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A least two common themes are exploited by secretory lysosomes that support their similarity to conventional lysosomes. Secretory lysosomes appear to utilise endosomal intermediates in their biogenesis, much like the biogenesis of

conventional lysosomes. The second theme is the commonality of sorting

pathways used to deliver membrane proteins to both lysosomes and their related organelles.

1.3.1 Lvsosome biogenesis

Models of conventional lysosomal biogenesis include ‘maturation’ of early

endocytic compartments to lysosomes, ‘vesicle-transport’ of carriers to lysosomes, and ‘kiss and run’ involving heterotypic fusion and reformation of late endosomes and lysosomes, with resulting hybrid intermediates (Bright et al. 1997, Mullock et al. 1998, reviewed in Storrie and Desjardins 1996, Luzio et al. 2000, Mullins and Bonifacino 2001a). Internal vesicles of multivesicular endosomes sort components that are to be recycled or to be delivered to lysosomes to be degraded (Gruenberg et al. 1989, Putter et al. 1996).

1.3.2 Secretorv lvsosome biogenesis - exploitation of inner vesicles of MVB

A number of functions have been attributed to the internal vesicles of MVB and late endosomes including sorting components for recycling, degradation and storage (reviewed in Gruenberg 2001, Piper and Luzio 2001, Murk et al. 2002). Lysosome- related organelles exploit these internal vesicles for the recycling, storage and

1.3.3 Recycling

Mannose-6-phosphate receptor is localised to internal vesicles of MVB and to the limiting membrane (Griffiths et al. 1988, Griffiths et al. 1990). This is a reversible association that allows for the regulated movement (in part mediated by

cholesterol) between inner vesicles and the limiting membrane, which results in the transit of this receptor into and out of MVB (Hirst et al. 1998, Kobayashi et al. 1999, Miwako et al. 2001). The recycling of CD63 in late endosomes of endothelial cells is also partially mediated by cholesterol (Kobayashi et al. 2000).

1.3.4 Storage

The localisation of MHC class II and antigen peptide fragments to internal vesicles of MIIC of dendritic cells is proposed to confer proteolytic resistance of these

molecules until a maturation signal is received to present antigen at the cell surface (Mellman and Steinman 2001, Inaba et al. 2000, Turley et al. 2000). Immature dendritic cells store more than 65% of their MHC class II in MIIC, of which 80% localises to internal vesicles. Upon an appropriate maturation signal, remodelling of MIIC ensues, with the formation of long tubules from inner vesicles, which fuse with and allow tubule outgrowth of the limiting membrane (Kleijmeer et al. 2001). The specific transfer of MIIC contents of inner vesicles via these long tubules to the plasma membrane to present MHC class II antigen complexes has recently been demonstrated by two groups with the use of GFP-MHC class II molecules and lysosomal markers (Chow et al. 2002, Boes et al. 2002). Thus, contrary to the proposed degradative fate of endosomal inner vesicles, MIIC internal vesicles serve as a temporary store/ reservoir of MHC class Il-antigen complexes, which confers upon them proteolytic resistance and allows for their rapid mobilisation to the cell surface upon a maturation signal.

Internal vesicles of MVB can also be secreted from cells upon the correct stimulus. Components of exosomes are sorted and stored within internal vesicles, which are then released when the limiting membrane of MVB fuses with the plasma

membrane (reviewed in Denzer et al. 2000, Stoorvogel et al. 2002). Exosomes have a unique protein and lipid composition, dependant on the cell type they

originate from. Cells that secrete exosomes include B lymphocytes (Escola et al. 1998, Raposo et al. 1996), dendritic cells (Thery et al. 1999, Zitvogel et al. 1998, Thery et al. 2001, Kleijmeer et al. 2001), mast cells (Raposo et al. 1997), platelets (Heijnen et al. 1999), and T lymphocytes (Peters et al. 1991). These are all cell types that appear to possess secretory lysosomes. Secreted exosomes are proposed to function in a wide variety of immune responses, such as antigen presentation, I cell activation and the transfer of membrane proteins from cell to cell, although their precise physiological role has not been shown in vivo.

1.3.5 Sorting

Multivesicular endosomal intermediates are used to differentially sort P-selectin and CD63 endocytic traffic to both WPB and lysosomes in human umbilical vein endothelial cells (HUVEC). Traffic of P-selectin and CD63 via the cell surface (in resting and stimulated cells) diverges upon entry into multivesicular endosomes, whereby CD63 is initially relatively resistant to proteolysis and is retained within late endosomes for 4-8 hours prior to traffic to WPB, whereas P-selectin is transported to WPB within 1-2 hours (Kobayashi et al. 2000, Arribas and Cutler 2000). P-selectin also traffics to lysosomes faster than CD63, probably due to the selective retention of CD63 in internal vesicles of late endosomes. It is not clear whether these proteins can cycle directly from late endosomes to WPB, or whether they have to use the TGN as an intermediate, but the MVB clearly plays an

important role in their differential traffic.

MVB also play a pivotal role in the maturation of platelet alpha and dense granules, and the sorting of their respective components (recently reviewed in King and Reed 2002). Immunogold staining of megakaryocytes reveal that vWF (alpha granule marker) and CD63 (dense granule marker) are predominantly localised to internal vesicles, while P-selectin (found in both types of granules) associates both with internal vesicles and the limiting membrane of MVB (Heijnen et al. 1998, Youssefian and Cramer 2000). These components appear to segregate to distinct internal vesicles within the same MVB, which may lead to the development of discreet organelles. Often, only dense granules of platelets are defective in the

unaffected, highlighting the efficient sorting and segregation that must occur within MVB.

1.3.6 Entry to internal vesicles

Mechanisms of entry into internal vesicles of endocytic compartments have been proposed to include the presence of polar residues in the TM domain (Reggiori et al. 2000), ubiquitination of cytosolic tails of membrane proteins (Strous et al. 1996, Govers et al. 1997, Hicke et al. 1998, Levkowitz et al. 1998, Reggiori and Pelham 2001, Katzmann et al. 2001), and the localised disassembly of “flat” or “bilayered” clathrin (Raposo et al. 2001, Sachse et al. 2002) to allow inward vésiculation. Disassembly of clathrin coat and inward vésiculation also involves Mrs (hepatocyte growth factor-regulated tyrosine kinase substrate), which binds to both clathrin and ubiquitin-tagged proteins (Sachse et al. 2002, Raiborg et al. 2002, Lloyd et al. 2002). A number of molecular players in the recognition of ubiquitinated cargo and inclusion into inner vesicles have been elucidated including TSG101 (Babst et al. 2000, Bishop and Woodman 2001) and homologues of the yeast ESCRT

complexes (Katzmann et al. 2001, Babst et al. 2002a, Babst et al. 2002b). In addition, inward vésiculation is thought to be facilitated by the physical

characteristics of lipids and proteins, including the cone-shaped structure of LBRA (lysobisphosphatidic acid) (Kobayashi et al. 1998b, Kobayashi et al. 2001) and the tertiary structure of tetraspanins, which are enriched in microdomains within

internal vesicles (Escola et al. 1998, Reggiori and Pelham 2001, Hemler 2001, Kropshofer et al. 2002).

1.3.7 Lvsosomal membrane protein sorting pathwavs

Lysosome-associated membrane proteins (lamp), also called lysosomal membrane glycoproteins (Igp) and lysosomal integral membrane proteins (limp) are

transported from the TGN to lysosomes via at least two distinct pathways

(Hunziker and Geuze 1996). Direct trafficking of lamp to lysosomes is thought to be directed by interactions with the adaptor complex AP3, while indirect traffic via the cell surface involves an internalisation step mediated by AP2 (Mullins and

Bonifacino 2001a). Study of the half-times of delivery of lamps to lysosomes reveals that lamp-1 and lamp-2 mainly use intracellular routes, deduced from the

half-time of 30-90 minutes (Barriocanal et al. 1986, D'Souza and August 1986, Green et al. 1987, Carlsson and Fukuda 1992), whereas lysosomal acid

phosphatase (LAP) is delivered with a half-time of 5-7 hours, mainly via the cell surface (Braun et al. 1989). The importance of AP3-mediated traffic to both lysosomes and their related organelles is highlighted by the genetic evidence of HPS2, pearl and mocha and is discussed further, below.

1.3.8 Exploitation of AP3-mediated sorting pathwav

AP3 one is one of four identified adaptor complexes that interact with a number of components at the cytoplasmic face of organelles, including specific cargo

receptors, clathrin, and accessory proteins (reviewed in Boehm and Bonifacino 2002, Robinson and Bonifacino 2001, Boehm and Bonifacino 2001, Kirchhausen 1999, Jackson 1998). Each mammalian adaptor complex is made up of 4 subunits, two large, one medium and one small (Table 1.2). Their heterotetrameric structure is illustrated in Figure 1.3.

Adaptor large medium small

API y1, y2 pi p.1A, a lA , alB , a lC

AP2 a l, a2 P2 li2 o2

AP3 Ô P3A, P3B |li3A, p3B o3A, o3B

AP4 E P4 |n4 o4

Table 1.2. Subunits of mammalian adaptor complexes.

AP3, like API and AP2, is thought to interact with clathrin in mammalian cells (DellAngelica et al. 1998, Drake et al. 2000, Liu et al. 2001), although it does not appear to be enriched in clathrin-coated vesicles (Newman et al. 1995, Simpson et al. 1996). AP4 is proposed to be part of a non-clathrin coat (Dell'Angelica et al. 1999b, Hirst et al. 1999) and indirect evidence suggests that AP4 functions in protein sorting to lysosomes (Aguilar et al. 2001).

Mutations in the subunits of AP3 lead to relatively mild phenotypes when compared to mutations with subunits of API. Disruption of the y1 gene (Zizioli et al. 1999) and

the |Li1 A gene (Meyer et al. 2000) cause embryonic lethality in mice. However, in

yeast, the function of AP1 is highly redundant with that of the G G As (Golgi-

localised, gamma-ear-containing, ADP-ribosylation factor-binding proteins), where they act in concert with clathrin to sort proteins in the late Golgi (Black and Pelham 2000, Costaguta et al. 2001, Dell'Angelica et al. 2000b, Hirst et al. 2001, Mullins and Bonifacino 2001b).

Components of the AP3 complex are mutated in human HPS2 (p3A), pearl mice (|33A), mocha mice (Ô), and also in Drosophila pigmentation mutants including garnet (6), ruby (P3), carmine (p3) and orange (o3), (Simpson et al. 1997, Ooi et al. 1997, Lloyd et al. 1999, Kretzschmar et al. 2000, Mullins et al. 1999, reviewed in Lloyd et al. 1998 and Mullins et al. 2000). The study of these naturally occurring mutations and wild-type AP3 function have identified a key role for AP3 in the trafficking of a subset of membrane proteins to lysosomes and their related organelles.

Immunofluorescence studies in wild-type cells have shown the subcellular localisation of AP3 at the TGN and in endosomal compartments (Simpson et al. 1996, Dell'Angelica et al. 1997, (Simpson et al. 1997, Dell'Angelica et al. 1998), suggesting that AP3 may function either at the TGN or endosomes. Fibroblasts cultured from HPS2 patients display increased surface expression of the lysosomal membrane proteins CD63, lamp-1 and lamp-2, in concert with the increased

internalisation of these proteins (Dell'Angelica et al. 1999a, Huizing et al. 2002). In contrast, transferrin receptor and mannose-6-phosphate receptor distribution and internalisation are unaffected. Inhibition of AP3 by antisense oligonucleotides to p3A in wild-type cells results in increased plasma membrane trafficking of lamp-1 and limp-II, but not of non-lysosomal membrane proteins (Le Borgne et al. 1998), thus mimicking the effect of HPS2.

AP2

AP3

#

p3

Figure 1.3. Heterotetrameric structure of the four known adaptor complexes.

A null allele for the P3A gene was created, due to the possibility that mutations in pearl mice might lead to truncated transcripts encoding residual mutant P3A protein (Yang et al. 2000). Residual p3A protein in pear/was later confirmed in Peden at al. (Peden et al. 2002). Null P3A leads to a more severe phenotype than seen in pearl fibroblasts, with increased cell surface staining and internalisation of lamp-1 and lamp-2. To confirm that the increased routing via the plasma membrane of lysosomal membrane proteins was a direct result of AP3 deficiencies, rescued phenotypes were shown in mocha cells transiently transfected with the Ô subunit and a stable pearl cell line expressing a wild-type copy of the p3A subunit (Peden et al. 2002). Furthermore, these studies show that the clathrin-binding site of p3A is not needed for AP3 function.

In addition to the increased cell surface localisation of lysosomal membrane

proteins in AP3-deficient cells, the distribution of other membrane proteins specific to their respective secretory lysosome is perturbed. Tyrosinase is mislocalised to MVB around the nucleus in HPS2 melanocytes (Huizing et al. 2001a) and a more lightened coat colour than pearl mice is exhibited in P3A null mice (Yang et al. 2000). HPS2-derived B-lymphoblast cells display a redistribution of CDIb, which functions in the antigen presentation of lipids, to the plasma membrane and early endocytic compartments, instead of its normal localisation in MIIC (Sugita et al. 2002). Pearl and mocha-6erWe6 cells reveal trafficking defects of P-selectin to Weibel-Palade bodies of endothelial cells but not to alpha granules of platelets (Daugherty et al. 2001). No phenotype for traffic of P-selectin in the dense granules of platelets has yet been reported. Thus, AP3 function is clearly not restricted to lysosomes and the trafficking of their resident membrane proteins, but extend to proteins destined for lysosome-related organelles, again highlighting the

relationship between these organelles.

Two models have been proposed for AP3 function in the trafficking of membrane proteins to lysosomes and their related organelles, both of which are consistent with the increase in plasma membrane staining and increased internalisation of lysosomal proteins in AP3 deficient cells (Starcevic et al. 2002). If AP3 functions at

TGN to endosome traffic, then impairment of this pathway would cause lysosomal proteins to enter the default secretory pathway to the cell surface. Any interaction with AP2 would then allow these proteins to become internalised and delivered to late endosomes and lysosomes. This increase of lysosomal proteins following the indirect pathway would result in increased staining at the cell surface and

increased internalisation via AP2. If, however, AP3 functions instead in traffic from early to late endosomes, then impairment would cause a hold up of lysosomal membrane proteins in early endosomes. This would result in increased cycling of proteins over the plasma membrane and back to early endosomes, leading to increased cell surface staining, and increased internalisation.

There is clearly redundancy in AP3-mediated trafficking steps to lysosomes and their related organelles, as membrane proteins can take the indirect alternative route to lysosomes via AP2. The diluted, rather than an albino pigmentation phenotype in AP3-deficiencies, and the almost steady state localisation of lysosomal proteins in lysosomes, although increased traffic via the cell surface, suggests a redundancy of function of AP3 (Dell'Angelica et al. 1999a). B- lymphoblast cell lines derived from HPS2 patients, and mocha mice, reveal no defects in the trafficking of MHC class II molecules to endosomal peptide-loading compartments, implying that AP3 is not necessary for their correct localisation (Caplan et al. 2000, Sevilla et al. 2001). Redundancy of function with AP3 may also be due to interactions of cytoplasmic tails with AP4 and other coat proteins in addition to that with AP2.