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The selective recruitment of membrane proteins into transport vesicles is mediated by the interaction of sorting signals in their cytoplasmic tails with components of membrane-bound coats, which results in the concentration of proteins into coated vesicles (Schekman and Orel 1996, Rothman and Wieland 1996, Kirchhausen et al. 1997). Cargo selection by adaptor complexes is mediated by the p. subunits.

which recognise both tyrosine and dileucine-based signals. pi and P2 have also been shown to interact with dileucine-based sorting signals for cargo selection (Rapoport et al. 1998, Greenberg et al. 1998). AP3 is able to bind to both tyrosine and dileucine-based sorting signals.

1.4.1 Tvrosine-based sorting signals

C-terminal YXX0 sorting motifs (where Y is tyrosine, X denotes any amino acid and O represents a bulky hydrophobic amino acid) mediate rapid internalisation from the cell surface (Trowbridge et al. 1993), basolateral targeting in polarised epithelial cells (Keller and Simons 1997), and lysosome and secretory lysosome targeting (reviewed in Kirchhausen et al. 1997, Marks et al. 1997). Yeast-2-hybrid assays with p chains show that the critical residues for function are Y and 0 (Ohno et al. 1995, Boll et al. 1996). The specificity for incorporation of the correct cargo lies in the residues XX and the amino acids that precede and follow the short sorting signal. The screening of combinatorial yeast-2-hybrid libraries of XXXYXXO with adaptor p subunits reveals preferences and common sequences between p i , p2 and p3 (Ohno et al. 1998). The identification of common sequences could

explain the redundancy in the function of AP3-mediated targeting to lysosomes and their related organelles. Amino acid preferences for the p3A and p3B subunits are shown in Table 1.3.

Signal position p3A p3B

X Y-3 R R X Y-2 (S) X Y-1 D, E, (G) (D) Y X Y+1 E E X Y+2 P _{(P). (E)} 0 1 _(L)

Table 1.3. Adapted from Ohno et al. 1998. Amino acids in parentheses represent residues that display a decreased preference for each subunit.

Both ^i3 subunits show a preference for glutamic acid (E) at Y+1, which is found in lamp-2 and CD63. In addition, |li3 A is the only \i subunit that shows a preference

for glycine (G) at Y-1, a feature that is characteristic of many lysosomal sorting signals (Ohno et al. 1996 and Table 1.4).

membrane orotein cvtoDlasmic tail

lamp-1 TM-GRKRSHAGYQTI lamp-2 TM-LKHHHAGYEQF LAP TM-RMQAQPPGYRHVADGEDHA CD63 TM-CCLVKSIRSGYEVM TRP-2 TM-YRRLRKGYTPLMETHLSSKRYTEEA tyrosinase TM-LLCRHKRKQLPEEKQPLLMEKEDYHSLYQSHL Table 1.4. Tyrosine-based sorting signals of membrane proteins destined for lysosomes and their related organelles (human sequences).

Direct interactions between ^i3A and the YXX0 motif of proteins destined for lysosomes and secretory lysosomes have been described for CD63 (Dell'Angelica et al. 1999a, Rous et al. 2002), lamp-1 (Stephens and Banting 1998, Dell'Angelica et al. 1999a), lamp-2 (Aguilar et al. 2001), tyrosinase (Honing et al. 1998) and CDIb (Sugita et al. 2002). Yeast-2-hybrid screening of interactions between n3A and mutations of the GYEVM motif of CD63 reveal the importance of G, Y and M, directly correlating the strength of interaction with AP3 to the degree of lysosomal localisation (Rous et al. 2002). This study also confirms the overlap in signal recognition by the adaptor complexes, as when interactions with ^i3A were

reduced, then so were interactions with \i2. An important finding is that mutation of O from M to I of CD63 results in increased binding to |li3 A than the wild-type motif,

but abolishes any interaction with \x2. This result is in accordance with the combinatorial peptide screen (Table 1.3) that shows a preference for |i3A interactions with isoleucine at position 0 (Ohno et al. 1998). This construct is entirely dependent on AP3-mediated traffic to lysosomes, highlighted by its cell surface localisation in pearl and mocha cells, and the rescue of lysosomal

localisation of CD63 in mocha cells expressing the Ô subunit of AP3 (Rous et al.

2002).

1.4.2 Dileucine-based sorting signals

C-terminal dileucine signals of lysosomal and secretory lysosomal membrane proteins can also mediate trafficking to their respective organelles (Table 1.5).

membrane orotein cvtoolasmic tail

limp II t m-g q g s m d e g t a d e r a p u r t

Pmel17 TM-(20aa)WLRLPRIFCSCPIGENSPy=SGQQV

TRP-1 t m-r a r r s m d e a n q p u=t d q y q c y a e e y e k l q n p n q s w

tyrosinase TM-LLCRHKRKQLPEEKQPU=MEKEDYHSLYQSHL QNR-71 TM-5aa-YKPI-29aa-ERNPLL-7aa

Table 1.5. Dileucine-based sorting signals of membrane proteins destined for lysosomes and their related organelles. Human sequences are shown, and aa represents amino acid residues.

A number of these dileucine signal motifs have been shown to interact with \i3A, again highlighting the role that AP3 plays in sorting proteins to lysosome-related organelles. The dileucine-sorting signal of limp II is critical for delivery of this

protein to lysosomes (Sandoval et al. 1994, Ogata and Fukuda 1994) and interacts with p3A in the yeast-2-hybrid system (Honing et al. 1998). TRP-1 also contains a dileucine signal in its C-terminal tail that mediates targeting to melanosomes of human melanomas (Vijayasaradhi et al. 1995, Xu et al. 1998b). In addition to the tyrosine-based sorting signal in tyrosinase, interactions with AP3 in vitro are also mediated by a dileucine signal (Honing et al. 1998). This dileucine motif is

predicted to play a predominant role, whereas the tyrosine-based sorting signal plays a secondary role in the sorting of tyrosinase to lysosomes (Simmen et al. 1999, Calvo et al. 1999). QNR-71 targeting to retinal-pigmented cells and to lysosomes of HeLa cells is dileucine signal-dependent and is perturbed by anti­ sense p3A, causing an increase in the cell surface localisation and internalisation

of QNR-71 (Le Borgne et al. 2001). This suggests that AP3 would normally mediate the internal traffic of QNR-71 to melanosomes.

1.4.3 Unconventional sorting signals

P-selectin localises to WPB of endothelial cells (Bonfanti et al. 1989, McEver et al. 1989) and to alpha and dense granules of platelets (Stenberg et al. 1985, Johnston et al. 1989, Israels et al. 1992). Heterologous expression studies have revealed that overlapping residues throughout the 35 amino acid cytoplasmic tail of P- selectin contribute to internalisation (Setiadi et al. 1995), and targeting to secretory granules (Disdier et al. 1992, Norcott et al. 1996, Modderman et al. 1998,

Blagoveshchenskaya et al. 1999a), synaptic-like microvesicles

(Blagoveshchenskaya et al. 1999a, Blagoveshchenskaya and Cutler 2000b) and lysosomes (Green et al. 1994, Blagoveshchenskaya et al. 1998b,

Blagoveshchenskaya et al. 1998a, Straley et al. 1998, Blagoveshchenskaya et al. 1999a), see Figure 1.4. The YGVF motif of P-selectin conforms to the YXX0 sorting signal, however, this sequence is often used in combination with other residues of the cytoplasmic tail. Targeting of P-selectin to WPB is dependent on the cytoplasmic tail (Hartwell et al. 1998) and is abolished in pearl cells with a mutation in P3A (Daugherty et al. 2001). Daugherty et al. also show that P-selectin binds p,3A in vitro and that this interaction is destabilised by mutation of L-A

(residue 768). In contrast, the cytoplasmic tail is not required for sorting of P- selectin to alpha granules of platelets (Hartwell et al. 1998) and localisation of P- selectin to alpha granules of pearl platelets appears unaffected (Daugherty et al.

2001).

Fas ligand is sequestered in the lytic granules of CTL and NK cells and is constitutively expressed on the cell surface of non-hemopoietic cells, in tissues such as the eye, testis and spleen, to maintain immune privilege of these tissues (Suda et al. 1993, Griffith et al. 1995). The N-terminal cytoplasmic domain of Fas ligand displays a proline-rich region, and positively charged residues flanking this sequence, which mediate the sorting of Fas ligand to lytic granules and to

secretory lysosomes of other cell types (Boss! and Griffiths 1999, Blott et al. 2001), see Figure 1.4.

1.4.4 Modification of sorting signals

Other features of membrane proteins may modify the effectiveness of interaction with adaptor complexes. These features include a defined distance of the sorting signal from the transmembrane (TM) domain (Rohrer et al. 1996), the identity of 0 of YXX0 (Gough and Fambrough 1997, Gough et al. 1999), use of proteolytic processing to modify sorting signals as they progress along their sorting pathway (Guarnieri et al. 1993, Akasaki et al. 1995) and the lumenal and TM domains (Reaves et al. 1998, Wimer-Mackin and Granger 1996, Fleming et al. 1998).

1.4.5 Universal signals

Many resident secretory lysosomal proteins are also able to localise to

conventional lysosomes when expressed in cells such as 3T3 Swiss fibroblasts, HeLa cells, H.Ep.2 and MDCK, a transport step that is dependent on the

cytoplasmic tails of these proteins. This has been shown for TRP-1 (Vijayasaradhi et al. 1995), tyrosinase (Bouchard et al. 1989, Winder et al. 1993, Simmen et al. 1999, Calvo et al. 1999), HLA-DM of MIIC (Marks et al. 1995, Copier et al. 1996) and P-selectin (Green et al. 1994, Blagoveshchenskaya et al. 1998b,

Blagoveshchenskaya et al. 1998a, Blagoveshchenskaya et al. 1999a). These results show that the cytoplasmic tails of these proteins possess the necessary sorting information to direct them to lysosomes, highlighting the similarity of sorting between lysosome-related organelles and conventional lysosomes. In addition, the dileucine signal of tyrosinase mediates targeting to synaptic-like microvesicles (SLMV) from the endocytic pathway of PC I2 cells (Blagoveshchenskaya et al. 1999b). Thus tyrosinase appears to possess the necessary sorting information for its localisation to secretory lysosomes, conventional lysosomes and SLMV.

However, Fas ligand is unable to localise to conventional lysosomes, but it is effectively targeted to cells that contain secretory lysosomes (Bossi and Griffiths 1999, Blott et al. 2001). This differential sorting of Fas ligand to secretory

P-selectin cytoplasmic tail — TM RKRFRQKDDGKCPLNPHSHLGTYGVFTNAAFDPSP -C

755 789

W e ib e l-P a la d e b o d ie s o f e n d o th e lia l c e lls (H a rtw e ll e t al. 1 993) RKRFRQ KDDGKCPLNPHSHLG TYGVFTNAAFDPSP

G ra n u le s o f A tT 2 0 c e lls (D is d ie r e t al. 1 992) A tT 2 0 ce lls (M o d d e rm a n e t al. 1998) P C I 2 c e lls (N o rc o tt e t al. 1 9 9 6 ) P C I 2 c e lls (B la g o v e s h c h e n s k a y a e t al. 1 9 9 9 a ) v W F p s e u d o -g ra n u le s in A tT 2 0 c e lls (B la g o v e s h c h e n s k a y a e t al. 2 0 0 2 ) S L M V o f P C I 2 c e lls (B la g o v e s h c h e n s k a y a e t al. 1999a, B la g o v e s h c h e n s k a y a e t al. 2 0 0 0 b ) L y s o s o m e s o f H .E p .2 c e lls (B la g o v e s h c h e n s k a y a e t al. 1 9 9 8 a ,b ) P C 1 2 c e lls (G re e n e t al. 1 9 9 4 ) P C I 2 c e lls (B la g o v e s h c h e n s k a y a e t al. 1 9 9 9 a ) PLNPHSHLG1FYGVFTNAAFDPSP LN YG F DG KCPLNPHSHLG TYG VFTNAAFDPSP YGVF YG VF KCPL YGVF DPSP KCPL DGKCPLNPHSI KCPL

Fas ligand cytoplasmic tail N- -GQRRPPPPPPPPPLPPPPPPPPLPPLPLPPLKKRGNHSTG TM

1 1 1 1 40 1 80 S e c re to ry ly s o s o m e s o f Rbl c e lls (B lo tt e t al. 2 0 0 2 ) _{RR PPPPPPPPPLPPPPPPPPLPPLPLPPL KKR}

lysosomes in cells that possess them, and to the plasma membrane in other cells may reflect the correct biological function of Fas ligand, both in cells of the

hemopoietic lineage that sequester this protein for polarised presentation at the cell surface upon a trigger, and other cells that constitutively express Fas ligand on their cell surface to induce cell death and prevent dangerous immune reactions, as part of the immune privilege of these cells (Suda et al. 1993, Suda et al. 1995, Griffith et al. 1995).