40%

Linking cargo to vesicle formation: receptor tail interactions with coat Tomas Kirchhausen*, Juan S Bonifacinot and Howard RiezmanS

How soluble cargo molecules concentrate into budding In this review, we focus primarily on the most recent vesicles is the subject of intensive current research. developments in the study of -coated pits and Clathrin-based vesiculation from the plasma membrane and vesicles, the best understood of the vehicles for moving the trans-Golgi network constitutes the best described system receptors and ligands from the plasma membrane and the that supports this sorting process. Soluble ligands bind to trans-Golgi network (TGN) to the endosome. We also sug- specific transmembrane receptors which have been shown to gest some speculative parallels of clathrin-coated vesicles interact directly with clathrin adaptor complexes, components with (ER)-derived COPII-coated of clathrin coats. At the same time, these clathrin adaptors vesicles, major vehicles in ER+Golgi vesicular traffic (see facilitate clathrin coat assembly and probably regulate the this issue, Kuehn and Schekman, pp 477-483). recruitment of the rest of the coat components. Recent studies have looked at both the interaction of receptor tails Clathrin-coated pits and vesicles with adaptors and the assembly of the clathrin coat. Progress The main structural component on clathrin-coated vesicles has also been made in elucidating how soluble cargo is clathrin, a trimeric scaffold , which organizes molecules may be concentrated for exit from the endoplasmic itself into cagelike lattices (reviewed in [ 1I). Clathrin has reticulum. the shape of a triskelion, where each one of the three legs is made of a heavy and a light chain. The extended conformation of a clathrin leg allows it to pack along a Addresses lattice edge, forming the characteristic open hexagonal and ‘Harvard Medical School, Department of Cell Biology and Center for Blood Research, 200 Longwood Avenue, Boston, MA 02115, USA pentagonal facets of the coat. The assembly of a clathrin Kell Biology and Metabolism Branch, National Institute of Child lattice on the cytosolic side of the plasma membrane or Health and Human Development, National Institutes of Health, TGN membrane occurs during the formation of a coated Building 18T, Room 101, Bethesda, MD 20892-5430, USA pit, and a section of membrane is ultimately captured into $Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland a coated vesicle. Clathrin is thus an organizing framework for the proteins that carry out receptor sorting, membrane Current Opinion in Cell Biology 1997, 9:488-495 budding, and other steps in the cycle of vesicle assembly, http://biomednet.comlelecref/O955067400900488 uncoating and fusion.

0 Current Biology l_td ISSN 0955-0674 The major proteins that drive clathrin coat formation are Abbreviations the ‘clathrin AP (adaptor protein) complexes’ or ‘clathrin AP adaptor protein CTLA4 cytotoxic T lymphocyte antigen 4 adaptors’, heterotetramers that couple coated pit assembly ER endoplasmic reticulum to the entrapment of membrane receptors. Endocytic TGN trans-Golgi network coated pits and coated vesicles contain the AP-2 complex, while coated buds and coated vesicles derived from the TGN contain the related complex AP-1. AP-2 contains Introduction two large chains or ‘adaptins’ (one a chain and one pl Vesicular traffic between intracellular compartments re- or p2 chain), a medium chain @2), and a small chain quires special mechanisms to ensure the selective move- (02). AP-1 contains the adaptins y and pl together with ment of proteins and lipids from the donor to the acceptor the medium pl and small 01 chains (reviewed in [l-3]). organelle. In general, this problem of selection has been The first hint of the existence of a third AP complex solved by the concentration of specific cargo molecules came from the identification in the yeast genome of open into vesicles that are formed in a controlled way and reading frames whose sequences were highly related to the which then fuse with the target organelle. The first step known subunit sequences of AP-1 and AP-2 [4,5]. More in this form of traffic is the binding of cargo molecules to recently, cDNAs from mammalian sources corresponding the lumenal or extracellular domain of a transmembrane to related AP sequences have also been isolated. The receptor. This is followed by concentration of receptors newest member of the family is AP-3, a complex found in through interaction with a protein coat that is also required mammalian cells that contains the 6 and p3 chains together for vesiculation of the membrane. It is likely that cargo with the smaller ~3 and 03 chains [6’,7*]. This complex, concentration and coat formation are linked in order to however, is not thought to interact with clathrin. ensure efficient cargo loading into the assembling vesicles. The vesicles then pinch off from the source membrane in Clathrin coat assembly the budding step. Finally, the vesicles are targeted to, and The current view of the recruitment of clachrin coat fuse with, the acceptor compartment. components to membranes is that APs are first recruited Linking cargo to vesicle formation Kirchhausen, Bonifacino and Riezman 489

from the cytosol to the membrane. Little is known about that Epsl5 might undergo cycles of binding to and release the requirements for recruitment of AP-2 to the plasma from APs during coat assembly. Phosphorylation of Epsl5 membrane. Recruitment of APs to the TGN is influenced in response to epidermal growth factor stimulation of by ADP-ribosylation factor (ARF) and GTP@ [8-10]. The cells does not affect its intracellular distribution [22]; presence of receptor tails, known binding partners for whether or not the function of EpslS is linked to signal APs (see below), also stimulates AP-1 recruitment to the transduction pathways and receptor downregulation is TGN [lO,ll]. However, receptor tails are not likely to clearly a question for future studies. Another protein that represent the sole determinant responsible for targeting is highly related to Epsl5 has been found [28] and it may APs to membranes because the former are known to be be that related proteins are required at different locations present in compartments to which APs are not normally of clathrin-coated-vesicle formation. recruited. Therefore, a membrane-bound, high-affinity docking apparatus has been postulated (Figure 1). The Internalization signals identity of the putative AP docking apparatus and how The components of clathrin coats are in a position it could work remain unknown. Two studies [l&13] to interact with the cytosolic tails of transmembrane have identified membrane proteins that bind AP-1, but receptors, which have been shown to carry specific signals their relevance to AP recruitment has not yet been that direct both their rapid internalization and other demonstrated. intracellular targeting steps. The signals are sequences or structural motifs, many of which have either a critical The probable next step in clathrin coat assembly is the tyrosine residue or a pair of leucine or bulky hydrophobic binding of clachrin to membrane-bound AP complexes. residues and are accordingly known as ‘tyrosine-based’ Assembly of the coat is highly coordinated, involving the or ‘dileucine-based’ signals (reviewed in [29-311) (see recruitment of at least 60 clathrin trimers and 20-30 APs. Table 1 [32-381). There is now extensive evidence Early evidence based on reconstructed images obtained indicating that tyrosine-based signals bind directly to the by electron microscopy of coats suggested that the most AP-2 complex and that this binding is the event that distal portion of the clathrin leg, known as the terminal mediates the concentration of certain plasma membrane domain, is in contact with APs [14]. The p chains of proteins within clathrin-coated pits ([39]; reviewed in AP-1 and AP-2 are sufficient to interact with clathrin and [31]). Recent experiments suggest that dileucine-based drive the formation of coats [15]. These chains of the signals also interact with AP-2 [40*], although they AP complex contain an amino-terminal core domain and most likely have a binding site different from that of a carboxy-terminal ‘ear’, which are linked by a hinge. The tyrosine-based signals [41*]. As would be expected for interaction with clathrin is mediated through the hinge steps dependent on interaction with a limited number [16]. Phosphorylation of the hinge seems to prevent the of recognition molecules, internalization mediated by association of AP-2 with clathrin and this may be part both tyrosine-based and dileucine-based signals is a of the mechanism by which APs initiate and coordinate saturable process [41*]. An important characteristic of clathrin coat assembly [17-l. Evidence for a relatively both tyrosine-based and dileucine-based signals is that high-affinity interaction between the a chain of AP-2 and subsets of these signals are involved in additional sorting clathrin has also been reported [18] but the biological processes such as targeting to lysosomes, specialized significance of this association is not clear. Interestingly, endosomal/lysosomal compartments, the TGN or the AP-3, which is found in clusters associated with endosomal basolateral plasma membrane of polarized epithelial cells membranes, lacks a recognizable clathrin-binding motif in (reviewed in [30,31]). Thus, some of these signals are the p3 hinge region [19]. likely co be recognized at intracellular sites other than the plasma membrane, probably by adaptor complexes such as A recently discovered component of endocytic clathrin- AP-1 or AP-3. coated pits and vesicles is Epsl5, originally defined as a substrate for phosphorylation by the epidermal growth One development in the past few years has been the factor receptor [ZO]. Epsl5 binds to the a chain of AP-2 realization that internalization signals are much more [Zl] and colocalizes with clathrin at the plasma membrane diverse than was originally thought. This is true not (Z&23]. The role of Epsl5 in coat formation remains to only of tyrosine-based and dileucine-based signals, which be established. Its carboxy-terminal segment has a binding are known to be highly degenerate, but also of an site for the a ear of AP-2 (21,241. Its amino-terminal assortment of other cytosolic domain sequences that segment contains three Eps homology (EH) domains, bear no obvious resemblance to classical internalization modules of 70-90 amino acids that are also present in signals (Table 1). Signal diversity may be a critical several yeast proteins including End3p and Panlp; these feature of the clathrin coated vesicle sorting machinery. proteins are required for endocytosis and organization of As the ability to concentrate in clathrin-coated pits may the accin cytoskeleton [25-271. The preferred localization not require high affinity, it is possible that even weak of Epsl5 to the rims of coated pits and not in other regions interactions of rather nondescript sequences with AP-2 or of the coat [22] was unexpected as AP-2, its binding other components of the clathrin coats may be sufficient partner, is located throughout the coat. This result suggests co effect internalization. The organization of the coat, 490 Membranes and sorting

Figure 1

The recognition of tyrosine-based endocytic signals in transmembrane cargo receptors by AP-2 complexes is enhanced by clathrin coats and by 3' phosphoinositides. The figure shows proposed models for the capture of membrane-bound cargo receptors by clathrin-coated pits. (a) (I) AP-2 complexes are first targeted from the cytosol to the plasma membrane by interaction with their putative membrane-bound 'docking' complex. (ii) At this point, AP-2 interacts with cytosolic clathrin to form the lattice of coated pits. 0ii) AP-2 located in partially formed coated pits displays a clathrin-dependent increase in the affinity of the p.2 chain of AP-2 for transmembrane receptor tails. (This increase is represented by the change from a triangular to a rectangular 'gap' in AP-2.) (iv) This increase leads to the capture of mobile cargo receptors (v) into the coated pit. It is proposed that the linkage between clathrin binding to APs and the increased affinity of the APs for receptors ensures that coated pit assembly is coupled to receptor sorting. (b) (i) AP-2 complexes targeted to the plasma membrane by the putative membrane-bound AP docking complex can (ii) interact with membrane-bound 3' phosphoinositides (Ptdlns 3-P), leading to an increase in the affinity of the g2 chain of AP-2 for the tyrosine-based endocytic signal located in the cytoplasmic tail of transmembrane cargo receptors. The receptor is shown as bound to AP-2 at this point. (ill) The AP-2-receptor complex recruits cytosolic clathrin to form a coated pit or can be captured by available clathrin already located at the edge of a coated pit. Reproduced with permission from [45"].

with multiple copies of AP-2 being immobilized on same cytosolic tail may also allow multivalent attachment a clathrin lattice, and the tendency of some plasma to AP-2, thus providing for stronger interactions with membrane proteins to oligomerize may provide the the internalization or intracellular sorting machineries. conditions for the generation of strong avidities from Indeed, tyrosine-based signals, dileucine-based signals and interactions that are weak at a bimolecular level. The acidic clusters are often found in combinations; the most combination of diverse internalization signals within the notable examples of this occurrence are found in the two Linking eargo to veside formation Kirchhausen, Bonifacino and Riezman 491

Table 1 Internalization signals.

Signals’ Example of signal sequences’ Proteins containing the signal References

Tyrosine-based (NPXY-type) FDNPW LDL receptor? [321 Tyrosine-based (YXXB-type) YKYSKV Cl mannose-&phosphate receptor* 1331 Diieucine-based DKQTLL CD3-y [341 Acidic clusters WQEECPSDSEEDEGRGER Furin 1351 Dilysine (KKFF-type) KRFY VIP368 1361 Ubiquitin addition DAKSS Yeast a-factor receptor [371 targeting EWDIMRVNV VAMP-2/-2# 1361

7he single-letter amino acid code is used in these columns. X represents any amino acid and 0 represents a bulky hydrophobic amino acid. Critical residues are shown in bold tvPe. tLDL, low-densiw- lipoprotein.. %I, cation-independent. pVIP36, vesicle integral protein 36. WAMP-2, vesicle-associated membrane protein-?. mannose-6-phosphate receptors which have all three types the known sequence requirements for function of the of signal (reviewed in [42]). signals in vivo (reviewed in [29,30]). All of these studies have demonstrated that ~2 is capable of binding to Recognition of tyrosine-based signals by AP-2 many different tyrosine-based signals, although it has a Despite the growing diversity of known internalization sig- preference for signals that have basic residues at the X nals, tyrosine-based signals continue to attract the greatest positions [46*]. The context in which the signals are found attention, not only because of historical reasons- they in the cytoplasmic domain also appears to be an important, were the first to be discovered- but also because they albeit less predictable, determinant of interactions [47*]. are the most commonly found among rapidly internalized proteins. Tyrosine-based signals are characterized by the Regulation of the interaction of tyrosine-based presence of a critical tyrosine residue within an otherwise signals with AP-2 degenerate sequence context (reviewed in [31]). Exten- The entrapment of plasma membrane proteins within sive analyses of the functional importance of residues clathrin-coated pits can be subject to regulation by neighboring the critical tyrosine residue have established modifications of both the tyrosine-based signals and the that tyrosine-based signals conform to various sequence AP-2 complex. With regard to the tyrosine-based signals, motifs, the most common being NPXY and YXXB phosphorylation of the critical tyrosine residue has been (single-letter code for amino acids, where X represents any shown to abrogate interaction with p2/AP-2, [46’,48*]. This amino acid and 0 represents a bulky hydrophobic amino modification is thought to play a role in the regulation of acid; see Table 1). Because of their variability, the residues the internalization of the T cell co-receptor cytotoxic T at the 8 and X positions may determine the affinity and lymphocyte antigen 4 (CTLA4) [48’]. Phosphorylation of fine specificity of the interaction of the signals with the other residues outside the signal could modify the local different adaptors and, consequently, may determine the conformational context, making the signals more or less rates of internalization of different proteins as well as the accessible for interaction with AP-2. Another important likelihood that the proteins will undergo sorting at some determinant of interactions could be the oligomeric state intracellular compartment. of the plasma membrane proteins, as the linking of two or more signals would be expected to increase dramatically Recent advances in the study of protein-protein inter- the avidity for the immobilized AP-2 complexes. All of actions have allowed detailed analyses of the specificity these processes could be triggered by binding of ligands of recognition of tyrosine-based signals by clathrin coat to the endocytic receptors, thus providing a means of components. In v&o binding assays have demonstrated regulating receptor concentration within clathrin-coated a direct interaction of tyrosine-based signals with AP-2 pits on the basis of the occupancy state of the receptors. [40’,43,44*]. Screening of cDNA libraries using the yeast two-hybrid system has identified ~2, the medium Modification of AP-2 might be another way to regulate chain of the AP-2 complex, as a recognition molecule the recognition of tyrosine-based signals. Binding of for tyrosine-based signals [43]. This observation was AP-2 to clathrin cages, for instance, has been shown confirmed by various in vim binding assays, including to increase the affinity of AP-2 for peptides encoding binding of in vitro translated, labeled ~2 to glutathione- tyrosine-based signals [45*]. This increase is probably due S-transferase (GST)-tyrosine-based-signal fusion proteins to a conformational change in the AP-2 complex induced [43] and photoaffinity labeling of the AP-2 complex with by interaction with clathrin [49]. A consequence of this tyrosine-based signals [45’]. In addition, ~2 is capable of affinity modulation might be that endocytic receptors selecting peptides encoding tyrosine-based signals from are preferentially recruited to AP-2 that is pre-assembled combinatorial peptide libraries [46*]. Finally, the fine with clathrin (see Figure 1). The AP-2 complex has also specificity of interaction of tyrosine-based signals with ~2 been shown to undergo phosphorylation in vivo [ 17.1, correlates with the interaction of tyrosine-based signals and it would be interesting to examine whether this with the complete AP-2 complex [46*] as well as with phosphorylation affects the recognition of tyrosine-based 492 Membranes and sorting

signals. The APs are large protein complexes that may re- the COP11 vesicle coat [54]. On the basis of indirect data, spond to additional signals. It has recently been observed it was concluded that Emp24p is required for efficient that 3’-phosphorylated phosphoinositides enhance the vesicle formation [SS], but a subsequent study showed interaction of AP-2 with tyrosine-based endocytic signals directly that ER membranes without Emp24p formed (45.1. This finding may help to explain the importance of vesicles efficiently [56*]. phosphoinositide 3’ kinases in membrane traffic (Figure 1). Several other members of this family have been found; p-arrestin as a clathrin adaptor the first, gp25L, co-purified with calnexin [57]. Other Until recently, all accumulation of endocytic proteins members have been isolated from COPII-coated vesicles within clathrin-coated pits was explained on the basis of [56*] and COPI-coated vesicles [55,58*1. In all cases, the interactions with AP-2. A study published last year [SO’] Emp24 family members that have been found are major offers an example of another way of linking endocytic components of the vesicles, suggesting that they have an proteins to clathrin lattices. The study shows that binding important function. of agonist to the &-adrenergic receptor causes phosphory- lation of the receptor followed by binding of the cytosolic All eight members of the yeast Emp24 family are protein p-arrestin to the cytosolic domain of the receptor. type I membrane proteins with overall sequence identity p-arrestin then interacts directly with clathrin, leading to of about 20-25%. Two pairs of these sequences (one concentration of the receptor in clathrin-coated pits and pair encoded by open reading frames YAR002A and its subsequent internalization. These observations suggest YGL002W, and the other by YAL007C and YOR016C) that binding to AP-2 is not an obligatory requirement for show much more extensive homology between members concentration within clathrin-coated pits and that other of each pair, suggesting overlapping functions. The most ways of linking receptors to clathrin may be equally variable region is found at the amino-terminal end of capable of mediating internalization. This mechanism may the molecule and, as a result of this diversity, could be relevant for other G-protein-coupled receptors and be involved in selective binding of cargo. There are signal transducing receptors that bind cytosolic proteins two highly conserved cysteine residues in this domain upon activation. Moreover, it is conceivable that receptors that are characteristic of protein trafficking receptors, with large cytoplasmic domains may be able to reach but no direct evidence for cargo binding has yet been into the clathrin lattice without a need for intermediary obtained. Following this domain is a region of predicted molecules. coiled-coil structure which could be involved in a protein assembly reaction. Clear evidence has been presented Parallels of clathrin-coated-vesicle transport that at least two members of the yeast family, Emp24p with transport between the ER and the Golgi and ErvZSp, are functionally and physically associated It has been known for several years that different secretory with each other [56*]. The membrane-proximal portion proteins exit the ER at distinct rates. Several attempts to of the lumenal domain is highly conserved, suggesting find signals within secretory proteins that could account that it may play a common role among the different for their disparate exit rates met with failure. With the members. The transmembrane domains of Emp24 family discovery of the quality control system in the ER lumen, members are also conserved, and typically contain polar it was thought that these different transport rates could be residues. These relatively polar transmembrane domains explained by retention of proteins due to their differences are likely to be associated with transmembrane domains in processing, folding, or assembly in the ER. However, of other proteins, perhaps other Emp24 family members, recently it has become clear that cargo proteins are to mask these polar residues. Finally, the cytoplasmic concentrated in exit sites or vesicles budding from the ER tails of these proteins are variations on a common (51,52]. More recently, a new class of small proteins with theme. All of them have a conserved glutamine which homology to the yeast protein Emp24p has been found may be found at the membrane-cytosol interface. The that could play a role in this concentration event. membrane-proximal sequence could form an amphipathic helix, with a hydrophobic residue three residues after The primary evidence for such a role came from studies the glutamine and one or two conserved phenylalanine showing that the emp24 mutation in Saccharomyces cenvisiae residues a turn of the helix later. Three members of the had a deleterious effect on the ER+Golgi transport of Emp24 family contain a typical dilysine ER-localization only some secretory proteins. For one of the affected sequence of the KXKXX (single-letter code for amino proteins, invertase, it was shown that this defect was not acids) type; four contain basic residues near the carboxyl due to a delay in folding or oligomerization. Emp’24p terminus, but would not be predicted to be efficient was concentrated in ER-derived COPII-coated vesicles; dilysine-type signals; and one, Emp24p, contains no basic this..fact led to the hypothesis that the Emp24p-related residues near the carboxyl terminus. The role of the proteins act to concentrate cargo molecules into budding cytoplasmic tail is very likely to be in the binding of vesicles [53]. The emp24 mutation also caused secretion cytoplasmic coat proteins (see below), probably both COP1 of KarZp, a lumenal ER chaperone, and could suppress a and COPII, as members of the Emp24 family are found deletion of the SEC13 which encodes a component of in both vesicle populations. If these proteins act as cargo Linking cargo to vesicle formation Kirchhausen, Bonifacino and Riezman 493

receptors for traffic between the ER and the Golgi, each National Science Foundation). T Kirchhausen would like to thank the members of his lab for putting up with him while he was having fun writing one would have to cycle between the two organelles. this review.

Recent experiments have studied the interaction of cytoplasmic tails of different Emp24 family proteins References and recommended reading with COPI proteins [.59]. This study suggested that the Papers of particular interest, published within the annual period of review, have been highlighted as: different tail sequences bind to different subcomplexes of COPI. Tails with dilysine motifs bound a, p’, and E COP . of special interest l * of outstanding interest (B subcomplex), while sequences without the dilysine motif bound /3, y, and 5 COP (F subcomplex). The latter 1. Kirchhausen T: Coated pits and vesicles-sorting it all out Gun binding was dependent upon the FF (single-letter code Opin Struct Biol 1993, 3:162-l 66. for amino acids) sequence in the tails while the former was 2. Robinson MS: Adaptins. Trends Cell Biol 1992, 2:293-297. not. The physiological relevance of these data remains to 3. Robinson MS: The role of clathrin. adaptors and in be shown because the binding efficiency was extremely endocytosis. Gun Opin Cell Biol 1994, 6:536-544. low (

However, this work is at a very early stage and more 16. Shih W, Gallusser A, Kirchhausen T: A clathrin-binding site in direct experiments will be necessary to examine cargo the hinge of the beta-2 chain of mammalian AP-2 complexes. J Biol Chem 1995, 270:31063-31090. concentration in this system. 1 7. Wilde A, Brodsky FM: In viva phosphorylation of adaptors Acknowledgements . regulates their interaction with clathrin. J Cell Biol 1996, 135:635-645. The writing of this review was supported by grams to T Kirchhausen Shows that cytosolic but not membrane-bound subunits of AP-2 (subunits IX, (from the National lnsticutes of Health) and to H Riezman (from the Swiss p2 and ~2) are phosphorylated in viva. Phosphorylated AP-2 failed lo bind 494 Membranes and sorting

in vitro to preformed clathrin cages, suggesting that phosphorylation may 37 Hicke L. Rierman t-t: Ubiauitinatton of a veast olasma regulate recruitment of clathrin to the plasma membrane. membrane receptor signals its ligand-siimulakd endocytosis. Cell 1996, 6:1721-1742. 16. Goodman OB Jr, Keen JH: The a chain of the AP-2 adaptor is a clathrin binding subunit J B/o/ Chem 1995, 270:23768-23773. 38. Grote E. Kelk RB: Endocvtosis of VAMP is facilitated by a synaptih vesicle targeting signal. J Cell Biol 1996, 19. Dell’Angelica EC, Ooi CE, Bonifacino JS: @A-adaptin: a 132~537-547. subunit of the adaptor-like complex AP-3. J Biol Chem 1997, 272:15078-l 5004. 39. Glickman JN, Conibear E, Pearse BM: Specificity of binding of clathrin adaptors to signals on the mannose-6- 20. Fazioli F, Minichiello L, Matoskova B, Wong WT, DiFiore PP: phosphate/insulin-like growth factor II receptor. EMS0 J 1989, EpslS. a novel tyrosine kinase substrate, exhibits transforming 8:1041-1047. activity. MO/ Cell Biol 1993, 13:5814-5828. 40. Heilker R, Manning-Krieg U, Zuber JF, Spiess M: In vitro 21. Benmerah A, Begue B, Dautry-Varsat A, Cerf-Bensussan N: The . binding of clathrin adaptors to sorting signals correlates ear of alpha-adaptin interacts with the COOH-terminal domain with endocytosis and basolateral sorting. EMS0 J 1996, of the Epsl5 protein. J Biol Chem 1996, 271 :1211 l-l 2116. 15:2893-2899. Shows that both tyrosme-based and drleucine-based receptor signals inter- 22. Tebar F, Sorkina T, Sorkin A, Ericsson M, Kirchhausen T: Epsl5 act with the AP-1 and AP-2 adaptors. is a component of clathrtn-coated pits and vesicles and is located at the rim of coated pits. J Biol Chem 1996, 41. Marks MS, Woodruff L, Ohno H, Bonifacino JS: Protein targeting 271:20727-28730. . by tyrosine- and di-leucine-based signals: evidence for distinct saturable components. J Cell Biol1996, 136:341-354. 23. Vandelft S, Schumacher C, Hage W, Verkleij At, Henegouwen P: Demonstrates that sorting processes mediated by both tyrosine-based and Association and colocalization of Epsi 5 with adaptor protein-2 dileucine-based signals are saturable in vivo. However, tyrosine-based and and clathrin. J Cell Biol 1997, 136:81 l-82 1. dileucine-based signals do not compete with one another, suggesting that they bind to different sites of the sorting machinery. 24. larmolo G, Salcini AE, Gaidorov I, Goodman 08, Baulida J, Carpenter G, Pelicci PG, DiFiore PP, Keen JH: Mapping of the 42. Kornfeld S: Structure and function of the mannose 6- molecular determinants involved in the interaction between phosphate/tnsulinlike growth factor II receptors. Annu Rev Epsl5 and AP-2. Cancer Res 1997, 57:240-245. Biochem 1992, 61:307-330.

25. Benedetti H, Raths S, Crausaz F, R&man H: The END3 gene 43. Ohno H, Stewart J, Fournier MC, Bosshart H, Rhee I, Miyatake S, encodes a protein that is required for the internalization step Saito T, Gallusser A, Kirchhausen T, Bonifacino JS: Interaction of endocytosis and for actin cytoskeleton organization in yeast of tyrosine-based sorting signals with clathrin-associated MO/ B/o/ Cell 1994, 5:1023-l 037. proteins. Science 1995, 269:1872-l 875.

26. Wendland B, McCaffery JM, Xiao Q, Emr SD: A novel 44. Honing S, Griffith J, Geuze HJ, Hunziker W: The tyrosine- . fluorescence-activated cell sorter-based screen for yeast based lysosomal targeting signal in lamp-l mediates sorting endocytosis mutants identifies a yeast homologue of into Golgi-derived clathrin-coated vesicles. EMBO J 1996, mammalian Epsl5. J Cell Biol 1996, 135:1465-l 500. 16:5230-5239. The cytosolic tail of the lysosomal membrane protein lamp-l, which contains 27. Tang HY, Cai M: The EH-domain-containing protein pan1 is a tyrosine-based sorting signal, is shown to interact with both the AP-1 and required for normal organization of the actin cytoskeleton in the AP-2 adaptors. In addition, the study shows that lamp-l is concentrated Saccharomyces cerevisiae. MO/ Cell Biol 1996, 16:4897-4914. in AP-l-containing vesicles in the TGN, suggesting that the protein can be transported to lysosomes from the TGN. 28. Schumacher C, Knudsen BS, Ohuchi T, DiFiore PP, Glassman RH, Hanafusa H: The SH3 domain of Crk binds specifically to a 45. Rapoport I, Miyaxali M, Boll W, Duckworth B, Cantley LC, . conserved proline-rich motif in Epsl6 and Epsl5R. J Biol Shoelson S, -Kirchhausen T: Regulatory interactions in the Chem 1995, 270:15341-l 5347. recognition of endocytic sorting signals by AP-2 complexes. EMBO J 1997,9:2240-2250. 29. Trowbridge IS, Collawn JF, Hopkins CR: Signal-dependent Demonstrates two ways by which the interaction of AP-2 with tyrosine-based membrane protein trafficking in the endocytic pathway. Annu sorting signals can be regulated. Phosphoinositide-3’-phosphate association Rev Cell Biol 1993, 9129-l 61. with AP-2 or co-assembly of AP-2 with clathrin to form coats increases the affinity of AP-2 for tyrosine-based endocytic signals. 30. Mellman I: Endocytosis and molecular sorting. Annu Rev Cell Dev Biol 1996, 12:575-625. 46. Boll W, Ohno H, Songyang 2, Rapoport I, Cantley LC, . Bonifacino JS, Kirchhausen T: Sequence requirements for the 31. Marks MS, Ohno H, Kirchhausen T, Bonifacino JS: Protein sorting recognition of tyrosine-based endocytic signals by clathrin by tyrosine-based signals: adapting to the Ys and wherefores. AP-2 complexes. E MB0 J 1996, 16:5789-5795. fiends Cell Viol 1997, 7:124-l 28. Screening of combinatorial libraries shows that both ~2 and AP-2 prefer to bind to YXXB-type sorting signals that have arginine residues at the X 32. Chen WJ, Goldstein JL, Brown MS: NPXY, a sequence often positions. The sequence requirements for binding to ~2 and AP-2 are similar, found in cytoplasmic tails is required for coated pit-mediated further suggesting that ~2 is the signal-recognition component of AP-2. internalization of the low density lipoprotein receptor. J Biol Chem 1990, 265:3116-3123. 47. Ohno H, Fournier MC, Pay G, Bonifacino JS: Structural . determinants of interaction of tyrosine-based sorting 33. Canfield WM, Johnson KF, Ye RD, Gregory W, Kornfeld S: signals with the adaptor medium chains. J Biol Chem 1996, Localization of the signal for rapid internalization of the bovine 271:29009-29015. cation-independent mannose B-phosphate/insulin-like growth The authors of this paper used a yeast two-hybrid screen to examine the factor-h receptor to amino acids 24-29 of the cytoplasmic tail. sequence factors that determine the avidity and specificity of interactions of J Biol Chem 1991,266:5682-5688. tyrosine-based signals with isolated ul, 12, and p3A. The authors demon- strate that both the exact sequence of the signal and its position within the 34. Letourneur F, Klausner RD: A novel di-leucine motif and a receptor tail are important determinants of this interaction with isolated u tyrosine-based motif independently mediate lysosomal chains. targeting and endocytosis of CD3 chains. Cell 1992, 69:1143-l 157. 48. Shiratori T, Miyatake S, Ohno H, Nakaseko C, Bonifacino JS, . Saito T: Tyrosine phosphorylation controls the internalization of 35. Voorhees P, Deignan E, van Donselaar E, Humphrey J, Marks MS, CTLM by regulating its interaction with the clathrin-associated Peters PJ, Bonifacino JS: An acidic sequence within the complex AP-2. immunity 1997, in press. cytoplasmic domain of furin functions as a determinant of Shows that a tyrosine-based signal in the cytosolic tail of the CTLA4 T cell trans.Golgi network localization and internalization from the co-receptor can function as either an internalization signal recognized by cell surface. EMBO J 1995, 14:4961-4975. u2 or a binding site for SHZ-confaining signal transduction molecules de- pending on whether it is unphosphorylated or phosphorylated, respectively. 36. ltin C, Kappeler F, Linstedt AD, Hauri HP: A novel endocytosis Suggests that phosphorylation of the critical tyrosine residue functions as signal related to the KKXX ER-retrieval signal. EMBO J 1995, a switch to determine either internalization of CTLA4 or coupling of CTLA4 14:2250-2256. activation to signal transduction pathways. Linking cargo to vesicle formation Kirchhausen, Bonifacino and Riezman 495

49. Matsui W, Kirchhausen T: Stabilization of clathrin coats by 55. Stamnes MA. Craiohead MW. Hoe MH. Lamoen N. Geromanos S. the core of the clathrin-associated protein complex AP-2. Tempst P RcthmG JE: An integral membrane component Biochemistry 1990, 29:10791-l 0796. of -coated transport vesicles defines a family of proteins involved in budding. Proc Nat/ Acad Sci USA 1995, 50. Goodman OB Jr, Krupnick JG, Santini F, Gurevich W, Penn RB, 92:601 l-601 5. . GaQnon AW, Keen JH, Banovic JL: 5-arrestin acts as a clathrin adaptor in endocytosis of the j32-adrenergic receptor. Nature 56. Belden WJ, Barlowe C: Erv26p, a component of COPII-coated 1996, 303447-450. . vesicles, forms a complex with Ernp24p that is required for Describes an AP-P-independent but clathrin-dependent mechanism of endo- efficient endoplasmic reticulum to Golgi transport. J Biol Chem cytosis. Binding of isoprenaline to the j31-adrenergic receptor triggers phos- 1996. 271:26939-26946. phorylation of @arrestin and its recruitment from a cytosolic pool to the cy- This paper shows that two members of the Emp24 family form a complex tosolic domain of the receptor. 5-arrestin then interacts directly with clathrin, and that deletion of EMP24 causes another member, Et-v25p, to become leading to concentration of the pa-adrenergic receptor within clathrin-coated unstable. The authors also show directly that membranes without Emp24p pits and subsequent internalization of the receptor. or Erv25p form ER-derived COPlI-coated vesicles as well as do wild-type membranes. 51. Mizuno M, Singer SJ: A soluble secretory protein is first concentrated in the endoplasmic reticulum before transfer 57. Wada I, Rindress D, Cameron PH, Ou WJ, Doherty JJ, Louvard D, to the . Proc Nat/ Acad Sci USA 1993, Bell AW, Dignard D. Thomas DY, Bergeron JJ: SSR alpha and 90:5732-5736. associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J Biol Chem 1991, 52. Batch WE, McCaffery JM, Plutner H, Farquhar MG: Vesicular 266:19599-l 9610. stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Ce// 1994, 56. Sohn K, Orci L, Ravszzola M, Amherdt M, Bremser M, Lottspeich F, 76:641-652. . Fiedler K, Helms JB, Wieland FT: A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in 53. Schimmliller F, Singer-KriJQer B, Schrijder S, Kriiger U, Barlow% C, coatomer binding. J Ce// Biol 1996, 136:1239-l 246. Riezman H: The absence of Emp24p. a component of ER- Shows that a member of the Emo24 familv is found in COPI-coated struc- derived COPII-coated vesicles, causes a defect in transport of tures near the Golgi. Demonstrates the b&ding of the tail sequence of the selected proteins to the Golgi. EMBO J 1995, 14:1329-l 339. Emp24 family member to COPI proteins and dependence of this binding on the Phe-Phe motif in the tail. 54. Elrod-Erickson MJ, Kaiser CA: that control the fidelity of endoplasmic reticulum to Golgi transport identifled as 59. Fiedler K. Veit M, Stamnes MA, Rothman JE: Bimodal interaction suppressors of vesicle budding mutations. MO/ Viol Cell 1996, of coatonier with the p24 family of putative cargo receptors. 7:1943-l 056. Science 1996,273:1396-l 399.