Lysosomes in Health and Disease
The role of ESCRT proteins in fusion events
involving lysosomes, endosomes and
autophagosomes
Daniel Metcalf and Adrian M. Isaacs1
Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London WC1N 3BG, U.K.
Abstract
ESCRT (endosomal sorting complex required for transport) proteins were originally identified for their
role in delivering endocytosed proteins to the intraluminal vesicles of late-endosomal structures termed
multivesicular bodies. Multivesicular bodies then fuse with lysosomes, leading to degradation of the
internalized proteins. Four ESCRT complexes interact to concentrate cargo on the endosomal membrane,
induce membrane curvature to form an intraluminal bud and finally pinch off the bud through a membranescission event to produce the intraluminal vesicle. Recent work suggests that ESCRT proteins are also required
downstream of these events to enable fusion of multivesicular bodies with lysosomes. Autophagy is a related
pathway required for the degradation of organelles, long-lived proteins and protein aggregates which also
converges on lysosomes. The proteins or organelle to be degraded are encapsulated by an autophagosome
that fuses either directly with a lysosome or with an endosome to form an amphisome, which then fuses with
a lysosome. A common machinery is beginning to emerge that regulates fusion events in the multivesicular
body and autophagy pathways, and we focus in the present paper on the role of ESCRT proteins.
These fusion events have been implicated in diseases including frontotemporal dementia, Alzheimer’s
disease, lysosomal storage disorders, myopathies and bacterial pathogen invasion, and therefore further
examination of the mechanisms involved may lead to new insight into disease pathogenesis and
treatments.
Introduction
ESCRT (endosomal sorting complex required for transport)
proteins were initially described in yeast as having key and
distinct roles in the biogenesis of MVBs (multivesicular
bodies) [1–3]. MVBs are late-endosomal structures involved
in the degradation of endocytosed proteins such as cellsurface receptors and transmembrane proteins [4]. MVBs are
characterized by the presence of ILVs (intraluminal vesicles)
which form by the inward budding and eventual pinching
off (or scission) of the endosomal membrane. Four ESCRT
complexes (ESCRT-0–ESCRT-III) act to recruit and deliver
endocytosed proteins to ILVs of MVBs [5–7]. The ESCRT
pathway is also responsible for two topologically similar
membrane-scission events: the budding of viruses out of a cell
[8–10] and the separation of mother and daughter cells at the
end of cytokinesis [11]. In the present paper, we concentrate
on the role of ESCRT proteins in the MVB and autophagy
pathways.
Key words: autophagosome, endosomal sorting complex required for transport (ESCRT),
endosome, fusion, lysosome.
Abbreviations used: CHMP, charged multivesicular body; EGF, epidermal growth factor; EGFR,
EGF receptor; ESCRT, endosomal sorting complex required for transport; GFP, green fluorescent
protein; HOPS, homotypic vacuole fusion and vacuole protein sorting; ILV, intraluminal vesicle;
LC3, light chain 3; MVB, multivesicular body; SNARE, soluble N-ethylmaleimide-sensitive fusion
protein-attachment protein receptor; Tsg101, tumour susceptibility gene 101; UVRAG, UVradiation-resistance-associated gene; VPS, vacuolar protein sorting.
To whom correspondence should be addressed (email [email protected]).
1
Biochem. Soc. Trans. (2010) 38, 1469–1473; doi:10.1042/BST0381469
Endocytosed proteins are tagged for degradation in the
MVB–lysosome pathway by the addition of polyubiquitin
chains linked through Lys48 of the ubiquitin protein, or
multiple mono-ubiquitins [12]. This is distinct from the
signal for proteasomal degradation, which is a Lys63 -linked
polyubiquitin chain. The roles of each of the ESCRTs is
relatively well defined in yeast and has been illuminated
further by reconstitution of the ESCRT pathway in vitro
[13–15]. The ESCRT-0 complex [also termed the Hse (has
symptoms of class-E mutants)/VPS (vacuolar protein sorting)
27 complex] has both membrane- and ubiquitin-binding
properties, allowing the concentration of ubiquitinated cargo
on the endosomal membrane [6,7,14]. The cargo is then
passed on to the ESCRT-I and -II complexes, which also have
ubiquitin-binding domains [6,7,12]. Recent work suggests
that ESCRT-I and -II are responsible for deforming the MVB
membrane inwards, into a bud containing the cargo [14],
although high concentrations of ESCRT-III proteins can also
cause membrane curvature [13,15,16], suggesting that they
could also play a role. ESCRT-III is recruited last and is
required for scission of the bud neck, completing formation
of the internal vesicle [15]. ESCRT-III also recruits VPS4,
an AAA (ATPase associated with various cellular activities)
protein required for disassembly of the ESCRT machinery
[6,7,17], allowing further rounds of membrane scission [15].
Deubiquitination of cargo is also required for efficient MVB
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Figure 1 Fusion events in the MVB and autophagy pathways
potentially involving ESCRT proteins
(1) Autophagosome–MVB fusion. (2) Amphisome–lysosome fusion.
(3) Autophagosome–lysosome fusion. (4) MVB–lysosome fusion. The
terminal compartment in the autophagy pathway is termed the autolysosome, whereas the result of fusion of a lysosome with an MVB
leads to a terminal degradative compartment termed a hybrid organelle.
ESCRT proteins appear to play a key role in fusion events in the MVB
and autophagy pathways, but the precise step(s) (1–3 above) at which
they act in the autophagy pathway remain to be delineated. Green
bars represent a transmembrane protein degraded through the MVB
pathway.
sorting and is achieved by the recruitment of a deubuitinating
enzyme by ESCRT-III [6,7,18].
Recent work has suggested that ESCRT proteins may also
be involved downstream of the membrane-scission event in
the MVB pathway and play a role in the fusion of MVBs
and lysosomes [19,20] (Figure 1). Parallel data have also
implicated the ESCRTs in fusion events in the autophagy
pathway [21–23], which converges on the MVB–lysosome
pathway (Figure 1). We will review the evidence that ESCRT
proteins play a role in vesicular fusion events and discuss
potential mechanisms.
ESCRT proteins and endosome–lysosome
fusion
The EGFR (epidermal growth factor receptor) is the
canonical target of the MVB–lysosome pathway [4]. Upon
EGF (epidermal growth factor) binding, the ligand–receptor
complex is endocytosed and EGFR signals in the cytoplasm
through its tyrosine kinase domain until it is internalized
on to the ILVs of MVBs, silencing signalling [4]. The EGF–
EGFR complex is then degraded through the fusion of
MVBs with lysosomes. The first evidence that late-acting
ESCRT proteins could be involved in endosome–lysosome
fusion came from the discovery that EGFR signalling and
degradation can be uncoupled [20]. Knockdown of ESCRT0 or ESCRT-I in mammalian cells prolongs EGF-induced
signalling and inhibits EGFR degradation as EGFR is not
internalized on to ILVs [20,24]. However, knockdown of
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Authors Journal compilation the ESCRT-III component CHMP (charged multivesicular
body protein) 3 (also termed VPS24) delayed degradation
of EGFR, but did not prolong EGF-induced signalling [20].
The simplest explanation for this result is that EGFR was
internalized on to ILVs, silencing signalling, but that MVBs
did not then fuse with lysosomes, leading to impaired EGFR
degradation. A similar uncoupling of EGFR signalling
and degradation was subsequently observed for the ESCRTII subunit VPS22 [24].
We have examined the ESCRT-III component CHMP2B,
mutations in which cause frontotemporal dementia [25–27].
The mutations lead to C-terminal truncations that are
likely to impair the ESCRT pathway through a dominantnegative or toxic gain-of-function effect [28]. The CHMP2B
C-terminal truncations, which we will refer to as mutant
CHMP2B, lead to the formation of aberrant enlarged
endosomes in cell culture, patient fibroblasts and patient
brains [19] and this appears to be due to impaired
endosome–lysosome fusion. Similarly to the knockdown of
CHMP3/VPS24 and VPS22, expression of mutant CHMP2B
leads to delayed EGFR degradation with no effect on
EGF-induced signalling [19]. Analysis of patient fibroblasts
carrying the CHMP2B mutations showed that, although
MVBs looked abnormal, they could still internalize EGFR on
to ILVs, again implicating fusion rather than ILV formation
[19].
Similar results were reported in knockout mice for the
ESCRT-III protein CHMP5 [29]. Homozygous CHMP5knockout mice die at embryonic day 10 due to gross
developmental abnormalities. The embryos accumulated
aberrant enlarged MVBs that were packed with ILVs, showing
that ILV formation was still intact. Analysis of primary
embryonic cultures from the knockout mice as well as
CHMP5-knockdown cells showed impaired degradation of
TGFβ (transforming growth factor β) receptors and HRP
(horseradish peroxidase) (both of which are degraded through
the MVB–lysosome pathway), even though they were still
delivered to MVB ILVs, pointing to an impairment of MVB–
lysosome fusion [29].
ESCRT proteins and autophagosome fusion
Autophagy is responsible for the degradation of long-lived
proteins, organelles and protein aggregates, and converges
on the MVB–lysosome pathway. The proteins/organelles to
be degraded are first encapsulated by an autophagosome,
which then fuses either directly with a lysosome [30] or
with endosomes to form a hybrid compartment termed an
amphisome, which then fuses with a lysosome to allow
degradation [31–34].
Evidence from several experimental paradigms has now
shown that impairing ESCRT function leads to an accumulation of autophagosomes. Expression of mutant CHMP2B or
knock down of another ESCRT-III protein, CHMP4B
in primary rodent neurons led to an accumulation of
autophagosomes [22]. Expression of the same mutant
CHMP2B protein or knock down of ESCRT-I, -II or -III
Lysosomes in Health and Disease
proteins in Drosophila also led to an accumulation of
autophagosomes [22,23]. Expression of mutant CHMP2B
or knock down of CHMP3/VPS24 or the ESCRT-I protein
Tsg101 (tumour susceptibility gene 101) in HeLa cells
similarly led to an increase in autophagosomes [21]. An
increase in autophagosomes could be due to either increased
autophagy induction or decreased autophagosome fusion.
Degradation of normal autophagy substrates was impaired
in HeLa cells when either Tsg101 or CHMP3/VPS24 was
depleted, implicating decreased fusion [21]. Fusion was
assessed further using the mCherry–GFP (green fluorescent
protein)–LC3 (light chain 3) autophagosome fusion reporter
construct. mCherry–GFP–LC3 fluoresces both red and green
in autophagosomes, but only red in lysosomes, owing to the
acid-quenching of the GFP signal and thus can give a readout of autophagosome fusion with acidic endosomes and
lysosomes [35]. Expression of mCherry–GFP–LC3 in cells
depleted of Tsg101 or CHMP3/VPS24 revealed a reduction
in autophagosome fusion [21]. Consistent with these data,
accumulation of autophagosomes, impaired degradation of
autophagy substrates and impaired delivery of endosomal
components to autophagosomes were observed in HeLa cells
expressing a dominant-negative form of VPS4 [36]. Loss
of the ESCRT-0 protein Hrs (hepatocyte growth factorregulated tyrosine kinase substrate) also appears to impair
autophagosome fusion with endosomes or lysosomes [37].
These data could be explained by ESCRT proteins
affecting any of three fusion events in the autophagy
pathway (Figure 1): (1) autophagosome–endosome fusion;
(2) amphisome–lysosome fusion; or (3) autophagosome–
lysosome fusion.
It is not clear whether just one or all three are blocked.
When ESCRT-I, -II and -III subunits were depleted in Drosophila, electron microscopy showed that autophagosomes
accumulated [23], indicating that ESCRTs are required for
either (1) or (3). However, in HeLa cells, knock down of
ESCRT-I and -III led to accumulation of amphisomes [21],
suggesting that (2) is blocked. The discrepancy could be due
to differing functions between mammals and Drosophila, or
an incomplete knockdown in the HeLa cells. As discussed
in more detail in the next section, we have shown that
mutant CHMP2B leads to impaired recruitment of Rab7,
which is required for autophagosome-fusion events [38–40].
Impairing Rab7 function did not block (2) [39,41], implicating
a role in (1) and/or (3). It is also possible that ESCRTs affect
processes other than fusion in the autophagy pathway, such
as the closure of the autophagosome [42].
Potential mechanisms of ESCRT-mediated
vesicular fusion
The initial interaction between two vesicular membranes
that will ultimately fuse is termed tethering. Tethering of
lysosomes to endosomes and autophagosomes is mediated
by the GTPase Rab7 [38,39,43] and the HOPS (homotypic
vacuole fusion and vacuole protein sorting) complex
[44,45], which bring the membranes in close proximity and
recruit SNARE (soluble N-ethylmaleimide-sensitive fusion
protein-attachment protein receptor) proteins which are
required for the fusion event [46,47]. We showed that late
endosomes/lysosomes in cells expressing mutant CHMP2B
proteins recruited endogenous Rab7 less effectively than
cells expressing wild-type CHMP2B, and that the effect
was not rescued by overexpression of Rab7 [19]. These data
suggest two potential mechanisms to explain the effects of
ESCRT proteins on vesicular fusion: (i) they are required for
recruiting the vesicular fusion machinery to late endosomes;
or (ii) they are required for delivery of the fusion machinery
to lysosomes or autophagosomes.
Consistent with (i) is the suggestion that ESCRT proteins
can interact directly with the HOPS complex [48], which
binds Rab7 [44,45]. Consistent with (ii) are data showing that,
in the presence of dominant-negative VPS4, late-endosome
traffic to autophagosomes was impaired [36]. It is possible
that both mechanisms contribute to the impaired fusion
observed. Two further proteins, UVRAG (UV-radiationresistance-associated gene) and Rubicon, also regulate both
autophagosome and endosome–lysosome fusion events
[49–51], so could also interact with the ESCRT pathway.
UVRAG is a positive regulator of fusion shown to bind the
HOPS complex [49], and Rubicon is a negative regulator
of fusion [50,51]. Therefore the exact sequence of events
required for HOPS and Rab7 delivery, recruitment and
activation is unclear and it will be important to establish
where ESCRT proteins act in the process.
A number of other proteins have also been implicated
in autophagosome fusion with endosomes/lysosomes, including LAMP2 (lysosome-associated membrane protein
2) [52], Rab11 [41], HDAC6 (histone deacetylase 6) [53],
the ubiquitin-binding proteins ubiquilin [54] and VCP
(p97/valosin-containing protein) [55,56], SNARE proteins
[57–59] and dynein [60,61], which probably reflects the
requirement for intact microtubule-based transport [60,62].
The lipid content of autophagosomes is also important for
fusion [63]. Whether ESCRT proteins have a role in these
effects is yet to be addressed.
It is intriguing that ESCRT-II and -III have been implicated
in endosome–lysosome fusion in mammalian cells and
ESCRT-0, -I, -II and -III in autophagosome–endosome
fusion in Drosophila and mammals, despite no evidence that
ESCRT proteins have such roles in yeast. It is not clear
how ESCRT proteins have taken on these additional roles,
but it is possible that the fusion machinery has evolved to
utilize the ESCRTs, potentially providing further regulation
of the fusion event. It also remains to be determined whether
ESCRT-0 and -I are also involved endosome–lysosome
fusion, or whether their role in autophagosome fusion events
reflects a genuine difference in requirements for the ESCRTs
in the two processes.
Conclusions
Various lines of evidence suggest that ESCRT proteins
are involved in fusion events in the autophagy and
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endosome–lysosome degradation pathways. Impaired fusion
events in these pathways have been implicated in a number
of neurodegenerative diseases, including frontotemporal
dementia [19,21,22,55], Alzheimer’s disease [64–66] and
lysosomal storage disorders [67], as well as in myopathy
[52,68] and in the clearance of bacterial pathogens [69].
Further dissection of the roles of ESCRT proteins will shed
new light on the basic mechanism of vesicular fusion and may
also give novel insight into disease pathogenesis.
Acknowledgements
We thank the FReJA (Frontotemporal Dementia Research in
Jutland Association) Consortium for ongoing collaboration into
frontotemporal dementia caused by CHMP2B mutations and Ray
Young for help with preparation of the Figure.
Funding
Our work is supported by the Medical Research Council.
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Received 17 June 2010
doi:10.1042/BST0381469
C The
C 2010 Biochemical Society
Authors Journal compilation 1473