Adaptor Protein Complexes As the Key Regulators of Protein Sorting in the Post-Golgi Network

Adaptor Protein Complexes as the Key Regulators of Protein Sorting in the Post-Golgi Network

Fubito Nakatsu1,2* and Hiroshi Ohno1,2 1Division of Molecular Membrane Biology, Cancer Research Institute, Kanazawa University, 13-1 Takara- machi, Kanazawa 920-0934, Japan and 2Laboratory for Epithelial Immunobiology, Research Center for Allergy and Immunology, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan

ABSTRACT. Adaptor protein (AP) complexes are cytosolic heterotetramers that mediate the sorting of membrane proteins in the secretory and endocytic pathways. AP complexes are involved in the formation of clathrin-coated vesicles (CCVs) by recruiting the scaffold protein, clathrin. AP complexes also play a pivotal role in the cargo selection by recognizing the sorting signals within the cytoplasmic tail of integral membrane proteins. Six distinct AP complexes have been identified. AP-2 mediates endocytosis from the plasma membrane, while AP- 1, AP-3 and AP-4 play a role in the endosomal/lysosomal sorting pathways. Moreover, tissue-specific sorting events such as the basolateral sorting in polarized epithelial cells and the biogenesis of specialized organelles including melanosomes and synaptic vesicles are also regulated by members of AP complexes. The application of a variety of methodologies have gradually revealed the physiological role of AP complexes.

Key words: adaptor protein complex/clathrin/membrane traffic/vesicular transport/sorting signals/post-Golgi network

Introduction brane, transports across the cytosol, and tether and fuse with the target organelle membrane. This process involves a vari- Protein transport between the organelles of secretory and ety of cytosolic factors. Among the important proteins that endocytic pathways is mainly mediated by membrane- regulate vesicular transport are the adaptor protein (AP) bound carriers, the transport vesicles. Cargo proteins are complexes. Growing evidence supports the view that AP concentrated in a specialized region called coated pits at the complexes play important roles in cargo selection as well as donor organelle membrane and packed into a nascent vesi- vesicle formation. In this review, we will focus on the cle. The vesicle eventually pinches off from the donor mem- advances in vesicular trafficking regulated by AP complexes. *To whom correspondence should be addressed: Fubito Nakatsu, Ph.D., Division of Molecular Membrane Biology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan, and Identification of AP complexes the Laboratory for Epithelial Immunobiology, Research Center for Allergy and Immunology, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, In 1969, Kanaseki et al. morphologically identified Japan. coated vesicles for the first time in guinea pig brain synapto- Tel: +81–76–265–2724, Fax: +81–76–234–4519 somes. They found a vesicle in a spherical “basketwork” E-mail: [email protected]/[email protected] Abbreviations: AAK1, adaptor-associated kinase 1; ALP, alkaline that is composed of regular pentagons and hexagons with phosphatase; AP, adaptor protein; ARF-1, ADP-ribosylation factor-1, sides of equal length (Kanaseki and Kadota, 1969). Several BFA, brefeldin A; CCP, clathrin-coated pit; CCV, clathrin-coated vesicle; years later, Pearse purified the coated vesicles biochemi- CI-MPR, cation-independent mannose 6-phosphate receptor; EGFR, epider- cally and showed that the coat is made of an approximately mal growth factor receptor; GAK, cyclin G-associated kinase; GGA, Golgi-localizing, -adaptin ear homology domain, ARF-binding proteins; 180 kD protein (Pearse, 1975). In that paper she proposed to HPS, Hermansky-Pudlak syndrome; Ii, invariant chain; LDLR, low-den- call it “clathrin.” Soon after clathrin was identified, other sity lipoprotein receptor; LRP, low-density lipoprotein receptor-related ~100 kD molecules were identified in coated vesicles (Keen protein; PI[4,5]P2, phosphatidylinositol (4,5)-bisphosphate; PI[3,4,5]P3, phosphatidylinositol (3,4,5)-triphosphate; PP2A, protein phosphatase 2A; et al., 1979). It was also shown that unknown ~100 kD mol- siRNA, small interfering RNA; SPD, storage pool deficiency; SPR, surface ecules are involved in the clathrin binding to the vesicles plasmon resonance spectroscopy; TfR, transferrin receptor; TGN, trans- (Unanue et al., 1981; Vigers et al., 1986). In subsequent Golgi network; -APP, -amyloid precursor protein.

419 F. Nakatsu and H. Ohno studies, biochemical experiments showed that the unknown Recognition of sorting signals by AP complexes ~100 kD molecules were the components of heterotet- Transmembrane proteins often contain sorting signals rameric protein complexes, and these complexes were that define their localization in the cell. Sorting signals con- termed AP-1 and AP-2 (Keen, 1987). sist of a short amino acid stretch usually located within the To date, four ubiquitously expressed AP complexes have cytoplasmic region of membrane proteins. It is known that been identified in human and mouse, AP-1~4 (Fig. 1) in the secretory and endocytic pathways, AP complexes (Boehm and Bonifacino, 2001; Kirchhausen, 1999). In addi- selectively recognize and directly bind to the sorting sig- tion to these ubiquitous AP complexes, AP-1 and AP-3 have nal(s) (Bonifacino and Traub, 2003; Bonifacino and a cell-type specific isoform. AP-1B is expressed in polar- Dell’Angelica, 1999). A number of sorting signals have ized epithelial cells and mediates basolateral sorting, while been identified in the last decade. Some of them listed AP-3B is expressed in neurons and involved in synaptic below have been well characterized as the targets of AP vesicle biogenesis. All six AP complexes are heterotetram- complexes. ers which consist of two large subunits (/1, /2, /3 and /4, 100~140 kD), one medium subunit ( 1–4, ~50 (i) Tyrosine signals kD) and one small subunit (1–4, ~20 kD). One of the large subunits in each AP complex (, , and ) mediates bind- In 1986, Goldstein et al. demonstrated that the low-den- ing to the target membrane. The other large subunits (1–3) sity lipoprotein receptor (LDLR) from a patient suffering recruit clathrin through the clathrin binding sequence (the from hypercholesterolemia had a mutation which replaced clathrin box) (Brodsky et al., 2001), although it is not the Y residue with C, and that rapid internalization of the recep- case for 4 which lacks the clathrin box (Boehm and Boni- tor was abolished due to the mutation (Davis et al., 1986). facino, 2001; Dell’Angelica et al., 1999a). Medium sub- This discovery led to the identification of an endocytosis units ( 1A/B, 2, 3A/B and 4) are responsible for cargo signal that contains the NPXY sequence as a consensus recognition. subunits directly recognize tyrosine-based amino acid residue in some transmembrane proteins such as sorting signals (for tyrosine signals, see below) within the low-density lipoprotein receptor-related protein (LRP), cytoplasmic tail of cargo membrane proteins (Bonifacino megalin and -amyloid precursor protein (-APP) (Bonifa- and Traub, 2003). The small subunits (1, 2, 3A/B and cino and Traub, 2003). Biochemical and ultrastructural 4) are thought to be involved in the stabilization of the studies have shown that the NPXY signal is involved only complex based on yeast two-hybrid analyses and X-ray in endocytosis from the plasma membrane but not in sorting crystal structure (Collins et al., 2002). to endosomes/lysosomes at the trans-Golgi network (TGN).

Fig. 1. Schematic representation of the AP complexes. Six distinct AP complexes have been identified so far. AP-1A, AP-2, AP-3A and AP-4 are expressed ubiquitously, whereas AP-1B and AP-3B are expressed exclusively in epithelia and neuron, respectively. Organelle(s) on which the complex is localized is shown under the schema of each complex.

420 Adaptor Protein Complexes and Protein Sorting

These data led us to the idea that AP-2 directly recognizes binding to the cytoplasmic domain of membrane protein. the NPXY signal. Boll et al. demonstrated that AP-2 could Substitution of leucine residues impede the signal activity, bind, albeit weakly, to the FDNPVY peptide from LDLR by although the second of the two leucines could be changed to surface plasmon resonance spectroscopy (SPR) (Boll et al., isoleucine without loss of the activity. Amino acid position 2002). Recently, however, PTB domain-containing mole- –5 from the first L is, in some cases, serine that can be cules Dab2 and ARH were reported to interact with the sig- phosphorylated. For instance, CD3 undergoes rapid endo- nal and be involved in the endocytosis of LDLR family cytosis when the serine residue of SDKQTLL is inducibly members. The key molecule(s) responsible for the recogni- phosphorylated upon stimulation (Dietrich et al., 1994; tion of the signals remains uncertain. Dietrich et al., 1997). A similar downregulation of CD4 is Two years after the discovery of the NPXY signal, observed upon phosphorylation of the serine residue in its another type of tyrosine signal YXXØ (where Y is tyrosine, SQIKRLL signal (Pitcher et al., 1999). D/EXXXLL signals X is any amino acid and Ø is an amino acid with a bulky mediate the targeting to lysosomes and lysosome-related hydrophobic side chain) was also reported to mediate organelles as well as endocytosis (Bonifacino and Traub, endocytosis (Canfield et al., 1991; Jadot et al., 1992). In 2003). contrast to the NPXY signal, YXXØ signal appears more Several lines of evidence clearly showed that AP com- frequently in membrane proteins that undergo rapid endo- plexes are involved in the recognition of D/EXXXLL sig- cytosis. Moreover, the YXXØ signal has been shown to nals (Heilker et al., 1996; Honing et al., 1998; Le Borgne mediate targeting to the basolateral plasma membrane, et al., 1998; Peden et al., 2001). In vitro binding studies lysosomes and lysosome-related organelles such as melano- demonstrated that AP complex weakly interacted with the somes and antigen-processing compartments (Matter and D/EXXXLL signals. However, the subunit responsible for Mellman, 1994; Mellman, 1996). YXXØ signals can be the recognition of the signals has been controversial. Interac- found in the cytosolic region of type I, type II and multi- tion of subunits with the D/EXXXLL signals from Ii was spanning membrane proteins. reported by phage display system and SPR (Craig et al., It has been difficult to identify the molecule(s) which rec- 2000; Rodionov and Bakke, 1998), while binding of ognize the YXXØ signal since the affinity of the interaction subunits to the signals was also detected by photoaffinity is so low. In order to overcome this problem, we took labeling (Greenberg et al., 1998; Rapoport et al., 1998). advantage of the yeast two-hybrid system to identify the Further experiments will be required to clarify the precise receptor for the signals. Using the YQRL signal from binding site for D/EXXXLL signals in AP complexes. TGN38 as a bait, we identified the medium subunit ( 2) of AP-2 as the recognition molecule for the signals (Ohno et al., 1995). Subsequent studies including combinatorial Function of the AP complexes library screening have demonstrated that the interaction of In higher eukaryotic, especially in mammalian, cells, 2 critically depends on Y and Ø residues, and that the many specialized organelles exist in the secretory and affinity of interaction is influenced by the residues sur- endocytic pathways. In order to interact with extracellular rounding the YXXØ sequence (Ohno et al., 1996). More- milieu in addition to keeping the cells alive, these organelles over, all the subunits were found to bind to a distinct but are interconnected by membrane traffic to constitute the so- overlapping subset of YXXØ signals (Aguilar et al., 2001; called “post-Golgi network”. AP complexes are among the Ohno et al., 1998). most important players that support the sophisticated post- Golgi network to make it function properly by regulating (ii) Di-leucine signals the protein traffic. Six distinct AP complexes participate in the sorting event along different pathways such as endocy- Di-leucine signal was first discovered by Letourneur and tosis, and endosomal/lysosomal and basolateral targeting Klausner in the cytoplasmic tail of CD3 subunit of T cell (Fig. 2). In this chapter, we will review recent advances in receptor. They defined the DKQTLL sequence as a minimal the function of each complex in the cells, and discuss the sequence that directs rapid endocytosis (Letourneur and physiological roles of the complexes in organisms. Klausner, 1992). After the discovery, other membrane proteins, such as the cation-independent mannose 6-phosphate (i) AP-1A receptor (CI-MPR) and the invariant chain (Ii) which also contain the di-leucine signal, were reported whose consen- AP-1A is isolated biochemically as a component of sus sequence turned out to be D/EXXXLL (where D/E is CCVs (Keen, 1987). Morphological studies including aspartate or glutamate, X is any amino acid and L is leucine) immunofluorescence staining and electron microscopy indi- (Bonifacino and Traub, 2003). In contrast with the YXXØ cated that AP-1 was involved in the formation of CCVs signal, the D/EXXXLL signal is found in the cytosolic pro- from the TGN (Traub and Kornfeld, 1997). Recruitment of teins such as Nef (Greenberg et al., 1998) and ubiquitin AP-1 to the TGN membrane is regulated by a small GTPase, (Nakatsu et al., 2000), and function as sorting signals upon ADP-ribosylation factor 1 (ARF-1) (Stamnes and Rothman,

421 F. Nakatsu and H. Ohno

Fig. 2. Sorting pathways mediated by AP complexes. Newly synthesized proteins are transported along the biosynthetic pathway to the TGN where initial selection of cargo proteins by AP complexes occurs. Some membrane proteins destined for endosomes, lysosomes, and/or lysosome-related organelles such as melanosomes and synaptic vesicles are sorted by AP-1, AP-3A/B and/or AP-4. Other membrane proteins reach the plasma membrane along the so-called ‘bulk-flow’ route, where some are subject to endocytosis by AP-2. In polarized epithelial cells, AP-1B and AP-4 is suggested to mediate the sorting to the basolateral plasma membrane.

1993; Traub et al., 1993), which cycles between an inactive especially required for the viability of a single cell since GDP-bound form in cytosol and an active GTP-bound form homozygous embryos die at as early as E3.5, at which stage that associates with the membrane like other small GTPases the maternal mRNAs and proteins generally dilute out from (reviewed in Boman and Kahn, 1995). There is also evi- the blastocyst cells (Zizioli et al., 1999). Embryonic let- dence that phosphorylation/dephosphorylation events are hality of -deficient mice also implies that the 2 sub- involved in the regulation of the function of AP-1. Ghosh unit, which shares 60% identity with and is expressed and Kornfeld demonstrated that 1 subunit of AP-1 is ubiquitously (Takatsu et al., 1998), could not substitute for dephosphorylated by a Golgi-associated isoform of protein the function of . On the other hand, 1A-deficient mice phosphatase 2A (PP2A) upon translocation to TGN, which died at E13.5, indicating that 1A is not essential for survival allows AP-1 to initiate clathrin assembly (Ghosh and Korn- of the cells (Meyer et al., 2000). This result raised the possi- feld, 2003). They also showed that 1A undergoes phos- bility that 1B could compensate for the 1A-deficiency phorylation, most likely by cyclin G-associated kinase until E13.5. In that paper, Meyer et al. demonstrated that (GAK/auxilin 2) (Umeda et al., 2000), which results in the in embryonic fibroblast cells derived from 1A-deficient conformational change associated with increased affinity to mice, MPRs were accumulated in endosomes instead of the sorting signals. AP-1A was also shown to be recruited to TGN. This result suggested that AP-1A is physiologically immature secretory granules (Dittie et al., 1996; Dittie involved in the sorting of MPRs back to the TGN. Recently, et al., 1997; Klumperman et al., 1998; Tooze and Tooze, novel coat proteins named Golgi-localizing, -adaptin ear 1986). homology domain, ARF-binding proteins (GGA) were Recently, Schu and colleagues have established - and identified. Subsequent studies have shown that GGAs play a 1A-deficient mice using gene targeting. Both mutant mice role in the sorting of several proteins including MPRs to suffer from embryonic lethality, indicating that AP-1A is endosomes from the TGN (for further details, see the review essential for the development of the mice. The subunit is by Nakayama and Wakatsuki in this issue). It had been sug-

422 Adaptor Protein Complexes and Protein Sorting gested, therefore, that AP-1A is responsible for retrograde and Robinson, 1998; Kirchhausen, 1999). Biochemical retrieval of MPRs from the endosomes to the TGN, while studies and structural analyses demonstrated that positively GGA is involved in the anterograde transport from the TGN charged residues located within the hinge region are to endosomes. However, Kornfeld and colleagues showed involved in the binding to phosphatidylinositol (4,5)- that GGAs directly interacted with the subunit of AP-1A bisphosphate (PI[4,5]P2) and/or phosphatidylinositol (3,4,5)- to form clathrin-coated buds at the TGN. In addition, bio- triphosphate (PI[3,4,5]P3) (Collins et al., 2002; Gaidarov et chemical and ultrastructural analyses suggested that AP-1A al., 1996). Once the critical residues for PI[4,5]P2 and/or and GGAs cooperate to package MPRs into the TGN- PI[3,4,5]P3 binding were mutated, the subunit exhibited derived intermediates (Doray et al., 2002). Furthermore, neither plasma membrane localization nor incorporation time-lapse recordings demonstrated that AP-1A labeled into the CCV (Gaidarov and Keen, 1999). Phosphorylation with green fluorescence protein or its spectral variants actu- of the subunit is also involved in the localization to the ally moved from the TGN toward the periphery of cells plasma membrane. Recognition of the sorting signals is a (Puertollano et al., 2003; Reusch et al., 2002). Altogether, quite important step for cargo selection. 2 is responsible the precise sorting pathways regulated by AP-1A still for the recognition of YXXØ signals. Yeast two-hybrid remain a matter of controversy. analyses indicated that the C-terminal 2/3 of 2 is important for YXXØ recognition, while the N-terminal 1/3 is respon- (ii) AP-1B sible for assembly with 2 (Aguilar et al., 1997). Subse- quent crystallography of the C-terminal region of 2 dem- In the search of the EST database, we isolated a new onstrated that four amino acid residues well-conserved in all subunit homolog, 1B, which shared 80% of amino acid members of subunit family formed the binding pocket for sequence with 1A. Northern blotting and in situ hybridiza- the Y residue of YXXØ signals (Owen and Evans, 1998). tion revealed that 1B was specifically expressed in polar- Indeed, mutagenesis of these critical residues in 2 as well ized epithelial cells (Ohno et al., 1999). Biochemical analy- as 1B has been shown to interfere with the sorting of sis showed that 1B formed a novel, epithelium-specific AP YXXØ-containing proteins in the cell (Nesterov et al., complex, AP-1B, with 1, 1 and 1 subunits, all of which 1999; Sugimoto et al., 2002). However, the atomic structure were shared with AP-1A (Folsch et al., 1999). We further of the AP-2 complex revealed that the C-terminal region of demonstrated that AP-1B is involved in the basolateral sort- 2, which directly binds the YXXØ signal, seemed to be ing. A porcine kidney cell line LLC-PK1 exhibited the mis- buried in the core structure of AP-2 (Collins et al., 2002). localization of the LDLR and transferrin receptor (TfR) to From the data obtained with the structural analysis, Collins the apical plasma membrane, and that the missorting was et al. proposed that phosphorylation of T156 in the connec- due to the lack of 1B because introduction of 1B to LLC- tion loop of the N-terminal region of 2 induces a confor- PK1 reconstituted the expression of AP-1B and restored the mational change that permits it to access the YXXØ signal basolateral localization of these proteins. 1B also mediates (Collins et al., 2002). The report that phosphorylation YXXØ signal-dependent basolateral sorting, although the of T156 by adaptor-associated kinase 1 (AAK1) results in basolateral sorting signal of LDLR and TfR did not belong increased affinity for the YXXØ signal supports this to the tyrosine signal. Mutagenesis experiment of 1B indi- hypothesis (Ricotta et al., 2002). In addition, 2 was also cates that the binding site for the signals of LDLR and TfR shown to be able to bind phosphoinositides, in particular is distinct from that for YXXØ signals (Sugimoto et al., PI[4,5]P2, within the C terminal region, while the expres- 2002). Morphologically, 1B was shown to be localized in sion of a mutant 2 lacking the PI[4,5]P2-binding site hin- the CCV near the TGN by immunoelectron microscopy, dered the receptor-mediated endocytosis (Rohde et al., which supports the involvement of 1B in the basolateral 2002), suggesting that the binding of PI[4,5]P2 to 2 is also sorting from TGN (Folsch et al., 2001). 1B was also involved in the recognition process of the tyrosine signals. reported to be involved in basolateral recycling of LDLR According to the clathrin box motif in the hinge region and TfR from the sorting endosomes (Gan et al., 2002). (Dell’Angelica et al., 1998), in conjunction with a second clathrin-binding site in the ear domain (Owen and Luzio, (iii) AP-2 2000), in the 2 subunit, clathrin is recruited to the complex AP-2 is a well-defined AP complex that regulates recep- to form the CCV. tor-mediated endocytosis. Until now, a number of plasma The work by Brown and Goldstein that demonstrated that membrane proteins including the receptors, transporters, rapid internalization of LDLR containing the Y to C muta- adhesion molecules and virus products are known to be tion in NPXY signal was hampered was quite important as a internalized in AP-2-dependent fashion. Fig. 2 depicts the breakthrough for understanding the mechanism of signal- molecular mechanism of endocytosis. Recruitment of AP-2 mediated endocytosis. Moreover, it also suggested that from cytosol to the plasma membrane is the initial step of endocytic defect by this mutation resulted in familial hyper- endocytosis. Several lines of evidence suggest that the cholesterolemia. Therefore, these results indicate that AP-2 subunit mediates the binding to the plasma membrane (Hirst has a physiologically essential role. Recently, the roles of

423 F. Nakatsu and H. Ohno

Fig. 3. Molecular mechanism of endocytosis. Biochemical, morphological and structural analyses have contributed to the molecular dissection of endocytosis mediated by AP-2. Endocytosis begins with the targeting of AP-2 to the plasma membrane mainly by the subunit. After the translocation to the plasma membrane, the 2 subunit is phosphorylated at T156. In conjunction with the binding of 2 to the PI[4,5]P2, this phosphorylation is thought to induce the conformational change of the C-terminal half of 2 to be able to bind the tyrosine signal of cargo membrane proteins. Recruitment of clathrin to the 2 subunit promotes the formation of CCV.

AP-2 in cultured cell lines have been investigated by several that, in the absence of AP-2, clathrin-mediated endocytosis different approaches. Nesterov et al. established a domi- can still occur. Several endocytic accessory proteins, includ- nant-negative mutant of 2, in which critical residues for ing Dab2, ARH, Hip1 and epsin have recently been shown the recognition of the tyrosine signals were changed to to be able to bind not only to AP-2, but also to plasma alanine, and showed that HeLa cells overexpressing the membrane lipids and clathrin (reviewed in Brodsky et al., dominant negative mutant 2 exhibited impairment of 2001; Conner and Schmid, 2003b; Slepnev and De Camilli, the endocytosis of TfR, whereas epidermal growth factor 2000). It is possible that epsin may facilitate the clathrin- receptor (EGFR) was efficiently internalized (Nesterov et mediated endocytosis of EGFR by interacting with ubiq- al., 1999). Motley et al. took advantage of small interfering uitin that is conjugated to EGFR, and that Dab2 and/or ARH RNA (siRNA) to knock down the AP-2 in HeLaM cells. may function as a clathrin-adaptor to incorporate the LDLR They demonstrated that both EGFR and LDLR were inter- into the CCVs. It was also shown that clathrin- as well as nalized normally even in the AP-2-depleted cells (Motley et AP-2-depleted cells seemed to be viable and did not show al., 2003). They also showed that clathrin-coated pits any apparent increase in apoptosis although some abnor- (CCPs) still exist in AP-2-depleted cells although CCPs at malities such as growth retardation, impairment of cyto- the plasma membrane were found to be 12-fold less abun- kinesis and vacuolation were seen in the clathrin-depleted dant than those in control cells, and that epsin colocalized cells (Motley et al., 2003). However, chicken B cell line with the remaining CCPs. As was the case for the dominant- DT40 cells deficient in clathrin gene underwent apoptosis negative mutant of 2, however, endocytosis of TfR was because of the dysfunction of the anti-apoptotic survival severely impaired in the AP-2-depleted cells. Likewise, pathway (Wettey et al., 2002). Furthermore, mice deficient Conner and Schmid reached the same conclusion with the in 2 showed embryonic lethality around E3.5, and that AP-2-inactivated cells by overexpression of AAK1 (Conner homozygous mutant blastocysts could not survive unlike and Schmid, 2003a). These results taken together suggest wild-type blastocysts (HO, FN and J. S. Bonifacino, unpub-

424 Adaptor Protein Complexes and Protein Sorting lished observation). Taken together, the cells may not sur- mouse named pearl has been reported to have a mutation in vive in the complete absence of clathrin as well as AP-2. 3A subunit (Feng et al., 1999). In fibroblast cells derived from the HPS patients, some lysosomal proteins were trans- (iv) AP-3A ported from the TGN to lysosomes via the plasma membrane, instead of directly targeted intracellularly AP-3A was identified as an AP-related complex with (Dell’Angelica et al., 1999b). Likewise, perturbation of the homology search of cDNA libraries as well as database 3A expression by introduction of antisense oligonucle- (Dell’Angelica et al., 1997; Pevsner et al., 1994; Simpson et otides to the cells exhibited plasma membrane appearance al., 1996). AP-3A formed a heterotetramer like AP-1 and of the lysosomal membrane proteins en route to lysosomes AP-2, which consists of , 3A, 3A and 3 subunits. (Le Borgne et al., 1998; Le Borgne et al., 2001). These Immunofluorescence and immunoelectron microscopy results indicate that AP-3 is involved in the sorting of localized AP-3A on the TGN membrane as well as membrane proteins to lysosomes and lysosome-related peripheral membranes in which endocytosed materials were organelles at TGN and/or endosomes. contained (Dell’Angelica et al., 1997). By analogy to the AP-1 and AP-2, AP-3 was thought to be involved in CCVs. (v) AP-3B Biochemical and morphological studies indicated that clathrin was not involved in the AP-3-containing vesicles, AP-3B is a neuron-specific AP-3 complex (Hirst and and that AP-3 might associate with another scaffolding Robinson, 1998; Le Borgne and Hoflack, 1998; Robinson protein that is structurally different from clathrin (Hirst and and Bonifacino, 2001). AP-3B consists of , 3B (also Robinson, 1998; Simpson et al., 1996). Genetic studies in called -NAP), 3B and 3. Both 3B and 3B are neu- yeast also suggested that AP-3 function without clathrin ronal homologs of 3A and 3A, respectively (Boehm and because transport of alkaline phosphatase (ALP) to the Bonifacino, 2001). Based on the expression pattern, AP-3B vacuole, which is dependent on AP-3, was not impaired in was thought to be involved in neuron-specific sorting event. clathrin mutant (Vowels and Payne, 1998). However, Dell’- In 1998, Faundez et al. showed that synaptic vesicles can be Angelica et al. clearly showed that 3A subunit could inter- coated in vitro in ARF-, ATP- and temperature-dependent act with clathrin through the conserved clathrin-binding manner, and that AP-3B is involved in the synaptic vesicle domain (Dell’Angelica et al., 1998). They also demonstrated formation from PC12 endosomes (Faundez et al., 1998). that AP-3 colocalized with clathrin on the endosomal Furthermore, by using this system, we demonstrated that membrane in HeLa cells. Thus, it is likely that AP-3 is synaptic vesicle formation from PC12 endosomes was dra- functionally associated with clathrin to form CCVs in vivo. matically impaired when brain cytosol prepared from 3B- AP-3A-dependent sorting pathways were identified from deficient mice were used as a source of the coat (Blumstein genetic studies. In fruit fly, a homolog of the mammalian et al., 2001). These results clearly indicate that AP-3B is subunit was the garnet gene product that is involved in the involved in the synaptic vesicle biogenesis from endo- biosynthesis of the pigment granules. In the eye color somes. mutant garnet, a decreased level of pigment granules was As is the case for AP-3A, AP-3B also has important roles observed, indicating that AP-3 is involved in the sorting in organisms. Mocha mice have been known to have similar pathway to pigment granules (Ooi et al., 1997). In yeast, phenotypes seen in pearl mice such as hypopigmentation deletion of any of the AP-3 subunit caused the missorting of and prolonged bleeding (Swank et al., 1998). However, the vacuolar protein ALP and vacuolar t-SNARE Vam3p mocha mice additionally exhibit neuronal disorders includ- without affecting the localization of a soluble vacuolar ing deafness, balance problem and seizure. It has been protease carboxypeptidase Y (Cowles et al., 1997; Stepp et shown that mocha mice have a large deletion of the gene al., 1997; Vowels and Payne, 1998). Since pigment granules encoding subunit of AP-3, which leads the depletion of are related to the lysosomes in their origin, these results both AP-3A and AP-3B since subunit is commonly used strongly suggest that AP-3 transports lysosomal membrane in both isoforms (Kantheti et al., 1998). Therefore, neuronal proteins. defect seen in mocha mice were thought to be attributable of In mammals, AP-3A deficiency was found in human AP-3B depletion. In order to investigate the physiological and mice (Boehm and Bonifacino, 2002). Dell’Angelica role of AP-3B in detail, we have established 3B-deficient et al. discovered the subunit mutation in a patient suffer- mice. As expected, 3B-deficient mice showed spontaneous ing from Hermansky-Pudlak syndrome (HPS) type 2 seizure, which is also seen in mocha mice. However, neither (Dell’Angelica et al., 1999b). HPS is characterized as a deafness nor balance problem was seen in the 3B-deficient storage pool deficiency (SPD), which accompanies defects mice, indicating that these phenotypes were caused by AP- in the function of lysosomes and lysosome-related 3A/B double deficiency (Nakatsu et al. submitted). Thus, organelles, such as prolonged bleeding, eye color dilution AP-3B probably plays an important role in synaptic trans- and few melanosomes (reviewed in Dell’Angelica et al., mission in the central nervous system. 2000; Swank et al., 1998). In addition, a typical SPD mutant Although some of the cargo molecules of AP-3A have

425 F. Nakatsu and H. Ohno been established, an AP-3B-dependent cargo proteins has YXXØ signals that is only recognized by 4 is localized in yet to be identified. Since 3B was also capable of binding late endosomes and/or lysosomes (Aguilar et al., 2001). to a subset of tyrosine signals (Ohno et al., 1998), AP-3B Since this chimera did not undergo endocytosis, 4 is complex might mediate the sorting of certain membrane involved in the direct sorting to these organelles. In contrast, protein to the synaptic vesicles from endosomes in neurons. Simmen et al. proposed that AP-4 binds basolateral sorting Kantheti et al. showed that ZnT3, a member of vesicular signals from furin, LDLR, MPR46 and TfR, and mediates zinc transporter family, was not detected in the hippocampal basolateral sorting of these proteins in MDCK cells (Sim- mossy fiber terminals in mocha mice (Kantheti et al., 1998). men et al., 2002). However, it is not sure at present how 4 ZnT3 could be a good candidate for AP-3B-specific cargo regulates there two apparently unrelated pathways and to since ZnT3 is expressed exclusively in neurons and found what extent 4 contributes to these pathways physiologi- on synaptic vesicles (Palmiter et al., 1996). As mentioned cally. The functional characterization of AP-4 has just above, however, mocha mice lack both AP-3A and AP-3B. started. Which complex as well as which subunit are responsible for the localization of ZnT3 are currently unknown. Further studies are required to identify the AP-3B cargo proteins. Concluding remarks In this review, we summarized recent advances of sorting (vi) AP-4 mechanisms regulated by AP complexes, especially in mammals. The human genome project tells us the fact that The AP-4 complex has also been identified recently by the AP complex family consists of four members and that BLAST search of the EST database (Dell’Angelica et al., no more such complexes exist. As shown in this review, 1999a). Unlike other AP complexes, AP-4 was found only much progress has been made since the AP complex was in mammals and plants (Boehm and Bonifacino, 2001). first isolated, but there are still plenty of unresolved ques- AP-4 consists of four subunits, , 4, 4 and 4. Northern tions. How is the di-leucine signal recognized? In which blotting showed that AP-4 is expressed ubiquitously direction does AP-1 transport? Is AP-2 essential for CCV (Dell’Angelica et al., 1999a; Hirst et al., 1999). Sequence formation? What is the cargo protein of AP-3B? What is the analysis demonstrated that 4 has a low homology to other physiological role of AP-4? In addition, how is the appar- subunits other than the N-terminal trunk domain, which is ently same pathway (i.e., AP-3A vs AP-4, and AP-1B vs thought to be involved in the association with other subunits AP-4) to be distinguished is also interesting question. of AP complexes (Matsui and Kirchhausen, 1990; Schroder Although not described in this article, novel coat proteins and Ungewickell, 1991). Although hinge-like and ear-like such as GGAs, Tip47 and PACS have been reported to be domains were found in 4 subunit, there seemed to be no involved in the sorting of certain membrane proteins. Coat clathrin-binding motif in it. Indeed, AP-4 was not associ- proteins constituting the sorting machinery seemed to be ated with clathrin by immunoelectron microscopy (Hirst et much more diverse than were originally thought. In addi- al., 1999). These results suggest that AP-4 might interact tion, more than 30 accessory proteins are thought to regulate with a non-clathrin coat. Immunofluorescence and immuno- the AP complex-driven CCP/CCV formation and cargo- electron microscopy showed that AP-4 was localized in sorting (reviewed in Brodsky et al., 2001; Conner and TGN and colocalized with TGN38 or furin (Dell’Angelica Schmid, 2003b; Slepnev and De Camilli, 2000). Unraveling et al., 1999a; Hirst et al., 1999). Brefeldin A (BFA) treat- the relationships between the AP complexes and these coat ment disrupted the punctate signal in TGN, indicating that proteins and/or accessory proteins may help us to better the membrane association of AP-4 was regulated by understand the overall sorting mechanisms in the secretory GTPase, possibly ARF-1 or a related protein (Boehm et al., and endocytic pathways. 2001).

As was the case for other subunits, 4 directly recog- Acknowledgments. The authors wish to thank Dr. Hiroyuki Takatsu nized a subset of tyrosine signals. Yeast two-hybrid analysis (Research Center for Allergy and Immunology, RIKEN) for helpful com- demonstrated that 4 only interacted with the tyrosine sig- ments on the manuscript. This work was supported in part by Grants-in- nals from human LAMP2, but not TGN38, CD68 and CD63 Aid for Young Scientists (B) (15700299) to FN, and Scientific Research (Aguilar et al., 2001). SPR analysis showed that the appar- (15370042) and Scientific Research in Priority Areas (15079203) to HO, ent dissociation constant for the interaction of 4 with the from the Ministry of Education, Culture, Sports, Science and Technology of Japan and also by the Uehara Memorial Foundation (HO). LAMP2 YXXØ signal was in the micromolar range (Agui- lar et al., 2001). There is also evidence that biochemically purified AP-4 by gel filtration bind the basolateral sorting References signals from furin, LDLR, MPR46 and TfR by SPR analysis Aguilar, R.C., Boehm, M., Gorshkova, I., Crouch, R.J., Tomita, K., Saito, (Simmen et al., 2002) T., Ohno, H., and Bonifacino, J.S. 2001. Signal-binding specificity of Two laboratories have reported on the function of AP-4. the mu4 subunit of the adaptor protein complex AP-4. J. Biol. Chem., Aguilar et al. showed that the Tac chimera bearing the 276: 13145–13152.

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