CELL STRUCTURE AND FUNCTION 28: 419–429 (2003) REVIEW © 2003 by Japan Society for Cell Biology 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 com- plexes. *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.