REVIEWS
THE ENDOCYTIC PATHWAY:
A MOSAIC OF DOMAINS
Jean Gruenberg
Organelles in the endocytic pathway are composed of a mosaic of structural and functional
regions. These regions consist, at least in part, of specialized protein–lipid domains within the
plane 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, a
given combination seems to be unique to each compartment, indicating that membrane
organization might be modular.
CLATHRIN
Large protein, which
polymerizes into a triskelion,
comprising three heavy chains
and three light chains.
Triskelions assemble into
polyhedral lattices to form
clathrin coats.
Department of
Biochemistry, University of
Geneva, 1211-Geneva-4,
Switzerland.
e-mail: jean.gruenberg@
biochem.unige.ch
Over the past 30 years, much work has been devoted to
the description of organelles, and consequently the
pathways that are followed by cargo proteins during
secretion or endocytosis. Many key processes have been
analysed at the molecular and atomic levels, with the
current challenges being to understand how molecular
machines regulate each transport step and then how
these different mechanisms are integrated. However, the
principles that guide the movement of proteins and
lipids, and the specific organization of each compartment, are still poorly understood; in particular, how is
the linear organization of the genome translated into a
three-dimensional cellular architecture?
In the endocytic pathway, some compartments can
be easily identified because of their characteristic multivesicular or multilamellar appearance. Similarly, the
Golgi ribbon or the endoplasmic reticulum network
can usually be easily identified by their typical organization and topology. And yet, the boundaries between two
easily distinguishable compartments in the same pathway are blurred at the molecular level, in part because
key proteins that regulate membrane transport are
often found in more than one compartment. Different
membrane domains, which might show defined biophysical properties, probably coexist in each endocytic
compartment. The dynamic interplay between these
domains might provide a driving force that is responsible both for the specific organization of each compartment and for the movement of cargo molecules. So, one
of the questions we now face is: do endocytic organelles
with a homogeneous membrane composition exist in
the strict sense of the word?
A mosaic of structural and functional domains
In higher eukaryotic cells, internalization of proteins
and lipids is mediated by CLATHRIN-coated vesicles, and
other less characterized pathways. Typically, endocytosed molecules, including recycling receptors with
their bound ligands and downregulated receptors, are
delivered to early endosomes, where efficient sorting
occurs (FIG. 1). After receptor–ligand uncoupling at the
mildly acidic lumenal pH, recycling receptors are rapidly (t1/2 ~ 2.5 min) segregated away from their ligand and
transported along the recycling route, whereas ligands
follow the degradation pathway together with downregulated receptors. Hence, it is generally accepted that
early endosomes represent both the single entry point
for internalized molecules, and the first sorting station
in the pathway.
The early endosome is a dynamic compartment
with a high homotypic fusion capacity1. But its elements display a highly complex and pleiomorphic organization that consists of cisternal regions from which
thin tubules (~ 60 nm diameter) and large vesicles (~
300–400 nm diameter) seem to emanate (FIG. 2). The
vesicles contain membrane invaginations, and are
therefore described as multivesicular — although it is
not clear to what extent these invaginations detach from
the limiting membrane and form free vesicles in the
lumen. Tubular elements closely resemble the tubules of
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Plasma membrane
Recycling
endosome
Early endosome
ECV/MVB
MTOC
Late endosome
Lysosome
Figure 1 | Outline of the endocytic pathway. The main
routes of endocytic membrane transport are indicated, with
the recycling pathway in green and the degradation pathway
in red. Microtubules and the microtubule-organizing centre
(MTOC) are in blue. In higher eukaryotic cells, internalization
of receptors and other cell-surface components occurs
through clathrin-mediated endocytosis, although other less
characterized pathways are also involved. Internalized
molecules are then delivered to early endosomes, where
efficient sorting occurs. Some receptors are recycled back to
the plasma membrane to be reused, at least in part through
recycling endosomes, whereas downregulated receptors are
transported to late endosomes and lysosomes for
degradation. Late endosomes provide the last sorting
station in the pathway, whereas lysosomes are generally
believed to represent the end station. Transport routes also
connect the biosynthetic and endocytic pathways, and are in
particular responsible for the delivery of lysosomal enzymes
and membrane proteins. ECV, endosomal carrier vesicle;
MVB, multivesicular body.
LIPID RAFTS
Dynamic assemblies of
cholesterol and sphingolipids in
the plasma membrane,
probably involved in cell
signalling.
CAVEOLA
Specialized raft that contains
the protein caveolin, and forms
a flask-shaped, cholesterol-rich
invagination of the plasma
membrane that might mediate
the uptake of some extracellular
materials, and is probably
involved in cell signalling.
CAVEOSOME
A recently discovered organelle
that is involved in the
intracellular transport of SV40
from caveolae to the
endoplasmic reticulum.
722
recycling endosomes, and multivesicular elements
resemble the endosomal carrier vesicles/multivesicular
bodies (ECV/MVBs) of the degradation pathway (FIG.
3). These tubular and multivesicular regions can therefore be considered as the trans face of the organelle. It
could be that the central cisternal region functions as
the entry or cis region, receiving incoming vesicles from
the plasma membrane or trans-Golgi network.
It is not easy to imagine how early endosomal
membranes can be shaped into such different structures. Selective changes must occur in the curvature
and organization of the bilayer, for example, during
membrane invagination in nascent ECV/MVBs or
tubule biogenesis2. Morphogenesis in the endocytic
pathway must involve the action of molecular
machines and the segregation of proteins and lipids in
the plane of the membrane.
surface occurs by default. This view is difficult to reconcile with the situation in epithelial cells, in which transcytosed and recycling receptors transit through a common
recycling endosome before being transported to opposite plasma membrane domains3. Lysosomal targeting
signals have been identified, but it is not always clear at
which transport step these operate. Sorting motifs have
been found in the cytoplasmic domains of P-selectin4,
the interleukin-2 (IL-2) receptor β chain5, in the viral
protein Nef during CD4 downregulation6, and in the
epidermal growth factor receptor (EGFR)7, but these
motifs bear little resemblance to each other. Recent studies indicate that ubiquitylation of the cytoplasmic
domain might contribute to lysosomal sorting8,9.
Transient protein monoubiquitylation is also believed to
act at the cell surface as an internalization signal, and it is
possible that a similar mechanism operates in sorting
elsewhere in the cell10.
This apparent lack of recycling signals and diversity
in degradation signals indicates that different sorting
principles might operate in early endosomes. Lipids are
not distributed randomly within endosomal membranes, and different lipid domains might coexist at
each step of the pathway. Protein and lipid sorting — for
example, during biogenesis of tubules and ECV/MVBs
— could be coupled to selective incorporation into specialized protein–lipid environments.
Attractive candidates for early endosomal sorting
functions include LIPID RAFTS, as these are believed to show
sorting functions in the trans-Golgi network and at the
plasma membrane11. Although the fate of rafts in endocytosis is not clear, it has been suggested that cell-surface
rafts can act as internalization platforms12,13, a route presumably followed by downregulated IL-2 receptors on
their way to endosomes and then lysosomes14. In addition, SV40 was recently found to enter cells through CAVEOLAE and then to reside within CAVEOSOMES, which do not
Sorting in early endosomes
Recycled and downregulated receptors are efficiently
sorted from one another in early endosomes. But it has
been particularly difficult to identify sorting signals in
the cytoplasmic domains of cargo proteins. This seems
surprising as such signals have been found for most, if
not all, other transport steps of the biosynthetic and
endocytic pathways. No recycling motif has been identified, leading to the proposal that recycling to the cell
Figure 2 | The early endosome. The figure shows an early
endosome containing low-density lipoprotein (LDL)–gold
particles endocytosed for 5 minutes (gold particles are
visualized as white spots, as contrast was reversed). After
internalization, cells were homogenized, crude fractions
prepared and deposited on mica plates. Samples were
analysed by freeze-etch electron microscopy. (Courtesy of J.
Heuser, Washington University, Missouri, USA).
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GPI ANCHOR
The function of this posttranslational modification is to
attach proteins to the
exoplasmic leaflet of
membranes, possibly to specific
domains therein. The anchor is
made of one molecule of
phosphatidylinositol to which a
carbohydrate chain is linked
through the C-6 hydroxyl of the
inositol, and is linked to the
protein through an
ethanolamine phosphate
moiety.
SNARES
(Soluble N-ethylmaleimidesensitive factor attachment
protein receptor). A family of
membrane-tethered coiled-coil
proteins that regulate fusion
reactions and target specificity
in the vacuolar system. They
can be divided into vesicleSNAREs and target-SNAREs on
the basis of their localization, or
into Q-SNAREs and R-SNAREs
on the basis of a highly
conserved amino acid.
YEAST CLASS E MUTANTS
One class of vacuolar protein
sorting (VPS) mutants in yeast.
Class E genes are involved in the
delivery of both newly
synthesized vacuolar enzyme
carboxypeptidase Y (CPY) and
endocytosed proteins to the
vacuole from the prevacuolar
compartment. Mutations in any
of the class E VPS gene products
causes an accumulation of
cargo in an aberrant endosomelike structure termed the class E
compartment.
Figure 3 | Endosomal carrier vesicles/multivesicular
bodies. ECV/MVBs containing endocytosed glycoprotein-G
of vesicular stomatitis virus (VSV) were purified and the
distribution of G-protein was analysed by immunogold
labelling of cryosections1. (Reproduced from The Journal of
Cell Biology, 1989, 108, 1301–1316 by copyright permission
of The Rockerfeller University Press.)
contain endocytosed tracers15. Whether a functional
relationship exists between caveosomes and endosomes
remains to be shown. Raft components, such as cholesterol and sphingomyelin, are abundant in recycling
endosomes, at least in Chinese-hamster ovary (CHO)
and Madin–Darby canine kidney (MDCK) cells16–18,
indicating that lipid rafts might contribute to endosomal
sorting. Indeed, GPI-ANCHORED proteins, which preferentially partition into rafts at the cell surface, follow the
same route as the rafts themselves in CHO cells18.
Whether raft components show the same distribution in
all cell types is not known. More importantly, it remains
to be shown whether these components actually assemble into rafts within endosomes — endocytosed lipid
analogues with a preference for ordered membrane
domains are targeted to late endosomes/lysosomes,
rather than recycling endosomes, in the same CHO
cells19. Whatever the mechanism and molecular components involved, protein–lipid sorting and membrane
organization seem to be intimately coupled in early
endosomal membranes.
Molecular architecture of early endosomes
In addition to the morphologically visible mosaic of
early endosomal domains, it is becoming apparent
that key components that regulate membrane organization and protein transport are distributed in a nonrandom manner on membranes, defining functional
domains (FIG. 4). Whether vesicles arriving from the
plasma membrane or the trans-Golgi network can
dock and fuse anywhere on these membranes is not
known, but if early endosomes display the cis–trans
polarity discussed above, docking/fusion sites might
not be randomly distributed.
Docking/fusion proteins of the SNARE family have
been identified along both recycling and degradation
pathways20. Although a given SNARE will be most
abundant in a certain compartment, SNAREs
inevitably spread through several compartments during vesicular transport. So, docking/fusion specificity
cannot be solely determined by the distribution of
endosomal SNAREs.
Much effort has been devoted to the study of small
GTPases of the Rab family, which are considered as
organelle markers due to their restricted distribution.
When in the active GTP-bound state, Rab5, which is
involved in early endocytic transport, can interact with
several effectors, such as other Rab proteins21. Through
these interactions, Rab5 probably builds a specific effector platform on the membrane, which could integrate
different mechanisms that regulate transport, including
membrane fusion, membrane budding and interaction
with cytoskeletal components21. Rab5 might also contribute to the spatial organization of docking/fusion
sites for vesicles arriving from different pathways, as its
effector EEA1 can interact with syntaxin 6 (REF. 22),
which has been implicated in the trans-Golgi network
to early endosome transport, and syntaxin-13, which is
required for endosome fusion23 and recycling24.
However, like other key regulators, Rab5 is not only
found in a single compartment, but is also associated
with the plasma membrane, where its activity, perhaps
related to clathrin-coated vesicle formation25, seems to
be regulated by the EGFR signalling pathway26. The
findings that the Rab5 effector rabaptin 5 also interacts
with Rab4 (REF. 27) — which is involved in recycling —
and that the Rab4 effector RABIP4 contains a FYVE
motif and localizes to early endosomes28, raise the possibility that these Rab proteins are functionally coupled,
perhaps reflecting the existence of a physical link
between different platforms. It will be interesting to
determine whether crosstalk between Rab effectors also
exists along other pathways.
Several proteins interact specifically with phosphatidylinositol-3-phosphate (PtdIns(3)P) through a
conserved FYVE motif29, including the Rab5 effectors
EEA1 and rabenosyn-5, which are both required for
early endosome fusion30,31. EEA1 (REFS 16,32,33) — and
perhaps other FYVE proteins28,34,35 — is, in fact, restricted to early endosomes. The presence of both phosphatidylinositol 3-kinase (PI3K) and PtdIns(3)P-binding proteins among Rab5 effectors would facilitate the
formation of microdomains or platforms that contain
Rab5 and PtdIns(3)P on early endosomal membranes21.
Although PtdIns(3)P–FYVE interactions are sufficient
for early endosomal targeting36, the distribution of
FYVE proteins also depends on other components. The
FYVE protein, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) — a homologue of the YEAST
35
CLASS E protein Vps27 — is found on early endosomes ,
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)P2, is essential for
maintenance of normal vacuolar morphology and was
proposed to regulate cargo-selective sorting into the
vacuole lumen38. In addition to its function in early
endosomal dynamics, PtdIns(3)P might also function
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Recycling
Degradation
PtdIns(3)P signalling pathway, FYVE and PHOX proteins
Rab5 and effectors
Endosomal COPs
Annexin II–cholesterol
RETROMER COMPLEX
Protein complex consisting of
Vps35, Vps26, Vps29, Vps17
and Vps5, which was discovered
through genetic screens in
Saccharomyces cerevisiae. It
functions in the retrieval of
proteins from the prevacuolar
compartment and transport to
the Golgi.
COPI COAT
Complex consisting of α-, β-,
β’-, γ-, δ-, ε- and ζ-COP, also
called coatomer. This coat
complex functions in
anterograde transport within
the Golgi and in retrograde
transport from the Golgi to the
endoplasmic reticulum.
COPII COAT
Complex consisting of Sec13,
Sec31, Sec23 and Sec24. This
coat complex functions in
anterograde transport from the
endoplasmic reticulum to the
Golgi.
LDLF CELLS
A mutant Chinese-hamster
ovary cell line that was
identified on the basis of its
defect in low-density
lipoprotein (LDL) transport.
The mutation causing the
phenotype was later identified
as a deletion of ε-COP.
724
Figure 4 | Lipid–protein microdomains and molecular
machines. A schematic early endosome is represented. A
cisternal region is represented with a thick black line,
recycling tubules are in yellow, whereas a forming MVB/ECV
is shown with a black limiting membrane and purple
invaginations. PtdIns(3)P is represented as yellow dots.
Regions of membrane constriction or invagination, which
presumably involve specialized lipids, are circled.
Lipid–protein domains and complexes that might function as
modular elements of early endosomal membranes are
represented. The precise function of PtdIns(3)P signalling and
class E VPS proteins in MVB/ECV formation is not clear in
mammalian cells (BOX 1). MVB/ECV, multivesicular
body/endosomal carrier vesicle.
in the biogenesis of multivesicular endosomes29, underscoring the close connection between early endosome
function and morphogenesis.
Recent studies have also uncovered the existence of
another phosphoinositide-binding domain shared by
several proteins, called the PX- or PHOX-homology
domain. This was first found in p40phox and p47phox —
two subunits of the neutrophil oxidase39,40. The PX
domain of p40phox binds PtdIns(3)P selectively, and the
presence of this domain is required for the endocytic
function and/or localization of the t-SNARE VAM7
(REF. 41) and the sorting nexin SNX3 (REF. 42).
Interestingly, the PX protein, SNX1, interacts with the
FYVE protein Hrs43, and is involved in EGFR degradation44, whereas the SNX1 yeast homologue, Vps5, is a
component of the RETROMER COMPLEX that is necessary for
endosome–trans-Golgi network recycling45. In addition,
SNX3 and SNX15 are involved in endosomal transport,
and their overexpression disrupts endosome morphology42,46. The relationships between PtdIns(3)P and PX
proteins or FYVE proteins are still unclear, but should
be of high interest in understanding the organization of
endosomal membranes.
Other proteins that might function in organizing
functional regions in the membrane are annexins47.
These proteins seem to have the intrinsic ability to selforganize at the membrane surface into bidimensional
ordered arrays48. It is attractive to speculate that annex-
ins, using a variety of membrane-association mechanisms, might form platforms on the membrane of different sub-cellular compartments, which would specifically interact with cytosolic components, including the
cytoskeleton.
Annexin II localizes to the plasma membrane and on
early endosomes, and it was shown to be involved in the
dynamics of early and/or recycling endosomes 49,50.
Annexin II binding to endosomes51, but perhaps not
plasma membrane52, seems to use an unconventional,
cholesterol-dependent — but calcium-independent —
mechanism. Annexin II also interacts with proteins of
the cortical actin cytoskeleton, and it seems to be nonrandomly distributed on early endosomal membranes,
and concentrated in areas from which actin-like filaments seem to emanate51. It is therefore possible that
cholesterol-rich regions of early endosomal membranes
that interact with annexin II are important in the general organization and dynamics of this compartment.
The COPI coat complex regulates retrograde transport
from the Golgi to the endoplasmic reticulum, but several lines of evidence indicate that an endosomal COPI
complex, lacking β- and γ- subunits, functions in endosomal transport53 — in particular in ECV/MVB biogenesis54 and Nef-mediated CD4 downregulation6. COPI
has also been implicated in transport from the phagosomal membrane55. Like biosynthetic COPI, endosomal
COPI interacts with membranes through the small
GTPase, ADP-ribosylation factor 1 (ARF1) (REF. 56).
However, recruitment of ARF1 and COPI onto endosomes, but not biosynthetic membranes, depends on the
acidic lumenal pH, possibly through a pH sensor54,56, as
does binding of ARF6 and the ARF 57 exchange factor,
ARNO. As is the case for other coat components, including COPII, as well as clathrin and some of its partners58,
COPI can also interact with membrane lipids59. Such
weak interactions could facilitate membrane targeting
and association, and concentrate coat components
locally.
The close relationship between early endosome functions in transport and morphogenesis is also apparent in
COPI functions. COPI inactivation in LDLF CELLS with a
temperature sensitive-defect in ε-COP60 inhibits transport to late endosomes and interferes with transferrin
receptor recycling61, but not with bulk62 recycling, and
also disrupts early endosomes62. These then become
tubular or cisternal clusters without multivesicular
domains — a phenotype reminiscent of class E VPS
mutants in yeast63. Identical perturbations are caused by
neutralization of the endosomal pH54,64,65, which inhibits
ARF1 and COPI binding.
Tubules of the recycling endosome
The two main circuits of recycling and degradation are
well separated, both topologically and functionally, to
ensure that proteins that need to be reused at the cell
surface remain separate from those that are destined to
be degraded (FIG. 1). However, compartment boundaries
along the recycling or degradation pathways seem
blurred at the molecular level, in particular when following a single component. It is probably the interplay
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LBPA
Lysobisphosphatidic acid
(LBPA) is a phospholipid,
structurally analogous with
phosphatidylglycerol. LBPA is
poorly degradable, presumably
because of its unusual
stereoconfiguration, and is
abundant within internal
membranes of late endosomes.
between key components that defines the functional
boundaries in each pathway.
After leaving early endosomes, recycling molecules
are found in distinct tubular structures that correspond to recycling endosomes. These do not contain
ligands and receptors that are destined to be degraded,
are less acidic than early endosomes, and are found
close to the centrioles in some, but not all, cell
types16,66,67. These tubules form dynamic networks68
and are collectively referred to as recycling endosomes.
But the distinction between transport intermediates
and compartments is not clear.
Early endosomal Rab5 and two small GTPases
involved in recycling, Rab4 and Rab11, show a distinct,
but partially overlapping, distribution in vivo, which
presumably corresponds to different effector
platforms69. However, the precise functions of Rab4 and
Rab11 are not clear. SNARE family members have been
found in recycling endosomes and were reported to
function in the pathway, including the v-SNAREs cellubrevin70 and endobrevin/VAMP8 in the apical pathway
of 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 transport steps to the plasma membrane, each with a distinct
molecular machinery; for example, from early endosomes or from recycling endosomes (FIG. 1).
Alternatively, fast and slow transport could reflect the
existence of a gradient of molecules on their way to the
cell surface within early and recycling endosomes. This
issue is not easily addressed. Drugs or expression of
mutant proteins might unbalance transport along
either route, perhaps explaining why recycling kinetics
are hardly affected by microtubule depolymerization,
although the pericentriolar distribution of the recycling
endosome depends on microtubules.
Transport along the recycling pathway depends on
the actin cytoskeleton and unconventional myosin
motors76–78, which might have a mechanical role in
tubule biogenesis and dynamics. Crosstalk between
transport, actin remodelling and Rac-mediated signalling could depend on the small GTPase ARF6 and its
possible partners79–82. Clathrin-coated buds have been
observed on recycling endosomes83, but whether they
mediate transport back to the cell surface83 or to the
trans-Golgi network remains to be fully established84. As
clathrin coats usually have sorting functions, their presence on recycling endosomes strengthens the view that
protein sorting occurs within recycling endosomes. A
genetic screen for endocytosis mutants in
Caenorhabditis elegans recently identified RME-1 (REFS
85,86) — a new member of the conserved family of
Eps15-homology (EH)-domain proteins87, which show
characteristics of an endocytic accessory protein. RME1 is associated with recycling endosomes and might be
involved in the exit of membrane proteins from this
compartment. Beyond these observations, little is
known about the molecular mechanisms that drive the
dynamics of tubular recycling endosomes.
Transport in the degradation pathway
Although 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 mammalian
cells, ECV/MVBs are clearly distinct from both early
and late endosomes. In particular, ECV/MVBs do not
contain early endosome-specific proteins or recycling
receptors, nor do they contain the main lipid and protein constituents of late endosomal membranes. Once
formed, ECV/MVBs move towards late endosomes in a
microtubule- and motor-dependent fashion, and
acquire the capacity to dock onto and fuse selectively
with late endosomes, a step which depends on the conventional docking/fusion machinery20,21,88. In axons,
ECV/MVBs also function as intermediates from early
endosomes at the presynaptic membrane to late endosomes in the cell body 67. Whether ECV/MVBs change
in composition as they undergo a maturation process,
or whether they mediate transport between two stable
compartments, has been the subject of much debate,
and so far, it has not been possible to solve this issue by
following single components at the boundary between
early and late endosomes.
ECV/MVBs, like late endosomes, contain two morphologically visible membrane domains, internal
invaginations and a limiting membrane. The composition of the ECV/MVB limiting membrane is not
known, except that it seems to lack early endosomal
proteins, and is different from the late endosomal membrane, which contains high amounts of LAMP1.
Similarly, little is known about the composition of
ECV/MVB invaginations. They accumulate downregulated receptors, in particular the EGFR89, which has led
to the idea that internal membranes are destined to be
degraded in lysosomes — a view partially challenged in
mammalian cells by recent observations (see the next
section). Electron-microscopy studies showed that
PtdIns(3)P is abundant within ECV/MVB internal
membranes, in addition to early endosomes, and that
it is not detected within late endosomes that contain
36
LYSOBISPHOSPHATIDIC ACID (LBPA) . However, it is not
clear whether the presence of PtdIns(3)P on these internal membranes reflects a role for this lipid in the biogenesis of invaginations, or simply the metabolism of
the lipid.
Wortmannin, a drug that inhibits PI(3)K was
reported to inhibit multivesicular body formation in
mammalian cells90. At the same time, evidence is accumulating in yeast that signalling through the PI(3)K
Vps34 and PtdIns(3)P is crucial in vacuole transport of
the trans-Golgi network29. Considering the close relationship that exists between ECV/MVB biogenesis and
early endosome organization, we might expect some of
the components that regulate early endosomal functions to also be important in the invagination process.
In addition, yeast class E VPS mutants are all defective
in MVB biogenesis, as they form stacks of tubules or
curved cisternae63, perhaps in a ‘frustrated invagination’
process. Several class E mutants have a mammalian
homologue that has been implicated in transport,
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Box 1 | The endocytic pathways in yeast and mammals
A degree of caution is needed when extrapolating from mammalian to yeast endocytic
pathways and vice versa, with respect to the specific structure and organization of
endosomal membranes. One of the most striking features of mammalian endocytosis,
without discussing features of polarized or other differentiated cells, is the capacity to
reuse cell-surface components efficiently and rapidly through recycling routes;
whereas equivalent recycling routes, if they exist, do not seem to be as efficient in yeast
cells. Although extensive sorting already occurs in early endosomes of mammalian
cells, it has been proposed that a prevacuolar compartment equivalent to late
endosomes functions as the main sorting station in the yeast pathway94,128. In
addition, yeast cells do not seem to share the striking appearance of mammalian
endosomes within the degradation pathway, with a complex network of membrane
invaginations. In yeast cells, small and regularly shaped vesicles can be observed in the
vacuole of strains with impaired vacuolar hydrolase activity129. In addition to the
basic machinery of yeast cells, mammalian cells are likely to have evolved a more
elaborate endosomal membrane system to ensure optimal regulation and reutilization
of proteins and lipids.
TETRASPANIN FAMILY
The tetraspanin family contains
proteins that span the
membrane four times with two
exoplasmic loops, and that can
be found at the cell surface.
Although some are highly
restricted to specific tissues,
others are widely distributed.
Members of this family have
been implicated in cell
activation and proliferation,
adhesion, motility,
differentiation and cancer.
ANTIGEN-PRESENTING CELLS
A cell, most often a B
lymphocyte, macrophage or
dendritic cell, that is specialized
in the generation of epitopes
that are presented through
major histocompatibility
complex (MHC) class I or II to
T lymphocytes.
DENDRITIC CELLS
‘Professional’ antigenpresenting cells found in T-cell
areas of lymphoid tissues, but
also as a minor cellular
component in most tissues.
They have a branched or
dendritic morphology and are
the most potent stimulators of
T-cell responses.
MANNOSE 6-PHOSPHATE
RECEPTOR
These receptors transport
soluble lysosomal hydrolases to
late endosomes by cycling
between the trans-Golgi
network and late endosomes.
They bind in the trans-Golgi
network to mannose 6phosphate moieties on Nlinked glycans of the hydrolases.
They release the hydrolases in
late endosomes and return to
the trans-Golgi network for
another round of transport.
726
including the Vps27 (REF. 91), Vps23 (REF. 92) and the
Vps4 homologues (REF. 93), indicating that the function
of these proteins might be conserved in mammalian
cells (BOX 1). Whereas there is compelling evidence in
yeast that class E proteins have an essential role in the
endocytic pathway, their precise functions remain to be
established. It has been proposed that the yeast class E
compartment — or prevacuolar compartment — corresponds to a late endosome94, but its mammalian
counterpart remains mysterious (BOX 1). In mammalian
cells, a phenotype that is morphologically similar to the
class E phenotype has not been well characterized, with
the possible exception of early endosome after COP
inactivation in LDLF cells62, or Hrs disruption in mice91.
The pomegranate and the onion
Much like early endosomes, late endosomal elements
are very dynamic95,96, with a highly complex and
pleiomorphic organization, containing cisternal, tubular and vesicular regions with numerous membrane
invaginations97. Their limiting membrane, similar to
that of lysosomes, contains high amounts of LAMP1,
which is believed to be protected from the degradative
milieu of the compartment because of its high glycosylation state97. The limiting membrane of late endosomes
also contains MLN64, a homologue of the mitochondrial
steroidogenic acute regulatory protein (StAR)98.
Internal membranes in higher eukaryotic cells undergo
much remodelling and accumulate large amounts (~
15% of total phospholipids) of LBPA99, which is a poor
substrate for phospholipase and therefore resistant to
degradation100. LBPA has not been detected on the outer
face of the limiting bilayer101,102. To what extent the biogenesis and dynamics of late endosome inner membranes are regulated by the same mechanisms as those
operating during budding of ECV/MVBs is not clear.
LBPA is presumably synthesized in situ103, and has an
inverted cone shape. This structure could facilitate the
formation of the invaginations that form the multivesicular elements of late endosomes101.
Proteins that are destined to be degraded accumulate within ECV/MVB internal membranes, leading to
the idea that the fate of internal membranes is degradation in lysosomes. Sandhoff and collaborators104 have
demonstrated, using an in vitro liposome assay, that negatively charged phospholipids, in particular LBPA, greatly facilitate the degradation of several glycolipids. As
LBPA itself is poorly degradable, one function of LBPA
membranes could be to present lipids and proteins that
need to be degraded to the hydrolytic machinery.
However, internal membranes of late endosomes
do not only contain proteins that are destined to be
degraded. Members of the TETRASPANIN FAMILY, including
CD63/LAMP3, have been shown to accumulate within
internal membranes105, in addition to their presence on
the cell surface. In ANTIGEN-PRESENTING CELLS, major histocompatibility complex (MHC) class II compartments
(MIIC) share most characteristics of late endosomes
and lysosomes, including a complex system of internal
membranes106, although they are specialized compartments that contain specific components, such as the
cathepsin S protease required for antigen processing107
and the human leukocyte antigen DM (HLA–DM)
complex108. MHC class II molecules are abundant in
MIIC internal membranes, both in B lymphocytes and
in immature DENDRITIC CELLS. MHC class II molecules
colocalize with LBPA in B lymphocytes, and they are
also found in LBPA-containing microvesicles, presumably secreted by antigen-presenting cells109. Finally,
MANNOSE 6-PHOSPHATE RECEPTOR (M6PR) molecules are
transported between the trans-Golgi network and late
endosomes to deliver lysosomal enzymes to endosomes and lysosomes. While in transit through late
endosomes, M6PR is found predominantly within
internal membranes97,99.
LBPA membranes probably have an important
function in transport through late endosomes. When
endocytosed in vivo, antibodies against LBPA accumulate in late endosomes, and specifically inhibit M6PR
transport99. Conversely, loss of M6PR expression promotes LBPA accumulation in multilamellar bodies110.
Endocytosed anti-LBPA antibodies also cause cholesterol accumulation in late endosomes, mimicking the
cholesterol-storage disorder Niemann–Pick type C
(NPC), leading to the idea that LBPA membranes serve
as a collecting and distribution device for low-density
lipoprotein (LDL)-derived cholesterol111. In turn, cholesterol accumulation in late endosomes interferes with
the M6PR cycle in several cell types111,112, and also with
CD63 and p-selectin cycling to WEIBEL–PALADE bodies in
endothelial cells113. Sphingolipids accumulate in multivesicular compartments in tissue from NPC patients,
and, conversely, cholesterol accumulates in late endocytic compartments in several sphingolipid storage
disorders114,115.
The function of LBPA membranes in protein and
lipid transport indicates that some membrane proteins
and lipids do not only enter internal membranes, but
could subsequently exit and return to other cellular
destinations. Incorporation within internal membranes can be explained easily by the selective partitioning of some molecules within LBPA invaginations,
or the selective exclusion from the limiting membrane.
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WEIBEL–PALADE BODIES
Morphologically unique
secretory structures of
endothelial cells, which store
von Willebrand factor — a
protein involved in blood
clotting — for eventual release.
If so, export from late endosomes should be slow, as
internal membranes are abundant, and only molecules
that are present on the limiting membranes would be
available for transport (FIG. 5) — this could account for
the slow cycle of CD63 in endothelial cells113. Other
mechanisms, however, must be evoked to account for
efficient export — for example, M6PR transport or
MHC class II export to the plasma membrane in activated dendritic cells. One possibility is that transport
between the internal and the limiting membranes is
regulated by several mechanisms, including posttranslational modifications and, in particular, ubiquitylation9. It is also possible that late endosomes in
higher eukaryotic cells might contain more than one
type of internal membrane, such as those specialized
in protein degradation or recycling. This is consistent
with the finding that ECV/MVBs and late endosomes
differ in their LBPA content, although both have
abundant internal membranes. Moreover, late endocytic compartments in mammalian cells can appear
like a pomegranate (multivesicular) or like an onion
(multilamellar). Essentially nothing is known about
the functional significance of these different types of
inner membranes, or about the differences in their
biophysical state, organization and composition. It is
tempting to speculate that, in addition to the basic
machinery that is responsible for the formation of
intralumenal vesicles in yeast — which could depend
on class E VPS proteins and PtdIns(3)P signalling38 —
mammalian cells have evolved more efficient recycling
mechanisms, perhaps LBPA dependent, for proteins
that need to be exported from internal membranes of
late endocytic compartments.
The end point or a dynamic network?
If it has been difficult to draw the line between early and
late endosomes, the boundary between late endosomes
and lysosomes is even more elusive, despite much work
from many groups. Both compartments contain lysosomal enzymes, their pH is similarly acidic (~ 5.5), and
their limiting membrane is primarily composed of the
same glycoproteins. Lysosomes can only be identified
by their physical properties on gradients and their electron-dense appearance, and by the fact that they lack
proteins found in late endosomes, including M6PR in
transit, Rab7 and Rab9, or phosphorylated hydrolase
precursors116. So far, neither proteins nor lipids have
been found that would only be present in lysosomes,
but not in endosomes, and the basic docking/fusion
machinery seems to be shared by both compartments20,117,118. Moreover, late endosomes and lysosomes
can exchange content and membrane proteins rapidly
and efficiently, and they probably interact dynamically
to form a hybrid intermediate119,120.
It is tempting to speculate that late endosomes and
lysosomes correspond to separate elements of a common, but dynamic, network. Such a sub-compartmentalization into regions and membrane domains that differ both structurally and functionally is reminiscent of
the early endosome organization. Highly motile tubular
lysosomes in macrophages121 might represent a more
A
1
2
?
3
B
Figure 5 | Degradation or recycling? The different
mechanisms that could account for protein recycling from
late endosome internal membranes are represented. A
canonical vesicle (A) delivers proteins, which will be
incorporated into the limiting membrane of late endosomes
(yellow), or into internal membrane invaginations (green and
blue). The latter proteins are destined to be degraded (green)
or recycled (blue). Proteins destined to be degraded could
simply partition preferentially into membrane invaginations
containing LBPA (red), or might be excluded from the limiting
membrane. The same mechanism might apply to some
proteins that need to be recycled (1). However, other
mechanisms must be evoked to account for efficient
incorporation of some proteins into vesicles budding from
late endosomes (B), including regulation by post-translational
modifications (2) or by the existence of more than one type of
internal membrane (3).
stable or extreme version of this network, which is consistent with the fact that they share some late endosomal
characteristics122,123. LBPA membranes could have turnpike functions in the network, as they seem to be
involved both in protein and lipid transport99,111, and
lipid degradation104. Other specialized regions might
exist. These could account for the idea that different late
endosome populations sort and package cargo molecules through Rab9–TIP47 or through the retromer
complex124, and also for the presence of specialized late
endosomes or lysosomes in highly differentiated cells,
including MIIC in antigen-presenting cells106.
Cholesterol export might occur in specialized elements, as the NPC1 protein — which is involved in
intracellular cholesterol transport — seems to distribute
to a subset of lysosomes125. Transport defects in
NPC101,111, and perhaps other lipid-storage disorders115,
could result from a collapse of the mosaic architecture
of endosomes, leading to mixing of lipid–protein
domains and eventually transport inhibition114.
Conclusions
Although individual machines and regulatory components in the endocytic pathway might be loosely
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
VOLUME 2 | OCTOBER 2001 | 7 2 7
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REVIEWS
distributed throughout several compartments, a certain
combination is unique to each compartment. Early endosomes, for example, contain Rab5 and its effectors,
PtdIns(3)P and partners, endosomal COPs, raft components and annexin II, which all function in transport
through this compartment. Each of these molecules is also
found on other membranes, but this particular combination is present only on early endosomes. It is attractive to
speculate that endosomal membranes, and probably the
membranes of other organelles, are built from modular
elements. This could explain the difficulties encountered
in defining organelle boundaries at the molecular level,
particularly when tracking a single component.
An important challenge will be to understand how
these different membrane machines and dynamic
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
728
Gruenberg, J., Griffiths, G. & Howell, K. E. Characterisation
of the early endosome and putative endocytic carrier
vesicles in vivo and with an assay of vesicle fusion in vitro.
J. Cell Biol. 108, 1301–1316 (1989).
Mukherjee, S. & Maxfield, F. R. Role of membrane
organization and membrane domains in endocytic lipid
trafficking. Traffic 1, 203–211 (2000).
Apodaca, G., Katz, L. A. & Mostov, K. E. Receptormediated transcytosis of IgA in MDCK cells is via apical
recycling endosomes. J. Cell Biol. 125, 67–86 (1994).
Together with reference 95 and studies from the Kai
Simons group, this paper shows the organization of
the apical and basolateral endocytic pathways in
epithelial cells along the degradation, recycling and
transcytotic routes.
Blagoveshchenskaya, A. D., Norcott, J. P. & Cutler, D. F.
Lysosomal targeting of P-selectin is mediated by a novel
sequence within its cytoplasmic tail. J. Biol. Chem. 273,
2729–2737 (1998).
Subtil, A., Rocca, A. & Dautry-Varsat, A. Molecular
characterization of the signal responsible for the targeting
of the interleukin 2 receptor β chain toward intracellular
degradation. J. Biol. Chem. 273, 29424–29429 (1998).
Piguet, V. et al. Nef-induced CD4 degradation: a diacidicbased motif in Nef functions as a lysosomal targeting
signal through the binding of β-COP in endosomes. Cell
97, 63–73 (1999).
Kil, S. J. & Carlin, C. EGF receptor residues Leu679, Leu680
mediate selective sorting of ligand-receptor complexes in
early endosomal compartments. J. Cell Physiol. 185,
47–60 (2000).
Rocca, A., Lamaze, C., Subtil, A. & Dautry-Varsat, A.
Involvement of the ubiquitin/proteasome system in sorting
of the interleukin 2 receptor β chain to late endocytic
compartments. Mol. Biol. Cell 12, 1293–1301 (2001).
Lemmon, S. K. & Traub, L. M. Sorting in the endosomal
system in yeast and animal cells. Curr. Opin. Cell Biol. 12,
457–466 (2000).
Hicke, L. Protein regulation by monoubiquitin. Nature Rev.
Mol. Cell Biol. 2, 195–201 (2001).
Ikonen, E. Roles of lipid rafts in membrane transport. Curr.
Opin. Cell Biol. 13, 470–477 (2001).
Parton, R. G., Joggerst, B. & Simons, K. Regulated
internalization of caveolae. J. Cell Biol. 127, 1199–1215
(1994).
Provides the first evidence that caveolae can be
endocytosed in non-endothelial cells.
Verkade, P., Harder, T., Lafont, F. & Simons, K. Induction of
caveolae in the apical plasma membrane of Madin–Darby
canine kidney cells. J. Cell Biol. 148, 727–739 (2000).
Lamaze, C. et al. Interleukin 2 receptors and detergentresistant membrane domains define a clathrinindependent endocytic pathway. Mol. Cell 7, 661–671
(2001).
A receptor known to follow the classical endocytic
pathway is shown to be internalized by a mechanism
— perhaps raft-dependent — that does not depend
on the clathrin machinery.
Pelkmans, L., Kartenbeck, J. & Helenius, A. Caveolar
endocytosis of simian virus 40 reveals a new two-step
vesicular-transport pathway to the ER. Nature Cell Biol. 3,
473–483 (2001).
Gagescu, R. et al. The recycling endosome of MDCK cells
is a mildly acidic compartment rich in raft components.
domains are integrated to support protein and lipid
transport, as well as organelle structure and functions.
Some clues are being provided from recent studies that
show the existence of a close interplay between signalling and transport pathways through protein networks126. Moreover, several components discussed in
this article are linked to human diseases and involved in
development127. We might expect that beyond protein
complexes and molecular machines with well defined
roles, organellar functions also require specific sets of
effectors that mediate cross-interactions and thereby
articulate a modular combination. These could control
the formation and maintenance of different membrane
domains, more or less transient in nature, providing the
architectural framework for a given compartment.
Mol. Biol. Cell 11, 2775–2791 (2000).
17. Mukherjee, S., Zha, X., Tabas, I. & Maxfield, F. R.
Cholesterol distribution in living cells: fluorescence imaging
using dehydroergosterol as a fluorescent cholesterol
analog. Biophys. J. 75, 1915–1925 (1998).
18. Mayor, S., Sabharanjak, S. & Maxfield, F. R. Cholesteroldependent retention of GPI-anchored proteins in
endosomes. EMBO J. 17, 4626–4638 (1998).
19. Mukherjee, S., Soe, T. T. & Maxfield, F. R. Endocytic sorting
of lipid analogues differing solely in the chemistry of their
hydrophobic tails. J. Cell Biol. 144, 1271–1284 (1999).
Shows that hydrophobic tails are important for lipid
sorting in early endosomes.
20. Chen, Y. A. & Scheller, R. H. SNARE-mediated membrane
fusion. Nature Rev. Mol. Cell Biol. 2, 98–106 (2001).
21. Zerial, M. & McBride, H. Rab proteins as membrane
organizers. Nature Rev. Mol. Cell Biol. 2, 107–117 (2001).
Following the work of Zerial and his group with Rab5
and its effectors, this review presents and discusses
the concept of Rab platforms.
22. Simonsen, A., Gaullier, J. M., D’Arrigo, A. & Stenmark, H.
The RAB5 effector EEA1 interacts directly with syntaxin-6.
J. Biol. Chem. 274, 28857–28860 (1999).
23. McBride, H. M. et al. Oligomeric complexes link RAB5
effectors with NSF and drive membrane fusion via
interactions between EEA1 and syntaxin 13. Cell 98,
377–386 (1999).
24. Prekeris, R., Klumperman, J., Chen, Y. A. & Scheller, R. H.
Syntaxin 13 mediates cycling of plasma membrane
proteins via tubulovesicular recycling endosomes. J. Cell
Biol. 143, 957–971 (1998).
25. McLauchlan, H. et al. A novel role for RAB5-GDI in ligand
sequestration into clathrin-coated pits. Curr. Biol. 8, 34–45
(1998).
26. Lanzetti, L. et al. The EPS8 protein coordinates EGF
receptor signalling through Rac and trafficking through
RAB5. Nature 408, 374–377 (2000).
27. Stenmark, H., Vitale, G., Ullrich, O. & Zerial, M. Rabaptin-5
is a direct effector of the small GTPase RAB5 in endocytic
membrane fusion. Cell 83, 423–432 (1995).
28. Cormont, M., Mari, M., Galmiche, A., Hofman, P. & Le
Marchand-Brustel, Y. A FYVE-finger-containing protein,
RABIP4, is a RAB4 effector involved in early endosomal
traffic. Proc. Natl Acad. Sci. USA 98, 1637–1642 (2001).
29. Simonsen, A., Wurmser, A. E., Emr, S. D. & Stenmark, H.
The role of phosphoinositides in membrane transport.
Curr. Opin. Cell Biol. 13, 485–492 (2001).
30. Simonsen, A. et al. EEA1 links PI(3)K function to RAB5
regulation of endosome fusion. Nature 394, 494–498
(1998).
31. Nielsen, E. et al. Rabenosyn-5, a novel RAB5 effector, is
complexed with hVPS45 and recruited to endosomes
through a FYVE finger domain. J. Cell Biol. 151, 602–612
(2000).
32. Mu, F.-T. et al. EEA1, an early endosome-associated
protein. EEA1 is a conserved α-helical peripheral
membrane protein flanked by cysteine ‘fingers’ and
contains a calmodulin-binding IQ motif. J. Biol. Chem.
270, 13503–13511 (1994).
33. Verges, M., Havel, R. J. & Mostov, K. E. A tubular
endosomal fraction from rat liver: biochemical evidence of
receptor sorting by default. Proc. Natl Acad. Sci. USA 96,
10146–10151 (1999).
34. Ito, K. et al. Molecular cloning of a novel 130-kDa
| OCTOBER 2001 | VOLUME 2
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
cytoplasmic protein, Ankhzn, containing Ankyrin repeats
hooked to a zinc finger motif. Biochem. Biophys. Res.
Commun. 257, 206–213 (1999).
Komada, M., Masaki, R., Yamamoto, A. & Kitamura, N.
Hrs, a tyrosine kinase substrate with a conserved double
zinc finger domain, is localized to the cytoplasmic surface
of early endosomes. J. Biol. Chem. 272, 20538–20544
(1997).
Gillooly, D. J. et al. Localization of phosphatidylinositol 3phosphate in yeast and mammalian cells. EMBO J. 19,
4577–4588 (2000).
Hayakawa, A. & Kitamura, N. Early endosomal localization
of hrs requires a sequence within the proline- and
glutamine-rich region but not the FYVE finger. J. Biol.
Chem. 275, 29636–29642 (2000).
Odorizzi, G., Babst, M. & Emr, S. D. FAB1p PtdIns(3)P 5kinase function essential for protein sorting in the
multivesicular body. Cell 95, 847–858 (1998).
This paper describes the mechanisms that control
incorporation of proteins within the yeast
endosome/vacuole lumen, and the role of PtdIns(3)P
signalling at this step.
Kanai, F. et al. The PX domains of p47phox and p40phox
bind to lipid products of PI(3)K. Nature Cell Biol. 3,
675–678 (2001).
Ellson, C. D. et al. PtdIns(3)P regulates the neutrophil
oxidase complex by binding to the PX domain of p40phox.
Nature Cell Biol. 3, 679–682 (2001).
Cheever, M. L. et al. Phox domain interaction with
PtdIns(3)P targets the Vam7 t-SNARE to vacuole
membranes. Nature Cell Biol. 3, 613–618 (2001).
Xu, Y., Hortsman, H., Seet, L., Wong, S. H. & Hong, W.
SNX3 regulates endosomal function through its PXdomain-mediated interaction with PtdIns(3)P. Nature Cell
Biol. 3, 658–666 (2001).
Chin, L. S., Raynor, M. C., Wei, X., Chen, H. Q. & Li, L. Hrs
interacts with sorting nexin 1 and regulates degradation of
epidermal growth factor receptor. J. Biol. Chem. 276,
7069–7078 (2001).
Kurten, R. C., Cadena, D. L. & Gill, G. N. Enhanced
degradation of EGF receptors by a sorting nexin, SNX1.
Science 272, 1008–1010 (1996).
Seaman, M. N., McCaffery, J. M. & Emr, S. D. A
membrane coat complex essential for endosome-to-Golgi
retrograde transport in yeast. J. Cell Biol. 142, 665–681
(1998).
Barr, V. A., Phillips, S. A., Taylor, S. I. & Haft, C. R.
Overexpression of a novel sorting nexin, SNX15, affects
endosome morphology and protein trafficking. Traffic 1,
904–916 (2000).
Gerke, V. & Moss, S. E. Annexins and membrane
dynamics. Biochim. Biophys. Acta 1357, 129–154 (1997).
Oling, F., Bergsma-Schutter, W. & Brisson, A. Trimers,
dimers of trimers, and trimers of trimers are common
building blocks of Annexin a5 two-dimensional crystals. J.
Struct. Biol. 133, 55–63 (2001).
Shows that annexins tend to self-organize into
bidimensional ordered arrays.
Harder, T. & Gerke, V. The subcellular distribution of early
endosomes is affected by the Annexin II(2) p11(2)
complex. J. Cell Biol. 123, 1119–1132 (1993).
Emans, N. et al. Annexin II is a major component of
fusogenic endosomal vesicles. J. Cell Biol. 120,
1357–1370 (1993).
www.nature.com/reviews/molcellbio
© 2001 Macmillan Magazines Ltd
REVIEWS
51. Harder, T., Kellner, R., Parton, R. & Gruenberg, J. Specific
release of membrane bound Annexin II and cortical
cytoskeletal elements by sequestration of membrane
cholesterol. Mol. Biol. Cell 8, 533–545 (1997).
52. Babiychuk, E. B. & Draeger, A. Annexins in cell membrane
dynamics. Ca2+-regulated association of lipid
microdomains. J. Cell Biol. 150, 1113–1124 (2000).
53. Whitney, J. A., Gomez, M., Sheff, D., Kreis, T. E. &
Mellman, I. Cytoplasmic coat proteins involved in
endosome function. Cell 83, 703–713 (1995).
54. Aniento, F., Gu, F., Parton, R. & Gruenberg, J. An
endosomal bcop is involved in the pH-dependent
formation of transport vesicles destined for late
endosomes. J. Cell Biol. 133, 29–41 (1996).
References 53 and 54 show that an endosomal
version of the COPI coat — which regulates
retrograde transport in the early biosynthetic
pathway — functions in the early endocytic pathway.
55. Botelho, R. J., Hackam, D. J., Schreiber, A. D. & Grinstein,
S. Role of COP1 in phagosome maturation. J. Biol. Chem.
275, 15717–15727 (2000).
56. Gu, F. & Gruenberg, J. ARF1 regulates pH-dependent
COP functions in the early endocytic pathway. J. Biol.
Chem. 275, 8154–8160 (2000).
57. Maranda, B. et al. Intra-endosomal pH-sensitive
recruitment of the ARF-nucleotide exchange factor ARNO
and ARF6 from cytoplasm to proximal tubule endosomes.
J. Biol. Chem. 276, 18540–18550 (2001).
58. Kirchhausen, T. Three ways to make a vesicle. Nature Rev.
Mol. Cell Biol. 1, 187–198 (2000).
59. Spang, A., Matsuoka, K., Hamamoto, S., Schekman, R. &
Orci, L. Coatomer, ARF1p, and nucleotide are required to
bud coat protein complex I-coated vesicles from large
synthetic liposomes. Proc. Natl Acad. Sci. USA 95,
11199–11204 (1998).
60. Hobbie, L., Fisher, A. S., Lee, S., Flint, A. & Krieger, M.
Isolation of three classes of conditional lethal Chinese
hamster ovary cell mutants with temperature-sensitive
defects in low density lipoprotein receptor. J. Biol. Chem.
269, 20958–20970 (1994).
61. Daro, E., Sheff, D., Gomez, M., Kreis, T. & Mellman, I.
Inhibition of endosome function in CHO cells bearing a
temperature-sensitive defect in the coatomer (COP1)
component epsilon-COP. J. Cell Biol. 139, 1747–1759
(1997).
62. Gu, F., Aniento, F., Parton, R. & Gruenberg, J. Functionnal
dissection of COP1 subunits in the biogenesis of
multivesicular endosomes. J. Cell Biol. 139, 1183–1195
(1997).
63. Rieder, S. E., Banta, L. M., Kohrer, K., McCaffery, J. M. &
Emr, S. D. Multilamellar endosome-like compartment
accumulates in the yeast Vps28 vacuolar protein sorting
mutant. Mol. Biol. Cell 7, 985–999 (1996).
64. Clague, M., Urbé, S., Aniento, F. & Gruenberg, J. Vacuolar
ATPase activity is required for endosomal carrier vesicle
formation. J. Biol. Chem. 269, 21–24 (1994).
65. Presley, J. F., Mayor, S., McGraw, T. E., Dunn, K. W. &
Maxfield, F. R. Bafilomycin A1 treatment retards transferrin
receptor recycling more than bulk membrane recycling. J.
Biol. Chem. 272, 13929–13936 (1997).
66. Yamashiro, D. J., Tycko, B., Fluss, S. R. & Maxfield, F. R.
Segregation of transferrin to a mildly acidic (pH 6.4) paraGolgi compartment in the recycling pathway. Cell 37,
789–800 (1984).
Provides the initial characterization of recycling
endosomes.
67. Parton, R. G., Simons, K. & Dotti, C. G. Axonal and
dentritic endocytic pathways in cultured neurons. J. Cell
Biol. 119, 123–137 (1992).
68. Hopkins, C. R., Gibson, A., Shipman, M. & Miller, K.
Movement of internalized ligand-receptor complexes along
a continuous endosomal reticulum. Nature 346, 335–339
(1990).
69. Sonnichsen, B., De Renzis, S., Nielsen, E., Rietdorf, J. &
Zerial, M. Distinct membrane domains on endosomes in
the recycling pathway visualized by multicolor imaging of
RAB4, RAB5, and RAB11. J. Cell Biol. 149, 901–914
(2000).
70. Galli, T. et al. Tetanus toxin-mediated cleavage of
cellubrevin impairs exocytosis of transferrin receptorcontaining vesicles in CHO cells. J. Cell Biol. 125,
1015–1024 (1994).
71. Steegmaier, M., Lee, K. C., Prekeris, R. & Scheller, R. H.
SNARE protein trafficking in polarized MDCK cells. Traffic
1, 553–560 (2000).
72. Besterman, J. M., Airhart, J. A., Woodworth, R. C. & Low,
R. B. Exocytosis of pinocytosed fluid in cultured cells:
kinetic evidence for rapid turnover and compartmentation.
J. Cell Biol. 91, 716–727 (1981).
73. Dunn, K. W., McGraw, T. E. & Maxfield, F. R. Iterative
fractionation of recycling receptors from lysosomally
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
destined ligands in an early sorting endosome. J. Cell Biol.
109, 3303–3314 (1989).
Sheff, D. R., Daro, E. A., Hull, M. & Mellman, I. The
receptor recycling pathway contains two distinct
populations of early endosomes with different sorting
functions. J. Cell Biol. 145, 123–139 (1999).
Hao, M. & Maxfield, F. R. Characterization of rapid
membrane internalization and recycling. J. Biol. Chem.
275, 15279–15286 (2000).
Apodaca, G. Endocytic traffic in polarized epithelial cells:
role of the actin and microtubule cytoskeleton. Traffic 2,
149–159 (2001).
Durrbach, A., Raposo, G., Tenza, D., Louvard, D. &
Coudrier, E. Truncated brush border Myosin I affects
membrane traffic in polarized epithelial cells. Traffic 1,
411–424 (2000).
Huber, L. et al. Both calmodulin and the unconventional
myosin MYR4 regulate membrane trafficking along the
recycling pathway of MDCK cells. Traffic 1, 494–503
(2000).
Al-Awar, O., Radhakrishna, H., Powell, N. N. & Donaldson,
J. G. Separation of membrane trafficking and actin
remodeling functions of ARF6 with an effector domain
mutant. Mol. Cell Biol. 20, 5998–6007 (2000).
D’Souza-Schorey, C., Boshans, R. L., McDonough, M.,
Stahl, P. D. & Van Aelst, L. A role for POR1, a Rac1interacting protein, in ARF6-mediated cytoskeletal
rearrangements. EMBO J. 16, 5445–5454 (1997).
Tarricone, C. et al. The structural basis of Arfaptinmediated cross-talk between Rac and Arf signalling
pathways. Nature 411, 215–219 (2001).
Di Cesare, A. et al. p95-APP1 links membrane transport to
Rac-mediated reorganization of actin. Nature Cell Biol. 2,
521–530 (2000).
Stoorvogel, W., Oorschot, V. & Geuze, H. J. A novel class
of clathrin-coated vesicles budding from endosomes. J.
Cell Biol. 132, 21–33 (1996).
Meyer, C. et al. µ1A-adaptin-deficient mice: lethality, loss
of AP-1 binding and rerouting of mannose 6-phosphate
receptors. EMBO J. 19, 2193–2203 (2000).
Lin, S. X., Grant, B., Hirsh, D. & Maxfield, F. R. Rme-1
regulates the distribution and function of the endocytic
recycling compartment in mammalian cells. Nature Cell
Biol. 3, 567–572 (2001).
Grant, B. et al. Evidence that RME-1, a conserved C.
elegans EH-domain protein, functions in endocytic
recycling. Nature Cell Biol. 3, 573–579 (2001).
Santolini, E., Salcini, A. E., Kay, B. K., Yamabhai, M. & Di
Fiore, P. P. The EH network. Exp. Cell Res. 253, 186–209
(1999).
Gu, F. & Gruenberg, J. Biogenesis of transport
intermediates in the endocytic pathway. FEBS Lett. 452,
61–66 (1999).
Futter, C. E., Pearse, A., Hewlett, L. J. & Hopkins, C. R.
Multivesicular endosomes containing internalized
EGF–EGF receptor complexes mature and then fuse
directly with lysosomes. J. Cell Biol. 132, 1011–1023
(1996).
Fernandez-Borja, M. et al. Multivesicular body
morphogenesis requires phosphatidyl-inositol 3-kinase
activity. Curr Biol. 9, 55–58 (1999).
Komada, M. & Soriano, P. Hrs, a FYVE finger protein
localized to early endosomes, is implicated in vesicular
traffic and required for ventral folding morphogenesis.
Genes Dev. 13, 1475–1485 (1999).
Babst, M., Odorizzi, G., Estepa, E. J. & Emr, S. D.
Mammalian tumor susceptibility gene 101 (TSG101) and
the yeast homolog, Vps23p, both function in late
endosomal trafficking. Traffic 1, 248–258 (2000).
Scheuring, S. et al. Cloning, characterisation, and
functional expression of the Mus musculus SKD1 gene in
yeast demonstrates that the mouse SKD1 and the yeast
VPS4 genes are orthologues and involved in intracellular
protein trafficking. Gene 234, 149–159 (1999).
Mulholland, J., Konopka, J., Singer-Kruger, B., Zerial, M. &
Botstein, D. Visualization of receptor-mediated
endocytosis in yeast. Mol. Biol. Cell 10, 799–817 (1999).
Bomsel, M., Parton, R., Kuznetsov, S. A., Schroer, T. A. &
Gruenberg, J. Microtubule and motor dependent fusion in
vitro between apical and basolateral endocytic vesicles
from MDCK cells. Cell 62, 719–731 (1990).
Aniento, F., Emans, N., Griffiths, G. & Gruenberg, J.
Cytoplasmic dynein-dependent vesicular transport from
early to late endosomes. J. Cell Biol. 123, 1373–1388
(1993).
Griffiths, G., Hoflack, B., Simons, K., Mellman, I. &
Kornfeld, S. The mannose-6-phosphate receptor and the
biogenesis of lysosomes. Cell 52, 329–341 (1988).
The pre-lysosomal compartment (or late endosome)
is characterized as the compartment that contains
M6PR in transit within membrane invaginations, in
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
contrast to lysosomes.
98. Alpy, F. et al. The StAR homolog MLN64 a late endosomal
cholesterol binding protein. J. Biol. Chem. 276,
4261–4269 (2000).
99. Kobayashi, T. et al. A lipid associated with the
antiphospholipid syndrome regulates endosome
structure/function. Nature 392, 193–197 (1998).
References 99 and 111 show that late endosome
internal membranes form specialized domains that
contain high amounts of LBPA and that LBPA
membranes are involved in protein and lipid traffic.
100. Brotherus, J., Renkonen, O., Herrmann, J. & Fisher, W.
Novel stereochemical configuration in lysobisphosphatidic
acid of cultured BHK cells. Chem. Phys. Lipids 13,
178–182 (1974).
101. Kobayashi, T., Gu, F. & Gruenberg, J. Lipids and lipid
domains in endocytic membrane traffic. Semin. Cell Dev.
Biol. 9, 517–526 (1998).
102. Kobayashi, T., Startchev, K., Whitney, A. J. & Gruenberg,
J. Localization of lysobisphosphatidic acid-rich membrane
domains in late endosomes. Biol. Chem. 382, 483–485
(2001).
103. Amidon, B., Brown, A. & Waite, M. Transacylase and
phospholipases in the synthesis of
bis(monoacylglycero)phosphate. Biochemistry 35,
13995–14002 (1996).
104. Wilkening, G., Linke, T. & Sandhoff, K. Lysosomal
degradation on vesicular membrane surfaces. Enhanced
glucosylceramide degradation by lysosomal anionic lipids
and activators. J. Biol. Chem. 273, 30271–30278 (1998).
105. Escola, J. M. et al. Selective enrichment of tetraspan
proteins on the internal vesicles of multivesicular
endosomes and on exosomes secreted by human B
lymphocytes. J. Biol. Chem. 273, 20121–20127 (1998).
106. Geuze, H. J. The role of endosomes and lysosomes in
MHC class II functioning. Immunol. Today 19, 282–287
(1998).
107. Pierre, P. & Mellman, I. Developmental regulation of
invariant chain proteolysis controls MHC class II trafficking
in mouse dendritic cells. Cell 93, 1135–1145 (1998).
Shows that a specific protease–protease inhibitor
system regulates the fate of MHC class II in dendritic
cells.
108. Sanderson, F. et al. Accumulation of HLA-DM, a regulator
of antigen presentation, in MHC class II compartments.
Science 266, 1566–1569 (1994).
109. Denzer, K. et al. Follicular dendritic cells carry MHC class
II-expressing microvesicles at their surface. J. Immunol.
165, 1259–1265 (2000).
Together with previous studies from Hans Geuze’s
group, this paper shows that exosomes are
microvesicles derived from late endocytic
membrane invaginations, which are secreted
through a novel pathway.
110. Reaves, B. J., Row, P. E., Bright, N. A., Luzio, J. P. &
Davidson, H. W. Loss of cation-independent mannose 6phosphate receptor expression promotes the
accumulation of lysobisphosphatidic acid in multilamellar
bodies. J. Cell Sci. 113, 4099–4108 (2000).
111. Kobayashi, T. et al. Late endosomal membranes rich in
lysobisphosphatidic acid regulate cholesterol transport.
Nature Cell Biol. 1, 113–118 (1999).
112. Galve de Rochemonteix, B. et al. Interaction of antiphospholipid antibodies with late endosomes of human
endothelial cells. Arterioscler. Thromb. Vasc. Biol. 20,
563–574 (2000).
113. Kobayashi, K. et al. The tetraspanin CD63/LAMP3 cycles
between endocytic and secretory compartments in human
endothelial cells. Mol. Biol. Cell 11, 1829–1843 (2000).
114. Simons, K. & Gruenberg, J. Jamming the endosomal
system: lipid rafts and lysosomal storage diseases. Trends
Cell Biol. 10, 459–462 (2000).
115. Pagano, R. E., Puri, V., Dominguez, M. & Marks, D. L.
Membrane traffic in sphingolipid storage diseases. Traffic
1, 807–815 (2000).
116. Gruenberg, J. & Maxfield, F. Membrane transport in the
endocytic pathway. Curr. Opin. Cell Biol. 7, 552–563
(1995).
117. Martinez, O. & Goud, B. Rab proteins. Biochim. Biophys.
Acta 1404, 101–112 (1998).
118. Caplan, S., Hartnell, L. M., Aguilar, R. C., Naslavsky, N. &
Bonifacino, J. S. Human Vam6p promotes lysosome
clustering and fusion in vivo. J. Cell Biol. 154, 109–122
(2001).
119. Mullock, B. M., Bright, N. A., Fearon, C. W., Gray, S. R. &
Luzio, J. P. Fusion of lysosomes with late endosomes
produces a hybrid organelle of intermediate density and is
NSF dependent. J. Cell Biol. 140, 591–601 (1998).
120. Jahraus, A., Storrie, B., Griffiths, G. & Desjardins, M.
Evidence for retrograde traffic between terminal lysosomes
and the prelysosomal/late endosome compartment. J. Cell
VOLUME 2 | OCTOBER 2001 | 7 2 9
© 2001 Macmillan Magazines Ltd
REVIEWS
Sci. 107, 145–157 (1994).
121. Hollenbeck, P. J. & Swanson, J. A. Radial extension of
macrophage tubular lysosomes supported by kinesin.
Nature 346, 864–866 (1990).
122. Rabinowitz, S., Horstmann, H., Gordon, S. & Griffiths, G.
Immunocytochemical characterization of the endocytic
and phagolysosomal compartments in peritoneal
macrophages. J. Cell Biol. 116, 95–112 (1992).
123. Parton, R. G., Dotti, C. G., Bacallao, R., Simons, K. &
Prydz, K. pH-induced microtubule-dependent
redistribution of late endosomes in neuronal and epithelial
cells. J. Cell Biol. 113, 261–274 (1991).
124. Pfeffer, S. R. Membrane transport: retromer to the rescue.
Curr. Biol. 11, R109–R111 (2001).
125. Neufeld, E. B. et al. The niemann-pick C1 protein resides
in a vesicular compartment linked to retrograde transport
of multiple lysosomal cargo. J. Biol. Chem. 274,
9627–9635 (1999).
126. Cavalli, V., Corti, M. & Gruenberg, J. Endocytosis and
signaling cascades: a close encounter. FEBS Lett. 498,
190–196 (2001).
730
127. Aridor, M. & Hannan, L. A. Traffic jam: a compendium of
human diseases that affect intracellular transport
processes. Traffic 1, 836–851 (2000).
Offers a complete list — at the time of publication —
of human diseases that are linked to transport
defects.
128. Gerrard, S. R., Bryant, N. J. & Stevens, T. H. VPS21
controls entry of endocytosed and biosynthetic proteins
into the yeast prevacuolar compartment. Mol. Biol. Cell 11,
613–626 (2000).
129. Wurmser, A. E. & Emr, S. D. Phosphoinositide signaling
and turnover: PtdIns(3)P, a regulator of membrane traffic, is
transported to the vacuole and degraded by a process
that requires lumenal vacuolar hydrolase activities. EMBO
J. 17, 4930–4942 (1998).
Acknowledgements
I am very grateful to G. van der Goot and R. G. Parton for comments and suggestions. Support was by grants from the Swiss
National Science Foundation and the International Human Frontier
Science Program.
| OCTOBER 2001 | VOLUME 2
Online links
DATABASES
The following terms in this article are linked online to:
Flybase: http://flybase.bio.indiana.edu/
Rab11 | Hrs
Locuslink: 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 | TIP47
Mouse Genome Informatics: http://www.informatics.jax.org/
εCOP | RME-1
OMIM:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM
Niemann–Pick type C
SGD: http://genome-www.stanford.edu/Saccharomyces/
Fab1 | VAM7 | Vps4 | Vps5 | Vps27 | Vps34
Swiss-Prot: http://www.expasy.ch/
CD4 | EGFR | FYVE domain | syntaxin-13
www.nature.com/reviews/molcellbio
© 2001 Macmillan Magazines Ltd