Lysosome-Endosome Fusion and Lysosome Biogenesis

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Journal of Cell Science 113, 1515-1524 (2000) 1515 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0742

COMMENTARY Lysosome-endosome fusion and lysosome biogenesis

J. Paul Luzio1,*, Brian A. Rous1, Nicholas A. Bright1, Paul R. Pryor1, Barbara M. Mullock1 and Robert C. Piper2 1Department of Clinical Biochemistry and Wellcome Trust Centre for the Study of Molecular Mechanisms in Disease, Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2XY, UK 2Department of Physiology and Biophysics, University of Iowa, Iowa City, IA52242, USA *Author for correspondence

Published on WWW 6 April 2000

SUMMARY

Recent data both from cell-free experiments and from hybrid organelles and discuss the current status of our cultured cells have shown that lysosomes can fuse directly understanding of the mechanisms of fusion and lysosome with late endosomes to form a hybrid organelle. This has a re-formation. We also review lysosome biosynthesis, led to a hypothesis that dense core lysosomes are in essence showing how recent studies of lysosome-like organelles storage granules for acid hydrolases and that, when the including the yeast vacuole, Drosophila eye pigment former fuse with late endosomes, a hybrid organelle for granules and mammalian secretory lysosomes have digestion of endocytosed macromolecules is created. identiﬁed novel proteins involved in this process. Lysosomes are then re-formed from hybrid organelles by a process involving condensation of contents. In this Commentary we review the evidence for formation of the Key words: Lysosome, Endosome, Endocytosis, Membrane fusion

INTRODUCTION formed. We discuss the consequences of this hypothesis for our understanding both of how endocytosed material is delivered Lysosomes are membrane-bound organelles containing many to lysosomes and of how newly synthesised acid hydrolases hydrolytic enzymes, which are optimally active at an acid pH. and lysosomal integral membrane glycoproteins (also known They are distinguished from endosomes by the absence of the as lgps, LAMPs, lamps, LIMPs or limps) are delivered to two mannose-6-phosphate receptors (MPRs) and recycling lysosomes. We also discuss how the hypothesis leads us to a cell surface receptors. They are characteristically observed by view of lysosomes as being analogous to secretory granules electron microscopy to be organelles of ~0.5 µm diameter that store acid hydrolases in between fusion events with late and often have electron-dense cores (Holtzmann, 1989). endosomes. Finally, we review recent evidence from genetic Lysosomes are usually regarded as the terminal degradation studies of yeast, Drosophila, mice and humans that have led compartment of the endocytic pathway (Kornfeld and to a rapid advance in discovery of proteins involved in the Mellman, 1989), and play important roles in the degradation molecular machinery for delivery of macromolecules to of phagocytosed material (Funato et al., 1997), in autophagy lysosomes and lysosome biogenesis. (Lawrence and Brown, 1992), in crinophagy (Noda and The discovery of lysosomes almost 50 years ago was a Farquhar, 1992) and in the proteolysis of cytosolic proteins triumph of the then new technique of subcellular fractionation transported across the lysosomal membrane by a carrier- by ultracentrifugation (reviewed by de Duve, 1983; Bowers, mediated mechanism (Cuervo and Dice, 1996). However, the 1998). Endosomal compartments on the major endocytic view that lysosomes are simply a ‘garbage-disposal unit’ has pathway from clathrin-coated pits at the plasma membrane to been challenged, both by a better understanding of how lysosomes were discovered later and found to constitute a endocytosed material is delivered to them and also by the pleiomorphic, smooth-membrane system consisting of tubular identification, in many cell types, of lysosomes that secrete and vesicular elements, many of the latter containing intra- their contents after fusion with the plasma membrane organelle vesicles and often being described as multivesicular (Stinchcombe and Griffiths, 1999). bodies or MVBs (Hopkins, 1983; Hopkins et al., 1990; Here, we review the evidence for the hypothesis that Trowbridge et al., 1993). Despite this complexity a generalised lysosomes are able to fuse directly with late endosomes to route map for the passage of endocytosed material to form hybrid organelles in which degradation of endocytosed lysosomes has been established (Fig. 1). By definition, this material takes place and from which lysosomes can be re- material is delivered first to early endosomes, then to late 1516 J. P. Luzio and others endosomes and subsequently to lysosomes, the time of delivery Nevertheless, we were able to establish a cell-free system to to each endosomal compartment varying between cell types study content mixing between late endosomes and lysosomes and between cells in culture and in living tissue (Mullock et derived from rat hepatocytes, using a slowly digested ligand in al., 1994). The early endosome is the major sorting the lysosomes (Mullock et al., 1994, 1998). The demonstration compartment of the endocytic pathway, in which many ligands of content mixing between the organelles showed that the dissociate from their receptors in the acid pH of the lumen and earlier hypothesis of maturation of late endosomes to from which many of the receptors recycle to the cell surface lysosomes (Roederer et al., 1990; Murphy, 1991) could (Mellman, 1996; Mukherjee et al., 1997). The membrane completely explain neither endocytic delivery to lysosomes nor traffic pathway from early to late endosomes has been studied lysosome biogenesis. Experiments in which late endosomes extensively (reviewed by Gruenberg and Maxfield, 1995; Gu have been incubated with cytosol and ATP, although showing and Gruenberg, 1999). In contrast, much less work has been changes in density, have not shown increases in density carried out on membrane traffic between late endosomes and sufficient to be consistent with the formation of lysosomes lysosomes. (Mullock et al., 1994). Three alternative hypotheses are available to explain content mixing between late endosomes and lysosomes (Fig. 2). The THE DELIVERY OF ENDOCYTOSED MATERIAL first assumes vesicular transport between the two organelles Ð FROM LATE ENDOSOMES TO LYSOSOMES largely by analogy with the many membrane traffic steps on both the secretory and endocytic pathways in which such a In mammalian cells, the organelles of the late endocytic mechanism operates. The second hypothesis is termed ‘kiss and pathway interact with each other and are in dynamic run’ and proposes that late endosomes and lysosomes undergo equilibrium both in vivo and in vitro. Thus, content mixing continuous repeated transient fusion and fission (Storrie and and/or exchange of membrane proteins has been observed Desjardins, 1996). Storrie and Desjardins proposed this between late endosomes (Aniento et al., 1993), between hypothesis to explain data from experiments in which small lysosomes (Storrie and Desjardins, 1996; Bakker et al., 1997; soluble markers appeared to move more readily between Ward et al., 1997) and between late endosomes and lysosomes lysosomes and endosomes/phagosomes than did larger soluble (Mullock et al., 1998). The investigation of these dynamic markers and from experiments in which soluble cargo was equilibria, and in particular the delivery of endocytosed delivered to lysosomes faster than membrane proteins. The third macromolecules from late endosomes to lysosomes, has been hypothesis involves direct and complete fusion between late hindered by the problems inherent in the fact that many such endosomes and lysosomes with subsequent recovery of macromolecules are rapidly digested once they reach the lysosomes for re-use. This hypothesis, which could be regarded degradative environment in late endocytic compartments. as an extension of ‘kiss and run’, in which some kisses result in fusion, has been argued eloquently by Griffiths (1996). He pointed out that, morphologically, lysosomes resemble electron-dense secretory granules, speculated that their function is to store mature lysosomal enzymes and proposed that they Plasma fuse periodically with late endosomes in a regulated fashion to Membrane form a compartment, ‘the cell stomach’, in which degradation Early Endosome occurs. He also proposed that lysosomes subsequently bud off this compartment and are re-used as required. Although no evidence supporting vesicular transport between late endosomes and lysosomes has emerged, there is Late Endosome clear evidence for direct fusion of these organelles. In the rat liver cell-free system, the immunoprecipitable product of content mixing between late endosomes and lysosomes is present in a membrane-bound organelle whose density is intermediate between those of late endosomes and lysosomes TGN Lysosome (Mullock et al., 1998). This organelle, which we have termed Hybrid a late-endosome-lysosome hybrid organelle, contains markers of both lysosomes (cathepsin L) and late endosomes (endocytosed avidin-labelled asialofetuin and the cation Fig. 1. A model of endocytic traffic and biosynthetic delivery to independent MPR), has a mean diameter (0.96 µm) larger than lysosomes. The major endocytic traffic route to lysosomes from those of either rat liver lysosomes (0.38 µm) or late endosomes plasma membrane coated pits is shown, incorporating present views (0.34 µm) and is less electron dense than lysosomes (Mullock of lysosome-late endosome fusion and re-formation of lysosomes et al., 1998). This hybrid organelle has many of the from the resulting hybrid organelles. In addition, short arrows show characteristics of the ‘cell stomach’ proposed by Griffiths. membrane traffic pathways emanating from the TGN, which deliver The idea that endosomes fuse with lysosomes in vivo is not newly synthesised lysosomal membrane glycoproteins via the plasma membrane or endosomal compartments. Such pathways include the only supported by the observation that direct fusion between clathrin-mediated route for delivery of newly synthesised, mannose- late endosomes and lysosomes can occur in vitro, but also by 6-phosphate-tagged acid hydrolases. An arrow is also used to experiments showing that an organelle fraction that has indicate traffic routes back to the TGN from endosomal characteristic features of the late endosome-lysosome hybrid compartments. organelle can be isolated from rat liver and by electron Lysosome-endosome fusion and lysosome biogenesis 1517

Fig. 2. Three hypotheses to explain content mixing between late (i) Vesicular transport endosomes and lysosomes. (i) Vesicular transport, for which no evidence has been found. In this model retrograde traffic of v- SNAREs and other proteins to endosomal compartments after Endosome Lysosome fusion of vesicles with lysosomes is expected; (ii) kiss and run, in which transient fusion pores are formed (ii) Kiss and run between late endosomes and lysosomes; (iii) direct fusion between late endosomes and lysosomes to form hybrid organelles, followed by subsequent re-formation of lysosomes. The Endosome Lysosome latter should require recondensation of hybrid content as well as recycling of v-SNAREs and other (iii) Direct fusion with formation of a hybrid organelle proteins to endosomal compartments, presumably by vesicular transport.

Endosome Lysosome Lysosome Hybrid organelle

microscopic experiments on cultured cells. The isolation from the molecular mechanisms of fusion and lysosome re- tissue of a fraction enriched in organelles that have the formation. In our cell-free content-mixing assay, we found that characteristics of the hybrid helps explain two earlier fusion between late endosomes and lysosomes is ATP, cytosol observations: (1) the heterogeneity of late endocytic and temperature dependent (Mullock et al., 1998). Fusion also compartments; and (2) the start of proteolysis of endocytosed requires the presence of NSF (N-ethylmaleimide sensitive macromolecules in pre-lysosomal compartments (Casciola- factor) and SNAPs (soluble NSF attachment proteins) and is Rosen and Hubbard, 1991; Tjelle et al., 1996). inhibited by Rab-GDI (GDP dissociation inhibitor). This Electron microscopic morphological analysis has also implies a need for a Rab GTPase (see Rodman and Wandinger- suggested that direct fusion of lysosomes with late endosomes Ness, 2000, for review of endosomal Rabs). Together, these can occur. An elegant study on cultured HEp-2 cells by Futter characteristics are consistent with the action of the fusion et al. (1996) showed that transfer of complexes of epidermal machinery used at many other sites on the secretory and growth factor (EGF) and its receptor to a late endocytic endocytic pathways and with this machinery functioning degradative compartment involved maturation of a late according to the tenets of the SNARE hypothesis (i.e. specific endosomal MVB that fused directly with MPR-negative pairing between v- (vesicle) and t- (target organelle) SNAREs; lysosomes. Futter et al. pre-labelled these lysosomes by Rothman, 1994). SNAREs have recently been re-classified on loading with horse radish peroxidase in a 15-minute pulse, 4- the basis of sequence alignment and structural data as Q hour chase protocol. Activation of the lysosomal horse radish SNAREs and R SNAREs (Fasshauer et al., 1998), and it now peroxidase by diaminobenzidine and H2O2 before EGF uptake seems likely that the overall fidelity of fusion depends on prevented fusion of the lysosomes with the MVBs, but not SNARE-SNARE and other protein-protein interactions (Bock maturation of the latter or docking of the MVBs with and Scheller, 1999; Fasshauer et al., 1999; Yang et al., 1999). lysosomes. Our own data from cultured cells are also consistent In yeast, many gene products necessary for membrane traffic with the existence of the late endosome-lysosome hybrid to the vacuole, the yeast equivalent of the lysosome (Klionsky organelle in vivo. They indicate that, at any one time, ~15% of et al., 1990), have been identified. Much of this information lysosomes are fused with late endosomes to form the hybrid has come from genetic screens in Saccharomyces cerevisiae. compartment in NRK cells and that lysosomes can be re- In particular, the isolation of vps (vacuolar protein sorting) formed from this compartment (Bright et al., 1997). mutants, in which sorting and transport of newly synthesised hydrolases (e.g. carboxypeptidase Y) to the vacuole are defective, has proved valuable (Jones, 1977; Robinson et al., THE MECHANISM OF FUSION OF LATE 1988; Raymond et al., 1992; Stack and Emr, 1993; ENDOSOMES AND LYSOSOMES Horazdovsky et al., 1995). One of the most useful aspects of this system is that a fully functional post-Golgi/endosomal The ‘hybrid’ hypothesis provides a framework for studies of system is not strictly required for viability, thus making these 1518 J. P. Luzio and others pathways amenable to many genetic approaches. A variety of permeabilised cell assays, antibodies to specific SNAREs genetic screens have identified >60 non-essential genes that are inhibit degradation of endocytosed EGF; this implicates both required for efficient trafficking to the vacuole. Phenotypic VAMP-7 and syntaxin 8 in the pathway from early endosomes analysis has allowed the mutant phenotypes to be roughly to lysosomes, probably in trafficking from early to late divided into six classes (A to F), according to vacuolar endosomes rather than from late endosomes to lysosomes morphology (Raymond et al., 1992), and this has been largely (Advani et al., 1999; Prekeris et al., 1999). Very little is known successful in grouping gene products that function together about the role of SNAP-25 homologues in the endocytic along the vacuolar biogenesis pathway. Thus, mutations in pathway, other than observation that in biochemical assays Class E VPS genes lead to an exaggerated form of the some are able to bind to SNAREs in the pathway (Steegmaier endocytic/prevacuolar compartment (PVC), and many Class B et al., 1998; Valdez et al., 1999). At present there is no and Class C proteins are involved in traffic between the PVC convincing published evidence as to which specific SNAREs and vacuole by controlling fusion events with the latter. are required for fusion of late endosomes and lysosomes. Currently, yeast genetics has identified Vam3p (a t-SNARE), Rothman and co-workers have suggested that SNAREs are Ypt7p (a Rab7 homologue), Vps33p (a Sec1p-like protein membrane-fusion catalysts on the basis of data from a thought to control SNARE complex formation) and Vam7p (a liposome fusion assay (Weber et al., 1998; Nickell et al., 1999; SNAP-25 like protein), as well as a host of other proteins Parlati et al., 1999). Other workers studying yeast homotypic whose molecular functions remain a mystery, as being involved vacuole fusion have shown that Ca2+ release from the vacuole in PVC-to-vacuole traffic and vacuolar morphogenesis (Banta is required in a post-docking phase of fusion. The effects of et al., 1990; Wada et al., 1992, 1997; Schimmoller and released Ca2+ are mediated by calmodulin (Peters and Mayer, Riezman, 1993; Darsow et al., 1997; Rieder and Emr, 1997; 1998), and a downstream effector required prior to Pan and Goldfarb, 1998; Sato et al., 1998). An important phospholipid bilayer fusion is protein phosphatase 1 (Peters et complement to these genetic studies has been the development al., 1999). BAPTA efficiently inhibits vacuole fusion; however, of a cell-free homotypic vacuolar fusion assay. This assay has EGTA, a chelator that has a similar dissociation constant for shown the functions of several of these proteins with regard to Ca2+ but an on-rate much slower than BAPTA has little effect. vacuolar fusion and their temporal organization and also In vitro, BAPTA also inhibits homotypic fusion between early allowed the identification of other factors required (Nichols et endosomes from mammalian cells, which implies that Ca2+ al., 1997; Ungermann et al., 1998, 1999). plays a role in this fusion process (Holroyd et al., 1999). The Our understanding of lysosomal biogenesis in animal cells Ca2+ is derived from the lumen of the endosome, because has also benefited from elucidation of homotypic fusion events fusion can also be inhibited by pre-treatment with EGTA-AM, at the early endosome. The fusion of endocytic vesicles with a membrane-permeant, hydrolysable ester of EGTA. By early endosomes, and of early endosomes with each other, is analogy with the emerging view of yeast vacuole fusion, we mediated by a process involving Rab5 and the t-SNARE therefore propose that, both early and late in the mammalian syntaxin 13. SNARE complex formation appears to be endocytic pathway, membrane fusion occurs by a three-stage preceded by the recruitment of tethering proteins into an process involving tethering, SNARE assembly and a oligomeric assembly on the endosome membrane (McBride et subsequent Ca2+-mediated fusion of phospholipid bilayers, al., 1999). Tethering is defined as formation of links that extend which requires Ca2+ derived from the organelle lumen (Fig. 3). over distances >25 nm from a given membrane surface, and docking is defined as the holding of membranes within a bilayer’s width (<5-10 nm) of one another (Pfeffer, 1999). LYSOSOME RECOVERY FROM FUSED LATE Tethering in early endosome fusion is dependent on the ENDOSOME-LYSOSOME HYBRID ORGANELLES presence of Rab5 and also of PtdIns(3)P patches produced by PtdIns 3-kinase activity (Christoforidis et al., 1999; Simonsen Because the fusion of late endosomes with lysosomes to form et al., 1998; Stenmark and Aasland, 1999). Although no hybrid structures results in consumption of the starting specific tethering proteins have yet been identified for late- organelles, we have proposed that a recovery system involving endosome-lysosome fusion, the evidence for their existence is membrane retrieval and a lysosomal-content-recondensation strong. Several groups have observed fine striations between process must occur in vivo (Bright et al., 1997; Mullock et al., adjacent endosomes and lysosomes in morphological studies 1998). Condensation of content must occur, since lysosomes on cultured cells (van Deurs et al., 1995; Futter et al., 1996; are characterised both by their greater density compared with Bright et al., 1997). It has been shown that PtdIns 3-kinase endosomes, which is evident from analysis by isopycnic inhibitors block late-endosome-lysosome fusion (Mullock et centrifugation in a variety of density gradient media and from al., 1998). the electron-dense appearance of lumenal content in electron Although there is now good evidence that syntaxin 13 is a micrographs. Another mammalian cell organelle in which t-SNARE functioning early in the mammalian endocytic condensation of content has been well described is the pathway, the roles of specific SNAREs at other stages of this regulated dense-core secretory granule. In this case, protein pathway are poorly understood. Localisation studies have aggregation, under conditions of low intraorganelle pH and identified VAMP-3 (cellubrevin), VAMP-5, VAMP-7, VAMP- high Ca2+ concentration (reviewed by Bauerfeind and Huttner, 8 (endobrevin), syntaxin 7, syntaxin 8 and syntaxin 11 as other 1993; Arvan and Castle, 1998; Tooze, 1998), plays an SNAREs in the endocytic pathway, but there is a lack of important role in the formation of the mature secretory granule. functional data on most of these. The ability of VAMP-3 to There is also evidence for formation of clathrin/AP-1 adaptor form complexes with syntaxin 13 suggests that the former also coats on immature secretory granules, possibly for removal of has a role in the early endosome (Prekeris et al., 1998). In lysosomal enzymes, as these organelles mature. We propose Lysosome-endosome fusion and lysosome biogenesis 1519 that the re-formation of lysosomes from late-endosome- MPR in clathrin-coated vesicles budding from the TGN lysosome hybrid organelles occurs by an analagous process to (Honing et al., 1996). However, in a different study, which the formation of regulated secretory granules from the TGN. analysed TGN-derived vesicles, Karlsson and Carlsson (1998) The finding that a low lumenal pH late in the endocytic found that LAMP-1 and LAMP-2 were in separate vesicles pathway is required for lumenal content aggregation and from those containing MPR and AP-1. delivery to lysosomes is consistent with this hypothesis Recently, two groups identified another complex that has (Roederer et al., 1990; van Weert et al., 1995). Recruitment of homology to the adaptor complexes AP-1 and AP-2: AP-3 clathrin and AP-2 adaptors to late endocytic compartments, (Simpson et al., 1996, 1997; Dell’Angelica et al., 1997a,b). including lysosomes, has also been demonstrated (Traub et al., This is a candidate for a non-AP-1 coat that might bud from 1996; Arneson et al., 1999) and could help remove endosomal the TGN and transport lysosomal glycoproteins to the membrane proteins that are not present in lysosomes. lysosome. Whether AP-3 is clathrin associated in mammalian cells is disputed (Simpson et al., 1996, 1997; Dell’Angelica et al., 1998). A homologous AP-3 complex in yeast that is not DELIVERY OF NEWLY SYNTHESISED PROTEINS TO clathrin associated is important for trafficking of alkaline LYSOSOMES phosphatase and the t-SNARE Vam3p to the vacuole independently of the more widely studied route taken by The existence of a hybrid organelle from which lysosomes are carboxypeptidase Y (Cowles et al., 1997; Stepp et al., 1997; re-formed gives a new context in which to review our Piper et al., 1997), which transits via the PVC. Recently, knowledge of the delivery of newly synthesised proteins to evidence has emerged that, in yeast, Vps41p binds to the AP- lysosomes. In mammalian cells the bulk of newly synthesised 3 δ subunit and functions with AP-3 in the formation of lysosomal hydrolases is delivered to the lysosome after binding transport vesicles at the late Golgi (Rehling et al., 1999). to the MPRs in the TGN. It is now thought that the trafficking Intriguingly, Vps41p and its homologues in Caenorhabditis route of MPRs involves recruitment into AP-1 containing elegans, Drosophila melanogaster and various plant species clathrin-coated pits at the TGN, vesicle traffic and fusion with contains a clathrin-heavy-chain-homology region that is early endosomes, followed by transport to late endosomes thought to be involved in oligomerisation and formation of the before return to the TGN after hydrolase release (Feng et al., clathrin lattice. This raises the possibility that in an AP-3 1995; LeBorgne and Hoflack, 1998; Diaz and Pfeffer, 1998; vesicle coat Vps41p carries out the functions performed by Hirst et al., 1998). At first sight, the suggestion that lysosomes clathrin in AP-1 and AP-2 coats. Interestingly, mutations in fuse with late endosomes to create a hybrid organelle VPS41 cause phenotypes different from those caused by containing an efficient proteolytic environment is difficult to mutations in AP-3 and appear to exhibit defective vacuolar reconcile with the need to recycle MPRs from late endosomes fusion. One speculation is that Vps41p helps catalyze the to the TGN. However, in HEp-2 cells kinetic studies show that fusion of the very vesicles it forms. much of the MPR is retrieved before the late endosomes In Drosophila (garnet), mice (mocha and pearl) and humans fuse with lysosomes (Hirst et al., 1998). Thus, only a small (a subset of patients with Hermansky-Pudlak Syndrome, HPS), proportion of the MPR will be exposed to the proteolytic mutations in AP-3 subunits have been identified and result in environment of the hybrid. defects in membrane traffic to lysosome-like organelles In contrast to delivery of mannose-6-phosphate-tagged including Drosophila eye-pigment granules, mammalian hydrolases, the delivery of newly synthesised lgps is less well melanosomes and platelet granules (Ooi et al., 1997; Simpson understood. With the notable exception of lysosomal acid et al., 1997; Lloyd et al., 1998; Swank et al., 1998; phosphatase, which is delivered first from the TGN to the cell Dell’Angelica et al., 1999a). Furthermore, in yeast, AP-3 surface and then by endocytosis to lysosomes, lgps are thought function is quite specific to a particular class of transport to be delivered mostly via an intracellular route from the TGN vesicle and is not required for transport to and from the (Hunziker and Geuze, 1996). The two different routes are endosome/PVC: these functions remain intact even in the reflected in the half-times of delivery of newly synthesised lgps absence of all identifiable AP complexes (Rehling et al., 1999; from the TGN network to lysosomes: the half-time of delivery Huang et al., 1999). Although the evidence that AP-3 is of lysosomal acid phosphatase is ~6 hours (as a result of involved in targeting to lysosome-like organelles is strong, its multiple rounds of recycling between early endosomes and the role in targeting to lysosomes has been more difficult to cell surface before delivery to lysosomes); that for other lgps establish, and the endocytic organelle at which AP-3 vesicles is 0.5-2 hours (Braun et al., 1989; Green et al., 1987; dock and fuse is unknown. Fibroblasts transfected with Barriocanal et al., 1986). Two cytosolic tail motifs mediate antisense oligonucleotides to µ3A or fibroblasts from lysosomal targeting of lgps. One of these, the GYXX¯ motif individuals lacking β3A show increased trafficking of LAMP- (where X is any amino acid and ¯ is a bulky hydrophobic 1 via the cell surface (Le Borgne et al., 1998; Dell’Angelica et amino acid), contains the YXX¯ motif known to be important al., 1999a). Disappointingly, the absence of functional AP-3 in for interaction with the µ2 subunit of the AP-2 adaptor and these cells does not alter the steady-state localisation of several endocytosis via clathrin-coated pits (Bonifacino and lgps. Dell’Angelica, 1999). The other is a di-leucine motif. Two Although a fourth adaptor complex, AP-4, that has subunits findings suggest that some lgps containing a GYXXØ motif homologous to those of AP-1 and AP-2 has been discovered, traffic via AP-1-containing clathrin-coated vesicles from the its function is unknown (Dell’Angelica et al., 1999b; Hirst et TGN to endosomes: (1) the GYQTI motif present in LAMP-1 al., 1999). It is expressed ubiquitously but at low abundance. interacts with the µ1 subunit of AP-1 in the yeast two-hybrid Interactions between the µ4 subunit and lysosomal system (Ohno et al., 1995); and (2) LAMP-1 co-localizes with glycoprotein (lgp) tails in a yeast two-hybrid system have led 1520 J. P. Luzio and others

Fig. 3. The mechanism of late- v-SNARE tether endosome-lysosome fusion. The t-SNARE diagram shows the possible Late stages of fusion of late endosome endosomes with lysosomes and SNARE re-formation of lysosomes from tethering assembly fusion Ca2+ the subsequent hybrid organelles. We propose that tethering by unknown proteins precedes Ca2+ Ca2+ Ca2+ Ca2+ SNARE assembly and fusion and Rab that release of Ca2+ from the NSF calmodulin organelle lumens (for simplicity, shown here only from the Ca2+ endosome) is necessary for fusion.

Hybrid organelle Although yeast genetics has, to date, made the greatest Lysosome impact on our knowledge of the molecular mechanisms recondensation involved in targeting of proteins to lysosomes, the genetics of multi-cellular to speculation about a role in delivery to lysosomes, but there organisms is likely to provide equally important insights over is, as yet, no corroborative evidence for this. the next few years. Eye-colour mutants in Drosophila and mouse coat-colour mutants have been known and studied throughout the twentieth century. Over 80 mutations affecting THE FUTURE IMPACT OF GENETICS ON OUR eye colour in Drosophila have been described, and, although UNDERSTANDING OF LYSOSOME BIOGENESIS some of the eye colour genes encode pigment-synthesis enzymes and membrane transporters, a substantial group The study of membrane traffic to the yeast vacuole still has known as the ‘granule group’ encode proteins that function in much to offer. Thus, there are recent clues as to the molecular the delivery of proteins to lysosomes and pigment granules basis of retrograde traffic from the vacuole to the PVC (which (Lloyd et al., 1998). These include garnet, which encodes the implicate VAC7; Bryant et al., 1998) and from the PVC to the the AP-3 δ subunit (Simpson et al., 1997), light, carnation and late Golgi (Seaman et al., 1997, 1998). Yeast genetics has also deep orange, which are, respectively, the homologues of yeast recently provided an insight into the origin of intra-organelle VPS41,VPS33 andVPS18 (Lloyd et al., 1998; Sevrioukov et al., membrane in the PVC and vacuole and thus, by analogy, 1999). The protein products of deep orange and carnation, like the classical morphological appearance of late endocytic those of VPS18 and VPS33 (Rieder and Emr, 1997), function organelles as multi-vesicular bodies in mammalian cells. in large protein complexes required for traffic from late- Odorizzi et al. (1998) showed that the targeting of some endosome-like to lysosome-like organelles. Another Drosphila membrane proteins into the vacuole lumen for degradation mutant, hook, originally identified on the basis of a bristle requires the Class E VPS genes. This indicates that the phenotype, encodes a 679-residue cytoplasmic protein that has accumulation of exaggerated endosomal compartments in a mammalian homologue and appears to function late in the these mutants is due to defects in the ability of the cell to form endocytic pathway: the wild-type Hook protein inhibits internal membranes within endosomal compartments. Recent delivery to lysosomes (Kramer and Phistry, 1999; Sunio et al., analysis of the function of the homologue of the class E 1999). protein, Vps27p, in mice shows similar defects in the endocytic It is likely that many mouse coat-colour mutants (in addition pathway, and it will be interesting to examine the biogenesis to mocha and pearl) result from defects in proteins required for of MVBs within these cells (Komada and Soriano, 1999). membrane traffic in the endocytic pathway and delivery to Another process that appears to be required for MVB melanosomes. Melanosomes have been classified with a class formation is synthesis of PtdIns(3,5)P2, which is formed from of lysosome-like organelles known as secretory lysosomes PtdIns(3)P by the PtdIns(3)P 5-kinase encoded by FAB1 (Stinchcombe and Griffiths, 1999). These are, in particular, (Odorizzi et al., 1998). The substrate for this kinase is found in cells of the hemopoietic lineage, and the identification PtdIns(3)P formed by the unique yeast PtdIns 3-kinase, of genes associated with abnormalities in their function might Vps34p. Interestingly, Fernandez-Borja et al. (1999) report that also provide insights into the biogenesis and function of in a human melanoma cell line, the PtdIns 3-kinase inhibitor ‘normal’ lysosomes. In human Chediak-Higashi Syndrome wortmannin, which causes the appearance of swollen (CHS) and the mouse model of the disease, beige, all endocytic compartments, inhibits budding of membrane into lysosomes are abnormally enlarged, but only cells with the lumen of these structures. It will be important to establish secretory lysosomes are functionally impaired. The Chediak- whether this result can be repeated in other cell types. Higashi gene encodes a cytosolic ~400-kDa protein, Lysosome-endosome fusion and lysosome biogenesis 1521 designated LYST (for lysosomal trafficking regulator protein). metabolite concentrations? The most likely way forward to a When LYST was overexpressed in mutant fibroblasts, complete understanding of lysosome-endosome fusion and abnormally small lysosomes resulted, which suggests the lysosome biogenesis may well come from investigation of the protein has a role in lysosome fission (Perou et al., 1997). function of proteins, many of which will be identified by LYST shares limited sequence homology with yeast Vps15p, genetic approaches in cell-free systems. the regulatory subunit of the yeast Vps34p PtdIns 3-kinase, and more extensive homology with yeast BPH1, mutations in Experimental work in our laboratories was funded by the Medical which result in no discernible phenotype. LYST also contains Research Council and the Human Frontiers Science Program. 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