Lysosome Biogenesis and Lysosomal Membrane Proteins: Trafficking Meets Function

focuS on EndocytoREVIEWSSIS

Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function

Paul Saftig* and Judith Klumperman‡ Abstract | Lysosomes are the primary catabolic compartments of eukaryotic cells. They degrade extracellular material that has been internalized by endocytosis and intracellular components that have been sequestered by autophagy. In addition, specialized cells contain lysosome-related organelles that store and secrete proteins for cell-type-specific functions. The functioning of a healthy cell is dependent on the proper targeting of newly synthesized lysosomal proteins. Accumulating evidence suggests that there are multiple lysosomal delivery pathways that together allow the regulated and sequential deposition of lysosomal components. The importance of lysosomal trafficking pathways is emphasized by recent findings that reveal new roles for lysosomal membrane proteins in cellular physiology and in an increasing number of diseases that are characterized by defects in lysosome biogenesis.

Lysosome-related organelle Lysosomes are ubiquitous organelles that constitute the limiting membrane and have diverse functions, including (LRO). A cell-type-specific primary degradative compartments of the cell. They acidification of the lysosomal lumen, protein import from organelle belonging to a family receive their substrates through endocytosis, phago the cytosol, membrane fusion and transport of degrada that includes melanosomes, cytosis or autophagy (FIGS 1,2). The catabolic function of tion products to the cytoplasm8 (FIG. 1). The most abun platelet-dense bodies and lysosomes is complemented by lysosome-related organelles dant LMPs are lysosomeassociated membrane protein 1 cytotoxic T cell granules. LROs contain subsets of lysosomal (LROs), such as melanosomes, lytic granules, major histo (LAMP1), LAMP2, lysosome integral membrane pro proteins in addition to compatibility complex (MHC) class II compartments and tein 2 (LIMP2; also known as SCARB2) and the tetraspanin cell-type-specific proteins. plateletdense granules1. LROs share many properties CD63 (see Supplementary information S1 (table)). with lysosomes, but they also contain celltypespecific Lysosome biogenesis requires integration of the Tetraspanin (FIG. 2) A member of a conserved proteins and might require additional cellular machinery endocytic and biosynthetic pathways of the cell . 2,3 protein family with four for their biogenesis . Lysosomes and LROs are involved Lysosomal targeting of newly synthesized lysosomal pro transmembrane domains and in various physiological processes, such as cholesterol teins can be direct, from the trans-Golgi network (TGN) to two extracellular loops. homeostasis, plasma membrane repair, bone and tissue the endosomal system, or indirect, involving transport Tetraspanins act as scaffolding remodelling, pathogen defence, cell death and cell signal to the plasma membrane and subsequent endocytosis. The proteins, anchoring multiple (FIG. 1) proteins to a specific area at ling . These complex functions make the lysosome best understood direct pathway is the mannose6phosphate the plasma membrane. a central and dynamic organelle and not simply the dead receptor (M6PR)mediated transport of lysosomal hydro end of the endocytic pathway. lases9,10. By contrast, remarkably little is known about the

*Department of Two classes of proteins are essential for the function of structural and molecular machinery for the transport Biochemistry, Christian- lysosomes: soluble lysosomal hydrolases (also referred to of LMPs to lysosomes. The significance of tightly regu Albrechts University, as acid hydrolases) and integral lysosomal membrane pro lated LMP trafficking is illustrated by recent findings that Kiel, D-20498 Germany. teins (LMPs). Each of the 50 known lysosomal hydrolases describe new and unexpected roles for LMPs in cellular ‡Department of Cell Biology, University Medical Centre targets specific substrates for degradation, and their col physiology. It is becoming apparent that LMPs can impose Utrecht, 3584CX Utrecht, lective action is responsible for the total catabolic capac specific functions onto the organelles through which The Netherlands. ity of the lysosome. In addition to bulk degradation and they are transported or in which they reside, such as the e-mails: J.Klumperman@ proprotein processing, lysosomal hydrolases are involved endoplasmic reticulum (ER), lysosomes and the plasma umcutrecht.nl; in antigen processing, degradation of the extracellular membrane. Their importance is further highlighted by [email protected] 4 doi:10.1038/nrm2745 matrix and initiation of apoptosis . The mammalian the discovery of an increasing number of gene mutations 5 11 Published online lysosome contains ~25 LMPs , but additional LMPs are that lead to lysosomal dysfunction and disease (TABLE 1). 12 August 2009 being revealed5–7. LMPs reside mainly in the lysosomal In addition, various knockout mice and nonmammalian

Macroautophagy Isolation Autophagosome membrane Chaperone-mediated Autolysosome autophagy Macroparticle + Degraded Phagocytosis H protein ADP+Pi V-type Cytosolic protein ATP H+-ATPase H+ Lysosomal exocytosis and Phagosome plasma membrane repair LAMP CLC7 + + H Cl– H+ H + H+ Lysosome H+ H + + + H + H H H+ H LIMP2 SYT7 Phagolysosome CD63 NPC1 Cathepsin MHC class II Lysosomal cell death compartment H+ MHC class II Cholesterol Exosome release Amino acids Cholesterol homeostasis Plasma and hexoses membrane MHC class II-dependent antigen presentation Antigenic peptide Peptide Figure 1 | Major functions of lysosomal membrane proteins. The lysosome is a central, acidic organelle that is involved in the degradation of macromolecules through the activity of lysosomal hydrolases. LysosomesNature Re viearews crucial | Mol ecularfor the Ce ll Biology maturation of phagosomes to phagolysosomes in phagocytosis, which is important for cellular pathogen defence. The macroautophagy pathway mediates the turnover of cytoplasmic components, such as organelles and large complexes, and is involved in cell death and proliferation. Macroautophagy depends on the fusion of lysosomes with autophagosomes to create autolysosomes, in which degradation occurs. Macroautophagy and chaperone-mediated autophagy, a direct lysosomal transport process for cytosolic proteins, are regulated by lysosome-associated membrane proteins (LAMPs). Lysosomal exocytosis and plasma membrane repair are Ca2+ and synaptotagmin 7 (SYT7)-dependent fusion events, which are possibly involved in pathogen entry, autoimmunity and neurite outgrowth. The lysosomal cell death pathway is triggered by a release of lysosomal cathepsins through an unknown mechanism. Cellular cholesterol homeostasis is controlled by lysosomal cholesterol efflux through Niemann–Pick C1 protein (NPC1). Major histocompatibility complex (MHC) class II-dependent antigen presentation requires lysosomal proteases and membrane proteins. The release of exosomes is thought to be involved in adaptive immune responses. Lysosomal membrane proteins are also involved in the transport of newly synthesized hydrolases to the lysosome (for example, lysosomal integral membrane protein 2 (LIMP2)) and across the lysosomal membrane (for example, the V-type H+-ATPase complex and chloride channel protein 7 (CLC7)) .

model organisms have highlighted the role of LMPs in cell through vesicular transport carriers, tubular connections Trans-Golgi network 18–20 (TGN). A convoluted membrane physiology (see Supplementary information S1 (table)). and kissandrun fusion events . compartment at the trans side Here, we give an overview of the cellular pathways The widely used distinction between early endosomes of the Golgi complex that involved in lysosome biogenesis, with a focus on the bio (EEs) and late endosomes (LEs)12 is based on functional mediates sorting and transport synthetic pathways that are independent of M6PR func and biological characteristics, but oversimplifies the com of proteins to various cellular destinations. tioning. In addition, we discuss the putative and emerging plexity of the endocytic pathway. This was exempli fied roles of LMPs in the transport of proteins and organelles by a recent immunoelectron microscopy (IEM) study Rab protein and the consequences of their impaired trafficking for linking the molecular makeup of endosomes with their A member of a family of small human health. ultrastructural characteristics21. Distinct EE marker GTPases that, when associated proteins showed different distributions, ranging from a with the cytosolic leaflet of the endosomal limiting membrane, Endocytic pathways to the lysosome restricted localization on earlystage EEs (for example, can initiate the formation of The degradative endocytic pathway starts at the plasma early endosome antigen 1 (EEA1)) to a more widespread functional microdomains. membrane and ends in lysosomes. Between these two distribution on other EEs and on earlystage LEs (for ‘stations’, endocytosed cargo passes through a range of example, the Rab proteins RAB11 and RAB4, and HRS (also HRS (FIG. 2) (Hepatocyte growth endosomal intermediates that are distinguished known as ESCRT0)). These observations indicate that factor-regulated tyrosine by their content, molecular makeup, morphology and functionally different intermediates of EEs and LEs can kinase substrate; also known pH and by the kinetics by which endocytic tracers reach be distinguished and that the transition between EEs and as ESCRT0). A cytosolic protein them12 (TABLE 2). A constant exchange of incoming and LEs is gradual. The endocytic pathway is therefore best that is involved in the out going membranes and multiple fusion events result in regarded as a spatiotemporal continuum of inter mediates, recognition of ubiquitylated cargo at endosomes, which the gradual remodelling of an endosomal intermediate which continuously exchange their content while under 13–16 initiates the recruitment of the into a laterstage endosome , a process called matu going gradual molecular and structural remodelling and ESCRT complex. ration17. In addition, endosomes can exchange content functional transformation (TABLE 2).

Plasma membrane Rapid Endocytosis recycling pathway Constitutive Tubular sorting Bilayered clathrin coat secretory endosome Vacuolar sorting pathway endosome ILV Early ETC endosomes

Tubular sorting

Perinuclear y a endosome endosomal w th recycling a Clathrin p compartment R 6P Multivesicular body M (late endosome intermediate) TGN

Golgi

Ribosome Lysosome ER

Figure 2 | Possible interactions between the biosynthetic and endocytic pathways. Lysosome biogenesis requires the concerted involvement of biosynthetic and endocytic pathways. Lysosomes receive cargoNatur efor Re degradationviews | Molecular as well Ce asll Biolognewlyy synthesized lysosomal proteins by the endocytic pathway (green arrows). Lysosomal proteins are synthesized in the endoplasmic reticulum (ER) and transported through the Golgi complex to the trans-Golgi network (TGN). From the TGN, they can follow the constitutive secretory pathway (blue arrows) to the plasma membrane and subsequently reach lysosomes by endocytosis. In addition, lysosomal proteins can follow a direct intracellular pathway (red arrows) to the endo-lysosomal system. The best-characterized direct intracellular pathway is the clathrin-dependent transport of lysosomal hydrolases by mannose-6-phosphate receptors (M6PRs). The available literature suggests that there are multiple pathways for both lysosomal hydrolases and lysosomal membrane proteins (for example, lysosomal integral membrane protein 2-mediated transport of β-glucocerebrosidase), which may enter the endo-lysosomal pathways at distinct stages of maturation (grey arrows). The black arrows represent multiple retrograde pathways from endosomes. For more information on the molecular machinery involved in these pathways, see the main text. ETC, endosome-to-TGN carrier; ILV, intraluminal vesicle.

Sorting events in early endocytic compartments. EEs to be independent of clathrin, but requires the retromer (REFS 21,31–36) Clathrin are the main recipients of incoming endocytic vesicles subunit sorting nexin 1 (SNX1) and/or SNX2 . A protein that forms a coat from the plasma membrane. They receive cargo that Association of the retromer with EEs is regulated by the which has a major role in the cycles back to the plasma membrane, as well as material sequential action of RAB5 and RAB7 (REF. 37), which formation of transport vesicles. that is transported further along the endocytic pathway. are exchanged during the maturation of EEs to LEs38. The coat consists of multiple In addition, EEs receive endogenous proteins from the Interference with RAB7 function causes dissociation of the triskelions, which are 22,23 composed of three clathrin TGN, such as M6PRs carrying lysosomal hydrolases retromer vacuolar protein sortingassociated protein 26 heavy chains and three light (FIG. 2; TABLE 2). The main function of EEs is to sort this (vPS26)–vPS29–vPS35 trimer and also interferes with chains. cargo for recycling or degradation. EEs contain a vacu M6PR recycling. Notably, the retromer component vPS26 olar part (also referred to as the sorting endosome) from colocalizes with clathrin on TSEs39,40, suggesting that there Retromer 24,25 A heterotetrameric protein which a reticulum of multibranching tubules emerges are two retro merdependent pathways for EEtoTGN 26,27 complex that associates with (referred to as the tubular sorting endosome (TSE) recycling of M6PRs: through endosometoTGN carriers the cytosolic leaflet of the or tubular endosomal network)27,28 (FIG. 2). Generally, directly from the EE vacuole21 and from TSEs39, presumably endosomal limiting membrane. cargo destined for lysosomes remains in the EE vacuole, in a clathrindependent manner (FIG. 2). The mammalian retromer whereas cargo to be recycled enters the TSE. Proteins A TSE can contain proteins for recycling to the plasma consists of a sorting nexin dimer composed of a without a specific targeting signal enter the TSE by membrane or to the TGN, as well as low levels of LMPs 29,30 still-undefined combination default . In the EE vacuole, a few intraluminal vesicles (for example, LAMP1, LAMP2 and CD63) that are des of SNX1, SNX2, SNX5 or (ILvs) are present that form by inward budding of the EE tined for the lysosome26. TSEs have multiple membrane SNX6 and the cargo limiting membrane. buds that can contain the adaptor complex proteins AP1 recognition trimer 26 VPS26–VPS29–VPS35. In the mildly acidic environment of EEs, lysosomal and AP3, with or without the association of clathrin . hydrolases dissociate from M6PRs and remain in the AP1 has been implicated in the recycling of M6PRs to the AP1 endosomal lumen, and the M6PRs return to the TGN for TGN41. AP3 is required for the efficient transport of LMPs A member of the other rounds of transport. M6PRs can enter specialized from TSEs to lysosomes and LROs26,42. The importance of heterotetrameric family of recycling carriers — the endosometoTGN carriers that this pathway is illustrated by the pigmentation and bleed adaptor proteins involved in 21 (FIG. 2) membrane trafficking, which form directly from endosomal vacuoles — or can ing disorder Hermansky–Pudlak syndrome 2, in which 28 also includes AP2, AP3 and enter the TSEs, where additional recycling carriers exit . patients lack a functional AP3 complex, and this results AP4. Recycling of M6PRs by endosometoTGN carriers seems in a redistribution of LMPs to the plasma membrane and

Table 1 | Human diseases caused by mutations in genes that encode lysosomal membrane proteins* Disease clinical pathology lMP TM helices Presumed function Action myoclonus-renal Autosomal recessive progressive myoclonic LIMP2 (also 2 Transport of βGC to lysosomes, failure syndrome epilepsy associated with renal failure known as lysosomal biogenesis and sorting of SCARB2)119,120 vesicles to apical membranes Cobalmin F-type disease Developmental delay, stomatitis, glossitis, LMBRD1 9 Lysosomal export of cobalmin seizures and minimal methylmalonic aciduria Cystinosis Pathology of kidney, eye, liver, muscles, Cystinosin 7 Lysosomal H+ and l-cysteine symport pancreas, brain and white blood cells, as well as diabetes, hypothyroidism and end-stage kidney failure Danon disease X-linked vacuolar cardiomyopathy, LAMP2 (REF. 156) 1 Chaperone-mediated autophagy, myopathy and variable mental retardation macroautophagy, lysosomal fusion and motility Niemann–Pick type C Hepatosplenomegaly, thrombocytopenia, NPC1 13 Lysosomal lipid and cholesterol export ataxia, dysarthria, dysphagia, dystonia, dementia and seizures Mucolipidosis type IV Autosomal recessive genetic disorder with MCOLN1 6 Lysosomal cation (Na+, K+, Ca2+, Fe2+ and delayed psychomotor development and (REF. 126) H+) channel ocular aberrations Mucopolysaccharidosis Progressive dementia, mental deterioration HGSNAT 11 Transfer of cytosolic acetyl-CoA to type IIIC in childhood, hyperactivity, sleep disorders luminal α-glucosamine residues of and loss of speech heparan sulphate Malignant infantile Anaemia, thrombocytopenia, CLC7 18 H+ and anion (Cl–) antiport osteopetrosis granulo-cytopenia, blindness, deafness, fractures and infections OSTM1 1 CLC7 subunit Neuronal ceroid Ataxia and seizures with rapid mental CLN7 (also 12 Transport of sugars, drugs, inorganic lipofuscinosis (late deterioration known as MFSD8) and organic cations and other infantile) metabolites Neuronal ceroid Early progressive vision loss, seizures and CLN3 6 Membrane transport lipofuscinosis (juvenile) ataxia or clumsiness Salla disease Nystagmus, hypotonia, cognitive Sialin 12 H+ and sugar symport (resulting in the impairment and reduced muscle tone and export of sialic acids and acidic hexoses) strength and asparagine and glutamine import *A more detailed description and additional primary references for the different diseases can be found in the main text and in a recent review by Ruivo et al.11. βGC, β-glucocerebrosidase; CLC7, Cl– channel protein 7; CLN, ceroid-lipofuscinosis neuronal protein; HGSNAT, heparan-α glucosaminide N-acetyltransferase; LAMP2, lysosome-associated membrane protein 2; LIMP2, lysosome integral membrane protein 2; LMBRD1, Limb region 1 domain-containing protein 1; LMP, lysosomal membrane protein; MCOLN1, mucolipin 1; NPC1, Niemann–Pick C1 protein; OSTM1, osteopetrosis-associated transmembrane protein 1; TM, transmembrane.

impaired biogenesis, particularly of melanosomes and contains concentrated levels of HRS, which binds to plateletdense granules43–45. Therefore, following the initial both ubiquitylated cargo and clathrin, providing a link sorting in EE vacuoles, TSEs further sort proteins to the between cargo selection and coat formation55,56,58. At the plasma membrane, TGN or lysosomes. The perinuclear edges of the coat, small membrane invaginations bud endosomal recycling compartment46 can be thought of off into the endosomal lumen to generate the ILvs59. as a subdomain of the TSE28 or as a separate compart Notably, however, impairment of ILv formation by ment that emanates from the TSE after the removal of the phosphoinositide 3kinase (PI3k) inhibitor wort proteins that are targeted for destinations other than the mannin60 or ablation of clathrin by RNA interference61 plasma membrane26,27. does not abolish EGFR degradation in lysosomes, sug gesting that there are additional endosomal retention Sorting events in late endosomal compartments. Cargo mechanisms. 62 ESCRT proteins that are sorted into ILvs thereby remain in the CD63 and other tetraspanins are efficiently sorted (Endosomal sorting complexes endosomal lumen and are routed for lysosomal degrad into ILvs (FIG. 3). In LEs, this leads to a ~7fold enrich required for transport). ation. The ESCRT machinery has been identified as an ment of CD63 in the ILvs relative to the endosomal lim Endosomal sorting machinery 63 consisting of four complexes — important regulator for ubiquitindependent cargo sort iting membrane . Interestingly, in oligodendroglial cells, 47–52 ESCRT0, ESCRTI, ESCRTII and ing into ILvs as well as for ILv formation . A well which secrete CD63positive exosomes, CD63 sorting into ESCRTIII — plus several established cargo for the ESCRT pathway is epidermal exosomes and into their ILv precursors was found to be accessory components. ESCRT growth factor receptor (EGFR)53–56. Prior to sorting into independent of the ESCRT machinery, but required the components recognize ILvs, EGFR and growth hormone receptor are concen sphingolipid ceramide64. Recent studies in dendritic cells ubiquitylated cargoes, deform the endosomal membrane and trated in a subdomain of the limiting membrane of the revealed that the mechanism by which the LMP MHC 55,57 catalyse the formation of ILVs EE vacuole that bears a clathrin coat comprising two class II is sorted into ILvs varies with the activation state containing the sorted cargo. layers57 — a bilayered coat (FIG. 2). The bilayered coat of the cells. In immature dendritic cells, endocytosis

Table 2 | Properties of endo-lysosomal intermediates early endosomes late endosomes lysosomes Functions in • Receive incoming endocytic vesicles • Receive newly synthesized • Receive cargo from late endosomes transport and • Sort proteins for recycling from endo-lysosomal proteins from the TGN sorting proteins for degradation • Recycle proteins to the TGN • Receive newly synthesized • Continue the formation of ILVs and endo-lysosomal proteins from the TGN sort proteins between the limiting • Start the formation of ILVs membrane and ILVs cargo present • High levels of endocytosed recycling • Low levels of endocytosed recycling • Endocytosed proteins destined for proteins (for example, TFR and ASGPR) proteins degradation • Endocytosed proteins destined for • Concentrated levels of endocytosed • High levels of LMPs degradation (for example, EGFR and proteins destined for degradation • High levels of lysosomal hydrolases GHR) • Proteins that recycle to the TGN • Endocytic tracer arrives after 30 min • Proteins that recycle to the TGN (for • Newly synthesized LMPs en route to example, M6PRs and sortilin) lysosomes • Low levels of lysosomal hydrolases • Elevated levels of lysosomal hydrolases • Low levels of newly synthesized LMPs • Endocytic tracer arrives after 10–15 en route to lysosomes (for example, min LAMP1 and LAMP2) • Endocytic tracer arrives after 1–5 min Morphology • Electron-lucent vacuole lined with a • Globular vacuole lined with a • Heterogenously shaped vacuole bilayer clathrin coat and attached to an bilayer clathrin coat and with a few (often globular but can be tubular) elaborate network of electron-dense electron-dense tubules attached that has an electron-dense lumen with tubules (the tubular sorting endosome) • Contain >9 ILVs irregular content and often membrane • Contain 0–8 ILVs sheets • Some ILVs can be present pH ~6 5–6 4.5–5 Main interacting Plasma membrane, recycling endosomes TGN, late endosomes, lysosomes and Late endosomes, plasma membrane, compartments and late endosomes plasma membrane autophagosomes and phagosomes ASGPR, asialoglycoprotein receptor; EGFR, epidermal growth factor receptor; GHR, growth hormone receptor; ILV, intraluminal vesicle; LAMP, lysosome-associated membrane glycoprotein; LMP, lysosomal membrane protein; M6PR, mannose-6-phosphate receptor; TFR, transferrin receptor; TGN, trans-Golgi network.

of MHC class II from the plasma membrane as well as LEs form fewer and less extensive tubular extensions sorting of MHC class II into ILvs requires ubiquitylation than EEs and communicate with the TGN, other LEs and leads to lysosomal degradation. Maturation of the cells and lysosomes rather than with the plasma membrane20. leads to a decrease in the ubiquitylation of MHC class II In addition to the retromerdependent pathways for and an increase in the plasma membrane levels of MHC retrograde transport of M6PRs from the EE vacuoles class II65,66. In these maturated cells, endosomeassociated and TSEs, other transport proteins have been implicated MHC class II is still sorted into ILvs, not for degradation in M6PR retrieval from endosomes74: RAB9 (REF. 75), but for secretion by exosomes (FIG. 1). These exosomes the target (t)SNARE syntaxin 16 (REF. 76), phosphofurin also contain the tetraspanin CD9 (REF. 67), which had pre acidic cluster sorting protein 1 (PACS1)77, EpsinR (also viously been shown to be enriched on exosomes together known as clathrininteractor 1)78, the recently identi with CD63 (REFS 62,68). Because sorting to ILvs and fied GARP complex79 and tailinteracting protein of 75 Exosome exosomes in these conditions seems to be independent 47 kD (TIP47; also known as M6PRBP1) , although A small vesicle that initially of ubiquitin, an ESCRTindependent mechanism of sort the functioning of TIP47 protein in M6PR trafficking exists as an ILV in the lumen of ing tetraspanins into ILvs is implicated69. The possible was recently debated80. It is not clear how these proteins LEs or MVBs. They are called exosomes once released, involvement of other factors in CD63 transport, as well relate to each other and where they act in the endosomal following fusion of LEs and as the emerging roles of CD63 as a transport regulator of system. Because TSEs exhibit AP1 and clathrincoated MVBs with the plasma other proteins and as a determinant of kidney physiol buds26 and PACS1 and EpsinR can interact with AP1– membrane. Exosomes are ogy, was recently described62,70 and will not be discussed clathrin, these pathways might be active in TSEs. RAB9 thought to have key roles in further here. In contrast to CD63, LAMP1, LAMP2 and mainly mediates M6PR recycling from LEs81, suggesting antigen presentation, cell-to-cell communication, LIMP2 are predominantly localized at the endosomal that there are multiple retrograde pathways for M6PR 63,71 the pathogenesis of retroviral limiting membrane (FIG. 3), but their relative intra recycling at different places in the endocytic system, infections and prion disease. endosomal distribution can vary (FIG. 3). These observa including both EEs and LEs. tions indicate that the intraendosomal distributions of The sorting events in LEs eventually result in an Dendritic cell A potent antigen-presenting LMPs are subject to regulation, which might have major increase in the degradative capacity of these compart 38 cell that is part of the implications for their functioning. ments , as well as an increased ability for homotypic fusion mammalian immune system. LE intermediates13,38, often referred to as multivesicu with other LEs and heterotypic fusion with preexisting Its main function is to process lar bodies (MvBs), are globular vacuoles with numerous lysosomes15,20,69,82–84. Lysosomes are generally defined as antigen material and present it, ILvs (TABLE 2). LEs no longer contain significant amounts organelles of heterogeneous size and content that are in the context of the MHC class 12,15 II complex, on its surface to of cargo for recycling to the plasma membrane, but instead LMPrich but lack M6PRs and have a pH below five other cells of the immune have elevated levels of proteins that are destined for lyso (TABLE 2). The specific functional properties of lysosomes system. somes and substantial levels of lysosomal hydrolases72,73. are discussed below.

a LAMP1 c LIMP2–GFP and βGC The best understood pathways are those for the delivery of lysosomal hydrolases.

M6PR-dependent transport. Most lysosomal hydro lases acquire an M6P tag during transport through the Golgi complex85, which is recognized by M6PRs in LE LYS LE/LYS the TGN9,10. There are two types of M6PR, 300 kD cationindependent M6PR (CIM6PR; also known as IGF2R) and 46 kD cationdependent M6PR ILV (CDM6PR), both of which are ubiquitously expressed. The M6PR pathway is the major pathway for the lyso somal targeting of lysosomal hydrolases and is fairly b LAMP1 well understood. A recent review74 effectively summa rizes our know ledge of M6PRdependent transport and we refer to this paper for detailed reading. In the TGN, M6PRs bind to AP1 and/or to GGA proteins86–91, result LE/LYS ing in the formation of 60–100nm clathrincoated vesi LE/LYS cles22,27,89,92. In addition, livecell imaging has revealed ILV the existence of larger, pleiomorphic carriers that are positive for GGA1, AP1, clathrin or the cytosolic tail of CIM6PR23,91,93 that originate from the TGN and move towards the cell periphery23,94. It is not known N exactly where in the endocytic pathway the M6PRs deliver their bound ligands, but livecell imaging23 as d CD63 well as the relatively equal distribution of M6PRs on EEs and LEs22 suggests that EEs are the predominant site for entry. Most M6PRs travel through this direct, clathrindependent pathway to endosomes. It is not yet clear whether GGA proteins and AP1 generate distinct types of M6PR carriers, or whether GGA proteins facil LYS itate the entry of M6PR into clathrincoated vesicles by LE/LYS interacting with AP1 (REFS 89,90).

M6PR-independent transport. In patients with Icell ILV disease (also known as mucolipidosis type II), lyso somal hydrolases do not acquire M6P tags because of a deficiency in Nacetylglucosamine (GlcNAc) 95–97 Figure 3 | intra-endosomal distribution of lysosomal membrane proteins. Electron phosphotransferase activity . Nevertheless, in some micrographs of ultrathin cryosections immunogoldNa labelledture Re forviews the | Molindicatedecular Celysosomalll Biology Icelldiseased cells, such as hepatocytes, kupffer cells membrane proteins. a | Endogenous lysosome-associated membrane protein 1 (LAMP1) and lymphocytes, a significant portion of newly synthe localization in human HepG2 cells. LAMP1 (labelled by 10 nm gold particles) is sized lysosomal hydrolases do reach the lysosome96,98,99. predominantly found at the limiting membrane of the lysosome (LYS). b | Endogenous Similar observations were made in GlcNAc phospho LAMP1 localization in human HepG2 cells can vary between distinct endo-lysosomal transferaseknockout mice, a model system for Icell intermediates. The thin arrow points to an inward-budding vesicle of the late endosome disease100, and in mice deficient for both CIM6PR (LE) or lysosome. The nucleus (N) is indicated. c | Lysosomal integral membrane protein 2 and CDM6PR, inferring a complete ablation of the (LIMP2)-deficient fibroblasts co-transfected with LIMP2–green fluorescent protein (GFP) M6PR pathway101. The M6PRindependent pathways and -glucocerebrosidase ( GC). LIMP2 (labelled by 15 nm gold particles) is β β of lysosomal hydrolase transport are mostly unknown, predominantly found in the endo-lysosomal limiting membrane, whereas βGC (labelled by 10 nm gold particles) is found associated with the limiting membrane as well as in the with the exception of βglucocerebrosidase (βGC) endo-lysosomal lumen. d | Endogenous CD63 localization in human HepG2 cells. CD63 transport (FIG. 4). In the absence of a functional M6PR (labelled by 10 nm gold particles) is almost exclusively found in the endo-lysosomal pathway, newly synthesized lysosomal hydrolases can lumen, where it associates with intraluminal vesicles (ILVs). Scale bars represent 200 nm. follow the constitutive secretory pathway to the plasma Thick arrows point to BSA-coated 5 nm gold particles that were internalized for 3 hours. membrane and after secretion might be taken up by fluidphase endocytosis. The multiligandbinding mannose receptor that is preferentially expressed on Direct transport of lysosomal hydrolases subsets of dentritic cells, on liver sinusoidal endo In addition to the endocytic pathway, lysosome bio thelial cells and on tissue macrophages binds lysosomal genesis requires input from the biosynthetic pathway hydrolases102 and could mediate endocytosis in these for the delivery of newly synthesized lysosomal pro cell types103. Mannose receptormediated uptake is also teins. The complexity of the endosomal system allows important for the successful delivery of some recom for multiple sites at which the biosynthetic pathway binant enzymes for the treatment of lysosomal storage can intersect with endosomal intermediates (FIG. 2). disorders.

The VPS10 receptor family. Recently, members of the still unresolved in βGC119. It is not yet known whether mammalian vacuolar protein sorting (vPS10) domain LIMP2 only carries βGC or if it also sorts other lyso containing protein family of receptors have emerged as somal hydrolases. The observations that βGC and LIMP2 candidates for mediating direct transport of lysosomal associate, that they colocalize in lysosomes (FIGS 3,4) hydrolases to the lysosome104. vPS10 domaincontaining and that the activity, levels and localization of βGC proteins share homology with the luminal part of yeast correlate with the expression of LIMP2 imply that M6PR vps10, which mediates sorting of the soluble hydrolase independent sorting of βGC requires βGC to bind to carboxypeptidase Y to the vacuole105. In mammals, the LIMP2 (REF. 117). Furthermore, these data suggest that vPS10 domaincontaining family presently consists of the transport of LMPs can be linked to transport path the multiligand receptors sortilin, SorLA (also known ways of lysosomal hydrolases. Interestingly, deletion of as sortilinrelated receptor) and SORCS1–SORCS3 the Cterminal tail of LIMP2 did not affect its lysosomal (REFS 106,107). Sortilin is likely to be responsible for the localization. Moreover, the steadystate localization of direct lysosomal targeting of the acid sphingomyelinase LIMP2 and βGC remained unaltered in AP3deficient and sphingolipid activator proteins (SAPs)108,109, which mocha cells, suggesting that there are additional sort are nonenzymatic cofactors that are required for glyco ing motifs in the transmembrane or luminal domain of sphingolipid degradation in lysosomes110. In addition, LIMP2 that do not involve interactions with AP3. a substantial portion of the SAP precursor protein pro Transport of βGC is probably not the only function saposin is secreted, after which reuptake can be car of LIMP2. Overexpression of LIMP2 leads to enlarged ried out by the lowdensity lipoprotein receptorrelated EEs, LEs and lysosomes with impaired membrane proteins, the M6PRs and, as seen in macrophages, the trafficking out of the endolysosomal compartments, mannose receptors111. accumulation of free cholesterol in the limiting mem The carboxyl terminus of sortilin contains AP1 and branes and impaired transferrin receptor recycling120. GGAbinding motifs that are essential for trafficking. As In mice, LIMP2 deficiency causes ureteric pelvic junc shown by IEM, sortilin colocalizes with CIM6PR in the tion obstruction, peripheral neuropathy and deafness118, TGN21,112,113, indicating that these nonrelated proteins which is probably associated with impaired apical traf are transported by the same clathrindependent path ficking and distribution of k+ channels and megalin, way to the endolysosomal system. Moreover, sortilin a lowdensity lipoproteinlike endocytic receptor121. and CIM6PR share a retromerdependent pathway that Recently, LIMP2null mutations were found in patients recycles them to the TGN21,39,114. Interestingly, antibodies suffering from action myoclonusrenal failure syndrome to the lysosomal hydrolases cathepsin D and cathepsin H (AMRF)122,123, a severe, autosomalrecessive multisystem can coimmunoprecipitate sortilin115, but the implica disorder. It still needs to be determined whether possible tion of this finding and a role for sortilin in lysosomal truncated forms of LIMP2 are associated with the disease hydrolase sorting remain to be established. The crystal and to what extent they can bind βGC. Patient fibroblasts structure of human sortilin in complex with neurotensin show a severe enzymatic deficiency of βGC, whereas the revealed that sortilin binds its ligand in a tunnel formed βGC activity in patient leukocytes seems unaffected, by a βpropeller domain116. Hence, sortilin — similar to suggesting an additional βGC targeting mechanism123. LIMP2 (see below) — binds its ligands through proteina Finally, LIMP2 was shown to have an extralysosomal ceous interactions that, unlike the M6PR pathway, do not role in intercalated discs of cardiac myocytes124. require glycosylation. TGN sorting of lysosomal membrane proteins LIMP2-dependent transport of βGC. unlike most solu The studies on LIMP2 and βGC underline the central role ble lysosomal hydrolases, βGC does not obtain an M6P of LMPs in lysosome biogenesis and lysosome function SNARE tag, and in Icelldiseased cells or in cells lacking M6PR ing, and show that the lysosomal targeting pathways of (Soluble N-ethylmaleimide- βGC is normally transported to lysosomes (FIG. 4). For an LMP and a soluble lysosomal hydrolase can overlap. sensitive factor (NSF) attachment protein (SNAP) a long time it was not understood how βGC, of which Our general understanding of how LMPs are sorted to receptor). A member of a mutation causes the most common lysosomal stor lysosomes and other cellular compartments is still in its family of membrane-tethered age disorder, Type I Gaucher disease, is transported to infancy, but accumulating evidence suggests that multi coiled-coil proteins that lysosomes. In a recent study, LIMP2 was unexpectedly ple pathways might exist that can be independent of regulate fusion reactions and found to be a selective and specific binding partner for clathrin. target specificity in the 117 vacuolar system. They can be βGC . LIMP2 is a heavily glycosylated LMP that spans divided into vesicle (v)-SNAREs the membrane twice, with both the N and C termini Direct and indirect transport pathways. A substantial and target (t)-SNAREs on the facing the cytosol (FIG. 1) and with a putative sorting fraction of the LMPs that exit the TGN travel to the basis of their membrane signal in the C terminus (see Supplementary informa plasma membrane along the default secretory pathway localization. tion S1 (table)). Binding between LIMP2 and βGC is and subsequently reach lysosomes through the endocytic 125 GGA protein pH dependent, enabling these proteins to associate in pathway . In addition, LMPs can travel directly from (Golgi-localized, the ER and all the way to the lysosome, where they dis the TGN to the endolysosomal pathway. Increasing γ-ear-containing, Arf-binding sociate because of the acidic pH. In mice lacking LIMP2 evidence suggests that there are multiple TGN exits for protein). A member of a family (REF. 118), βGC is no longer sorted to lysosomes but is LMPs; this would allow for a targeted delivery to defined of monomeric adaptor (FIG. 4) proteins. In mammals there are instead secreted . The site of interaction between endosomal intermediates, including LEs (J.k., unpub three different GGA proteins: βGC and LIMP2 was mapped to a highly conserved lished observations). The relative contributions of each GGA1, GGA2 and GGA3. coiledcoil motif in the luminal loop of LIMP2, but is transport pathway might differ depending on the cell

a Wild-type cell type, the LMP26,125–127 and its expression level128 and the TGN cellular conditions. A particular LMP can contain sort Golgi ing motifs for distinct trafficking pathways. For example, ER the LMP mucolipin 1 (MCOLN1) is a putative ion chan nel with six predicted transmembrane domains that is defective in the lysosomal storage disorder mucolipidosis Lysosome type Iv129 (TABLE 1; see Supplementary information S1 (table)). A chimeric protein containing the Cterminal tail Nucleus of MCOLN1, fused to the luminal and transmembrane domains of the plasma membrane glycoprotein TAC, is transported to the plasma membrane and subsequently endocytosed. By contrast, a chimaera containing the Nterminal tail of MCOLN1 fused to TAC travels directly from the TGN to the endolysosomal system130.

Multiple TGN exits. Many LMPs contain dileucine or tyrosinebased sorting motifs131 (see Supplementary b M6PR-deficient cell information S1 (table)), which equip them to use the TGN AP1–clathrin and GGA–clathrindependent exits from Golgi the TGN. Indeed, LAMP1 was found in AP1–clathrin ER positive TGN membranes by both biochemical and IEM studies125,132,133. In addition, TGN exit and efficient lysosomal targeting of the LMP lysosomalassociated Lysosome protein transmembrane 5 (LAPTM5), which is specifically Nucleus expressed in haematopoietic cells, requires GGA3, the same component involved in the exit of M6PRs from the TGN134. However, in vitro studies indicated that LAMPs can be sorted into noncoated TGNderived vesicles, which are different from those containing the M6PRs135. Moreover, in mice lacking a functional AP1 complex, LAMP1 is still found in lysosomes without increased transport through the plasma membrane41, and when AP1 or clathrin is depleted from HeLa cells125 LAMP1 c LIMP2-deficient cell still travels to lysosomes. The transport of MHC class II to MHC class II compartments also seems to involve TGN sorting into noncoated TGNderived vesicles136 (FIG. 1). Golgi Biochemical and IEM studies in B lymphoblasts revealed ER that MHC class II enters TGN exit carriers that are devoid of AP1, clathrin and CDM6PR137. Together, these data Lysosome indicate that LMPs can exit the TGN in AP1–clathrin coated vesicles, but that additional TGN exit carriers must Nucleus exist for direct TGNtolysosome transport. Lysosomal targeting of multiprotein complexes, such as ion channels, exhibits an additional level of complexity in that different subunits may be targeted independ ently. A recent study showed that a mutation in the gene encoding vMA21, an assembly chaperone of the vtype H+ ATPase complex, causes Xlinked myopathy with excessive autophagy (XMEA). The vtype H+ ATPase Lysosomal hydrolase βGC complex is required for the import of protons into the M6P M6PR LIMP2 lysosomal lumen. In patients carrying a mutation in vMA21, only reduced levels of the vtype H+ ATPase Figure 4 | M6Pr-dependent and -independent targeting of lysosomal hydrolases complex reach the lysosomes. This in turn results in a to the lysosome. a | In wild-type cells, most lysosomalNatur hydrolasese Reviews are| Mol transportedecular Cell Biolog to y general increase in endolysosomal pH and a subsequent lysosomes by mannose-6-phosphate receptors (M6PRs) that bind their ligands in the decrease of autophagic degradation, resulting in the accu trans-Golgi network (TGN). -Glucocerebrosidase ( GC), however, does not follow β β autophagosomes the classical M6PR pathway but instead binds to lysosomal integral membrane protein 2 mulation of autolysosomes and . This (LIMP2) in the endoplasmic reticulum (ER), which directs it to the lysosome. b | In cells study illustrates the importance of tightly linked regula 138 that lack M6PRs (or in I-cell-diseased cells, which lack the phosphotransferase necessary tion of LMP assembly and transport . Other examples to make the M6P tag), most lysosomal hydrolases are secreted, but βGC continues to be of ion and protein transport over the lysosomal limiting targeted to the lysosomes. c | In LIMP2-deficient cells, most lysosomal acid hydrolases are membrane through channels or by transporters include delivered to lysosomes, whereas βGC is secreted. chloride transport through chloride channel protein 7

(CLC7), cystine transport through cystinosin, sialic significantly alter the functional properties of the underly acid transport through sialin, cobalamin transfer by the ing compartment or transport intermediate. This empha cobalamin transporter and cholesterol and lipid trans sizes the need to understand how LMPs are sorted to port through Niemann–Pick C1 protein (NPC1)11,139 (see various cellular compartments, and how their intracellular Supplementary information S1 (table)). Mutations in the transport and distribution relates to their functioning. genes encoding these proteins cause a block or retarda tion of the respective transport events and can cause vari LAMPs and lysosomal integrity. Although most LMPs ous lysosomal storage diseases, neurodegeneration and predominantly reside in lysosomes, their subcellular osteopetrosis11 (TABLE 1). distributions can change and are more dynamic than so The pathways used by LE and lysosomal SNARE far appreciated. This has become especially apparent for proteins — that is, syntaxin 7, syntaxin 8, vesicle trans LAMP1 and LAMP2. LAMPs are type1 transmembrane port through interaction with tSNAREs homologue 1B proteins with considerable sequence homology that con (vTI1B), vesicleassociated membrane protein 8 (vAMP8) tain a large, heavily glycosylated luminal domain and a and vAMP7 (REF. 20) — are virtually unknown. Although short cytosolic tail. For example, there are three LAMP2 the cytoplasmic domains of SNAREs are essential for sort isoforms with different transmembrane and cytosolic ing140, their transport is complicated by their association in domains, which show a preferential localization in either multiple cis or transSNARE complexes, which may also endosomes and lysosomes or the plasma membrane145. be required for sorting141. vAMP7 depends on its socalled More generally, the cell surface expression of LAMPs longin domain for delivery to lysosomes. Interestingly, this is increased in the activation of platelets, peripheral domain can bind AP3, linking the lysosomal targeting of blood monocytes and cytotoxic T cells146 and in highly vAMP7 to LMP transport rather than to the M6PR path malignant tumour cells147. way142. However, significant levels of vAMP7 are found we are only beginning to understand the significance at the plasma membrane, where endocytosis is mediated of local LAMP concentrations. The plasma membrane by a specific adaptor, HIv1 Revbinding protein (HRB; levels of CD63 are of major consequence for other local also known as AGFG1)143. It therefore remains to be estab protein concentrations62, but elevated plasma membrane lished whether vAMP7 reaches AP3positive TSE tubules levels of LAMP1 and LAMP2 have not yet been linked to a through a direct intracellular pathway, by passage over the specific phenotype. An important clue to the significance plasma membrane, or both. of sustained LAMP levels in lysosomes came from a recent study which showed that LAMP proteins are involved Luminal sorting determinants. An entirely different clue in sensitizing tumour cells to lysosomal cell death (LCD) to the mechanism of sorting comes from recent studies (FIG. 1). Oncogenic transformation of fibroblasts on the one in melanocytes. In addition to lysosomes, these cells con hand leads to a decrease in the levels of LAMP proteins tain melanosomes — LROs that store melanin pigment. in lysosomes and on the other hand increases the sus The melanosomal LMP tyrosinaserelated protein 1 ceptibility of these cells to the LCD pathway148. Likewise, (TYRP1) follows a direct TGNtomelanosome path decreased levels of LAMP1 and LAMP2 also contribute to way in melanocytes, but uses an indirect pathway via an enhanced sensitivity of transformed cells to anticancer the cell surface in cells that lack glucosylceramide127. drugs that trigger LCD. Overexpression of LAMPs had the unexpectedly, LAMP1 and LAMP2 behave in the oppo opposite effect, indicating that LAMPs can protect cells site manner and are transported indirectly in wildtype from the LCD pathway. In addition, LAMPdepleted cells cells and directly in the absence of glucosylceramide. showed a redistribution of lysosomes to the cell periph Because the level of glucosylceramide is in part regulated ery, pointing to a role for LAMPs in lysosomal dynamics. Autophagosome through the activity of βGC, which is transported to lyso Together with earlier studies correlating surface expres A double-membrane vesicle somes with the help of LIMP2 (see earlier), it is conceiv sion of LAMP proteins to metastatic potential of carcin that forms at an early stage of able that LIMP2 also indirectly controls the transport of oma cells147,149, these findings exemplify the importance of the autophagic pathway and can fuse with endosomes proteins to melanosomes. A chimeric protein compris LAMP targeting in maintaining lysosomal integrity and in and lysosomes for degradation ing the luminal domain of TYRP1 and the cytosolic tail regulating LCD pathways. They also underscore the need of its contents. of LAMP1 was transported directly to melanosomes, to understand the relationship between LAMP trafficking indicating that the luminal domain of TYRP1 contains and successful anticancer treatment. Lysosomal cell death sorting information to guide proteins into a direct TGN Apoptosis induced by 127 permeabilization of the tomelanosome pathway . Another melanosomal LMP, LAMPs and lysosomal dynamics. The significance of the lysosomal membrane and the PMEL17 (also known as SILv), is efficiently sorted into intracellular localization of LAMPs is further exemplified subsequent release of ILvs by a mechanism that does not require the cytosolic by the observation that in LAMPdeficient cells the distri cathepsins into the cytosol. domain of PMEL17 and is independent of ubiquitylation bution of lysosomes is more dispersed and peripheral, sug The mechanism of membrane permeabilization is not yet and the ESCRT machinery. Instead, PMEL17 requires gesting that lysosome migration towards the microtubule 150 known. its luminal domain for an unknown sorting mechanism organization centre is delayed . These findings resemble that is conserved in nonpigmented cells144. observations in AP3deficient cytotoxic T lymphocytes, in Immunological synapse which minusenddirected, microtubulemediated move The interface between an LMP trafficking and function ment of lytic granules towards the immunological synapse antigen-presenting cell and TABLE 1 151 a lymphocyte, consisting of a Because LMPs mediate a wide range of functions ( ; was impaired, resulting in a loss of cytotoxicity . Similar cluster of T cell receptors and see Supplementary information S1 (table)), the presence, to the lysosomes in LAMPknockout cells, lytic granules a ring of adhesion molecules. absence or mutation of a given LMP in a membrane can in AP3deficient cytotoxic T lymphocytes remained at

the cell periphery. These findings suggest that LAMPs strictly regulated lysosomal pathway that degrades cyto are required to move lysosomes because a reduction in plasmic material and organelles159 and is activated during LAMP, by genetic downregulation or impaired transport stress conditions, such as during amino acid starvation, the as a result of AP3 deficiency, leads to their redistribution. unfolded protein response or viral infection. Phagocytosis It is not yet clear whether and how the short (11 amino is also an evolutionarily conserved mechanism that medi acids) Cterminal tails of LAMP proteins can interact, ates the lysosomemediated degradation of large particles, directly or indirectly, with the dyneinmediated centripetal such as internalized pathogens. migration system of lysosomes. Deficiency of LAMP2 in mice160 recapitulates human In addition, a recent study has shown that LAMP1 Danon disease, a rare disorder characterized by LAMP2 participates in the regulation of lysosomal exocytosis152. mutations that lead to a clinical triad of hypertrophic unexpectedly, lysosomal exocytosis is increased in cardio myopathy, skeletal myopathy and mental retarda macro phages and fibroblasts that are defective in neu tion161 (TABLE 1). The pathological hallmark of LAMP2 raminidase, a model for the lysosomal disorder sialidosis, deficiency in both mice and humans is an accumulation which results in oversialylation of LAMP1. Lysosomal of autophagic compartments, which is most pronounced exo cytosis, which is involved in plasma membrane in muscle cells. The cellular pathology is explained by a repair, is Ca2+ dependent and requires the ubiquitously role of LAMP2, and possibly also of LAMP1 (REF. 162), expressed LMP synaptotagmin 7 (SYT7)153, a Ca2+ sen in mediating the fusion of lysosomes with autophago sor that cooperates with SNARE proteins to promote somes. The lack, or large reduction, of these fusion the fusion of the lysosomal and plasma membranes153. events would explain the abnormal accumulation of The lysosomal vesicle SNARE vAMP7 is also required autophagosomes and autolysosomes. Interestingly, analysis for Ca2+triggered lysosomal exocytosis154 and forms of LAMPdeficient cells revealed that RAB7 recruitment complexes with syntaxin 4 and synaptosomalassociated to autophagosomes and their fusion with lysosomes was protein 23 (SNAP23), two plasma membrane tSNAREs severely retarded150,163,164. A similar lysosome–phagosome that interact in a Ca2+dependent manner with SYT7 fusion defect is observed in neutrophils that lack LAMP2, (REF. 154). vAMP7 and SYT7 colocalize in LEs and lyso possibly explaining the reduced ability of these cells to somal compartments that exocytose after an increase in kill pathogens, which in LAMP2knockout mice leads 2+ periodontis165 Bacterial type III secretion the intracellular Ca concentration. Interestingly, genetic to . system ablation of SYT7 leads to a defect in neurite outgrowth, The Cterminal tail of LAMP2A is also implicated in A specialized, needle-like, suggesting that SYT7regulated exocytosis of LEs and the transport of cytosolic substrates across the lysosomal multiprotein structure in lysosomes plays a part in the addition of new membrane limiting membrane, a process referred to as chaperone- Gram-negative bacteria that is to developing neurites155. How LAMP1 fits into this pic mediated autophagy (CMA)166. CMA is important for involved in the direct secretion of proteins from the bacterial ture and how luminal alterations in LAMP1 give rise to different biological processes, such as the presentation of 167 cell to the host. altered lysosome dynamics need to be resolved. cytoplasmic antigens by MHC class II molecules , cell LAMPs150, vAMP7 (REF. 156) and SYT7 are also required ular ageing168 and neurodegeneration169. How the recog Phagosome for Ca2+dependent phagosome–lysosome fusion, a proc nition of CMAtargeting sequences and the translocation A vacuole formed around a particle — for example, a ess that is necessary for limiting the intracellular growth of into the lysosome occurs at the molecular level is not yet pathogenic microorganism pathogenic bacteria. Bacterial type III secretion systems can understood. absorbed by phagocytosis — permeabilize membranes and cause a Ca2+ influx in mam that can mature into a malian cells, thereby promoting lysosomal exocytosis. Conclusion and perspectives degradative compartment by Phagolysosome fusion is also a Ca2+dependent process. Each protein that enters the endocytic pathway passes fusion with lysosomes. Analysis of SYT7deficient cells revealed that the intra a number of decision stations that determine the rest of Autolysosome cellular survival of bacteria that have to evade lysosomes its journey. The exact sorting decisions that need to be A single-membrane vesicle that in order to replicate is dependent on SYT7. Most likely, taken depend on where the cargo enters the endosomal forms at a late stage of the shortly after invasion phagosomes are permeabilized, Ca2+ continuum. Apart from the M6PR pathway, only little is autophagic pathway by fusion of autophagosomes with influx into the cytosol is triggered and SYT7dependent known about the biosynthetic pathways to the lysosome. endosomes or lysosomes and phagolysosomal fusion and bacterial killing is initiated. Identification of alternative receptors and/or sorting that contains degradative Therefore, the lysosomal repair response is an evolutionar mechanisms for direct targeting of lysosomal hydrolases enzymes obtained after fusion. ily conserved protection mechanism against pathogens157. or LMPs to the lysosome is one of the major challenges Also, invasion of the protozoan Trypanosoma cruzi, in the lysosomal trafficking field. The recent availability Periodontis 100 A common inflammatory which causes the debilitating human Chagas disease, of a mouse model system for I cell disease provides an disease of the supporting requires host cell lysosomes that are recruited to the site exciting new opportunity to study M6PRindependent tissues of the teeth that leads of parasite entry and gradually fuse with the plasma mem mechanisms of lysosomal hydrolase sorting and may to resorption of alveolar bone brane, thereby providing the membrane for formation lead to new clues to the role of vps10 domaincontaining and eventually to tooth loss. of the parasitophorous vacuole158. receptors. Moreover, the LIMP2dependent transport Chaperone-mediated of βGC indicates that the M6PRindependent delivery of autophagy LAMPs in autophagy and phagocytosis. Apart from the lysosomal hydrolases might be linked to the lysosomal A direct pathway for documented role of lysosomal hydrolases in defence targeting pathways of LMPs. transporting cytosolic proteins against bacteria, there is an increasing body of evidence An important emerging theme is that the delivery of over the lysosomal limiting membrane and into the that suggests that LAMPs are of key importance for the LMPs can essentially change the properties of the target lysosome lumen for maturation of autophagosomes and phagosomes (FIG. 1). compartment; for example, acidity can be altered by the degradation. Autophagosomes mediate autophagy, a conserved and delivery of the vtype H+ ATPase complex, fusogenicity

can be altered by the delivery of vAMP7, catabolic capac tails can be involved in lysosomal targeting117,127,144 and the ity can be altered by the delivery of LIMP2, dynamics occurrence of posttranslational modifications (glycosyla and drug resistance can be altered by the delivery of tion, phosphorylation, palmitoylation and ubiquitylation) LAMP1 and LAMP2, and the availability of cytoplasmic that can finetune the sorting signals170. substrates can be altered by the delivery of LAMP2A. Multiple pathways for the delivery of newly syn Moreover, LMPs are increasingly being implicated as the thesized proteins to the endolysosomal system would causative genes for a range of human diseases. The increas require multiple TGN exits. Current data suggest the ing number of functions ascribed to the LAMP cytosolic existence of clathrinindependent TGN exit pathways tails and the presence of enlarged, more peripheral lyso that could be used by LMPs and even, to a minor extent, somes in LAMPdeficient cells raise the notion that LAMPs by lysosomal hydrolases. Additional evidence for multiple might be incorporated in functional microdomains at pathways comes from yeast, in which the lysosomal tar the lysosomal limiting membrane (allowing, for exam geting of the lysosomal hydrolase carboxypeptidase Y and ple, association with microtubules, lysosomal membrane the LMP alkaline phosphatase requires distinct pathways transporters or sorting determinants to the lysosomal and machinery. In Drosophila melanogaster, the granule pathway) that can act in events as diverse as lysosome group protein Deep Orange (DOR) is involved in the exocytosis, dynamics and protein translocation. lysosomal targeting of lysosomal hydrolases, but not of A timely and targeted delivery of LMPs to endosomal LMPs171. Because the mammalian endosomal system is intermediates at distinct stages of the endocytic pathway far more complex than in yeast, the presence of multiple might be an important mechanism for the gradual deliv TGNtoendosome pathways seems a realistic possibility. ery of essential building blocks to assemble a functional Elucidating these pathways is a major challenge for future lysosome. This scenario is supported by the presence studies and is of key importance to expand our knowledge of multiple sorting signals in the cytosolic tails of some of lysosome biogenesis and to understand the pathologies LMPs, the evidence that domains other than the cytosolic associated with lysosomal dysfunctioning.

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