Focus Review
Endosome maturation
Jatta Huotari and Ari Helenius*
Institute of Biochemistry, ETH Zurich, Zurich, Switzerland
Being deeply connected to signalling, cell dynamics,
growth, regulation, and defence, endocytic processes are
linked to almost all aspects of cell life and disease. In this
review, we focus on endosomes in the classical endocytic
pathway, and on the programme of changes that lead to
the formation and maturation of late endosomes/multi-
vesicular bodies. The maturation programme entails a
dramatic transformation of these dynamic organelles dis-
connecting them functionally and spatially from early
endosomes and preparing them for their unidirectional
role as a feeder pathway to lysosomes.
The EMBO Journal (2011) 30, 3481–3500.
doi:10.1038/emboj.2011.286
Subject Categories: membranes & transport
Keywords: degradation; endocytosis; intralumenal vesicle
(ILV); multivesicular body (MVB); sorting
Introduction
Endocytosis is the general term for internalization of fluid,
solutes, macromolecules, plasma membrane components,
and particles by the invagination of the plasma membrane
and the formation of vesicles and vacuoles through mem-
brane fission. In metazoan cells, endocytosed cargo includes
a spectrum of nutrients and their carriers, receptor–ligand
complexes, fluid, solutes, lipids, membrane proteins, extra-
cellular–matrix components, cell-debris, bacteria, viruses,
etc. By sorting, processing, recycling, storing, activating,
silencing, and degrading incoming substances and receptors,
endosomes are responsible for regulation and fine-tuning of
numerous pathways in the cell.
Having left endosome research after the early discovery
period in the 1980s, and returning to it only recently, one of
us (AH) has been impressed by the amount of information
that has become available in the meantime on most aspects
of endocytosis. Also, it is evident that the central role of
endocytosis in cell life and pathogenesis is now much more
fully appreciated. The reason for concentrating again on this
topic is our interest in host cell entry of animal viruses, the
majority of which enter cells by endocytosis and exploit
endosomes and the endocytic pathways for penetration into
the cytosol (Marsh and Helenius, 1989; Mercer et al, 2010).
While virus particles themselves are relative simple and in
many cases well characterized, the challenge is to understand
the cellular processes used by them, and the responses of
the cell to the invasion. Deeper knowledge of endocytosis is
urgently required. Incoming viral particles also provide a tool
to learn more about the endocytic machinery.
In this review, we focus on a relatively narrow topic; the
formation and maturation of late endosomes (LEs) in mam-
malian cells. They are also known as multivesicular bodies,
because—although heterogeneous and variable in size and
composition—most LEs have a multivesicular morphology,
that is, they contain intralumenal vesicles (ILVs). There are
many excellent reviews to recommend dealing with endo-
somes and different aspects of LE maturation. These include
the following (Mellman, 1996; Zerial and McBride, 2001;
Maxfield and McGraw, 2004; Piper and Katzmann,
2007; Luzio et al, 2007b; van Meel and Klumperman, 2008;
Woodman and Futter, 2008; Saftig and Klumperman, 2009;
Jovic et al, 2010; Von Bartheld and Altick, 2011).
The logistics of the endosome system
In a basic, ‘stripped-down’ representation, the classical
endocytic pathway has only a few elements (Figure 1). The
elements include a recycling circuit for plasma membrane
components and their ligands, a degradative system for
digestion of macromolecules, and a connecting, unidirec-
tional feeder pathway for transport of fluid and select ed
membrane components from the recycling circuit to the
degradative system. The feeder function is mediated by LEs.
LEs also function as a system for mediating transport of
lysosomal components from the trans-Golgi network (TGN)
to lysosomes. This allows maintenance, diversification, and
expansion of the recycling and degradative systems. Finally,
the cytosol must be included among the essential elements,
because it provides a spectrum of transiently associated,
peripheral membrane components that support, regulate,
and define the pathway (Figure 1).
Of the cargo internalized by ongoing endocytosis in
mammalian cells, the majority is recycled back to the
plasma membrane via early endosomes (EEs) (Figure 2).
In typical mammalian cells, the equivalent of 50–180% of
the surface area of the plasma membrane is cycled in and
out of the cell every hour (Steinman et al, 1983). The
amount of fluid internalized by macrophages corresponds
to some 30% of cell volume per hour of which about two-
thirds are returned to the extracellular space within about
10–15 min (Steinman et al, 1976, 1983; Besterman and
Low, 1983).
Received: 25 May 2011; accepted: 21 July 2011
*Corresponding author. Institute of Biochemistry, ETH Zurich,
ETH-Hoenggerberg, HPM E 6.3, Schafmattstrasse 18, Zurich CH-8093,
Switzerland. Tel.: þ 41 44 632 6817; Fax: þ 41 44 632 1269;
E-mail: ari.helenius@bc.biol.ethz.ch
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One of the consequences of the active recycling is that
transport to lysosomes via LEs is a side pathway limited to a
relatively small fraction of internalized fluid and especially
membrane components. To enter this side pathway,
membrane components undergo stringent selection so that
only a specific cohort is transported to lysosomes and
degraded. The majority of large particles (such as viruses
and ILVs) are also targeted to LEs. The bulk fluid and
solutes diverted into this side pathway are not specifically
sorted.
In addition to ferrying cargo for degradation, LEs transport
new lysosomal hydrolases and membrane proteins to lyso-
somes for the maintenance and amplification of the degrada-
tive compartment. Lysosomes depend on the influx of new
components, because without incoming endosomal traffic,
they loose their intactness, acidity, and perinuclear localiza-
tion (Bucci et al, 2000).
It is evident that the magnitude of the side pathway
from EEs to lysosomes is under regulation through some of
the cargo. Thus, formation of LEs and inward vesiculation to
form ILVs has been shown to increase upon signalling via
growth factor receptors. This suggests that the cell adjusts the
use of this side pathway according to need (White et al,
2006). In doing so, it probably also adjusts the size of the
degradative compartment. How such adjustment is achieved
and which factors influence it, is an interesting question that
deserves careful study.
Unlike the secretory pathway, the endocytic pathway has
the advantage that the starting compartment, the extracellu-
lar space, is open and accessible. This means that a variety of
ligands, fluid, solutes, and particles can be added to cells,
and their fate after endocytosis followed in different ways. In
mammalian tissue culture cells, the most commonly used
cargo markers today are transferrin (Tf) and its receptor
(TfR), which faithfully follow the recycling pathway, and
epidermal growth factor (EGF) and its receptor (EGFR),
which after ubiquitination of the receptor’s cytosolic domain
and inclusion in ILVs take the pathway to lysosomes
for inactivation and degradation. Fluid uptake is usually
followed using fluorescently-labelled dextran and other fluor-
escent solutes that do not adsorb to the cell surface. The
membrane as such can be followed using fluorescent lipid
markers (Maier et al, 2002). Viruses and bacterial toxins are
also useful tools.
In addition to tissue culture cells, the most valuable system
for endocytosis studies has been yeast, which seems to have
endosomal compartments comparable to animal cells (Lewis
et al, 2000; Pelham, 2002). More recently, filamentous fungi
such as Aspergillus nidulans, have proven to be excellent
systems to study endosomes and their maturation (Penalva,
2010). Caenorhabditis elegans and Drosophila melanogaster
also have an impact given the background of strong genetics
and the possibility of in situ studies in a multicellular organ-
ism (Grant and Sato, 2006; Michelet et al, 2010; Poteryaev
et al, 2010). Endocytosis in plant cells is actually quite active
(Irani and Russinova, 2009). The most important difference is
the apparent lack of an independent EE compartment. The
functions of EEs are carried out by an organelle that combines
EEs and the TGN (Dettmer et al, 2006; Niemes et al, 2010).
Conceptually, the situation in plants and fungi implies that by
participating actively in secretory functions, endosomes can
be viewed as an extension of the TGN.
Early endosomes
EEs provide the starting point for LE maturation. Defined
initially as the compartment that first receives incoming cargo
and fluid (Helenius et al, 1983), EEs are now recognized as
the main sorting station in the endocytic pathway. Exactly
how EEs arise is not entirely clear, but the membrane and
volume is mainly derived from primary endocytic vesicles
that fuse with each other. EEs receive endocytic cargo not
only through the clathrin-mediated pathway but several other
pathways including caveolar-, GEEC-, and ARF6-dependent
pathways (Mayor and Pagano, 2007). Typically, an EE accepts
incoming vesicles for about 10 min during which time mem-
RECYCLING
LE
DEGRADATION
Golgi
Plasma
membrane
Cytosol
BIOSYNTHESIS
Figure 1 The basic elements of the endocytic machinery. The membrane organelles involve a recycling circuit (the plasma membrane (PM),
the EEs, the recycling endosomes, and a variety vesicular carriers), a degradation cycle (lysosomes), and a connecting ‘feeder’ pathway (LEs)
from the recycling circuit to the degradative system. The main interacting partner in the Golgi providing lysosomal components is the TGN,
which communicates with the PM, EEs and LEs. The recycling circuit has functions independent of the degradative cycle. The degradative cycle
is, in turn, a shared ‘facility’ for degradation in the cell and is not only used for substrates delivered via endosomes. The cytosol has a central
role by providing peripheral proteins to all the membrane compartments. These proteins define functions such as molecular sorting, membrane
fusion and fission, compartment identity, and organelle motility.
Endosome maturation
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brane and fluid is rapidly recycled away, while some of the
incoming cargo is retained and accumulates over the lifetime
of the EE to be included in the LEs (Maxfield and McGraw,
2004).
Association of proteins from the cytosol to the cytosolic
surface of the EE membrane defines many of its functional
attributes. Rab5 is a key component together with its effector
VPS34/p150, a phosphatidylinositol 3-kinase (PI(3)K) com-
plex that generates the phosphoinositide (PI) PtdIns(3)P and
thus helps to manifest the identity of the organelle
(Christoforidis et al, 1999; Zerial and McBride, 2001; Behnia
and Munro, 2005). Rab5 follows the endocytic membrane
from the beginning through various stages of EE maturation,
and is later the main regulator of the conversion to LEs. EEs
communicate with the TGN through bidirectional vesicle ex-
change. The arrival of hydrolases gives them an initial degra-
dative identity further strengthened during maturation of LEs.
EEs are heterogenous in terms of morphology, localization,
composition, and function (Miaczynska et al, 2004; Lakadamyali
et al,2006;vanMeelandKlumperman,2008).Mostofthemare
relative ly small and patrol the peripheral cytoplasm close to the
plasma membrane through saltatory movement along microtu-
bules (Nielsen et al, 1999; Hoepfner et al, 2005). The overall
distribution of EEs is cell-type dependent.
Individual EEs have a complex structure with tubular and
vacuolar domains (Figure 3A). Most of the membrane surface
area is in the tubules, and much of the volume in the
vacuoles. The limiting membrane contains a mosaic of sub-
domains that differ in composition and function (Zerial and
McBride, 2001). They include domains enriched in Rab5,
Rab4, Rab11, Arf1/COPI, retromer, and caveolae
(Vonderheit and Helenius, 2005; Rojas et al, 2008; Hayer
et al, 2010). Many of the domains are located in the tubular
extensions where they provide for molecular sorting and
generate vesicle carriers targeted to distinct organelles, in-
cluding the plasma membrane, the recycling endosomes, and
the TGN (Bonifacino and Rojas, 2006) (Figure 2).
The formation of ILVs begins already in EEs. For this the
cytosolic surface of the EE membrane has characteristic
‘plaques’ containing clathrin and components of the endoso-
mal sorting complex required for transport (ESCRT), machin-
ery responsible for sorting of ubiquitinated membrane
proteins into ILVs (Raiborg et al, 2002; Sachse et al, 2002)
(Figure 3B). The lumen of the vacuolar EE domains often
contains several ILVs; in HepG2 cells there are 1–8 ILVs (van
Meel and Klumperman, 2008). EEs are weakly acidic (pH
6.8–5.9) (Maxfield and Yamashi ro, 1987), and contain a
relatively low Ca
2 þ
concentration (Gerasimenko et al, 1998).
The traffic between endosomes and the TGN is a continu-
ously ongoing process that has been extensively studied. It is
responsible for the delivery of lysosomal and removal
of endosomal components during endosome maturation. It
Plasma
membrane
Golgi
LE maturation
LE
fusion
LE/
lysosome
fusion
Lysosome
Recycling endosome
Endolysosome
EE
MT
TGN
Figure 2 The endosome/lysosome system. The primary endocytic vesicles deliver their contents and their membrane to EEs in the peripheral
cytoplasm. After a period of about 8–15 min during which the EEs accumulate cargo and support recycling to the plasma membrane (directly or
via recycling endosomes in the perinuclear region), conversion of the EEs to LE takes place. Thus, as the endosomes are moving towards the
perinuclear space along microtubules (MT), the nascent LE are formed inheriting the vacuolar domains of the EE network. They carry a
selected subset of endocytosed cargo from the EE, which they combine en route with newly synthesized lysosomal hydrolases and membrane
components from the secretory pathway. They undergo homotypic fusion reactions, grow in size, and acquire more ILVs. Their role as feeder
system is to deliver this mixture of endocytic and secretory components to lysosomes. To be able to do it, they continue to undergo a
maturation process that prepares them for the encounter with lysosomes. The fusion of an endosome with a lysosome generates a transient
hybrid organelle, the endolysosome, in which active degradation takes place. What follows is another maturation process; the endolysosome is
converted to a classical dense lysosome, which constitutes a storage organelle for lysosomal hydrolases and membrane components.
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occurs at the level of EEs, maturing LEs, and possibly for
some time after the fusion of LEs with lysosomes. At the
endosome level, the sorting and vesicle formation for trans-
port to the TGN depends on factors such as Rab7, Rab9, and
the retromer complex (Bonifacino and Hurley, 2008; Pfeffer,
2009). The retromer is a multimeric complex composed of
sorting nexins and other proteins recruited to the cytosolic
surface of EEs and maturing LEs.
LEs and lysosomes
Mature LEs are typically round or oval and have a diameter of
250–1000 nm (Figure 3C). They have a low bouyant density,
and a high negative surface charge (Bayer et al, 1998;
Falguieres et al, 2008). The limiting membrane contains
lysosomal membrane proteins such as LAMP1 and the
lumen contains a complement of acid hydrolases. The
lumen also has numerous ILVs (often up to X30) with a
diameter of about 50–100 nm. The pH ranges between pH
6.0–4.9 (Maxfield and Yamashiro, 1987).
LEs formed in the peripheral cytoplasm move to the peri-
nuclear area of the cell where they fuse with each other to
form larger bodies and to undergo transient (‘kiss-and-run’)
fusions and eventually full fusions with lysosomes and
pre-existing hybrid organelles between endosomes and
lysosomes (Luzio et al, 2007b) (Figure 2). By fusing with
lysosomes, the LEs follow what is essentially a unidirectional,
dead-end pathway. Whereas most of the components of LEs
are degraded in the lysosomal environment, others are longer
lived and contribute, as already mentioned, to the mainte-
nance and generation of lysosomes. It has been suggested
that a few components such as mannose-6-phosphate recep-
tors, tetraspanins, and SNAREs may escape degradation
through vesicle trafficking after LE fusion with lysosomes,
but this pathway is not well characterized.
To determine whether a multivesicular organelle is a LE, or
a fusion product between LEs and lysosomes is not always
straightforward because the hybrid organelles contain
components from both fusion partners (Figure 3D and E).
We will call these hybrid organelles endolysosomes,todistin-
guish them from classical dense, primary lysosomes and from
lysosomes generated through fusion with phagosomes, macro-
pinosomes, and autophagosomes. It is in the endolysosomes
that most of the hydrolysis of endocytosed cargo takes place.
The lysosomal compartment as a whole consists of a
collection of vacuoles of heterogeneous composition, mor-
phology, location, and density. The heterogeneity is due to
the diversity of cargo and the existence of several feeder
pathways of which the classical endosome pathway is a
major one. Heterogeneity is also generated by the varying
degree of cargo degradation within individual vacuoles, and
by fusion events between the various vacuoles. The classical
lysosomes of high buoyant density and high hydrolase con-
tent correspond to the end point of the degradation process.
They serve in part as storage vacuoles for lysosomal compo-
nents ready to be redeployed. These components include the
hydrolases, the limiting membrane protected by LAMPs, and
other substances resistant to degradation. They also contain
slowly degraded lumenal lipids often present as multilamellar
membrane whirls.
Taken together, the EEs, LEs, endolysosomes, and lyso-
somes provide a dynamic and adaptable continuum ( Von
Bartheld and Altick, 2011). The pathway is elusive because
the organelles are scattered, and undergoing continuous
maturation, transformation, fusion, and fission. Specific
protein and lipid components are only partially useful as
molecular markers because the majority is either transiently
associated with the organelles or follow the organelles
through several steps of transformation. In the pathway,
events occur, moreover, non-synchronously. A cohort of
cargo molecules simultaneously internalized from the plasma
membrane may, for example, arrive at a certain acidic
pH value in a time window spread over hours (Kielian
et al, 1986). The ambiguity, heterogeneity, and lack of
synchrony in the endocytic system may, in part, explain a
certain lack of common, universally agreed concepts and
models for endosome function and maturation.
Formation of LEs
LEs are derived from the vacuolar domains of EEs. By itself
this constitutes molecular sorting because in addition to a
Figure 3 Morphologies of endosomes and lysosomes at the ultra-
structural level. (A) Electron micrographs of peripherally located
EEs containing HRP-conjugated Tf. They contain vacuolar and
tubular domains. Courtesy of Tooze and Hollinshead (1992).
Electron micrographs of (B) EE with clathrin lattices and a few
ILVs; (C) LE, containing numerous ILVs; (D) endolysosome, with
partial electron dense areas; and (E) lysosomes, with electron dense
lumen. Images are all from HeLa cells that had been processed for
thin section EM. Scale bars in (A): 500 nm and (B–E): 100 nm.
Figure 3A is reproduced with kind permission from Rockefeller
University Press;r2009 Rockefeller University Press. Originally
published in J Cell Biol 118: 813–830. doi: 10.1083/jcb.118.4.813.
Endosome maturation
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large fraction of the fluid, the vacuolar domains of EEs have a
different composition than the tubules. In the lumen, they
contain ligands dissociated from their receptors as well as
proteins and solutes internalized as components of the bulk
fluid. The ILVs, and other large particles such as incoming
viruses, are also present in the vacuolar elements of EEs. The
membrane probably contains most of the cholesterol and
sphingolipid-rich lipid rafts, membrane protein aggregates, V-
ATPases, clathrin, ESCRT complexes, and a selected spectrum
of membrane proteins destined for degradation (Ukkonen
et al, 1986; Mukherjee et al, 1999; Mukherjee and Maxfield,
2004).
The formation of a new LE is preceded by the generation of
a Rab7 domain (Rink et al, 2005; Vonderheit and Helenius,
2005). This leads to the transient formation of a hybrid Rab5/
Rab7 endosome. As discussed below, Rab7 is recruited to the
EE by Rab5-GTP. How the tubules and the rest of the EE
domains are lost during the formation of an LE is not entirely
clear, but there are two possibilities. Evidence obtained by
analysing large, spherical, juxtanuclear Rab5-positive endo-
somes supports a mechanism in which Rab5 after recruiting
Rab7 is converted to the GDP-bound form and dissociates
together with its effectors. A process like this was observed
by live microscopy (Rink et al, 2005). Rab5 was lost within a
few minutes, and replaced with Rab7.
Another mechanism envisages a fission event that sepa-
rates parts of the hybrid endosome containing the nascent
Rab7 domain from the rest. Gruenberg and Stenmark (2004)
call the newly formed LEs endos omal carrier vesicles (ECVs),
and suggest that these serve as transporters of cargo to a
stable LE compartment before delivery to lysosomes. While
following viruses after endocytosis in small peripheral endo-
somes by live microscopy, we found support for such splitting
of the endosomes. We observed microtubule-dependent
events in which the virus and the Rab7 domain separated
from the rest of the endosome containing Rab5, Rab4, and
Arf1 (Vonderheit and Helenius, 2005). Consistent with this, it
has been shown that dynein-mediated pulling forces are
critical for the separation of endosomal elements containing
the recycling ligand Tf from the lysosome-targeted ligand EGF
(Driskell et al, 2007). Similar observations were recently
made by a group that proposed a role for dynamin in fission
of LEs from EEs (Mesaki et al, 2011). It is possible that
different mechanisms exist for the peripheral and perinuclear
populations of EEs, with gradual maturation occurring in
perinuclear endosomes and a fission-based mechanism in
peripherally located, relatively small and motile endosomes.
Maturation of LEs
Regardless of the mechanism of initial formation, the newly
formed LEs continue to undergo a multitude of changes
(Table I). As a result, by the time they fuse with lysosomes
some 10–40 min later, they have completed a remarkable
transformation leaving them with few similarities to EEs.
The maturation process involves exchange of membrane
components, movement to the perinuclear area, a shift in
choice of fusion partners, formation of additional ILVs, a drop
in lumenal pH, acquisition of lysosomal components, and a
change in morphology (Table I). The programme is closely
coordinated and regulated by factors recruited to the surface
of the limiting membrane from the cytosol.
Before considering the conversion in more detail, it is
important to ask why LEs are subject to such a dramatic
transformation. What is the reason for this complex matura-
tion programme? The general answer is that the programme
is in place to close down recycling and other functions of EEs
and to allow the union of LEs with the degradative compart-
ment. Similar maturation using some of the same factors
occurs in phagosomes, autophagosomes, and probably
macropinosomes before they fuse with LEs and lysosomes
(Eskelinen, 2008; Kinchen and Ravichandran, 2008; Kerr and
Teasdale, 2009).
Since lysosomes constitute a point-of-no-return for most
macromolecules and lipids, the cargo contents of LEs must
be narrowed down to molecules and particles that need to be
degraded, and to cargo that the LEs and lysosomes require for
their functionality. Some of the membrane-bound cargo,
moreover, needs to be presented in a form easily digested
by hydrolases, which may explain in part the formation and
Table I The endosome maturation programme
Rab switch. Rab5 is exchanged for Rab7. This switch reprograms the
association of effector proteins from the cytosol and redefines many
of the properties of the endosomes. Other Rabs, such as Rab4,
Rab11, are Rab22 also lost, while Rab9 is added.
Formation of ILVs. Ubiquitinated cargo recruits machinery from the
cytosol (ESCRT and other factors) that induce inward-budding of the
limiting membrane, and thus the formation of ILVs containing
membrane proteins and lipids targeted for lysosomal degradation.
Capacitation of microphagocytosis-like mechanisms for the inclu-
sion of cytoplasmic proteins and RNAs, of ILV backfusion with the
limiting membrane, and exosome release.
Acidification. The lumenal pH drops from values above pH 6 to pH
6.0–4.9.
PI conversion. PtdIns(3)P is converted to PtdIns(3,5)P(2), and some
of the PtdIns(3)P is sorted into the ILVs.
Change in size and morphology. The tubular extensions present on
EEs are lost and the endosomes acquire a round or oval shape and
grow in size.
Loss of recycling with the plasma membrane. Recycling receptors are
lost from the organelle and recycling of membrane and fluid to the
cell surface stops.
Gain of lysosomal hydrolases and membrane proteins. These lyso-
somal components are transported mainly from the TGN. Some of
them are active already in the maturing endosomes.
Switch in fusion specificity. The endosomes can no longer fuse
with EEs. Instead, they acquire the necessary tethering complexes
and SNAREs to fuse with each other, with lysosomes, and with
autophagosomes. The conversion of CORVET to HOPS complex on
membranes. The HOPS/CORVET complexes are involved in a
number of processes, including membrane tethering, the Rab5/7
switch, and mediating SNARE assembly.
A switch in cytoplasmic motility. The endosomes associate with a
new set of microtubule-dependent motors that allow them to move
into the perinuclear region of the cell.
Changes in lumenal ionic environment. In addition to the drop in
pH, there is an increase in Cl
, and changes in Ca
2 þ
,Na
þ
,K
þ
concentrations.
Change in temperature sensitivity. Unlike earlier steps in endocy-
tosis, LE formation or their fusion with lysosomes is blocked at
temperatures below 19–201C.
Decrease in buoyant density and increase in negative surface charge.
These properties are used to isolate LEs.
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