the endocytic pathway: a mosaic of domains · organelles in the endocytic pathway are composed of a...

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REVIEWS

Over the past 30 years, much work has been devoted tothe description of organelles, and consequently thepathways that are followed by cargo proteins duringsecretion or endocytosis. Many key processes have beenanalysed at the molecular and atomic levels, with thecurrent challenges being to understand how molecularmachines regulate each transport step and then howthese different mechanisms are integrated. However, theprinciples that guide the movement of proteins andlipids, and the specific organization of each compart-ment, are still poorly understood; in particular, how isthe linear organization of the genome translated into athree-dimensional cellular architecture?

In the endocytic pathway, some compartments canbe easily identified because of their characteristic multi-vesicular or multilamellar appearance. Similarly, theGolgi ribbon or the endoplasmic reticulum networkcan usually be easily identified by their typical organiza-tion and topology. And yet, the boundaries between twoeasily distinguishable compartments in the same path-way are blurred at the molecular level, in part becausekey proteins that regulate membrane transport areoften found in more than one compartment. Differentmembrane domains, which might show defined bio-physical properties, probably coexist in each endocyticcompartment. The dynamic interplay between thesedomains might provide a driving force that is responsi-ble both for the specific organization of each compart-ment and for the movement of cargo molecules. So, oneof the questions we now face is: do endocytic organelles

with a homogeneous membrane composition exist inthe strict sense of the word?

A mosaic of structural and functional domainsIn higher eukaryotic cells, internalization of proteinsand lipids is mediated by CLATHRIN-coated vesicles, andother less characterized pathways. Typically, endocy-tosed molecules, including recycling receptors withtheir bound ligands and downregulated receptors, aredelivered to early endosomes, where efficient sortingoccurs (FIG. 1). After receptor–ligand uncoupling at themildly acidic lumenal pH, recycling receptors are rapid-ly (t

1/2~ 2.5 min) segregated away from their ligand and

transported along the recycling route, whereas ligandsfollow the degradation pathway together with downreg-ulated receptors. Hence, it is generally accepted thatearly endosomes represent both the single entry pointfor internalized molecules, and the first sorting stationin the pathway.

The early endosome is a dynamic compartmentwith a high homotypic fusion capacity1. But its ele-ments display a highly complex and pleiomorphic orga-nization that consists of cisternal regions from whichthin tubules (~ 60 nm diameter) and large vesicles (~300–400 nm diameter) seem to emanate (FIG. 2). Thevesicles contain membrane invaginations, and aretherefore described as multivesicular — although it isnot clear to what extent these invaginations detach fromthe limiting membrane and form free vesicles in thelumen. Tubular elements closely resemble the tubules of

THE ENDOCYTIC PATHWAY:A MOSAIC OF DOMAINSJean Gruenberg

Organelles in the endocytic pathway are composed of a mosaic of structural and functionalregions. These regions consist, at least in part, of specialized protein–lipid domains within theplane of the membrane, or of protein complexes associated with specific membrane lipids.Whereas some of these molecular assemblies can be found in more than one compartment, agiven combination seems to be unique to each compartment, indicating that membraneorganization might be modular.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | OCTOBER 2001 | 721

Department ofBiochemistry, University ofGeneva, 1211-Geneva-4,Switzerland.e-mail: [email protected]

CLATHRIN

Large protein, whichpolymerizes into a triskelion,comprising three heavy chainsand three light chains.Triskelions assemble intopolyhedral lattices to formclathrin coats.

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surface occurs by default. This view is difficult to recon-cile with the situation in epithelial cells, in which transcy-tosed and recycling receptors transit through a commonrecycling endosome before being transported to oppo-site plasma membrane domains3. Lysosomal targetingsignals have been identified, but it is not always clear atwhich transport step these operate. Sorting motifs havebeen found in the cytoplasmic domains of P-selectin4,the interleukin-2 (IL-2) receptor β chain5, in the viralprotein Nef during CD4 downregulation6, and in theepidermal growth factor receptor (EGFR)7, but thesemotifs bear little resemblance to each other. Recent stud-ies indicate that ubiquitylation of the cytoplasmicdomain might contribute to lysosomal sorting8,9.Transient protein monoubiquitylation is also believed toact at the cell surface as an internalization signal, and it ispossible that a similar mechanism operates in sortingelsewhere in the cell10.

This apparent lack of recycling signals and diversityin degradation signals indicates that different sortingprinciples might operate in early endosomes. Lipids arenot distributed randomly within endosomal mem-branes, and different lipid domains might coexist ateach step of the pathway. Protein and lipid sorting — forexample, during biogenesis of tubules and ECV/MVBs— could be coupled to selective incorporation into spe-cialized protein–lipid environments.

Attractive candidates for early endosomal sortingfunctions include LIPID RAFTS, as these are believed to showsorting functions in the trans-Golgi network and at theplasma membrane11. Although the fate of rafts in endo-cytosis is not clear, it has been suggested that cell-surfacerafts can act as internalization platforms12,13, a route pre-sumably followed by downregulated IL-2 receptors ontheir way to endosomes and then lysosomes14. In addi-tion, SV40 was recently found to enter cells through CAVE-

OLAE and then to reside within CAVEOSOMES, which do not

recycling endosomes, and multivesicular elementsresemble the endosomal carrier vesicles/multivesicularbodies (ECV/MVBs) of the degradation pathway (FIG.

3). These tubular and multivesicular regions can there-fore be considered as the trans face of the organelle. Itcould be that the central cisternal region functions asthe entry or cis region, receiving incoming vesicles fromthe plasma membrane or trans-Golgi network.

It is not easy to imagine how early endosomalmembranes can be shaped into such different struc-tures. Selective changes must occur in the curvatureand organization of the bilayer, for example, duringmembrane invagination in nascent ECV/MVBs ortubule biogenesis2. Morphogenesis in the endocyticpathway must involve the action of molecularmachines and the segregation of proteins and lipids inthe plane of the membrane.

Sorting in early endosomesRecycled and downregulated receptors are efficientlysorted from one another in early endosomes. But it hasbeen particularly difficult to identify sorting signals inthe cytoplasmic domains of cargo proteins. This seemssurprising as such signals have been found for most, ifnot all, other transport steps of the biosynthetic andendocytic pathways. No recycling motif has been identi-fied, leading to the proposal that recycling to the cell

Figure 1 | Outline of the endocytic pathway. The mainroutes of endocytic membrane transport are indicated, withthe recycling pathway in green and the degradation pathwayin red. Microtubules and the microtubule-organizing centre(MTOC) are in blue. In higher eukaryotic cells, internalizationof receptors and other cell-surface components occursthrough clathrin-mediated endocytosis, although other lesscharacterized pathways are also involved. Internalizedmolecules are then delivered to early endosomes, whereefficient sorting occurs. Some receptors are recycled back tothe plasma membrane to be reused, at least in part throughrecycling endosomes, whereas downregulated receptors aretransported to late endosomes and lysosomes fordegradation. Late endosomes provide the last sortingstation in the pathway, whereas lysosomes are generallybelieved to represent the end station. Transport routes alsoconnect the biosynthetic and endocytic pathways, and are inparticular responsible for the delivery of lysosomal enzymesand membrane proteins. ECV, endosomal carrier vesicle;MVB, multivesicular body.

Plasma membrane

Recyclingendosome

Early endosome

ECV/MVB

Late endosome

Lysosome

MTOC

Figure 2 | The early endosome. The figure shows an earlyendosome containing low-density lipoprotein (LDL)–goldparticles endocytosed for 5 minutes (gold particles arevisualized as white spots, as contrast was reversed). Afterinternalization, cells were homogenized, crude fractionsprepared and deposited on mica plates. Samples wereanalysed by freeze-etch electron microscopy. (Courtesy of J.Heuser, Washington University, Missouri, USA).

LIPID RAFTS

Dynamic assemblies ofcholesterol and sphingolipids inthe plasma membrane,probably involved in cellsignalling.

CAVEOLA

Specialized raft that containsthe protein caveolin, and formsa flask-shaped, cholesterol-richinvagination of the plasmamembrane that might mediatethe uptake of some extracellularmaterials, and is probablyinvolved in cell signalling.

CAVEOSOME

A recently discovered organellethat is involved in theintracellular transport of SV40from caveolae to theendoplasmic reticulum.

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pathways20. Although a given SNARE will be mostabundant in a certain compartment, SNAREsinevitably spread through several compartments dur-ing vesicular transport. So, docking/fusion specificitycannot be solely determined by the distribution ofendosomal SNAREs.

Much effort has been devoted to the study of smallGTPases of the Rab family, which are considered asorganelle markers due to their restricted distribution.When in the active GTP-bound state, Rab5, which isinvolved in early endocytic transport, can interact withseveral effectors, such as other Rab proteins21. Throughthese interactions, Rab5 probably builds a specific effec-tor platform on the membrane, which could integratedifferent mechanisms that regulate transport, includingmembrane fusion, membrane budding and interactionwith cytoskeletal components21. Rab5 might also con-tribute to the spatial organization of docking/fusionsites for vesicles arriving from different pathways, as itseffector EEA1 can interact with syntaxin 6 (REF. 22),which has been implicated in the trans-Golgi networkto early endosome transport, and syntaxin-13, which isrequired for endosome fusion23 and recycling24.However, like other key regulators, Rab5 is not onlyfound in a single compartment, but is also associatedwith the plasma membrane, where its activity, perhapsrelated to clathrin-coated vesicle formation25, seems tobe regulated by the EGFR signalling pathway26. Thefindings that the Rab5 effector rabaptin 5 also interactswith Rab4 (REF. 27) — which is involved in recycling —and that the Rab4 effector RABIP4 contains a FYVEmotif and localizes to early endosomes28, raise the possi-bility that these Rab proteins are functionally coupled,perhaps reflecting the existence of a physical linkbetween different platforms. It will be interesting todetermine whether crosstalk between Rab effectors alsoexists along other pathways.

Several proteins interact specifically with phos-phatidylinositol-3-phosphate (PtdIns(3)P) through aconserved FYVE motif29, including the Rab5 effectorsEEA1 and rabenosyn-5, which are both required forearly endosome fusion30,31. EEA1 (REFS 16,32,33) — andperhaps other FYVE proteins28,34,35 — is, in fact, restrict-ed to early endosomes. The presence of both phos-phatidylinositol 3-kinase (PI3K) and PtdIns(3)P-bind-ing proteins among Rab5 effectors would facilitate theformation of microdomains or platforms that containRab5 and PtdIns(3)P on early endosomal membranes21.Although PtdIns(3)P–FYVE interactions are sufficientfor early endosomal targeting36, the distribution ofFYVE proteins also depends on other components. TheFYVE protein, hepatocyte growth factor-regulated tyro-sine kinase substrate (Hrs) — a homologue of the YEAST

CLASS E protein Vps27 — is found on early endosomes35,even when the FYVE motif is defective37. In addition,the yeast FYVE protein, Fab1, which is a PtdIns(3)P-5´-OH kinase that generates PtdIns(3,5)P

2, is essential for

maintenance of normal vacuolar morphology and wasproposed to regulate cargo-selective sorting into thevacuole lumen38. In addition to its function in earlyendosomal dynamics, PtdIns(3)P might also function

contain endocytosed tracers15. Whether a functionalrelationship exists between caveosomes and endosomesremains to be shown. Raft components, such as choles-terol and sphingomyelin, are abundant in recyclingendosomes, at least in Chinese-hamster ovary (CHO)and Madin–Darby canine kidney (MDCK) cells16–18,indicating that lipid rafts might contribute to endosomalsorting. Indeed, GPI-ANCHORED proteins, which preferen-tially partition into rafts at the cell surface, follow thesame route as the rafts themselves in CHO cells18.Whether raft components show the same distribution inall cell types is not known. More importantly, it remainsto be shown whether these components actually assem-ble into rafts within endosomes — endocytosed lipidanalogues with a preference for ordered membranedomains are targeted to late endosomes/lysosomes,rather than recycling endosomes, in the same CHOcells19.Whatever the mechanism and molecular compo-nents involved, protein–lipid sorting and membraneorganization seem to be intimately coupled in earlyendosomal membranes.

Molecular architecture of early endosomesIn addition to the morphologically visible mosaic ofearly endosomal domains, it is becoming apparentthat key components that regulate membrane organi-zation and protein transport are distributed in a non-random manner on membranes, defining functionaldomains (FIG. 4). Whether vesicles arriving from theplasma membrane or the trans-Golgi network candock and fuse anywhere on these membranes is notknown, but if early endosomes display the cis–transpolarity discussed above, docking/fusion sites mightnot be randomly distributed.

Docking/fusion proteins of the SNARE family havebeen identified along both recycling and degradation

Figure 3 | Endosomal carrier vesicles/multivesicularbodies. ECV/MVBs containing endocytosed glycoprotein-Gof vesicular stomatitis virus (VSV) were purified and thedistribution of G-protein was analysed by immunogoldlabelling of cryosections1. (Reproduced from The Journal ofCell Biology, 1989, 108, 1301–1316 by copyright permissionof The Rockerfeller University Press.)

GPI ANCHOR

The function of this post-translational modification is toattach proteins to theexoplasmic leaflet ofmembranes, possibly to specificdomains therein. The anchor ismade of one molecule ofphosphatidylinositol to which acarbohydrate chain is linkedthrough the C-6 hydroxyl of theinositol, and is linked to theprotein through anethanolamine phosphatemoiety.

SNARES

(Soluble N-ethylmaleimide-sensitive factor attachmentprotein receptor). A family ofmembrane-tethered coiled-coilproteins that regulate fusionreactions and target specificityin the vacuolar system. Theycan be divided into vesicle-SNAREs and target-SNAREs onthe basis of their localization, orinto Q-SNAREs and R-SNAREson the basis of a highlyconserved amino acid.

YEAST CLASS E MUTANTS

One class of vacuolar proteinsorting (VPS) mutants in yeast.Class E genes are involved in thedelivery of both newlysynthesized vacuolar enzymecarboxypeptidase Y (CPY) andendocytosed proteins to thevacuole from the prevacuolarcompartment. Mutations in anyof the class E VPS gene productscauses an accumulation ofcargo in an aberrant endosome-like structure termed the class Ecompartment.


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ins, using a variety of membrane-association mecha-nisms, might form platforms on the membrane of dif-ferent sub-cellular compartments, which would specifi-cally interact with cytosolic components, including thecytoskeleton.

Annexin II localizes to the plasma membrane and onearly endosomes, and it was shown to be involved in thedynamics of early and/or recycling endosomes49,50.Annexin II binding to endosomes51, but perhaps notplasma membrane52, seems to use an unconventional,cholesterol-dependent — but calcium-independent —mechanism. Annexin II also interacts with proteins ofthe cortical actin cytoskeleton, and it seems to be non-randomly distributed on early endosomal membranes,and concentrated in areas from which actin-like fila-ments seem to emanate51. It is therefore possible thatcholesterol-rich regions of early endosomal membranesthat interact with annexin II are important in the gener-al organization and dynamics of this compartment.

The COPI coat complex regulates retrograde transportfrom the Golgi to the endoplasmic reticulum, but sever-al lines of evidence indicate that an endosomal COPIcomplex, lacking β- and γ- subunits, functions in endo-somal transport53 — in particular in ECV/MVB biogen-esis54 and Nef-mediated CD4 downregulation6. COPIhas also been implicated in transport from the phagoso-mal membrane55. Like biosynthetic COPI, endosomalCOPI interacts with membranes through the smallGTPase, ADP-ribosylation factor 1 (ARF1) (REF. 56).However, recruitment of ARF1 and COPI onto endo-somes, but not biosynthetic membranes, depends on theacidic lumenal pH, possibly through a pH sensor54,56, asdoes binding of ARF6 and the ARF57 exchange factor,ARNO. As is the case for other coat components, includ-ing COPII, as well as clathrin and some of its partners58,COPI can also interact with membrane lipids59. Suchweak interactions could facilitate membrane targetingand association, and concentrate coat componentslocally.

The close relationship between early endosome func-tions in transport and morphogenesis is also apparent inCOPI functions. COPI inactivation in LDLF CELLS with atemperature sensitive-defect in ε-COP60 inhibits trans-port to late endosomes and interferes with transferrinreceptor recycling61, but not with bulk62 recycling, andalso disrupts early endosomes62. These then becometubular or cisternal clusters without multivesiculardomains — a phenotype reminiscent of class E VPSmutants in yeast63. Identical perturbations are caused byneutralization of the endosomal pH54,64,65, which inhibitsARF1 and COPI binding.

Tubules of the recycling endosomeThe two main circuits of recycling and degradation arewell separated, both topologically and functionally, toensure that proteins that need to be reused at the cellsurface remain separate from those that are destined tobe degraded (FIG. 1). However, compartment boundariesalong the recycling or degradation pathways seemblurred at the molecular level, in particular when fol-lowing a single component. It is probably the interplay

in the biogenesis of multivesicular endosomes29, under-scoring the close connection between early endosomefunction and morphogenesis.

Recent studies have also uncovered the existence ofanother phosphoinositide-binding domain shared byseveral proteins, called the PX- or PHOX-homologydomain. This was first found in p40phox and p47phox —two subunits of the neutrophil oxidase39,40. The PXdomain of p40phox binds PtdIns(3)P selectively, and thepresence of this domain is required for the endocyticfunction and/or localization of the t-SNARE VAM7(REF. 41) and the sorting nexin SNX3 (REF. 42).Interestingly, the PX protein, SNX1, interacts with theFYVE protein Hrs43, and is involved in EGFR degrada-tion44, whereas the SNX1 yeast homologue, Vps5, is acomponent of the RETROMER COMPLEX that is necessary forendosome–trans-Golgi network recycling45. In addition,SNX3 and SNX15 are involved in endosomal transport,and their overexpression disrupts endosome morpholo-gy42,46. The relationships between PtdIns(3)P and PXproteins or FYVE proteins are still unclear, but shouldbe of high interest in understanding the organization ofendosomal membranes.

Other proteins that might function in organizingfunctional regions in the membrane are annexins47.These proteins seem to have the intrinsic ability to self-organize at the membrane surface into bidimensionalordered arrays48. It is attractive to speculate that annex-

Figure 4 | Lipid–protein microdomains and molecularmachines. A schematic early endosome is represented. Acisternal region is represented with a thick black line,recycling tubules are in yellow, whereas a forming MVB/ECVis shown with a black limiting membrane and purpleinvaginations. PtdIns(3)P is represented as yellow dots.Regions of membrane constriction or invagination, whichpresumably involve specialized lipids, are circled.Lipid–protein domains and complexes that might function asmodular elements of early endosomal membranes arerepresented. The precise function of PtdIns(3)P signalling andclass E VPS proteins in MVB/ECV formation is not clear inmammalian cells (BOX 1). MVB/ECV, multivesicularbody/endosomal carrier vesicle.

PtdIns(3)P signalling pathway, FYVE and PHOX proteinsRab5 and effectorsAnnexin II–cholesterol

Recycling

Degradation

Endosomal COPs

RETROMER COMPLEX

Protein complex consisting ofVps35, Vps26, Vps29, Vps17and Vps5, which was discoveredthrough genetic screens inSaccharomyces cerevisiae. Itfunctions in the retrieval ofproteins from the prevacuolarcompartment and transport tothe Golgi.

COPI COAT

Complex consisting of α-, β-,β’-, γ-, δ-, ε- and ζ-COP, alsocalled coatomer. This coatcomplex functions inanterograde transport withinthe Golgi and in retrogradetransport from the Golgi to theendoplasmic reticulum.

COPII COAT

Complex consisting of Sec13,Sec31, Sec23 and Sec24. Thiscoat complex functions inanterograde transport from theendoplasmic reticulum to theGolgi.

LDLF CELLS

A mutant Chinese-hamsterovary cell line that wasidentified on the basis of itsdefect in low-densitylipoprotein (LDL) transport.The mutation causing thephenotype was later identifiedas a deletion of ε-COP.


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Transport in the degradation pathwayAlthough tubules mediate recycling to the cell surface,transport intermediates along the degradation pathway— from early to late endosomes — involve large (~300–400 nm diameter) ECV/MVBs. In mammaliancells, ECV/MVBs are clearly distinct from both earlyand late endosomes. In particular, ECV/MVBs do notcontain early endosome-specific proteins or recyclingreceptors, nor do they contain the main lipid and pro-tein constituents of late endosomal membranes. Onceformed, ECV/MVBs move towards late endosomes in amicrotubule- and motor-dependent fashion, andacquire the capacity to dock onto and fuse selectivelywith late endosomes, a step which depends on the con-ventional docking/fusion machinery20,21,88. In axons,ECV/MVBs also function as intermediates from earlyendosomes at the presynaptic membrane to late endo-somes in the cell body67. Whether ECV/MVBs changein composition as they undergo a maturation process,or whether they mediate transport between two stablecompartments, has been the subject of much debate,and so far, it has not been possible to solve this issue byfollowing single components at the boundary betweenearly and late endosomes.

ECV/MVBs, like late endosomes, contain two mor-phologically visible membrane domains, internalinvaginations and a limiting membrane. The composi-tion of the ECV/MVB limiting membrane is notknown, except that it seems to lack early endosomalproteins, and is different from the late endosomal mem-brane, which contains high amounts of LAMP1.Similarly, little is known about the composition ofECV/MVB invaginations. They accumulate downregu-lated receptors, in particular the EGFR89, which has ledto the idea that internal membranes are destined to bedegraded in lysosomes — a view partially challenged inmammalian cells by recent observations (see the nextsection). Electron-microscopy studies showed thatPtdIns(3)P is abundant within ECV/MVB internalmembranes, in addition to early endosomes, and thatit is not detected within late endosomes that containLYSOBISPHOSPHATIDIC ACID (LBPA)36. However, it is notclear whether the presence of PtdIns(3)P on these inter-nal membranes reflects a role for this lipid in the bio-genesis of invaginations, or simply the metabolism ofthe lipid.

Wortmannin, a drug that inhibits PI(3)K wasreported to inhibit multivesicular body formation inmammalian cells90. At the same time, evidence is accu-mulating in yeast that signalling through the PI(3)KVps34 and PtdIns(3)P is crucial in vacuole transport ofthe trans-Golgi network29. Considering the close rela-tionship that exists between ECV/MVB biogenesis andearly endosome organization, we might expect some ofthe components that regulate early endosomal func-tions to also be important in the invagination process.In addition, yeast class E VPS mutants are all defectivein MVB biogenesis, as they form stacks of tubules orcurved cisternae63, perhaps in a ‘frustrated invagination’process. Several class E mutants have a mammalianhomologue that has been implicated in transport,

between key components that defines the functionalboundaries in each pathway.

After leaving early endosomes, recycling moleculesare found in distinct tubular structures that corre-spond to recycling endosomes. These do not containligands and receptors that are destined to be degraded,are less acidic than early endosomes, and are foundclose to the centrioles in some, but not all, celltypes16,66,67. These tubules form dynamic networks68

and are collectively referred to as recycling endosomes.But the distinction between transport intermediatesand compartments is not clear.

Early endosomal Rab5 and two small GTPasesinvolved in recycling, Rab4 and Rab11, show a distinct,but partially overlapping, distribution in vivo, whichpresumably corresponds to different effectorplatforms69. However, the precise functions of Rab4 andRab11 are not clear. SNARE family members have beenfound in recycling endosomes and were reported tofunction in the pathway, including the v-SNAREs cellu-brevin70 and endobrevin/VAMP8 in the apical pathwayof polarized cells71, and the t-SNARE syntaxin 13 (REF.

24), which is also involved in endosome fusion23. Again,the precise function of these molecules is not clear.

Recycling can occur by a fast and a slow route72–75.These could correspond to at least two separate trans-port steps to the plasma membrane, each with a distinctmolecular machinery; for example, from early endo-somes or from recycling endosomes (FIG. 1).Alternatively, fast and slow transport could reflect theexistence of a gradient of molecules on their way to thecell surface within early and recycling endosomes. Thisissue is not easily addressed. Drugs or expression ofmutant proteins might unbalance transport alongeither route, perhaps explaining why recycling kineticsare hardly affected by microtubule depolymerization,although the pericentriolar distribution of the recyclingendosome depends on microtubules.

Transport along the recycling pathway depends onthe actin cytoskeleton and unconventional myosinmotors76–78, which might have a mechanical role intubule biogenesis and dynamics. Crosstalk betweentransport, actin remodelling and Rac-mediated sig-nalling could depend on the small GTPase ARF6 and itspossible partners79–82. Clathrin-coated buds have beenobserved on recycling endosomes83, but whether theymediate transport back to the cell surface83 or to thetrans-Golgi network remains to be fully established84. Asclathrin coats usually have sorting functions, their pres-ence on recycling endosomes strengthens the view thatprotein sorting occurs within recycling endosomes. Agenetic screen for endocytosis mutants inCaenorhabditis elegans recently identified RME-1 (REFS

85,86) — a new member of the conserved family ofEps15-homology (EH)-domain proteins87, which showcharacteristics of an endocytic accessory protein. RME-1 is associated with recycling endosomes and might beinvolved in the exit of membrane proteins from thiscompartment. Beyond these observations, little isknown about the molecular mechanisms that drive thedynamics of tubular recycling endosomes.

LBPA

Lysobisphosphatidic acid(LBPA) is a phospholipid,structurally analogous withphosphatidylglycerol. LBPA ispoorly degradable, presumablybecause of its unusualstereoconfiguration, and isabundant within internalmembranes of late endosomes.


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the idea that the fate of internal membranes is degrada-tion in lysosomes. Sandhoff and collaborators104 havedemonstrated, using an in vitro liposome assay, that neg-atively charged phospholipids, in particular LBPA, great-ly facilitate the degradation of several glycolipids. AsLBPA itself is poorly degradable, one function of LBPAmembranes could be to present lipids and proteins thatneed to be degraded to the hydrolytic machinery.

However, internal membranes of late endosomesdo not only contain proteins that are destined to bedegraded. Members of the TETRASPANIN FAMILY, includingCD63/LAMP3, have been shown to accumulate withininternal membranes105, in addition to their presence onthe cell surface. In ANTIGEN-PRESENTING CELLS, major histo-compatibility complex (MHC) class II compartments(MIIC) share most characteristics of late endosomesand lysosomes, including a complex system of internalmembranes106, although they are specialized compart-ments that contain specific components, such as thecathepsin S protease required for antigen processing107

and the human leukocyte antigen DM (HLA–DM)complex108. MHC class II molecules are abundant inMIIC internal membranes, both in B lymphocytes andin immature DENDRITIC CELLS. MHC class II moleculescolocalize with LBPA in B lymphocytes, and they arealso found in LBPA-containing microvesicles, presum-ably secreted by antigen-presenting cells109. Finally,MANNOSE 6-PHOSPHATE RECEPTOR (M6PR) molecules aretransported between the trans-Golgi network and lateendosomes to deliver lysosomal enzymes to endo-somes and lysosomes. While in transit through lateendosomes, M6PR is found predominantly withininternal membranes97,99.

LBPA membranes probably have an importantfunction in transport through late endosomes. Whenendocytosed in vivo, antibodies against LBPA accumu-late in late endosomes, and specifically inhibit M6PRtransport99. Conversely, loss of M6PR expression pro-motes LBPA accumulation in multilamellar bodies110.Endocytosed anti-LBPA antibodies also cause choles-terol accumulation in late endosomes, mimicking thecholesterol-storage disorder Niemann–Pick type C(NPC), leading to the idea that LBPA membranes serveas a collecting and distribution device for low-densitylipoprotein (LDL)-derived cholesterol111. In turn, cho-lesterol accumulation in late endosomes interferes withthe M6PR cycle in several cell types111,112, and also withCD63 and p-selectin cycling to WEIBEL–PALADE bodies inendothelial cells113. Sphingolipids accumulate in multi-vesicular compartments in tissue from NPC patients,and, conversely, cholesterol accumulates in late endo-cytic compartments in several sphingolipid storagedisorders114,115.

The function of LBPA membranes in protein andlipid transport indicates that some membrane proteinsand lipids do not only enter internal membranes, butcould subsequently exit and return to other cellulardestinations. Incorporation within internal mem-branes can be explained easily by the selective parti-tioning of some molecules within LBPA invaginations,or the selective exclusion from the limiting membrane.

including the Vps27 (REF. 91), Vps23 (REF. 92) and theVps4 homologues (REF. 93), indicating that the functionof these proteins might be conserved in mammaliancells (BOX 1). Whereas there is compelling evidence inyeast that class E proteins have an essential role in theendocytic pathway, their precise functions remain to beestablished. It has been proposed that the yeast class Ecompartment — or prevacuolar compartment — cor-responds to a late endosome94, but its mammaliancounterpart remains mysterious (BOX 1). In mammaliancells, a phenotype that is morphologically similar to theclass E phenotype has not been well characterized, withthe possible exception of early endosome after COPinactivation in LDLF cells62, or Hrs disruption in mice91.

The pomegranate and the onionMuch like early endosomes, late endosomal elementsare very dynamic95,96, with a highly complex andpleiomorphic organization, containing cisternal, tubu-lar and vesicular regions with numerous membraneinvaginations97. Their limiting membrane, similar tothat of lysosomes, contains high amounts of LAMP1,which is believed to be protected from the degradativemilieu of the compartment because of its high glycosy-lation state97. The limiting membrane of late endosomesalso contains MLN64, a homologue of the mitochondrialsteroidogenic acute regulatory protein (StAR)98.Internal membranes in higher eukaryotic cells undergomuch remodelling and accumulate large amounts (~15% of total phospholipids) of LBPA99, which is a poorsubstrate for phospholipase and therefore resistant todegradation100. LBPA has not been detected on the outerface of the limiting bilayer101,102. To what extent the bio-genesis and dynamics of late endosome inner mem-branes are regulated by the same mechanisms as thoseoperating during budding of ECV/MVBs is not clear.LBPA is presumably synthesized in situ103, and has aninverted cone shape. This structure could facilitate theformation of the invaginations that form the multi-vesicular elements of late endosomes101.

Proteins that are destined to be degraded accumu-late within ECV/MVB internal membranes, leading to

Box 1 | The endocytic pathways in yeast and mammals

A degree of caution is needed when extrapolating from mammalian to yeast endocyticpathways and vice versa, with respect to the specific structure and organization ofendosomal membranes. One of the most striking features of mammalian endocytosis,without discussing features of polarized or other differentiated cells, is the capacity toreuse cell-surface components efficiently and rapidly through recycling routes;whereas equivalent recycling routes, if they exist, do not seem to be as efficient in yeastcells. Although extensive sorting already occurs in early endosomes of mammaliancells, it has been proposed that a prevacuolar compartment equivalent to lateendosomes functions as the main sorting station in the yeast pathway94,128. Inaddition, yeast cells do not seem to share the striking appearance of mammalianendosomes within the degradation pathway, with a complex network of membraneinvaginations. In yeast cells, small and regularly shaped vesicles can be observed in thevacuole of strains with impaired vacuolar hydrolase activity129. In addition to thebasic machinery of yeast cells, mammalian cells are likely to have evolved a moreelaborate endosomal membrane system to ensure optimal regulation and reutilizationof proteins and lipids.

TETRASPANIN FAMILY

The tetraspanin family containsproteins that span themembrane four times with twoexoplasmic loops, and that canbe found at the cell surface.Although some are highlyrestricted to specific tissues,others are widely distributed.Members of this family havebeen implicated in cellactivation and proliferation,adhesion, motility,differentiation and cancer.

ANTIGEN-PRESENTING CELLS

A cell, most often a Blymphocyte, macrophage ordendritic cell, that is specializedin the generation of epitopesthat are presented throughmajor histocompatibilitycomplex (MHC) class I or II toT lymphocytes.

DENDRITIC CELLS

‘Professional’ antigen-presenting cells found in T-cellareas of lymphoid tissues, butalso as a minor cellularcomponent in most tissues.They have a branched ordendritic morphology and arethe most potent stimulators ofT-cell responses.

MANNOSE 6-PHOSPHATE

RECEPTOR

These receptors transportsoluble lysosomal hydrolases tolate endosomes by cyclingbetween the trans-Golginetwork and late endosomes.They bind in the trans-Golginetwork to mannose 6-phosphate moieties on N-linked glycans of the hydrolases.They release the hydrolases inlate endosomes and return tothe trans-Golgi network foranother round of transport.


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stable or extreme version of this network, which is con-sistent with the fact that they share some late endosomalcharacteristics122,123. LBPA membranes could have turn-pike functions in the network, as they seem to beinvolved both in protein and lipid transport99,111, andlipid degradation104. Other specialized regions mightexist. These could account for the idea that different lateendosome populations sort and package cargo mole-cules through Rab9–TIP47 or through the retromercomplex124, and also for the presence of specialized lateendosomes or lysosomes in highly differentiated cells,including MIIC in antigen-presenting cells106.

Cholesterol export might occur in specialized ele-ments, as the NPC1 protein — which is involved inintracellular cholesterol transport — seems to distributeto a subset of lysosomes125. Transport defects inNPC101,111, and perhaps other lipid-storage disorders115,could result from a collapse of the mosaic architectureof endosomes, leading to mixing of lipid–proteindomains and eventually transport inhibition114.

ConclusionsAlthough individual machines and regulatory compo-nents in the endocytic pathway might be loosely

If so, export from late endosomes should be slow, asinternal membranes are abundant, and only moleculesthat are present on the limiting membranes would beavailable for transport (FIG. 5) — this could account forthe slow cycle of CD63 in endothelial cells113. Othermechanisms, however, must be evoked to account forefficient export — for example, M6PR transport orMHC class II export to the plasma membrane in acti-vated dendritic cells. One possibility is that transportbetween the internal and the limiting membranes isregulated by several mechanisms, including post-translational modifications and, in particular, ubiqui-tylation9. It is also possible that late endosomes inhigher eukaryotic cells might contain more than onetype of internal membrane, such as those specializedin protein degradation or recycling. This is consistentwith the finding that ECV/MVBs and late endosomesdiffer in their LBPA content, although both haveabundant internal membranes. Moreover, late endo-cytic compartments in mammalian cells can appearlike a pomegranate (multivesicular) or like an onion(multilamellar). Essentially nothing is known aboutthe functional significance of these different types ofinner membranes, or about the differences in theirbiophysical state, organization and composition. It istempting to speculate that, in addition to the basicmachinery that is responsible for the formation ofintralumenal vesicles in yeast — which could dependon class E VPS proteins and PtdIns(3)P signalling38 —mammalian cells have evolved more efficient recyclingmechanisms, perhaps LBPA dependent, for proteinsthat need to be exported from internal membranes oflate endocytic compartments.

The end point or a dynamic network?If it has been difficult to draw the line between early andlate endosomes, the boundary between late endosomesand lysosomes is even more elusive, despite much workfrom many groups. Both compartments contain lysoso-mal enzymes, their pH is similarly acidic (~ 5.5), andtheir limiting membrane is primarily composed of thesame glycoproteins. Lysosomes can only be identifiedby their physical properties on gradients and their elec-tron-dense appearance, and by the fact that they lackproteins found in late endosomes, including M6PR intransit, Rab7 and Rab9, or phosphorylated hydrolaseprecursors116. So far, neither proteins nor lipids havebeen found that would only be present in lysosomes,but not in endosomes, and the basic docking/fusionmachinery seems to be shared by both compart-ments20,117,118. Moreover, late endosomes and lysosomescan exchange content and membrane proteins rapidlyand efficiently, and they probably interact dynamicallyto form a hybrid intermediate119,120.

It is tempting to speculate that late endosomes andlysosomes correspond to separate elements of a com-mon, but dynamic, network. Such a sub-compartmen-talization into regions and membrane domains that dif-fer both structurally and functionally is reminiscent ofthe early endosome organization. Highly motile tubularlysosomes in macrophages121 might represent a more

Figure 5 | Degradation or recycling? The differentmechanisms that could account for protein recycling fromlate endosome internal membranes are represented. Acanonical vesicle (A) delivers proteins, which will beincorporated into the limiting membrane of late endosomes(yellow), or into internal membrane invaginations (green andblue). The latter proteins are destined to be degraded (green)or recycled (blue). Proteins destined to be degraded couldsimply partition preferentially into membrane invaginationscontaining LBPA (red), or might be excluded from the limitingmembrane. The same mechanism might apply to someproteins that need to be recycled (1). However, othermechanisms must be evoked to account for efficientincorporation of some proteins into vesicles budding fromlate endosomes (B), including regulation by post-translationalmodifications (2) or by the existence of more than one type ofinternal membrane (3).

?

1

2

3

A

B

WEIBEL–PALADE BODIES

Morphologically uniquesecretory structures ofendothelial cells, which storevon Willebrand factor — aprotein involved in bloodclotting — for eventual release.


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domains are integrated to support protein and lipidtransport, as well as organelle structure and functions.Some clues are being provided from recent studies thatshow the existence of a close interplay between sig-nalling and transport pathways through protein net-works126. Moreover, several components discussed inthis article are linked to human diseases and involved indevelopment127. We might expect that beyond proteincomplexes and molecular machines with well definedroles, organellar functions also require specific sets ofeffectors that mediate cross-interactions and therebyarticulate a modular combination. These could controlthe formation and maintenance of different membranedomains, more or less transient in nature, providing thearchitectural framework for a given compartment.

distributed throughout several compartments, a certaincombination is unique to each compartment.Early endo-somes, for example, contain Rab5 and its effectors,PtdIns(3)P and partners, endosomal COPs, raft compo-nents and annexin II, which all function in transportthrough this compartment.Each of these molecules is alsofound on other membranes, but this particular combina-tion is present only on early endosomes. It is attractive tospeculate that endosomal membranes, and probably themembranes of other organelles, are built from modularelements. This could explain the difficulties encounteredin defining organelle boundaries at the molecular level,particularly when tracking a single component.

An important challenge will be to understand howthese different membrane machines and dynamic

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AcknowledgementsI am very grateful to G. van der Goot and R. G. Parton for com-ments and suggestions. Support was by grants from the SwissNational Science Foundation and the International Human FrontierScience Program.

Online links

DATABASESThe following terms in this article are linked online to:Flybase: http://flybase.bio.indiana.edu/Rab11 | HrsLocuslink: http://www.ncbi.nlm.nih.gov/LocusLink/Annexin II | ARF1 | ARF6 | ARNO | β-COP | CD63 | cellubrevin |EEA1 | endobrevin | endosomal COPI complex | γ-COP |interleukin-2 receptor β chain | LAMP1 | M6PR | MLN64 | NPC-1| P-selectin | RAB4 | RAB5 | RAB7 | RAB9 | Rabaptin-5 |Rabenosyn-5 | StAR | syntaxin-6 | SNX1 | SNX3 | SNX15 | TIP47Mouse Genome Informatics: http://www.informatics.jax.org/εCOP | RME-1OMIM:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIMNiemann–Pick type CSGD: http://genome-www.stanford.edu/Saccharomyces/ Fab1 | VAM7 | Vps4 | Vps5 | Vps27 | Vps34Swiss-Prot: http://www.expasy.ch/CD4 | EGFR | FYVE domain | syntaxin-13

the endocytic pathway: a mosaic of domains · organelles in the endocytic pathway are composed of a...

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