the association of AP-2 with the plasma membrane. In addition, the catalytic activity of the kinase that phosphorylates Thrl 56, termed AAKl, is stimulated by assembled clathrin.^^'^^ This suggests that AP-2 is focally activated to engage cargo as the coat assembles, possibly making cargo recognition a relatively late event. Indeed, \l2 subunit mutations that preclude YXX0 engagement do not prevent the proper deposition of AP-2 on the plasma membrane. ^^'^'^ None­ theless, cargo capture does appear to be a decisive step in the assembly process as live-cell imaging of clathrin dynamics in cultured cells shows that failure to concentrate cargo (transferrin) within an assembling lattice rapidly triggers catastrophic dissolution of the assemblage.^^ Mutation or targeted disruption of individual AP-2 subunits is lethal in mice, melanogaster and ^ (although not in Saccharomyces cerevisiae). ' In cultured mammalian cells, post-transcriptional silencing of AP-2 a or 112 subunit mRNA using small interfering RNA (siRNA) halts the internalization of the trans­ ferrin receptor ^ and reduces the number of morphologically discernable (brisde-like) clathrin coats at the surface more than tenfold. These results clearly highlight the pivotal contribution AP-2 makes to clathrin-mediated endocytosis. Diversity in Cargo Recognition Events AP-2 is structurally and functionally homologous to the AP-1 and AP-3 heterotetramers that operate at the trans-Golgi network and on endosomes. A nontyrosine sorting signal, the [DE]XXXL[LI]-type dileucine sequence (Table 1) binds to a hemicomplex of the yl/al or 5/ a3 subunits of AP-1 or AP-3, respectively, at a presently unknown location. The [DE]XXXL[LI] sequence also acts as an efFicient internalization motiP^ so it probable that AP-2 binds physically to this sorting signal as well. The capability of AP-2 to contact two discrete signals though separate subunits implies that individual coated vesicles can simulta­ neously carry multiple classes of cargo. In reality, endocytic clathrin coats do not only garner YXX0- and [DE]XXXL[LI]-harboring proteins. Other receptors with tyrosine-based FXNPXY-type internalization sequences, like the LDL receptor, likewise cluster at assembled clathrin structures at the cell surface. ^'^^ And still others, like the epidermal (EGF) receptor, also congregate in clathrin-coated regions following activation. '^^ Although the EGF receptor contains a YXX0 signal (YRAL) within the cytosolic domain and binds direcdy to AP-2, this segment is dispensable for internalization. Accordingly, point mutations within the |X2 subunit that prevent YXX0-sequence binding have little inhibitory effect on EGF receptor uptake.^^ In fact, ectopic receptor overexpression studies show unambiguously that saturating levels of FXNPXY or [DE]XXXL [LI]-containing proteins at the surface have negligible effect on the kinetics of transferrin or EGF receptor internalization and vice versa.^^'^^'^^ And each receptor type saturates the endocytic machinery at different surface densities.^ One explanation for these general findings could be that the internalization of each cargo type is overseen by distinct sets of clathrin-coated vesicle with nonoverlapping cargo selectivity. Ultrastructural analysis of surface clathrin-coated regions argue strongly against this notion. In electron micrographs, receptors for transferrin, LDL, EGF and and are clearly colocalized within individual coated profiles; ' each clathrin-coated vesicle contains numerous types of cargo utilizing different sorting signals which, moments later, are found in common early endosomes. ^^ Good coincidence of the transferrin and LDL receptor in individual punctate Clathrin Adaptor Proteins in Cargo Endocytosis 67

Figure 3. AP-2 does not regulate the sorting of all clathrin-dependent cargo. Single confocal optical sections of confluent HeLa cell monolayers treated either with mock (A-D) or AP-2 a subunit siRNA (E-H) and then double-labeled with fluorescent transferrin (A and E, red) and anti-LDL receptor (B and F, green), or stained with anti-AP-2 a subunit antibodies (D and H). In untreated cells, the transferrin and LDL receptors colocalize but after AP-2 silencing, only the transferrin receptor undergoes massive redistribution at the cell surface. Insets in A, B, and C show a magnified region to better judge the degree of colocalization between transferrin andtheLDL receptor.

(cladirin-containing) sites on the plasma membrane is also seen by light microscopy (Fig. 3A-D). But, while RNAi silencing of the AP-2 a subunit (Fig. 3H) causes the transferrin receptor to diffuse over the cell surface (Fig. 3E), the LDL receptor, strikingly, remains concentrated in punctate zones (Fig. 3F) and internalizes rather efficiently, even in the absence of AP-2. Entry of the LDL receptor is still clathrin dependent under these conditions because siRNA-mediated knock down of clathrin prevents the internalization of both transferrin and LDL receptors. This capability of receptors lacking a YXX0 -or [DE]XXXL[LI]-type internalization signal to enter the cell in the absence of functional AP-2 suggests that alternate adaptor proteins must regulate the internalization of some classes of cargo and these might correspond to the differen­ tially saturable connectors revealed in the overexpression studies. '^ Clathrin-Associated Sorting Proteins (CLASPs), the Alternate Adaptors Early clues to the nature of putative alternate adaptors came from studies on the seven-transmembrane-spanning G protein-coupled receptors (GPCRs). GPCRs are the most extensive superfamily of signaling receptors in and many, but not all, of these proteins are quickly downregiJated by clathrin-mediated endocytosis following agonist-induced activa­ tion. Ligand binding prompts phosphorylation of the cytosolic region of most GPCRs. This reversible modification acts as a tri^er for one of two ubiquitous arrestin family members (P-arrestin 1 or P-arrestin 2) to translocate onto the activated GPCR.^^"^^ The B- desensitize the receptor by uncoupling signaling events, but also direct the phosphorylated GPCR to clathrin-coated structures for prompt internalization. The capacity of p-arrestins to promote GPCR endocytosis depends upon four fundamental properties of these adaptor proteins: the ability to bind physically to the GPCR, to PtdIns(4,5)P2, to AP-2 and to clathrin.^'^ Interaction motifs 68 Endosomes arrayed tandemly within the carboxy-terminai segment of the p-arrestins specify direct interac­ tions with the clathrin heavy chain P-propeller^^*^^'^ and with the appendage of the AP-2 p2 subunit. All four interactions are required for efficient GPCR endocytosis, and p-arrestins work by melding the GPCR with preexisting clathrin coats by synchronously engaging both. These are essentially the properties of the archetypical adaptor, AP-2, but it is the P-arrestins, and not AP-2 direcdy, that are charged with overseeing the internalization of the largest family of recep­ tors known. There is also evidence that P-arrestin 2 handles the uptake of nonclassical seven-transmembrane-spanning smoothened and frizzled receptors that operate in the Wnt and Hedgehog signaling pathways.^ Also, p-arrestin 2 apparendy participates in the internaliza­ tion of transforming growth factor p (TGPP) by binding to the type III TGFp receptor (also termed betaglycan) in a phosphorylation-sensitive manner.'^^ And finally, P-arrestin nullizygous mice fed a high fat diet display defective lipoprotein metabolism and it appears that p-arrestin 2 may also modulate constitutive LDL receptor endocytosis by binding to the cytosolic domain of the receptor in a phosphorylation-dependent fashion.^ The cargo selective properties of the P-arrestins make them the founding members of the CLASP family. But certain other so-called endocytic ^accessory' proteins^^ also display the important binding properties typical of the P-arrestins (and AP-2).^^ These presumptive CLASPs bind to PtdIns(4,5)P2, albeit using different modular lipid-binding folds, bind to the clathrin B-propeller domain, and all can engage the appendages of the large AP-2 adaptor subunits ' '^ (Fig. 2). CLASPs appear to operate by contributing to the efficiency of clathrin lattice assembly while simultaneously expanding the sorting repertoire of the forming coat. ' Currendy, the experimental evidence for a set of dedicated, phosphotyrosine-binding (PTB) domain-containing CLASPs governing LDL receptor internalization is most compelling. PTB Domain CLASPs and FXNPXY Signal Endocytosis The PTB acronym is actually a misnomer. Although the domain was originally identified as a non-SH2 phosphotyrosine binding module, it is clear that is not a prerequisite for productive binding of the canonical PTB binding-partner sequence [FY]XNPXY.^^ A form of diis sequence positioned within the cytosolic domain of the LDL receptor (FDNPVY), in its nonphosphorylated form, was actually the first internalization signal ever discovered. Two PTB domain CLASPs, designated Disabled-2 (Dab2) and the autosomal recessive hypercholesterolemia (ARH) protein, appear to decode the FXNPXY internalization signal in the LDL and related receptors. Inherited mutations or targeted gene disruption of these proteins cause obvious pathological . For ARH, individuals with inactivating mutations in both ARH alleles have early onset hypercholesterolemia that is very difficult to distinguish from that typical of patients entirely lacking LDL receptor function.^^'^^" Thus, these unfortunate individuals present with a disease that closely mirrors a complete failure to bind and clear circulating LDL particles. In full agreement, ARH'^' mice show dramatic accumulation of LDL receptors at the sinusoidal plasma membrane of hepatocytes, the cell type responsible for clearing the bulk of circulating LDL from the plasma.^ In a similar fashion, Dab2 nullizygous mice display a proteinuria because of ineffective sorting of an LDL superfamily receptor termed megalin at the apical surface of renal proximal tubules.^^'^^ Megalin is a scavenger receptor that retrieves considerable amounts of albumin, vitamin- and lipid-binding proteins from the glomeridar filtrate to prevent protein excretion in the urine; the Dab2'' is actually a milder version of that seen upon megalin gene disruption.^^ ARH and Dab2 use a single folded PTB domain to bind the FXNPXY sequence and PtdIns(4,5)P2 noncompetitively.^^' ^'^^This promotes cargo recognition, while binding to the clathrin coat is controlled by the carboxy-terminal segment in both like the p-arrestins, ARH and Dab2 have tandemly arrayed interaction sequences that promote associations with the AP-2 appendages and the clathrin P-propeller (Fig. 2). That these pro­ teins can contribute to clathrin coat assembly is evidenced by a decrease in detectable clathrin-coated vesicles at the apical pole of Dab2-null proximal tubules.^^ Clathrin Adaptor Proteins in Cargo Endocytosis 69

Secondary structure predictions suggest that outside of the modular amino-terminal PTB domain, these proteins are largely unstructured. The reason for this may be that intrinsically disordered tracts of polypeptide are highly mobile and pliable, which allows a large ^capture radius' for binding partners. This property could draw receptor-CLASP complexes into a pre­ existing clathrin structure rather efFiciendy. A gross absence of secondary structural elements is also consistent with what is known about the mode of engagement of various interaction mo­ tifs with the clathrin p-propeller and the AP-2 appendages. "'^^"^'^ In fact, extended regions of unstructured random-coil polypeptide turns out to be an unexpected characteristic of the CLASP group of endocytic components. '^ Another very informative example of the endocytic action of a PTB-domain CLASP, termed Numb, is in the biogenesis of retro-orbital neurosensory bristles in Drosophila?^ The mature sensory brisde is composed of four cell types; hair, socket, and sheath cells. The four all arise from a single sensory organ precursor (SOP) cell by two rounds of asymmetric cell divi­ sion. ^'^^^ During SOP cell . Numb distributes asymmetrically in the mother cell so as to selectively partition into only one of the two daughter cells. ^^^ Mutant numb alleles lead to cell fate transformations, resiJting in only four socket cells being formed. ^'^^^ Mutations that dis­ rupt the activity of the appendage of the AP-2 a subunit, to which Numb binds direcdy, generate a phenotype equivalent to numb mutants.^ The simplest model for Numb activity is that asymmetric Numb-AP-2 association during cell division results in one of the two siblings having a considerably higher Numb/AP-2 complement, which then drives the removal of the transmembrane Notch receptor from the surface in this cells. This, then, changes the fate of the cell compared to the other daughter.^'^^ Numb binds to Notch, yet there are no obvious diflPer- ences in Notch expression level between the two cells that ultimately become quite different cell types. Instead, a Notch-binding accessory protein termed Sanpodo, seems to be the surface component downregulated in Numb-enriched daughter cells. ^ Sanpodo is a tetraspannin with a variant [YFjXNPXY sequence (YTNPAF) in the cytosolic portion and Numb-mediated clearance of Sanpodo from the surface is suggested to compromise Notch signaling, leading to a different cell fate.^^^ ENTH/ANTH-Domain Containing CLASPs Posttranslational conjugation of ubiquitin is another important method of generating sorting signals, as covered in several other chapters of this book (see chapters by Puertollano, Pattni and Stenmark, and Gur et al). A discussion of the nature and generation of this internalization signal is beyond the scope of this chapter, but the epsin family of CLASPs (together with epsl 5, an epsin binding partner) are good candidates for recognizing ubiquitinated cargo at the cell surface. ^^^'^^^ Again, the conserved modular architecture and binding properties of epsin are fully compatible with a role as a cargo-selective adaptor. The epsin N-terminal (ENTH) domain binds direcdy to PtdIns(4,5)P2/^ while two or three ubiquitin-interaction motifs (UIMs) enable the protein to bind physically to ubiquitin^^^ (Fig. 2). The UIMs are located between the ENTH domain and the unstructured carboxy-terminal region, housing standard interaction motifs that enable epsin to bind both clathrin and the AP-2 append- ages (rig. I). In DrosophiUy while a PTB-domain CLASP (Numb) attenuates Notch receptor signal transduction in certain SOP progeny, an ENTH-domain CLASP (epsin) actively promotes Notch signaling. ^^^'^^^ Notch is activated in a signal-receiving cell by engaging the transmem­ brane ligand Delta, presented at the surface of an adjacent signal-sending cell. The Notch transcriptional response is preceded by two ordered, ligand-induced cleavage events that sever Notch on both sides of the plasma membrane allowing the intracellular domain to translocate to the nucleus.^^^ It appears that the second proteolytic event that liberates the Notch intracel­ lular domain requires prior endocytosis of the extracellular region of Notch, bound to Delta, into the signal-sending cell. There are strong genetic interactions between Delta, two E3 ubiquitin ligases (Neuralized and Mind bomb), and the fly epsin, termed Liquid facets.^^^'^^^ Current 70 Endosomes evidence strongly suggests that the E3-mediated ubiquitination of Delta that allows Liquid facets to internalize the Delta/Notch complex is obligatory for Notch signaling. The pivotal role of Liquid facets in promoting Notch signaling is underscored by the participation of the deubiquitinating Fat facets. Fat facets appears to operate by buffering the intracellular epsin concentration by salvaging proteasome destined, polyubiquitinated Liquid facets by deubiquitinating the protein. In fact, expression of a single extra copy of Liquid facets abol­ ishes the requirement for Fat facets in the developing compound eye; there is no eye phenotype in Fat facets-null flies expressing an extra Liquid facets gene.^ The Drosophila data suggest that during development, the intracellular abundance of epsin is critical, presumably to effect the timely endocytosis of certain membrane proteins/receptors thereby facilitating appropriate cell fate determination. '^^^'^^^ This is in full accord with the concept of epsin functioning as a CLASP. Likewise, in , another ENTH domain protein, interacting protein 1 (HIPl) displays a variation on the general CLASP architecture (Fie. 2), and may manage the internalization of AMPA receptors in the central nervous system.^ ^ Finally, the structure of the API 80 N-terminal homology (ANTH) domain is highly related to the ENTH module and binds to Ptdlns(4,5)p2, although by a different molecular mechanism. API80 and the nonneuronal orthologue CALM both bind clathrin and AP-2. As for funaion, UNC-11, the C elegans APXSO, is abundandy expressed in and analysis of the effect of unc-11 mutants suggests that this protein regulates the retrieval of the v-SNARE from the presynaptic plasma membrane.^ Conclusions and Perspective The identification of cargo selective CLASPs allows several previously unexplained findings now to be resolved. General collaboration between AP-2 and CLASPs can adequately explain how Ptdlns(4,5)p2 within the cytosolic leaflet of the plasma membrane can act as a compartmental cue to direct the assembly of endocytic clathrin coats. The overall architectural similarity of the CLASP family is striking (Fig. 2), and the capability of numerous CLASPs to bind physically to both AP-2 and clathrin could account for the very rapid assembly of coated vesicles and the swift internalization of a diverse array of transmembrane proteins from the cell surface. Perhaps most importandy, the activity of these proteins can explain how internalization of select transmembrane proteins can continue in cells essentially lacking functional AP-2. Intriguingly, vesicle coats that operate at other sorting stations within the cell (the COPI and COPII coats, for example) do not appear to have diversified cargo capture operations to involve a host of CLASP-like components.'^^'^^'^ Thus a legitimate question is why there are so many endocytic CLASPs (and we may not yet have the complete list of cargo specific components operating at the cell surface). As discussed above, in Drosophila, the Notch pathway utilizes both PTB- (Numb) and ENTH- (Liquid facets) domain CLASPs to fine-tune signaling activity. But Notch/Sanpodo and the ligand Delta are endocytosed in different cells, the signal-receiving and signal-sending cell, respectively. So one obvious possibility is that the complexity of the endocytic CLASP network (Fig. 2) is substantially reduced in individual cell types. In other words, perhaps not all coated structures contain a fixll complement of CLASPs. This seems unlikely, however, because in SOP cell progeny for example, the daughter cell utilizing Numb to suppress intracellular Notch signal transduction is simultaneously using Liquid facets to promote effective Notch signaling in the adjacent sister cell. In fact, the vast majority of the core CLASP members are found both in brain and in cultured lines, like HeLa cells and fibroblasts. It therefore seems more probable that numerous different CLASPs populate a single clathrin structure assembling at the surface. The synchronous operation of a diverse group of CLASPs has the clear advantage of equipping individual clathrin coats with the capability of capturing an extended range of cargo molecules at the cell surface. But even with the incorporation of CLASP activity into models for clathrin-based sorting, our general understanding of the molecular events that underpin coat assembly is still rather rudimentary. A major challenge is now to begin to understand the temporal chronology and Clathrin Adaptor Proteins in Cargo Endocytosis 71

regulation of the numerous protein-protein interactions necessary for the successful fabrication of a clathrin-coated vesicle. While CLASPs clearly bind to the AP-2 appendages, there exists a diverse set of appendage binding sequences and two spatially separate contact sites upon each appendage. ^^'' Unfortunately, the static representation of dense network connectivity (Fig. 2) cannot adequately explain the assembly and cargo selection process chronologically; we do not understand the flow and processing of information. For instance, the biologic advantage of one CLASP displaying a particular set of appendage-binding sequences and not another is not at all clear. Presumably these sequence dictate the temporal behavior and possibly the rank of a particular CLASP, but there is a paucity of functional information on the individual contributions of these interactions to the coupled process of sorting and budding. It seems certain that the application of a wide array of experimental approaches, including live-cell imaging,^^'^ ^^ RNAi and gene replacement, time-resolved proteomics and, perhaps most importantly, computational analysis and network theory modeling will be necessary to unlock all the secrets of this highly efficient and sophisticated vesicular shutde. References 1. Le Roy C, Wrana JL. Clathrin- and nonclathrin-mediated endocytic regulation of ceil signalling. Nat Rev Mol Ceil Bioi 2005; 6:112-126. 2. Cadavid AL, Ginzei A, Fischer JA. The function of the Drosophiia fat facets deubiquitinating enzyme in limiting photoreceptor cell number is intimately associated with endocytosis. Develop­ ment 2000; 127:1727-1736. 3. Berdnik D, Torok T, Gonzalez-Gaitan M et al. The endocytic protein a-Adaptin is required for Numb-mediated asymmetric cell division in Drosophiia. Dev Cell 2002; 3:221-231. 4. Resh MD. Membrane targeting of lipid modified signal transduction proteins. Subcell Biochem 2004; 37:217-232. 5. Roper K, Corbeil D, Huttner WB. Retention of prominin in microvilli reveals distinct -based lipid micro-domains in the apical plasma membrane. Nat Cell Biol 2000; 2:582-592. 6. Brone B, Eggermont J. PDZ proteins retain and regulate membrane transporters in polarized epi­ thelial cell membranes. Am J Physiol Cell Physiol 2005; 288:C20-C29. 7. Bonifacino JS, Traub LM. Signals for sorting of transmembrane proteins to endosomes and lysos- omes. Annu Rev Biochem 2003; 72:395-447. 8. Sanan DA, Van der Westhuyzen DR, Gevers W et al. The surface distribution of low density lipoprotein receptors on cultured fibroblasts and endothelial cells. Ultrastructural evidence for dis­ persed receptors. Histochemistry 1987; 86:517-523. 9. Lobel P, Fujimoto K, Ye RD et al. Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis. Cell 1989; 57:787-796. 10. Nabi IR, Le PU. /raft-dependent endocytosis. J Cell Biol 2003; 161:673-677. 11. Brodsky FM, Chen CY, Knuehl C et al. Biological basket weaving: Formation and function of clathrin-coated vesicles. Annu Rev Cell Dev Biol 2001; 17:517-568. 12. Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature 2003; 422:37-44. 13. Damm EM, Pelkmans L, Kartenbeck J et al. Clathrin- and caveolin-1-independent endocytosis: Entry of simian 40 into cells devoid of caveolae. J Cell Biol 2005; 168:477-488. 14. Kirkham M, Fujita A, Chadda R et al. Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles. J Cell Biol 2005; 168:465-476. 15. Roth TF, Porter KR. Yolk protein uptake in the oocyte of the mosquito Aedes aegypti. L J Cell Biol 1964; 20:313-332. 16. Fotin A, Cheng Y, Sliz P et al. Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 2004; 432:573-579. 17. ter Haar E, Musacchio A, Fiarrison SC et al. Atomic structure of clathrin: A |3 propeller terminal domain joins an a zigzag linker. Cell 1998; 95:563-573. 18. Blondeau F, Ritter B, Allaire PD et al. Tandem MS analysis of brain clathrin-coated vesicles re­ veals their critical involvement in synaptic vesicle recycling. Proc Natl Acad Sci USA 2004; 101:3833-3838. 19. Owen DJ, Collins BM, Evans PR. Adaptors for clathrin coats: Structure and function. Annu Rev Cell Dev Biol 2004; 20:153-191. 72 Endosomes

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