Cell Science at a Glance
The ESCRT machinery
at a glance
Thomas Wollert, Dong Yang,
Xuefeng Ren, Hyung Ho Lee,
Young Jun Im and James H.
Hurley*
Laboratory of Molecular Biology, National Institute
of Diabetes and Digestive and Kidney Diseases,
National Institute of Health, US Department of
Health and Human Services, Bethesda, MD, USA
20892, USA
*Author for correspondence (e-mail:
[email protected])
Journal of Cell Science 122, 2163-2166
Published by The Company of Biologists 2009
doi:10.1242/jcs.029884
The ESCRT machinery consists of the five
protein complexes ESCRT-0, ESCRT-I,
ESCRT-II, ESCRT-III and Vps4-Vta1, and
several
ESCRT-associated
proteins
(Hurley, 2008; Saksena et al., 2007;
Williams and Urbe, 2007). ESCRT-0,
ESCRT-I, ESCRT-II and Vps4-Vta1 are
soluble complexes that move between
cytosolic and membrane-bound states. By
contrast, the subunits of ESCRT-III are
soluble monomers that assemble on
membranes into tightly bound filaments
(Ghazi-Tabatabai et al., 2008), tubes
(Hanson et al., 2008; Lata et al., 2008) and
spirals (Hanson et al., 2008) that can only
be released by the ATP-dependent action of
Vps4-Vta1.
In this article and its accompanying poster,
we focus on the fundamentals of the
structure of the ESCRT machinery and its
molecular
interactions
with
other
molecules that explain its function in the
Journal of Cell Science
This article is part of a Minifocus on the ESCRT
machinery. For further reading, please see related
articles: ‘No strings attached: the ESCRT machinery
in viral budding and cytokinesis’ by Bethan
McDonald and Juan Martin-Serrano (J. Cell Sci. 122,
2167-2177) and ‘How do ESCRT proteins control
autophagy?’ by Tor Erik Rusten and Harald
Stenmark (J. Cell Sci. 122, 2179-2183).
The endosomal sorting complex required
for transport (ESCRT) machinery is
conserved from Archaea to animals, and
catalyzes the scission of membrane necks
(Wollert et al., 2009) in the biogenesis of
multivesicular bodies (MVBs) (Gruenberg
and Stenmark, 2004; Hurley, 2008;
Piper and Katzmann, 2007), cytokinesis
(Carlton and Martin-Serrano, 2007;
Lindas et al., 2008; Morita et al., 2007;
Samson et al., 2008) and the budding of
enveloped viruses such as HIV-1
(Bieniasz, 2006; Fujii et al., 2007; Morita
and Sundquist, 2004). Cleavage occurs
from the surface of the membrane that is
contiguous with the inside of the
membrane neck. The topology of scission
mediated by the ESCRT machinery is
opposite to that of the dynamin family of
membrane-scission proteins, which cleave
membrane necks by constricting them
from the outside.
2163
(See poster insert)
2164
Journal of Cell Science 122 (13)
Journal of Cell Science
diverse biological processes of MVB
biogenesis, cytokinesis and virus budding.
We hope that this short overview will
provide a useful foundation for
developmental biologists, clinicians and
others that aim to explore the roles of
ESCRTs in their research.
ESCRTs in MVB biogenesis and
receptor downregulation
The ESCRT proteins were discovered and
characterized less than 10 years ago as
having a role in the sorting of ubiquitylated
cell-surface receptors and other proteins to
the vacuole in yeast (Saksena et al., 2007).
This sorting process occurs through the
formation of MVBs, which are endosomes
containing intralumenal vesicles (ILVs)
that are destined for degradation in the
yeast vacuole or in the mammalian
lysosome. The ESCRTs have a central role
in the sorting of ubiquitylated receptors, to
which they bind directly via their
ubiquitin-binding domains. In addition, the
ESCRTs also have a direct role in
the membrane-remodeling events that lead
to ILV formation.
In yeast, ILVs have a mean diameter of
24 nm, and are therefore thought to derive
from a roughly circular patch on the
limiting membrane with a radius of 24 nm
(Nickerson et al., 2006). This value is
similar to the dimensions of a single copy
each of ESCRT-0, ESCRT-I and ESCRT-II
when they are assembled together on the
limiting membrane. It is notable that
the size of the ESCRTs is on the same
order as the dimensions of the ILVs that
they produce, and this prompts speculation
that as few as one copy each of these
ESCRTs might be required to produce a
single ILV.
ESCRTs bind to ubiquitylated
proteins
Ubiquitylation is the main molecular signal
that sends cargo into the ESCRT pathway
for lysosomal degradation. The ESCRTs
interact with ubiquitylated receptors and
other ubiquitylated cargo through various
motifs. These include the ubiquitininteracting motif (UIM) and the doublesided ubiquitin-interacting motif (DUIM)
of the yeast and human Hrs subunits of
ESCRT-0; the VHS (Vps27, Hrs and
STAM) domain and UIM of the STAM
subunit of ESCRT-0; the ubiquitinconjugating enzyme E2 variant (UEV)
domain of the Vps23 subunit of ESCRT-I;
the GRAM-like ubiquitin binding in
EAP45 (GLUE) domain of the VPS36
subunit of human ESCRT-II; and the NZF2
domain of the Vps36 subunit of yeast
ESCRT-II (Hurley, 2008; Saksena et al.,
2007; Williams and Urbe, 2007). All of
these motifs bind to ubiquitin Ile44 and the
surrounding residues, which precludes the
simultaneous interaction of multiple
ESCRTs with a single ubiquitin moiety.
The affinities of the individual motifs for
monoubiquitin are very low (less than
100 μM) – probably too low to have an
important role in targeting ESCRTs to
membranes. We favor a model in which
multiple ESCRTs co-assemble on
membranes and cluster multiple cargo
molecules, which may be monoubiquitylated, or polyubiquitylated through
the Lys63 residue of ubiquitin.
Membrane lipids recruit ESCRTs to
endosomes
ESCRTs are targeted to endosomes through
a variety of specific, partially specific and
nonspecific interactions with membrane
lipids. The FYVE domain of the Hrs
subunit of ESCRT-0 is highly specific
for phosphatidylinositol 3-phosphate
[PtdIns(3)P], whereas the GLUE domain
of the Vps36 subunit of ESCRT-II binds
to PtdIns(3)P and also to other
phosphoinositides. In addition, the first
helix of the Vps22 subunit of ESCRT-II
binds to acidic phospholipids with no
apparent specificity, yet the gain in overall
membrane affinity is still important
for membrane targeting (Im and Hurley,
2008). Finally, ESCRT-III subunits also
bind to acidic lipids with little specificity
(Saksena et al., 2009). Membrane binding
promotes a conformational change in
ESCRT-III subunits that appears to favor
their functional polymerization (Saksena
et al., 2009). These specific and nonspecific interactions are collectively
central to the recruitment, assembly and
activating conformational changes of the
ESCRTs.
The ESCRT-III cycle
Vps4 and the subunits of ESCRT-III are the
most conserved elements in the pathway
and are directly responsible for membrane
cleavage. In the ESCRT-III cycle, soluble
ESCRT-III monomeric subunits are
recruited from the cytosol to their site of
action on the membrane, where they
undergo an activating conformational
change, recruit various effector proteins
and
catalyze
membrane
scission.
Subsequent to membrane scission, Vps4
hydrolyzes ATP to drive the recycling of
ESCRT-III subunits back to their soluble
monomeric form.
In yeast, the four essential ESCRT-III
proteins are Vps20, Snf7, Vps24 and Vps2,
which assemble in that order (Teis et al.,
2008). Two more ESCRT-III proteins,
Vps60 and Did2, join later during ESCRT
assembly, as does the ESCRT-III-like
MIM1 and MIM2-containing protein Ist1
(Agromayor et al., 2009; Bajorek et al.,
2009; Dimaano et al., 2008; Rue
et al., 2008). Upon membrane binding
(Saksena et al., 2009), motifs in the
C-termini of ESCRT-III subunits are
exposed, allowing them to bind to the
microtubule-interacting and transport
(MIT) domains of downstream effector
proteins (Hurley and Yang, 2008).
Together, the short C-termini of ESCRT-III
subunits make up a dense region of
multiple regulatory signals. Vps4 functions
as a 12- to 14-mer, which therefore
contains 12-14 MIT domains. One site in
the Vps4 MIT domain can bind to the
MIM1s (MIT-interaction motif 1) of Vps2
and Did2 (Obita et al., 2007; StuchellBrereton et al., 2007), while another site
can bind to the MIM2 of Vps20 (Kieffer
et al., 2008). The Vps4 MIT domain also
binds with very low affinity to the
C-termini of most, if not all, of the other
ESCRT-III proteins (Kieffer et al., 2008).
Vta1, the non-catalytic subunit of the
Vps4-Vta1 complex, contains two MIT
domains that bind to late-joining
ESCRT-III proteins (Azmi et al., 2008;
Shim et al., 2008; Xiao et al., 2008). The
full Vps4-Vta1 assembly can thus bind to
ESCRT-III with very high cooperativity.
The ATPase activity of Vps4, in concert
with the multiplicity of binding events
between ESCRT-III and the MIT domains
of Vps4 and Vta1, is instrumental to
ESCRT-III disassembly. The disassembly
activity of the Vps4-Vta1 complex allows
the subunits of ESCRT-III to be recycled,
thereby completing the ESCRT-III cycle
and sustaining membrane-scission activity
(Wollert et al., 2009).
ESCRTs in HIV-1 budding
The ESCRT proteins are essential for the
efficient budding of HIV-1 from cultured
cells. The main role of the ESCRTs in viral
budding is to cut the neck that connects the
virion to the plasma membrane, releasing
the virion from the host cell. The HIV-1
Gag p6 protein recruits the host-cell
Journal of Cell Science
Journal of Cell Science 122 (13)
ESCRTs via two late-domain (L-domain)
sequence motifs (Fujii et al., 2007;
Bieniasz, 2006; Morita and Sundquist,
2004; McDonald and Martin-Serrano,
2009). The most important motif in the
Gag p6 protein that mediates ESCRT
recruitment is an L-domain with the
sequence PTAP, which binds to the UEV
domain of the Vps23 subunit of ESCRT-I.
Additionally, a YPxL motif near the
C-terminus of HIV-1 Gag p6 directly
interacts with the V domain of the ESCRTassociated protein ALIX (also known as
PDCD6IP and AIP1). The Bro1 domain of
ALIX directly activates ESCRT-III by
recruiting and activating the Snf7 subunit,
which is crucial for ESCRT-III function
(McCullough et al., 2008). It is less clear
how ESCRT-I activates ESCRT-III;
however, it is thought that the main role of
ESCRT-I and ALIX is to recruit and
activate
ESCRT-III.
ESCRT-II
is
apparently not involved in HIV-1 budding,
nor is the Vps20 subunit of ESCRT-III,
which couples ESCRT-II and ESCRT-III in
MVB biogenesis. Ubiquitylation by the
ligase NEDD4L is also involved in
ESCRT-mediated HIV-1 budding (Chung
et al., 2008; Usami et al., 2008). In
summary, ESCRT-I and ALIX appear to
act as adaptors that connect the viral Gag
protein to ESCRT-III, which in turn is
directly responsible for membrane
cleavage and virion release.
ESCRTs in cytokinesis
During cytokinesis, the ESCRTs play a
crucial role in the scission of the membrane
neck connecting two daughter cells, both in
mammalian cells (Carlton and MartinSerrano, 2007; Morita et al., 2007;
McDonald and Martin-Serrano, 2009) and
in a subset of Archaea (Lindas et al., 2008;
Samson et al., 2008). In mammalian
cytokinesis, the two daughter cells are
tethered to one another by a dense
microtubule-rich structure known as the
midbody. As with HIV-1 budding,
ESCRT-I and ALIX are the key adaptors
for membrane scission, and ESCRT-II is
not involved. GPPx3Y motifs in ESCRT-I
and ALIX bind to CEP55, a protein at the
midbody (Lee et al., 2008) that is
responsible for recruiting the ESCRTs to
the site of membrane scission. In turn,
ALIX, and probably ESCRT-I, recruit
ESCRT-III subunits, which close the neck.
However, full closure of the membrane
neck is obstructed by the microtubules of
the central spindle. ESCRT-III has at least
one additional role in the process, which is
to recruit the microtubule-severing enzyme
spastin (Connell et al., 2008; Yang et al.,
2008), which probably cuts the central
spindle, together with the ESCRT-IIImediated membrane cleavage.
Perspectives
Recent discoveries have shown that
ESCRT-III and Vps4 make up a conserved
membrane-scission machine that functions
in many different contexts, including MVB
biogenesis,
HIV-1
budding
and
cytokinesis. The ESCRT machinery is
targeted to the site of action by different
means for each of these biological
processes: 3-phosphoinositides are central
in MVB biogenesis, Gag L-domains in
viral budding and CEP55 in cytokinesis. In
addition, ubiquitin is involved in most, if
not all, of these pathways. Each pathway
converges at the ESCRT-III cycle, which
probably functions in a similar way in each
setting. It will be interesting to see whether
the list of biological processes in which the
ESCRT machinery has a role expands; for
example, the possibility that ESCRTs have
a role in sealing autophagosomes is
intriguing (Rusten and Stenmark, 2009).
Although beyond the scope of this article,
the catalog of ESCRT functions that are
involved in health and disease continues to
grow rapidly (Saksena and Emr, 2009). In
addition to the role in HIV-1 budding
described
above,
genetic
defects,
alterations in gene expression, mutations or
altered interactions involving ESCRTs
have been implicated in several
pathological conditions, including cancer,
frontotemporal dementia, amyotrophic
lateral sclerosis, Huntington’s disease,
Parkinson’s disease, hereditary spastic
paraplegia and cataracts (Saksena and Emr,
2009). At the molecular level, the structure
and interactions of ESCRTs in solution are
now understood in great detail. However,
upcoming
challenges
for
ESCRT
biochemistry and structural biology
include gaining a better understanding of
the structure and interactions in the
membrane-bound setting in which
the ESCRTs function. As the fundamental
mechanisms of normal ESCRT function
become more clear, the long-term
challenges in this field will evolve towards
understanding how these functions are
perturbed in disease.
Research in the Hurley lab is funded by the intramural
program of the NIDDK, NIH, and the IATAP program
of the NIH. T.W. is an EMBO long-term fellow.
Deposited in PMC for release after 12 months.
2165
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