Delivery of Endocytosed Membrane Proteins to the Lysosome

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Biochimica et Biophysica Acta 1793 (2009) 615–624

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Review Delivery of endocytosed membrane proteins to the lysosome

Paul R. Pryor a,⁎, J. Paul Luzio b,⁎

a Department of Biology (Area 9), University of York, York, YO10 5YW, UK b Cambridge Institute for Medical Research and Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0XY, UK

article info abstract

Article history: The delivery of proteins from the plasma membrane to the lysosome for degradation is essential for normal Received 22 July 2008 cellular function. There is now a good understanding of the protein complexes involved in sorting proteins at Received in revised form 1 December 2008 the plasma membrane and into the intralumenal vesicles of the multi-vesicular body. A combination of cell Accepted 12 December 2008 free content mixing assays and live-cell imaging has dissected out the final step in delivery of Available online 8 January 2009 macromolecules to the lysosome from the multi-vesicular body and provided insights into the molecular mechanisms by which late endosomes and lysosomes exchange lumenal contents. The endocytic pathway Keywords: fi Endocytosis has provided a platform with which to understand the autophagic and phagocytic pathways, but the ne Late endosomes details of how traffic through these pathways is regulated remain to be determined. Lysosomes © 2008 Elsevier B.V. All rights reserved. Multivesicular body Membrane fusion

1. Introduction brane proteins destined to be delivered to mammalian lysosomes. It includes descriptions of sorting at the plasma membrane and in Lysosomes are highly dynamic membrane-bound organelles that endosomes and the plethora of proteins required to regulate and sort act as the terminal degradative compartment of the endocytic, cargo into the intralumenal vesicles (ILVs) of multivesicular bodies phagocytic and autophagocytic pathways (Fig. 1). Many of the (MVBs). Finally, as all membrane fusion events may be defined as proteins required for protein sorting and endocytic delivery to having tethering and docking and fusion steps, the review will lysosomes in mammalian cells are orthologues of those used by the indicate the key regulators in the membrane fusion between MVBs yeast Saccharomyces cerevisiae for delivery to the vacuole (the yeast and lysosomes. equivalent of the lysosome). Studies on vacuolar delivery and fusion have been extremely informative in providing insights to the universal 2. Clathrin mediated endocytosis at the plasma membrane mechanisms governing sorting and delivery in eukaryotic cells. Additionally, some cell types contain lysosome-related organelles A plasma membrane protein that is destined to be endocytosed (LROs) that include melanosomes, lytic granules, MHC-II compart- needs to be recognised by the endocytic machinery. Although other ments, platelet dense granules and neutrophil azurophil granules [1]. uptake pathways have been described [3], the best understood Some LROs are simply the modified lysosome, for instance in cytotoxic endocytic mechanism is clathrin mediated endocytosis. At its simplest, T lymphocytes the lytic granules are the only lysosome-type organelle this may be regarded as a co-ordinated series of events involving cargo present. In other cell types such as melanocytes, both the melano- recruitment into small regions of the plasma membrane, clathrin cage somes and the ‘conventional’ lysosomes co-exist. Studies of LROs, in assembly, membrane bending, vesicle formation, vesicle fission and particular from patients with Hermansky–Pudlak syndrome that have finally vesicle uncoating (for reviews see [4,5]). Cargo recruitment deficiencies in melanosomes and platelet-dense granules, have requires clathrin adaptors that interact with sequence motifs, identified mutations in genes encoding proteins (some that are not structural features or added ubiquitin molecules in the cytosolic tail found in yeast) that assemble into five distinct complexes (adaptor of the membrane protein. Some membrane proteins require the protein-3 (AP-3), homotypic fusion and vacuole protein sorting binding of extracellular ligand before rapid internalisation proceeds, (HOPS) complex and biogenesis of lysosome-related protein complex but others do not need the addition of ligand. In the latter case, (BLOC)-1, -2 and -3) that have provided further insights into delivery endocytosis occurs to maintain low levels of the protein at the plasma of endocytic cargo to the lysosome [2]. This review therefore membrane and/or to redirect mis-sorted proteins to intracellular sites. summarises recent insights into endocytic sorting of plasma mem- Efficient incorporation of some endocytosed membrane proteins into clathrin- coated-pits and -vesicles (CCVs) is mediated by short linear amino acid sequences, such as the YXXΦ and [DE]xxxLL motifs, that are ⁎ Corresponding authors. P.R. Pryor is to be contacted at tel.: +44 1904 328563. – J.P. Luzio, tel.: +44 1223 336780. recognised by the clathrin adaptor AP2 (reviewed in [6 8]). Other E-mail addresses: [email protected] (P.R. Pryor), [email protected] (J.P. Luzio). membrane proteins, including several growth factor receptors, are

Fig. 1. Intracellular pathways to the lysosome. Endocytic cargo is internalised from the plasma membrane and delivered to early endosomes. Maturation of the early endosome gives rise to a late endosome/multi-vesicular body where the cargo destined for degradation has been sorted into intralumenal vesicles. The limiting membranes of late endosomes and lysosomes can fuse to form a hybrid organelle where degradation of endocytosed macromolecules commences. Lysosomes are reformed from the hybrid organelle by membrane retrieval and condensation reactions. Autophagic and phagocytic pathways both feed into the endocytic pathway with the lumenal contents of the autophagosome and phagosome eventually being delivered to the lysosome for degradation.

ubiquitylated, such that they carry multiple single ubiquitins (multiple of the clathrin adaptor HIV Rev-binding protein (HRB) allowing monoubiquitylation), or short polyubiquitin chains, added to specific endocytosis of VAMP7, but only when VAMP7 is incorporated into a lysines in the cytosolic tail (reviewed in [9]). The presence of many cis-SNARE complex [11]. VAMP7 is found on lysosomes and is required ubiquitins allows efficient capture into CCVs by specific clathrin for lysosome fusion with the plasma membrane. Its folded N-terminal adaptors such as EPS15 and epsin, despite the relatively low affinity of domain thus acts as a retrieval signal from the cell surface and the ubiquitin binding domains in these adaptors for single ubiquitin interaction with an unstructured region of HRB has provided an molecules. insight into the potential role of the unstructured regions of clathrin Recently, a third mechanism for efficient incorporation into CCVs adaptors in cargo recognition. has been described, and is used by at least one soluble N- It should be noted that clathrin-mediated endocytosis may play an ethylmaleimide-sensitive factor (NSF) attachment protein receptor important role in lysosome biogenesis. In most cell types studied, (SNARE) at the plasma membrane. SNAREs are critical for driving many newly synthesised acid hydrolases are delivered to lysosomes membrane fusion events on the secretory and endocytic pathways via an intracellular route that requires addition of mannose 6- (reviewed in [10]). However, the mechanisms by which SNAREs are phosphate and binding to mannose 6-phosphate receptors, which actually sorted to different membranes are largely unknown. Most of traffic between the trans-Golgi network and endosomes. However, in the SNAREs do not possess short linear endocytic sequence motifs and proximal kidney tubule cells the lysosomal enzyme cathepsin B is are not known to be ubiquitylated. However, the folded N-terminal delivered to lysosomes following secretion of non-mannose 6- domain of the SNARE VAMP7 binds to a predicted unstructured region phosphate-labelled pro-cathepsin B, that then binds to the cell surface P.R. Pryor, J.P. Luzio / Biochimica et Biophysica Acta 1793 (2009) 615–624 617 receptor megalin, which is then internalised by clathrin mediated components are directly involved in the generation of ILVs. However, endocytosis [12]. Some newly synthesised lysosomal membrane depletion of the ESCRT-III component VPS24 inhibits EGFR degrada- proteins, such as lysosomal acid phosphatase are also mainly tion but not the kinetics of signal termination [26]. Similarly a CHMP5 delivered to lysosomes by an indirect route via the plasma membrane. knock-out mouse forms MVBs, but degradation of receptors and fluid However, the major lysosomal membrane proteins LAMP-1 and phase markers is reduced [27,28]. These latter observations suggest LAMP-2, that together account for over 50% of the membrane protein that some of the components of ESCRT-III may be dispensable for ILV in a lysosome, are mainly delivered to endosomes/lysosomes from the formation but may have a role in downstream events. trans-Golgi network by a direct intracellular route. Whilst LAMP-1 and Rab interacting lysosomal protein (RILP) is a RAB7 binding protein LAMP-2, like lysosomal acid phosphatase, contain cytosolic tail YXXΦ known to act in events late on in the delivery of cargo to the lysosome. motifs and are efficiently endocytosed by clathrin mediated endocy- However, it has also been observed that RILP interacts with the tosis, this is thought to be mainly a retrieval route for these two mammalian ESCRT-II proteins VPS22 (EAP30) and VPS36 (EAP45) proteins (reviewed in [13]). [29,30]. Depletion of RILP by RNA interference reduces ILV formation and degradation of EGF receptors in a manner similar to VPS22 3. Sorting at the recycling endosome depletion [31]. Depletion of RILP causes a reduction of cellular VPS22 concentration and vice versa, so some of the effects of RILP depletion Endocytosed cargo is first delivered to early endosomes, then to might be related to the accompanying loss of VPS22. However, RILP late endosomes, which have the morphology of MVBs and finally to depleted endosomes are morphologically different to VPS22-depleted lysosomes. In the early endosome some bound ligands dissociate from endosomes [31]. Given that RILP also interacts with the dynein– their receptors in the acidic lumen allowing recycling of the receptors dynactin motor complex [32,33], it has been suggested that RILP to the cell surface. Recycling may also occur via the tubular recycling might co-ordinate the formation of MVBs with dynein-mediated endosome derived from the early endosome (reviewed in [14]). It is motility along microtubules. also likely that there are retrieval routes from the recycling endosome to the late endosome/lysosome. One membrane protein that appears 5. Delivery to the lysosome to use such a route is the SNARE VAMP7. In addition to binding HRB, its N-terminal folded longin domain also binds to the clathrin adaptor Several hypotheses have been proposed for the mechanism of AP3 and this interaction is required for delivery to late endosomes/ transfer of endocytosed material from endosomes to lysosomes lysosomes [15]. Given the localisation of AP3 to recycling endosomes (reviewed in [34]). These include a maturation model where the [16], an attractive hypothesis is that VAMP7 endocytosed from the cell endosome ‘matures’ into a lysosome [35], a vesicular transport model surface is delivered to early endosomes, sorted into recycling where vesicles carry cargo from the endosome to the lysosome [36],a endosomes and then retrieved by AP3 to late endosomes/lysosomes. kiss-and-run model where endosomes and lysosomes engage in Although the precise mechanism of binding of VAMP7 to AP3 has not repeated transient fusions [37] and a model where endosomes and been resolved. There is evidence that in the direct route of lysosomes completely fuse giving rise to a hybrid compartment from intracellular delivery of newly synthesised LAMP-1 and LAMP-2 to which lysosomes reform [13,38–41]. The maturation model cannot lysosomes one sorting step is an AP3 dependent sorting at tubular explain how lysosomes remain accessible to endocytic enzyme recycling endosomes [16]. replacement therapies [42] nor how they undergo content mixing with late endosomes [38,39]. Whilst there is no formal evidence for a 4. Multi-vesicular body formation vesicular transport model, cell-free content mixing assays [38,39] and more recently time-lapse confocal microscopy experiments [43] have The multivesicular body (MVB) or late endosome is characterised provided evidence for both kiss-and-run and direct fusion events as by intralumenal vesicles (ILVs). These ILVs are formed through inward the major mechanism for delivery of endocytosed macromolecules to budding of the endosomal membrane, a process that commences at the lysosome. the early endosome. The sorting of cargo from the endosomal limiting Fusion of membranes in the secretory and endocytic pathways membrane to these ILVs eventually allows endocytosed cargo destined requires tethering, SNARE complex formation and then phospholipid for degradation to be efficiently degraded by lysosomal enzymes. The bilayer fusion (recently reviewed in [44]). There is a high degree of best characterised signal for sorting of proteins into the internal complexity in the molecular events preceding phospholipid bilayer vesicles is ubiquitylation. Hence, ubiquitylation not only represents an fusion and the clear distinction between initial tethering events and endocytosis signal but it also acts as a signal for degradation. membrane fusion is becoming less apparent as the molecular Originally identified in yeast, the soluble class E vacuolar protein mechanisms of these processes are unravelled. Because there is a sorting (Vps) proteins organise into four endosomal sorting com- high degree of conservation in the mechanism of membrane fusion plexes required for transport (ESCRT) complexes, namely ESCRT-0, between yeast and higher eukaryotes, studies of vacuolar fusion in ESCRT-I, ESCRT-II and ESCRT-III (reviewed in [17]). Current models Saccharomyces cerevisiae have aided our understanding of the suggest that the ESCRT complexes are required for both formation of molecular events required for delivery of endocytic cargo to the the ILVs and for sorting ubiquitylated cargo into them. Certainly lysosome. The following sections summarise our current under- depletion of components of ESCRT-0 and ESCRT-I in mammalian cells standing of tethering, SNARE complex formation and fusion events reduces ILV formation [18–20]. The role of ESCRT-III proteins in ILV between the late endosome/MVB and the mammalian lysosome and formation is not so clearly understood. Recent structural studies have provide a model of delivery to the mammalian lysosome (Fig. 2). shown that components of ESCRT-III can form lattices on membranes [21,22] and, in the presence of a dominant negative VPS4, over- 5.1. Tethering complexes expression of VPS32 (CHMP4) in mammalian cells can promote negative membrane curvature [23]. In addition, it has been shown in Homotypic vacuolar tethering in yeast is mediated by the HOPS vitro that filaments formed by ESCRT-III proteins VPS2 (CHMP2) and complex (reviewed in [45]). This complex consists of six subunits, four VPS24 (CHMP3) or a chimeric VPS2–VPS24 protein can be disas- class C proteins Vps11p,16p,18p and 33p and two class B proteins sembled by VPS4 [24,25] suggesting that helical VPS2 and VPS24 Vps41p/Vam2p and Vps39p/Vam6p (vps mutants are divided into six structures could assemble at the base of the neck of an inwardly classes, A–F, based upon several criteria, including the morphology of the budding vesicle leading to membrane constriction and eventually mutant vacuoles [46,47]). The individual HOPS complex subunits have abscission when coupled to VPS4. These data suggest that ESCRT-III been studied in detail but their precise functions still require further 618 P.R. Pryor, J.P. Luzio / Biochimica et Biophysica Acta 1793 (2009) 615–624

Fig. 2. The molecular machinery required for delivery of endocytosed material to lysosomes. The delivery of endocytosed material to the lysosome involves maturation of the early endosome to form a MVB/late endosome. Maturation events involve several complexes whose activity must be co-ordinated to give rise to an organelle with a membrane that is fusion competent with the lysosome. Complexes involved in priming the endosomal membrane ready for delivery to the lysosome include tethering factors such as the Class-C VPS complex, which in yeast can interact with different proteins to form a CORVET or HOPS complex. These complexes regulate early Vps21p-dependent (mammalian RAB5-dependent) or later Ypt7p-dependent (mammalian RAB7-dependent) events, respectively. Sorting of membrane proteins into lumenal vesicles, a pre-requisite for efficient degradation by the lysosome, requires ESCRT machinery. The ESCRT machinery is recruited to membranes by cargo and specific phosphoinositides, but probably also by other proteins such as the lysosomal protein RILP that interacts with components of ESCRT-II. The ESCRT machinery is also likely to act as a platform for recruitment of other proteins to the limiting membrane. All the complexes are likely to be integrated with each other. Interactions include, Class C-VPS subunits and ESCRT subunits (unpublished observations); RILP with ESCRT-II; RAB7 with RILP and the HOPS complex. Endosomal maturation is likely to occur at the same time as traffic along the microtubules towards the microtubule organising centre. Finally, membrane fusion between the MVB and the lysosome occurs as a SNARE-dependent event. Yeast vacuolar fusion studies indicate that this too might be subjected to a proof-reading by the HOPS complex. Both membrane fusion to form a hybrid organelle and reformation of the lysosome are calcium-dependent processes where the calcium is derived from the lumen of the organelles.

elucidation. Vam6p has been observed to have guanyl-nucleotide causes organelle dispersion [58]. However, it is not only in delivery exchange factor (GEF) activity for Ypt7p (the yeast equivalent of to the vacuole/lysosome where components of the HOPS complex RAB7) [47] and binding of the HOPS to Ypt7p occurs when it is in its appear to be important, and in both yeast and mammalian cells GTP-bound form [48]. Furthermore, binding of the HOPS complex to the components of the HOPS complex also play a role early in the vacuole is diminished in the absence of Ypt7p [49]. HOPS binding to the endocytic pathway [61–63]. Recent work in yeast has helped to vacuolar membrane is also likely to mediated by an affinity of the HOPS resolve how components of the HOPS complex can act at both early complex for both phospholipids [50] and vacuolar SNAREs [50–53]. and late endosomes. Ungermann and colleagues have shown that the At the core of every membrane fusion reaction is the formation of a four Class C proteins Vps11p,Vps16p,Vps18p and Vps33p can specific tetrameric trans-SNARE complex. Each type-II trans-mem- differentially interact with two other subunits [64]. At the endosome brane SNARE protein contains a 16 turn ‘SNARE’ helix and can be the Class C Vps proteins interact with Vps8p and Vps3p to form a defined as either a Q- or R-SNARE depending on the residue at layer 0 class C core vacuole/endosome tethering complex (CORVET), in the SNARE helix [54]. The Q-SNAREs can be further sub-divided into whereas at the vacuole they interact with Vam6p and Vps41p to Qa, Qb and Qc SNAREs with the tetrameric trans-SNARE complex form the HOPS complex. Intermediate protein complexes between consisting of one of each class of Q-SNARE and a single R-SNARE [55]. CORVET and HOPS have been isolated but the mechanisms that Like other membrane fusion reactions vacuolar fusion requires three regulate CORVET to HOPS conversion, and vice versa remain to be Q-SNAREs (Vti1p, Vam3p, Vam7p) and one R-SNARE (Nyv1p), but in determined. Interestingly, Vps3p binds to Vps21p (yeast RAB5) in vitro vacuolar fusion can still occur when the Q or R residues are both its GTP and GDP-bound forms and may therefore be a Vps21p mutated to give a 4Q or 2Q:2R complex [56]. It has been shown that GEF [64]. As described above, Vam6p is a GEF for Ypt7p. This vacuolar fusion is inhibited upon the addition of exogenous HOPS to corresponds with mammalian cells in which RAB5 is required for SNARE complexes with altered 0-layers, or containing truncated early endocytic traffic [65] and RAB7 in late endocytic traffic [66–68]. Vam7p, This has suggested a ‘proofreading’ role for the HOPS complex, Taken together with data where RAB5 and RAB7 associate with the but precisely how this is achieved is unclear [57]. same endosome [69] it suggests that the transition from an early In mammalian cells, there are orthologues of the HOPS subunits endosome to a mature MVB ready for fusion with the lysosome is and there is considerable evidence that they also serve a role in regulated by tethering proteins and RABs (Fig. 2). Another link delivery of endocytosed macromolecules to the lysosome. Thus, between RAB5 and the mammalian orthologues of yeast class C VPS overexpression of VPS18, VAM6 and VPS33B cause lysosomal proteins has also been described. The autophagy protein UVRAG clustering [58–60], whereas depletion of VPS18 by RNA interference (also known as beclin-1 binding autophagy protein), which is also P.R. Pryor, J.P. Luzio / Biochimica et Biophysica Acta 1793 (2009) 615–624 619 important for endocytic traffic [70], binds to both class C VPS form indicates a defect in recycling of the pro-alpha factor-processing proteins and GTP-bound Rab5 [71]. A further example of the multi- enzyme Kex2p between the Golgi and the endosome and sensitivity to functional role of the mammalian class C VPS proteins is the chemicals, such as caffeine and SDS. In this screen, known complexes observation that class C VPS proteins can interact with ESCRT regulating endocytosis were found including the ESCRT complexes, subunits (unpublished observation from the Luzio laboratory), but a new complex consisting of two proteins Vps55p and Vps68p was consistent with tethering proteins also regulating MVB formation. also identified [83]. Both Vps55p and Vps68p are predicted to contain This is not surprising, since MVB formation needs to be co-ordinated four transmembrane domains, and in yeast both proteins localise to with organelle maturation. When other accessory proteins are added the vacuole limiting membrane, their stability depending upon each into the system, including RILP that binds both ESCRT subunits other, suggesting that they form a stable protein complex. Mutation of [29,30] and RAB7 [72], it becomes difficult to assign a linear order of either protein slows the traffic of both endocytic and biosynthetic molecular events to endosome maturation. cargo through the MVB, but does not alter MVB formation itself [83]. In mammalian cells there is more than one homologue of both These data suggest that Vps55p and Vps68p regulate endosome Vps16p and Vps33p, namely VPS16A, VPS16B and VPS33A and maturation at a step downstream of MVB formation. Both Vps55p and VPS33B. The function of these is also related to the function of Dro- Vps68p have two human homologues, OB-RGRP and LEPROTL1 for sophila orthologues. Drosophila eye pigment granules and mammalian Vps55p and SMP1 and C21ORF4 for Vps68p [83]. OB-RGRP expression melanosomes are both lysosome related organelles (LRO) and defects in yeast can complement the carboxypeptidase Y trafficking defect in a in trafficking to these organelles results in changes in eye colour in the vps55 mutant [84], but a role of OB-RGRP in delivery of macro- fly and coat colour changes in mice [1]. Mutations in DmVps33 results molecules, or Vps68p orthologues, to the mammalian lysosome is yet in a mutant fly called carnation [73], whereas in the mouse mutations to be established. in VPS33A result in a buff mouse [74]. Genomic analyses have suggested that the carnation gene is more similar to VPS33A than 5.2. trans-SNARE complexes VPS33B [75]. Overexpression of human VPS33B in cultured cells causes lysosomal clustering, but lysosomes are not clustered by In mammalian cells a trans-SNARE complex consisting of Q- overexpression of VPS33A or DmVps33 [75]. These data suggest that SNAREs SYNTAXIN7, VTI1B and SYNTAXIN8 together with the R- VPS33A may be important for LRO biogenesis and VPS33B for delivery SNARE VAMP8 has been shown to be responsible for homotypic fusion to the lysosome. Additionally, phagocytosis and endocytosis share of late endosomes [85]. But by exchanging the R-SNARE VAMP8 for common intracellular machinery for delivery of material to the VAMP7 (also known as tetanus neurotoxin insensitive VAMP (TI- lysosome and a recent report has shown that the phagocytosed VAMP) or synaptobrevin-like-1 (SYBL1)), heterotypic membrane pathogen Mycobacterium tuberculosis, which delays progression to the fusion between late endosomes and lysosomes is achieved (Fig. 3) lysosome and thereby improves its ability to survive, secretes the [41]. To date, all lysosomal fusion events in which the trans-SNARE tyrosine phosphatase PtpA into its host cell cytosol which depho- complex has been defined, appear to require the R-SNARE VAMP7 sphorylates VPS33B [76]. VPS33B is a self-phosphorylating tyrosine- (reviewed in [34]), thus the high concentration of VAMP7 on the kinase and the phosphorylation state of VPS33B may help to explain lysosomal membrane may help to define the lysosome. VAMP7, yeast older observations showing that yeast Vps33p binds ATP and its Nyv1p [86] along with the yeast and mammalian orthologues of association with membranes is regulated by cellular ATP levels [77]. Sec22p [87] and Ykt6p [88] comprise the ‘longin’ sub-family of R- One caveat to VPS33B being more important for delivery to the SNAREs since they possess an N-terminal, profilin-like, longin domain lysosome compared to VPS33A is that a proteomic study of lysosome (LD) [89]. As described above the LD of VAMP7 is important for its membrane proteins identified the presence of VPS33A but not VPS33B intracellular trafficking, because it binds the endocytic adaptor HRB at [78]. VPS33B is not the only HOPS subunit to undergo phosphoryla- the plasma membrane [11], thus enabling SNARE endocytosis, and AP3 tion, because yeast Vps41p is also phosphorylated by the casein kinase at the recyling/sorting endosome [15] to sort the SNARE to lysosomes. Yck3p [79], Yck3p deletion leads to an increase of Vps41p at the The LD of VAMP7 can bind back to its own SNARE helix but not when vacuole and an increase in vacuolar fusion, whereas Yck3p over- the SNARE helix is complexed to other SNAREs [11]. VAMP7 is the only expression reduces fusion [79]. protein known to be differentially required for heterotypic late In yeast, a complex of two proteins, Mon1p and Ccz1p, appears to endosome–lysosome fusion when compared to homotypic late also bind the Class C Vps subunits of the HOPS complex, with vacuole endosome fusion. Thus, the LD of VAMP7, that will be free to bind fusion reduced if either protein is missing [80]. In mammalian cells other proteins when a SNARE complex has formed, may also serve as a there has been a gene duplication event resulting in two orthologues domain to recruit further proteins to regulate the final stages of of Mon1p, MON1A and MON1B. A knock-out mouse for MON1A shows lysosomal fusion. trafficking defects through the secretory pathway. However, no The interaction of tethers and SNAREs may play a role in changes in endocytic trafficking to the lysosome, lysosome size or regulating fusion because there is good evidence to suggest that lysosome distribution were observed [81]. A proteomic study has tethering complexes are capable of directly binding the SNAREs identified MON1B associated with lysosomes [78], suggesting that in involved in lysosomal fusion. In both yeast and mammalian cells mammalian cells MON1B may be important for traffic to the lysosome. Class C VPS complex subunits have been found to bind to the The precise roles of MON1B and its lysosomal target in mammalian vacuolar SNARE Vam3p and its mammalian orthologue SYNTAXIN7 cells are yet to be established. There is no obvious orthologue of Ccz1p, [53,63,90]. It is possible that the binding of the Class C VPS complex however Ccz1p does have an N-terminal CHiPS domain that has to SYNTAXIN7 may keep SYNTAXIN7 in a closed conformation or in a homology to the Hermansky–Pudlak protein HPS4, one of the conformation that prevents trans-SNARE pairing. The interaction subunits of BLOC-3 [82]. between SYNTAXIN7 and the Class C VPS complex interaction may Recently a large phenotypic screen was undertaken in yeast to be regulated by phosphorylation since a recent report in yeast has identify proteins on the endosomal pathway based upon the shown that both Vps41p and Vam3p may be phosphorylated [91]. hypothesis that genes with similar functions on a particular pathway The phosphorylation of Vps41p and Vam3p is reduced in the give a similar phenotype when mutated [83]. To assess whether presence of GTP-bound Ypt7p, the RAB7 orthologue [91]. Ypt7p has endocytosis had been perturbed various criteria were used including also been shown to bind Vps41p in addition to Vps39p (the Ypt7p trafficking of the vacuolar hydrolase carboxypeptidase Y (CPY) which GEF) [91]. One hypothetical scenario for how tethering and trans- is secreted when traffic to the vacuole is impaired, processing of the SNARE pairing may be co-ordinated is shown by Fig. 4. Here, yeast hormone alpha-factor which if secreted in its pro-alpha-factor exchange of RAB5 for RAB7 would allow for recruitment of VPS39 620 P.R. Pryor, J.P. Luzio / Biochimica et Biophysica Acta 1793 (2009) 615–624

Fig. 3. Schematic model of SNARE dependent fusion between late endosomes and lysosomes. Tethering reactions between organelles are likely to precede trans-SNARE pairing. Tethers between late endosomes and lysosomes are likely to be the HOPS complex and may regulate the fusion process itself. Fusion between a late endosome and a lysosome requires the R-SNARE VAMP7 and three Q-SNAREs (SYNTAXIN7, SYNTAXIN8 and VTI1B) on the opposing membrane. VAMP7 has an N-terminal longin domain (green oval) that binds back to its SNARE domain. Upon trans-SNARE pairing the longin domain is released, but regulatory proteins may bind the longin domain to regulate the fusion process. Following trans-SNARE pairing membrane fusion occurs allowing content mixing. Membrane retrieval and condensation reactions allow reformation of the lysosome. Homotypic late endosome fusion uses the same three Q-SNAREs as late endosome–lysosome fusion but a different R-SNARE, namely VAMP8. and VPS41. VPS39 and VPS41 could bind to the Class C VPS complex, remain to be determined. In mammalian late endosome–lysosome bound to SYNTAXIN7, on an opposing membrane. The GEF activity of fusion microcystin-LR does not inhibit content mixing (unpublished VPS39 would activate RAB7 leading to de-phosphorylation events observations from authors' research). and release of the Class C VPS complex from SYNTAXIN7 and thereby allow for trans-SNARE pairing and membrane fusion. 6. Lysosome reformation

5.3. Organelle fusion Delivery of endocytic material to the lysosome results in a hybrid organelle, that has markers of both late endosomes and lysosomes and In mammalian cells there are events after trans-SNARE pairing that is of intermediate density [38]. We have proposed that it is in the are necessary for content mixing to occur, as there are for yeast hybrid organelle where degradation of endocytosed macromolecules homotypic vacuole fusion. In a similar manner to homotypic vacuole commences and not the lysosome per se, which may be regarded as fusion [92], late endosome–lysosome content mixing can be inhibited storage organelles of mature lysosomal enzymes. Once digestion of by the calcium chelator BAPTA [40]. The terminal step of late lumenal material has occurred there needs to be a mechanism endosome–lysosome fusion is also inhibited by calmodulin antago- whereby limiting membrane proteins, such as the SNAREs, are nists, which implies that calcium/calmodulin is required for comple- recycled for further rounds of fusion with the lysosome and a tion of the fusion process. However, BAPTA inhibition of yeast vacuole mechanism for lysosome re-formation/content condensation. Hybrid fusion can be relieved by MgCl2 [93] and this has led to the suggestion organelles both from rat liver and from late endosome–lysosome that in yeast the BAPTA effects may not entirely be due to calcium content mixing assays can be isolated on discontinuous Nycodenz/ chelation but that divalent cations may have a more direct Ficoll gradients and lysosome reformation/content condensation ‘biophysical’ role in the membrane fusion process [94]. Using an examined [38]. It has been shown that content condensation is immobilised phosphatase inhibitor (microcystin LR) as a bait, protein independent of cytosol, but is dependent upon ATP. Furthermore, the phosphatase-1 (PP-1) in yeast, Glc7p, was identiﬁed as being use of baﬁlomycin A1 and EGTA-AM showed that lysosome reforma- important for vacuole fusion [95], and the use of microcystin LR tion is dependent upon lumenal pH and Ca2+ [40]. Evidence for implied that the Glc7p reaction was post SNARE pairing. However, it membrane retrieval from hybrid organelles has come from live-cell has subsequently been shown that microcystin-LR inhibits the vacuole studies where vesicles have been observed budding from hybrid fusion assay but not vacuole fusion itself [96,97], so the substrate of compartments [43,98]. Machinery that may be involved in membrane Glc7p and the point in the vacuole fusion process where it is involved retrieval could be the retromer complex, which mediates retrograde P.R. Pryor, J.P. Luzio / Biochimica et Biophysica Acta 1793 (2009) 615–624 621

Fig. 4. Hypothetical model for regulation of tethering and SNARE pairing. Exchange of RAB5 for RAB7 and binding of RAB7 to RILP on membranes leads to the recruitment of VPS39 and VPS41. VPS39 and VPS41 can bind to Class C VPS subunits on an opposing membrane that are themselves are bound to the SNARE SYNTAXIN7. Activation of RAB7 by the GEF activity of VPS39 leads to changes in protein phosphorylation of both VPS41 and SYNTAXIN7 (based upon data in yeast and changes in phosphorylation of Vps41p and Vam3p) which in turn allows SYNTAXIN7 to form a trans-SNARE complex with VTI1B, SYNTAXIN8 and VAMP7. transport of transmembrane cargo from endosomes to the trans-Golgi and -7 gives phenotypes similar to a subset of human lysosomal network (recently reviewed in [99,100]). Whilst there is little further storage diseases grouped as neuronal ceroid lipofuscinosis (NCL) information on how lysosomes are regenerated from hybrid orga- [114–116]. It has recently been shown that ClC-7 is the major chloride nelles, there is a clear need to understand further the lysosome transporter in lysosomes and the transporter acts as a Cl−/H+ reformation process, since a failure to re-form lysosomes may underlie antiporter [117]. Depletion of ClC-7 by RNA interference strongly some diseases. For instance, mutations in the lysosomal ion channel diminishes the ability of lysosomes to acidify [117]. Together these mucolipin-1 results in the debilitating disease mucolipidosis type-IV data indicate that chloride transport in the endocytic pathway is (MLIV) [101–104], a rare lysosomal storage disease, where unlike critical for normal endocytic functions and membrane dynamics. other lysosomal storage diseases there does not appear to be a defect In yeast, of the 18 proteins encoded by the class E VPS genes, which in lysosomal enzymes but a defect in trafficking of material out of the includes the ESCRT proteins, only one, Vps44p/Nhx1p, is a transmem- hybrid organelle [105–108]. brane protein. Nhx1p shows high homology to mammalin Na+/H+ exchangers of the NHE family and is thought to transport sodium ions 7. Intracellular ions into the yeast prevacuolar compartment in exchange for protons [118]. It is unclear which isoform of NHE is important for transport through As has been mentioned above, fusion between the late endosome the endocytic pathway and whilst no NHE isoform has been found in and the lysosome and lysosome reformation are dependent upon lysosomes, NHE6 has been localised to recycling endosomes and NHE9 lumenal calcium concentrations [40]. Precisely what channels to late recycling endosomes [119]. regulate lumenal calcium are unknown but possible lysosomal calcium channels include mucolipin-1 [106], an NAADP regulated 8. Conclusions calcium channel [109] and the ATP-regulated P2X4 cation channel [110]. Whilst mucolipin-1 does not appear to be the lysosomal There is now a wealth of information on the molecular mechan- receptor for NAADP a recent study has shown that antibodies to isms of delivery of endocytosed molecules to the lysosome. However, mucolipin-1 can inhibit NAADP-induced calcium release from lyso- whilst we have a greater understanding of these mechanisms there is somes suggesting that the NAADP-sensitive calcium release channel is still some way to go to understand precisely how these protein associated with mucolipin-1 [111]. complexes integrate with one another. Additionally, it is clear that the The increased lumenal acidity in the endocytic pathway is mainly proteins regulating protein sorting and membrane fusion are accomplished by V-type H+-ATPases. As these pumps are electro- themselves subject to regulation by phosphorylation/de-phosphor- genic they would create a lumenal-positive voltage in the absence of a ylation (as described above for VPS33B and Vps41p). Indeed, a study neutralising current. A family of 9 chloride channels has been silencing each kinase of the human kinome using siRNA was shown to identified [112], four of which (ClC-3, -4, -5, -6 and -7) reside on the have profound effects on the endocytic pathway [119]. A review of the endocytic pathway (reviewed in [113]). Disruption in mice of ClC-3, -6 role of kinases on regulating the endocytic machinery has recently 622 P.R. Pryor, J.P. Luzio / Biochimica et Biophysica Acta 1793 (2009) 615–624 been published [120] and clearly we still have much to learn on how [19] M. Razi, C.E. Futter, Distinct roles for Tsg101 and Hrs in multivesicular body formation and inward vesiculation, Mol. Biol. Cell 17 (2006) 3469–3483. endocytosis is regulated by phosphorylation and de-phosphorylation [20] A. Doyotte, M.R. Russell, C.R. Hopkins, P.G. Woodman, Depletion of TSG101 forms events. 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