Download The molecular basis for selective assembly of the UBAP1

ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 663–672 doi:10.1242/jcs.140673
RESEARCH ARTICLE
The molecular basis for selective assembly of the
UBAP1-containing endosome-specific ESCRT-I complex
ABSTRACT
ESCRT-I is essential for the multivesicular body (MVB) sorting of
ubiquitylated cargo such as epidermal growth factor receptor, as well as
for several cellular functions, such as cell division and retroviral budding.
ESCRT-I has four subunits; TSG101, VPS28, VPS37 and MVB12.
There are several members of VPS37 and MVB12 families in
mammalian cells, and their differential incorporation into ESCRT-I
could provide function-specific variants of the complex. However, it
remains unclear whether these different forms of VPS37 and MVB12
combine randomly or generate selective pairings within ESCRT-I, and
what the mechanistic basis for such pairing would be. Here, we show
that the incorporation of two MVB12 members, UBAP1 and MVB12A,
into ESCRT-I is highly selective with respect to their VPS37 partners.
We map the region mediating selective assembly of UBAP1–VPS37A
to the core ESCRT-I-binding domain of VPS37A. In contrast, selective
integration of UBAP1 requires both the minimal ESCRT-I-binding region
and a neighbouring predicted helix. The biochemical specificity in
ESCRT-I assembly is matched by functional specialisation as siRNAmediated depletion of UBAP1, but not MVB12A and MVB12B, disrupts
ubiquitin-dependent sorting at the MVB.
KEY WORDS: EGFR, Endosome, TSG101, ESCRT-I, UBAP1, VPS37,
MVB12
INTRODUCTION
Activated epidermal growth factor receptor (EGFR) is
ubiquitylated, then internalised and sorted to the multivesicular
body (MVB) en route to its degradation in the lysosome (Eden
et al., 2012; Sorkin and Goh, 2009). MVB sorting of ubiquitylated
EGFR involves several ubiquitin-binding protein complexes,
termed endosomal sorting complex required for transport
(ESCRT)-0, -I and -II (Bishop et al., 2002; Doyotte et al.,
2005; Garrus et al., 2001; Henne et al., 2011; Hurley and
Stenmark, 2011; Komada and Soriano, 1999; Malerød et al.,
2007; Raiborg and Stenmark, 2009; Raiborg et al., 2002; Razi and
Futter, 2006; Ren and Hurley, 2010; Slagsvold et al., 2005;
Wollert and Hurley, 2010). Although the function of ESCRT-II is
less well characterised, ESCRT-0 and ESCRT-I combine with the
scaffold protein, his domain protein tyrosine phosphatase (HDPTP, also known as PTPN23) (Ali et al., 2013; Doyotte et al.,
Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
*These authors contributed equally to this work
{
Author for correspondence ([email protected])
This is an Open Access article distributed under the terms of the Creative Commons Attribution
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Received 14 August 2013; Accepted 30 October 2013
2008; Stefani et al., 2011) and the deubiquitylating enzyme,
UBPY/USP8 (Alwan and van Leeuwen, 2007; Bowers et al.,
2006; Pareja et al., 2012; Row et al., 2006), to facilitate the
transfer of EGFR to a further complex, ESCRT-III. ESCRT-III
drives the formation of the intralumenal vesicles (ILVs) that
capture the receptor within the MVB (Henne et al., 2012; Wollert
and Hurley, 2010).
Although the ESCRT pathway was first identified in budding yeast
in the context of MVB sorting of ubiquitylated cargoes, it supports
several other unrelated cellular processes found in fission yeast,
archaea and metazoans (Hanson and Cashikar, 2012; Peel et al.,
2011). These include mitotic spindle maintenance (Frost et al., 2012;
Morita et al., 2010), cytokinesis (Caballe and Martin-Serrano, 2011;
Carlton and Martin-Serrano, 2007; Morita et al., 2007b) and viral
budding (Garrus et al., 2001; Martin-Serrano and Neil, 2011). In
addition, some MVB cargoes are sorted in ESCRT-dependent but
ubiquitin-independent pathway(s) (Dores et al., 2012). These
functionally distinct ESCRT pathways each have a unique
complement of ESCRT complexes and accessory proteins
associated with them (Bajorek et al., 2009; Carlton et al., 2008;
Carlton et al., 2012; Morita et al., 2010; Peel et al., 2011). Hence,
a crucial goal towards understanding the mechanism of EGFR
downregulation is to identify the ESCRT pathway components that
act selectively in this process.
The ESCRT-I complex is central to all ESCRT pathways so far
characterised. Yeast ESCRT-I is a heterotetramer, comprising
vacuolar protein sorting protein 23, (Vps23p), Vps28p, Vps37p
and MVB protein of 12 kDa (Mvb12p) (Katzmann et al., 2001;
Kostelansky et al., 2007; Martin-Serrano and Neil, 2011). A
core complex assembles from Vps23p, Vps28p and Vps37p,
comprising a stalk region that involves helices from Vps23p and
Vps37p, and a head region that includes part of Vps28p as well as
Vps23p and Vps37p. Mvb12p associates primarily with the
Vps23p–Vps37p stalk (Kostelansky et al., 2007). Mammals
possess a single orthologue of Vps23p, tumor susceptibility gene
101 (TSG101) (Babst et al., 2000) and a single VPS28 (Bishop
and Woodman, 2001), albeit with splice isoforms. Both proteins
are evolutionarily conserved. In contrast, there are four VPS37encoding genes expressed in mammalian cells (VPS37A–
VPS37D) (Bache et al., 2004; Eastman et al., 2005; Stuchell
et al., 2004) that resemble each other and Vps37p only within the
ESCRT-I-core-forming domain, called the Mod(r) domain
(Bache et al., 2004; Stuchell et al., 2004). Although the Mod(r)
domains of VPS37B and VPS37C are highly similar (47% amino
acid identity), those of VPS37A and VPS37D are more divergent
(29% and 39% identity with VPS37B, respectively). VPS37A is
particularly divergent in its stalk-forming region (22% identity
compared with VPS37B versus 49% for VPS37C compared with
VPS37B). VPS37A also has an N-terminal ubiquitin enzyme
variant (UEV) domain, whereas VPS37B–VPS37D have
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Lydia Wunderley*, Kim Brownhill*, Flavia Stefani, Lydia Tabernero and Philip Woodman{
RESEARCH ARTICLE
C-terminal proline-rich regions of differing lengths (see Fig. 1)
(Bache et al., 2004; Eastman et al., 2005; Stuchell et al., 2004).
There are three known mammalian proteins that serve as
equivalents to Mvb12p based on their ability to integrate into
ESCRT-I: MVB12A, MVB12B (Konishi et al., 2006; Morita et al.,
2007a; Tsunematsu et al., 2010) and UBAP1 (Agromayor et al., 2012;
Stefani et al., 2011). However, there is no sequence conservation
between any of these proteins and Mvb12p. MVB12A and MVB12B
each utilise conserved regions (EBB1 and EBB2) towards their Ctermini to bind to the VPS37–TSG101 stalk (Morita et al., 2007a)
(Fig. 1B). EBB2 resembles an N-terminal region within UBAP1,
termed the UBAP1-MVB12-associated (UMA) domain (Agromayor
et al., 2012; de Souza and Aravind, 2010; Stefani et al., 2011). Aside
from this, UBAP1 is quite distinct from MVB12A and MVB12B. It
contains a predicted coiled-coil-forming helix (residues 59–92)
(Fig. 1C), a central region (residues 159–308), which binds the
ESCRT accessory protein HD-PTP (Stefani et al., 2011), and a Cterminal arrangement of three overlapping UBA domains (residues
381–502) termed a solenoid of overlapping UBA domains (SOUBA)
(Fig. 1B) (Agromayor et al., 2012; de Souza and Aravind, 2010;
Stefani et al., 2011).
UBAP1 is important for the MVB sorting and degradation of
ubiquitylated endosomal cargoes, including EGFR, but not for
cytokinesis or retroviral budding (Agromayor et al., 2012; Stefani
et al., 2011). By contrast, modulating the levels of MVB12A or
MVB12B profoundly affects the assembly of infective retroviral
particles (Morita et al., 2007a), whereas siRNA-mediated
depletion of MVB12A or MVB12B only mildly affects the
stability of EGFR, as assessed by western blotting (Tsunematsu
et al., 2010). Previous studies from our laboratory have provided
evidence that UBAP1 selectively associates with ESCRT-I
complexes containing VPS37A, supporting a model of
functionally distinct ESCRT-I types of defined MVB12 and
VPS37 composition (Stefani et al., 2011). In contrast, however,
several other studies based on expression of epitope-tagged
components have found no such selectivity (Agromayor et al.,
2012; Morita et al., 2007a; Tsunematsu et al., 2010), suggesting
that the incorporation of VPS37 and MVB12 family members
might be stochastic. To further test the selectivity of ESCRT-I
function and assembly, we have compared the function of UBAP1
with that of MVB12A and MVB12B during ubiquitin-dependent
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EGFR sorting to the MVB. In conjunction, we have used cellular
and in vitro systems in order to determine the selectivity of pairings
of VPS37 and MVB12 family members within ESCRT-I. We
demonstrate that the generation of at least a subset of ESCRT-I
types is highly selective, matching their functional specialisation,
and we have mapped the principal determinants that underlie this
selectivity.
RESULTS
UBAP1, but not MVB12A or MVB12B, is crucial for ubiquitindependent EGFR sorting
To test for specialisation in ESCRT-I function, we transfected cells
with siRNA directed either against UBAP1 (Stefani et al., 2011)
(supplementary material Fig. S1A), or against both MVB12A and
MVB12B (supplementary material Fig. S1B), and directly
compared the effects on EGFR trafficking. Note that because we
failed to detect MVB12B in HeLaM cells, in line with previous
reports that this protein has a restricted expression (Morita et al.,
2007a), RNA interference was assessed by a failure to detect
exogenously expressed protein. Cells were examined for transport
of fluorescent EGF to lysosomes (assessed by loss of fluorescence
signal), because disruption of this pathway, and a consequent
build-up of fluorescent EGF in ubiquitin-rich early endosomes, is
an excellent reporter of loss of ESCRT-I and UBAP1 function at
the endosome (Doyotte et al., 2005; Stefani et al., 2011). As shown
in Fig. 2A, depletion of UBAP1 arrested a pulse of fluorescent
EGF in clustered compartments that labelled strongly for
ubiquitylated proteins. Staining for early endosome antigen 1
(EEA1) confirmed that fluorescent EGF accumulated in clustered
early endosomes in UBAP1-depleted cells (Fig. 2B), in agreement
with an earlier report (Stefani et al., 2011). In contrast, cells
depleted for MVB12A and MVB12B showed no apparent
impairment in EGF degradation, as assessed by the loss of
fluorescent EGF during a 3-hour chase. Images taken after a 30minute chase confirmed that these cells internalised fluorescent
EGF as well as control cells did (supplementary material Fig. S1C).
In addition, the distribution of early endosomes and ubiquitylated
proteins was similar in cells depleted of MVB12A and MVB12B to
that in control cells. Depletion of UBAP1, but not MVB12A and
MVB12B, caused the swelling of lysosomes (supplementary
material Fig. S1D). In summary, these data indicate that UBAP1,
and MVB12A and MVB12B are functionally distinct.
Fig. 1. Domain organisation and conservation of VPS37 and MVB12
families. (A) Domain organisation of VPS37 family members. (B) Structural
organisation of UBAP1- and MVB12-specific ESCRT-I complexes.
(C) Coiled-coil prediction of the first 140 residues of UBAP1.
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Our previous work, focussed on UBAP1 and based largely on
native biochemistry, led us to conclude that MVB12 and VPS37
family members might combine selectively to assemble discrete
ESCRT-I complexes (Stefani et al., 2011). Because these
conclusions differ from those of other studies (Agromayor et al.,
2012; Morita et al., 2007a; Tsunematsu et al., 2010), it was
important to re-examine the subunit composition of ESCRT-I
complexes in further detail. An inherent problem with coexpression studies is that all four ESCRT-I subunits are
expressed at non-physiological levels, which might favour the
efficient assembly of complexes that are otherwise rare. To
minimise this risk, we transfected cells with one ESCRT-I subunit
only and examined how this associated with native partners within
ESCRT-I. As demonstrated previously (Stefani et al., 2011),
epitope-tagged UBAP1 was efficiently incorporated into ESCRT-I,
as judged by its ability to be immunoprecipitated with antibodies
against TSG101 or VPS28 (Fig. 3A). Prior depletion of VPS37A
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The assembly of ESCRT-I complexes containing either UBAP1 or
MVB12 is highly selective
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Fig. 3. Exogenously expressed
UBAP1 selectively incorporates
into VPS37A-containing ESCRT-I.
(A) UBAP1–Myc was transiently
expressed and cell lysates
immunoprecipitated (IP) with antiTSG101 or anti-VPS28 antibodies.
(B) Left panel: cells were siRNAdepleted for control (cont kd) or
VPS37A (VPS37A kd) and western
blotted for VPS37A and dynactin
p150, as a loading control. Right
panel: UBAP1–Myc was expressed in
control- or VPS37A-depleted cells,
and lysates were immunoprecipitated
with anti-TSG101 or anti-VPS28
antibodies. (C) Full-length UBAP1–
strep, or the UBAP1–strep N-terminal
(1–95) or central (122–308)
fragments, were transiently
expressed in control- or VPS37Adepleted cells. Cell lysates were
applied to Strep-Tactin beads, and
samples were western blotted
as indicated.
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Fig. 2. UBAP1, but not MVB12A or MVB12B, regulates ubiquitin-dependent EGFR trafficking. HeLaM cells were depleted of UBAP1 (UBAP1 kd), or of
MVB12A and MVB12B (MVB12A/B kd). Cells were incubated with fluorescent EGF for 3 hours, then fixed and stained for ubiquitylated protein (A), or the early
endosome marker EEA1 (B). Scale bars: 20 mm.
alone completely abolished the ability of UBAP1–Myc to interact
with TSG101 or VPS28 (Fig. 3B). Furthermore, upon expression
of UBAP1–strep, pull-down using Strep-tactin beads recovered
UBAP1–strep, TSG101 and VPS37A, but neither VPS37B nor
VPS37C (Fig. 3C, left panel). The recovery of TSG101 with
UBAP1–strep was completely abolished by depletion of VPS37A,
even though total levels of endogenous TSG101, VPS37B and
VPS37C were unaffected (Fig. 3C, left panel). Hence, even when
VPS37A is absent, UBAP1 cannot utilise endogenous alternative
VPS37 family members to incorporate into ESCRT-I. UBAP1–
GFP, which is expressed at high levels, was also selectively
incorporated into ESCRT-I-containing VPS37A, although a trace
amount (over background levels) of VPS37B was also detected in
UBAP1–GFP pull-downs (supplementary material Fig. S2). We
could not find reagents against VPS37D that detected the
endogenous protein.
Incorporation of UBAP1 into ESCRT-I requires the UMA
domain (amino acids 17–63) (Agromayor et al., 2012; de Souza
and Aravind, 2010; Stefani et al., 2011). Although we failed to
express UBAP1(1-63)–strep (data not shown) a slightly longer Nterminal fragment, UBAP1(1-95)–strep, that incorporates an
additional predicted a-helix (Fig. 2B), could be generated.
Like full-length UBAP1–strep, this fragment was efficiently
incorporated into ESCRT-I containing VPS37A, but not
into ESCRT-I containing VPS37B or VPS37C. In addition,
association of this fragment with TSG101 was absolutely
dependent on the presence of VPS37A (Fig. 3C, centre panel).
In contrast, a central fragment of UBAP1 known to associate with
HD-PTP (Stefani et al., 2011), UBAP1(122-308)–strep, did not
bind either TSG101 or VPS37A (Fig. 3C, right panel). Hence, we
identified a minimal region of UBAP1 that retains the ability to
selectively incorporate into an ESCRT-I complex within cells.
We next examined whether MVB12 incorporation into
ESCRT-I was also selective, concentrating on MVB12A, the
isoform expressed in HeLaM cells. MVB12A–strep was
incorporated into ESCRT-I, shown by its ability to pull down
endogenous TSG101 (Fig. 4A). It associated equally well with
complexes containing VPS37B and VPS37C, but was not
incorporated into ESCRT-I complexes containing VPS37A.
Consistent with this, depletion of VPS37A did not influence the
efficiency of MVB12A–ESCRT-I assembly (Fig. 4A). Hence, in
contrast to other studies (Morita et al., 2007a; Tsunematsu et al.,
2010), we find that although MVB12 shows no strong preference
between VPS37B and VPS37C, it does not readily associate with
VPS37A in cells. We then asked whether exogenously expressed
VPS37 family members were also selectively incorporated into
native ESCRT-I complexes. As shown in Fig. 4B, VPS37A–Myc,
but neither VPS37B–Myc nor VPS37C–Myc, efficiently coimmunoprecipitated endogenous UBAP1.
Selective assembly of ESCRT-I complexes in vitro
To further validate our findings, we expressed ESCRT-I
components in vitro. Although human ESCRT-I can assemble
when co-expressed in bacteria (Agromayor et al., 2012), we
decided to generate recombinant proteins by translation in rabbit
reticulocyte lysates. This system has the advantages that
eukaryotic folding factors are present during protein synthesis
and that labelled proteins are generated at nanomolar levels
(Jackson and Hunt, 1983). Hence, ESCRT-I assembly conditions
should more closely mirror those within the cell. We first found
conditions that allowed UBAP1–strep, Flag–VPS37A, VPS28–
Myc and HA–TSG101 to be co-translated in roughly equal
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Fig. 4. MVB12A and VPS37C form a selective ESCRT-I complex.
(A) Strep-MVB12A was transiently transfected in control cells (cont kd) or
cells depleted of VPS37A, VPS37B or VPS37C (VPS37A kd, VPS37B kd
and VPS37C kd, respectively). Cell lysates were applied to Strep-Tactin
beads, and samples were western blotted as indicated. (B) Lysates made
from control cells (-) and cells expressing VPS37A–Myc, VPS37B–Myc or
VPS37C–Myc (A, B and C, respectively) were immunoprecipitated with antiMyc antibody and samples were western blotted as indicated. The asterisk
indicates a non-specific band.
amounts (Fig. 5A, left panel, lane 2). With the exception of a
distinct doublet for VPS37A, the translation mixes were generally
free of proteolytic fragments and/or incomplete translation
products that might complicate analysis, although minor
products were inevitably present. ESCRT-I was efficiently coassembled, as judged by the ability to pull down each component
on anti-HA beads (Fig. 5A, lane 10). As a control, no other
components associated with anti-HA beads in the absence of HA–
TSG101 (supplementary material Fig. S3A).
ESCRT-I assembly was coordinated correctly, given that
efficient association of VPS28–Myc or Flag–VPS37A with
HA–TSG101 occurred in the absence of additional ESCRT-I
components in the translation mix (Fig. 5A, lanes 14 and 15),
whereas UBAP1 associated with anti-HA beads only when Flag–
VPS37A was present (Fig. 5A, lanes 10 and 12). In addition, no
association was seen between VPS37A and any other ESCRT-I
components in the absence of translated TSG101 (Fig. 5A, lane
13; supplementary material Fig. S3B). These findings are fully
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consistent with previous studies examining the assembly pathway
of mammalian ESCRT-I (Bache et al., 2004; Bishop and
Woodman, 2001; Eastman et al., 2005; Morita et al., 2007a;
Stuchell et al., 2004) in which VPS28 and VPS37 can associate
independently with TSG101, and MVB12 binds to a TSG101–
VPS37 dimer. In addition, the data show that ESCRT-I assembly
in reticulocyte lysates occurs exclusively between translation
products, with no evidence for the involvement of any free
endogenous ESCRT-I components that might be present in the
lysates. Binding of UBAP1 to HA beads still occurred when
VPS28 was absent, although it was significantly reduced (33% as
efficiently as controls, 63.0% s.e.m., n55; see supplementary
material Fig. S3C for a further example). Previous work has
shown that MVB12A and MVB12B assembly into ESCRT-I does
not require VPS28 (Morita et al., 2007a), which fits well with the
crystal structure of yeast ESCRT-I (Kostelansky et al., 2007), in
which minimal contacts between Vps28p and Mvb12p occur. The
orientation of UBAP1 with respect to VPS28 within the complex
might therefore be slightly different to that of MVB12A and
MVB12B. Importantly, the selectivity of ESCRT-I assembly was
largely maintained under these conditions (Fig. 5B, compare
lanes 17 and 18). Although UBAP1 was incorporated efficiently
into complexes co-assembled with VPS37A, only a small amount
of UBAP1 bound to complexes co-assembled with VPS37C
(7.0%61.23% s.e.m. compared to with VPS37A; n54), despite
VPS37C binding to TSG101 as efficiently as VPS37A
(117%630.0% s.e.m., n57).
A further difference between bacterial expression and in vitro
translation in rabbit reticulocyte lysates is the potential presence
in the latter of post-translational modifications that might enhance
the selectivity of ESCRT-I assembly. Of particular note,
both MVB12A and MVB12B are subject to serine/threonine
(Morita et al., 2007a) and tyrosine (Tsunematsu et al., 2010)
phosphorylation. Hence, to rule out protein phosphorylation as a
major determinant of selective ESCRT-I assembly, in vitro
translations were also performed using wheat germ lysates
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Fig. 5. Assembly of ESCRT-I complexes in vitro. (A) The indicated combinations of UBAP1–strep, Flag–VPS37A, VPS28–Myc and HA–TSG101 were
translated in rabbit reticulocyte lysates (left), and samples were immunoprecipitated (IP) as shown (right). (B) In vitro translation mixtures as indicated (left) were
immunoprecipitated with anti-HA beads (right).
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because plant protein kinase systems are highly divergent from
those of mammals. Additionally, no obvious UBAP1 orthologue
is present in plants, and VPS37A is only poorly conserved [the
most highly conserved region, the Mod(r) domain, is only 53%
similar between human and wheat]. Therefore, assembly of the
human ESCRT-I in wheat germ lysates minimises the possibility
that endogenous proteins in the lysate aid selective ESCRT-I
assembly. Indeed, assembly of wheat germ lysate translation
products into ESCRT-I was both efficient and selective with
respect to the fate of UBAP1 (supplementary material Fig. S3D,
lanes 17 and 18).
Mapping the determinants of selective ESCRT-I assembly
Biochemical specificity in the binding of EBB and/or UMA
domains to Mod(r)–TSG101 dimers could explain the selective
ESCRT-I assembly we observe because these regions are known
to drive assembly of MVB12A and MVB12B into ESCRT-I.
These regions are divergent in UBAP1 and VPS37A compared to
MVB12A and MVB12B, and VPS37B and VPS37C. We first
tested which minimal region of VPS37A was required for
selective incorporation of UBAP1 into ESCRT-I. Co-translations
were performed using either full-length VPS37A, or either the
UEV or Mod(r) domains. In contrast to full-length Flag–VPS37A,
Flag–VPS37A(UEV) did not co-immunoprecipitate with HA–
TSG101 from translation mixtures (Fig. 6A, compare lanes 10
and 11), despite a very weak association of the VPS37A UEV
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domain with TSG101 having been reported previously (Bache et al.,
2004). As expected, almost no UBAP1 was co-immunoprecipitated
from this mixture. Furthermore, no direct association between
translated Flag–VPS37A(UEV) and UBAP1–strep could be detected
(data not shown). Instead, UBAP1 co-immunoprecipitated with
HA–TSG101 in the presence of Flag–VPS37A(Mod(r)) as
efficiently as with full length Flag–VPS37A (Fig. 6A, compare
lanes 10 and 12). The amount of VPS37A(Mod(r)) recovered in
HA–TSG101 immunoprecipitates relative to full-length VPS37A
was 77%615% s.e.m. (n58), whereas the recovery of UBAP1 was
76%65% (n56). In contrast, strep–MVB12A did not associate
with complexes containing full-length Flag–VPS37A or Flag–
VPS37A(Mod(r)), but associated strongly with those containing
Flag–VPS37C (Fig. 6B). Hence, as described for MVB12A and
MVB12B (Morita et al., 2007a), UBAP1 associates with a Mod(r)domain–TSG101 dimer, but in this case only those containing the
VPS37A Mod(r). Moreover, the Mod(r) domain of VPS37A does
not readily bind MVB12A, consistent with the selective assembly of
MVB12 into ESCRT-I that we observe in cells.
Next, the UMA domain of UBAP1 (amino acids 1–63), and the
EBB1–EBB2 region of MVB12A were co-translated with other
ESCRT-I components. UBAP1(UMA) incorporated into ESCRTI efficiently relative to full-length UBAP1 (Fig. 7A). Hence, the
minimal information necessary for assembly of UBAP1 with
TSG101 is UBAP1(1-63) and VPS37(Mod(r)) (supplementary
material Fig. S4A). Strikingly, however, the selectivity of this
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Fig. 6. The VPS37A Mod(r) domain confers
selective assembly of UBAP1 into ESCRTI. (A) Domains of Flag–VPS37A were
translated with or without other ESCRT-I
components (left) and immunoprecipitated
(IP) with anti-HA beads (right). FL, full length.
(B) Translations performed as indicated, with
or without MVB12A, (left) were
immunoprecipitated with anti-HA beads
(right). Note that MVB12A translations were
consistently lower in the presence of the
VPS37A(Mod(r)) domain.
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Fig. 7. Mapping the regions of
UBAP1 and MVB12A that confer
selective ESCRT-I assembly. (A) In
vitro translations containing the
indicated fragments of UBAP1 and
epitope-tagged ESCRT-I subunits
(left) were immunoprecipitated (IP)
with anti-HA (TSG101) beads (right).
The asterisks denote a background
band (probably label bound to
globin). A section of the image is
shown at higher exposure to highlight
the behaviour of the short UBAP1
fragments, which each contain a
single methionine residue. An
independent experiment is shown in
supplementary material Fig. S4. FL,
full length. (B) Translations as
indicated (left), with full-length
MVB12A (filled circle) or the EBB1–
EBB2 fragment (open circle), were
immunoprecipitated with anti-HA
beads (right).
binding. In summary, we have identified peptide regions within
MVB12 and VPS37 family members that confer their selective
assembly into functionally distinct ESCRT-I complexes (Fig. 8).
DISCUSSION
Our studies reveal that selective association between MVB12 and
VPS37 subunit family members generates distinct ESCRT-I
complexes that correlate with different biological activities.
Hence, although stochastic association of known MVB12 and
VPS37 family members would generate up to 12 different
ESCRT-I complexes, our studies suggest that the cellular
ESCRT-I complement is rather more restricted. Discrimination
between UMA and Mod(r) domains contributes to the selectivity
of ESCRT-I assembly, although ‘unmatched’ UMA and Mod(r)
domain pairings might bind each other with low affinity and
hence support less-selective assembly of ESCRT-I when multiple
subunits are co-expressed. The Mod(r) domains of all VPS37
family members are predicted to form similar, largely helical,
structures, though it is noteworthy that the level of conservation
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Journal of Cell Science
association was almost completely lost, because UBAP1(UMA)
also associated readily with VPS37C-containing ESCRT-I
(Fig. 7A, compare lanes 11 and 15). In contrast, a fragment of
UBAP1 containing the UMA domain and the neighbouring
predicted helical region (amino acids 1–95) was incorporated into
ESCRT-I as selectively as the full-length protein (Fig. 7A,
compare lanes 10 and 14). No binding could be detected between
the UBAP1 predicted helix region alone (amino acids 45–95) and
ESCRT-I (Fig. 7A, lane 12). Note that the closeness of a nonspecific band to the UBAP1(1-95) fragment prevented reliable
quantification (an independent experiment is shown in
supplementary material Fig. S4B). Hence, the UMA domain
might fully account for the efficiency of UBAP1 incorporation
into ESCRT-I, but the neighbouring region helps restrict its
association to ESCRT-I containing VPS37A. MVB12A(EBB1EBB2) bound VPS37C-containing ESCRT-I efficiently compared
with full-length MVB12A, but did not associate with VPS37Acontaining ESCRT-I (Fig. 7B, compare lanes 11 and 12). Hence,
this region of MVB12A is sufficient to confer selective ESCRT-I
RESEARCH ARTICLE
Journal of Cell Science (2014) 127, 663–672 doi:10.1242/jcs.140673
between VPS37A and other family members diminishes within
the stalk-forming region of the Mod(r) domains compared to the
head-forming region. Based on the structure of yeast ESCRT-I
(Kostelansky et al., 2007), it is the stalk-forming region that is
likely to form the principal interface with MVB12. We provide
evidence that other domains might regulate the binding reactions.
Specifically, a putative helix contiguous with the UMA domain of
UBAP1 appears important for preventing the non-selective
association of UBAP1 with VPS37C. Although this region
might also enhance the avidity of UBAP1 for VPS37A, we
found no direct evidence of this. The EBB1 and EBB2(UMA)
regions of MVB12A and MVB12B each contain sufficient
information to bind ESCRT-I (Morita et al., 2007a) and, in
combination, are likely to form a binding surface that is more
selective than the EBB2(UMA) sequence alone, although we
have not tested this possibility.
Although the incorporation of UBAP1 into ESCRT-I appears
to be highly selective, other ESCRT-I complexes might well form
in a more stochastic fashion. For example, the lack of preference
of exogenous MVB12A for integrating into ESCRT-I complexes
containing native VPS37B or VPS37C is in keeping with the
greater similarity in sequence and domain organisation of these
VPS37 family members. Hence, MVB12A and MVB12B,
and VPS37B and VPS37C, might integrate into ESCRT-I
interchangeably, and perhaps function in related processes. We
have not examined the potential for known MVB12 proteins to
associate with VPS37D, which is less well characterised.
Additionally, there might be further MVB12 family members
that await identification. It is also noteworthy that our previous
study suggested that a minor portion of VPS37A might lie outside
of an UBAP1-containing ESCRT-I (Stefani et al., 2011). Clearly,
further work is required to fully characterise the range of cellular
ESCRT-I complexes.
The insensitivity of ubiquitin-dependent EGFR sorting to the
combined depletion of MVB12A and MVB12B is consistent with
the distinct ESCRT-I assembly pathway that we report here.
However, it remains quite possible that MVB12A and MVB12B
have some role at the endosome, and/or in event(s) downstream
of EGFR distinct from UBAP1. Both MVB12A and MVB12B
associate with EGFR, and their patterns of tyrosine
phosphorylation alter upon EGFR activation (Tsunematsu et al.,
2010). Also consistent with an endosomal function, MVB12A
binds CD2AP and CIN85, known regulators of EGFR signalling
and endosomal trafficking (Konishi et al., 2006). Furthermore,
loss of TSG101 causes more severe defects in endosome
morphology than loss of UBAP1 (Doyotte et al., 2005; Stefani
et al., 2011). This might point to the presence of additional
ESCRT-I complexes acting alongside that containing UBAP1,
though it is also possible that a TSG101–VPS28–VPS37A
complex lacking UBAP1 retains some sorting activity. The
documented interaction between VPS37C and the ESCRT-0
component, hepatocyte growth factor receptor substrate (Hrs)
670
(Eastman et al., 2005), is also consistent with the possibility that
other ESCRT-I complexes have some endosomal function. By
analogy, ESCRTs perform two roles during cytokinesis;
they drive membrane scission but also promote microtubule
disassembly by recruiting the microtubule severing protein,
spastin (Caballe and Martin-Serrano, 2011; Yang et al., 2008).
The finding that ubiquitin-dependent EGFR sorting requires an
ESCRT-I complex of unique composition extends the conclusions
of several studies identifying ESCRT components or ESCRTinteracting proteins that are involved only in certain cellular
processes. For example, although ESCRT-0 initiates the pathway
at the endosome (Hurley and Stenmark, 2011; Komada and
Soriano, 1999; Raiborg and Stenmark, 2009; Raiborg et al., 2002;
Razi and Futter, 2006; Ren and Hurley, 2010; Wollert and
Hurley, 2010), the centrosomal protein CEP55 and the p6 late
domain of Gag perform this role during cytokinesis and HIV
budding, respectively (Caballe and Martin-Serrano, 2011; Carlton
and Martin-Serrano, 2007; Morita et al., 2007b; Peel et al., 2011;
von Schwedler et al., 2003; Strack et al., 2003). These
interactions would negate the need for ESCRT-0 in these
events, although interestingly, binding does occur between
CEP55 and ESCRT-0 (Morita et al., 2007b). Bro1 proteins
support ESCRT function in various cellular contexts and, like
ESCRTs, are divergent in higher eukaryotes. One Bro1 protein,
Alix promotes CHMP4B recruitment during cytokinesis and viral
budding (Caballe and Martin-Serrano, 2011; Fisher et al., 2007),
and is required for sorting of some endocytic cargo (Baietti et al.,
2012; Dores et al., 2012; Hanson and Cashikar, 2012; Matsuo
et al., 2004; Peel et al., 2011). Meanwhile, another Bro1 protein,
HD-PTP, plays a part in CHMP4 recruitment during EGFR
sorting (Ali et al., 2013). The selective role that HD-PTP appears
to have over Alix in ubiquitylated MVB cargo sorting (Bowers
et al., 2006; Cabezas et al., 2005; Doyotte et al., 2008) is
underlined by its selective binding to the ESCRT-0 subunit signal
transducing adaptor molecule (STAM), and to UBAP1 (Ali et al.,
2013; Stefani et al., 2011). A further ESCRT component with
selective function is IST1, an ESCRT-III subunit that is essential
for cytokinesis but not for EGFR sorting or virus budding
(Agromayor et al., 2009; Bajorek et al., 2009). IST1 binds a
further effector, MITD1, which also acts selectively during
cytokinesis (Hadders et al., 2012; Lee et al., 2012). Hence, our
understanding of the divergence between ESCRT pathways
continues to expand. This study elucidates the molecular basis
for one such specialisation.
MATERIALS AND METHODS
Reagents
Antibodies against the following proteins were used. Mouse: EEA1
(Transduction Labs, Oxford, UK); LAMP1 (Developmental Studies
Hybridoma Bank, University of Iowa); UBAP1 (Abnova Corporation);
ubiquitin-protein conjugates (FK2; Enzo Life Sciences, Exeter, UK);
TSG101, tubulin and actin (AbCam; Cambridge, UK); Myc (Millipore,
Journal of Cell Science
Fig. 8. A summary of selective ESCRT-I assembly. The TSG101
core can recruit VPS37B and/or VPS37C in combination with
MVB12A or MVB12B (upper interaction), or VPS37A in combination
with UBAP1 (lower interaction). Integration of UBAP1 or MVB12A or
MVB12B requires recognition of the UMA and EBB2 domains by
corresponding Mod(r) domains in VPS37, probably in the stalk
regions, but additional interactions involving the UBAP1 helix (and
perhaps the EBB1 region of MVB12) confer specificity of assembly.
RESEARCH ARTICLE
DNA
MVB12B and VPS37B with C-terminal Myc-Flag tags in pCMV6-Entry were
purchased from Origene (Rockville, MD, USA). VPS37A–Myc and
VPS37C–Myc were in pcDNA5. For in vitro translations, the following
constructs were used: HA–TSG101 in pGADT7, full-length and the Mod(r)
domain (amino acids 229–397) of VPS37A, and VPS37C were in pcDNA3.1+
containing an N-terminal Flag tag. VPS28–Myc was in pcDNA3.1+
containing a C-terminal Myc tag. Full-length MVB12A, and fragments 1–
63, 1–95, 44–95 and 122–308 of UBAP1 were in pTriEX-5 with C-terminal
strep tags. The coiled coil prediction for UBAP1 was performed by COILS
(http://www.ch.embnet.org/software/COILS_form.html) (Lupas et al., 1991).
Cell culture
For siRNA experiments, 20 nM of each siRNA duplex was transfected using
INTERFERin (Polyplus; Illkirch, France), as specified by the manufacturer,
and samples were harvested after 72 hours. To confirm the knockdown of
MVB12A and MVB12B, cells were also transiently transfected with both
DNA constructs after 48 hours. Sequences for the UBAP1 duplex are as
described previously (Stefani et al., 2011), and for MVB12A+B knockdowns,
SMARTpools containing four duplexes were purchased from Thermo Fisher
(catalogue numbers L-026207 and L-015563; Loughborough, UK). AllStars
negative control siRNA was from Qiagen (Manchester, UK). For uptake of
Alexa-Fluor-488-conjugated EGF (Invitrogen), cells were grown on glass
coverslips and incubated with labelled EGF for 5 minutes at 37˚C in binding
medium [L-15 medium (Invitrogen) containing 0.2% (w/v) BSA], then chased
in binding medium containing unlabelled EGF before fixation. Times refer to
total pulse-chase times. For MG132 treatments cells were incubated with
1 mM for 18 hours.
Immunofluorescence and imaging
For immunofluorescence, cells were fixed in 3% formaldehyde and
quenched with glycine and permeabilised for 3 minutes in 0.1% (w/v)
Triton X-100. For LAMP staining, cells were fixed in methanol at
220 ˚C. DNA was stained using 49,6-diamidino-2-phenylindole (DAPI).
Fluorescence was examined using a wide-field Olympus BX-60
microscope fitted with a 606 1.40 N.A. PlanApo objective and a
CoolSnap ES camera (Roper Scientific, Ottobrunn, Germany), with
images captured using MetaVue. Images were opened as 16-bit greyscale images and scaled using linear transformations in ImageJ, then
converted to 24-bit RGB files in PhotoShop CS before being placed in
Adobe Illustrator. For EGF trafficking experiments, all samples were
imaged and adjusted for image contrast using identical settings.
Co-immunoprecipitation
At 24 hours post-transfection cells were lysed in IP buffer (10 mM TrisHCl pH 7.4, 150 mM NaCl, 1 mM EDTA) supplemented with protease
inhibitor cocktail, 1 mM PMSF and 0.5% CHAPS. After incubation at
4 ˚C for 1 hour, insoluble material was removed by centrifugation at
14,000 g for 10 minutes. A sample of the total cell lysate (input) was
retained for analysis, and immunoprecipitations were performed at 4 ˚C
for 18 hours, before the resin was washed with IP buffer and SDS-PAGE
sample buffer was added. UBAP–strep was immunoprecipitated using
Strep-Tactin beads (Thermo Fisher, UK), and UBAP–GFP was
immunoprecipitated using GFP–TRAP (ChromoTek, Martinsred, Germany).
Protein-A–Sepharose (Genscript, Piscataway, NJ, USA) with the appropriate
antibody, was used for all other immunoprecipitations.
In vitro translation
RNAs were synthesised from PCR products using T7 RNA polymerase
(Promega, Southampton, UK). To generate the EBB fragment of
MVB12A (amino acids 192–273) a T7 promoter sequence was
included in the forward primer and the full-length DNA was used as a
template. Proteins were translated in 15 ml reactions of nuclease-treated
rabbit reticulocyte lysate (Promega) containing 35S-methionine (Perkin
Elmer, Buckinghamshire, UK), and 100 units of RNasin (Promega) for
1 hour at 30 ˚C. Samples were then incubated at 30 ˚C with 1 mM
puromycin for 10 minutes. Alternatively, wheat germ translations were
incubated at 25 ˚C, and an additional incubation with 0.5 mg/ml RNaseA
for 5 minutes at 37 ˚C was included post-translation, followed by
centrifugation at 10,000 g for 5 minutes. A 10% sample of the input
was retained for analysis, and IP buffer (10 mM Tris-HCl pH 7.4,
150 mM NaCl, 1 mM EDTA) supplemented with protease inhibitor
cocktail, 1 mM PMSF and 0.1% Triton X-100 was used to dilute the
remaining sample tenfold. A pre-clearing step was performed using 20 ml
Protein-A–Sepharose (Genscript), 10 mM cold L-methionine, 1 mM
PMSF and protease inhibitor cocktail for 1 hour at 4 ˚C. For translations
of small fragments, an additional pre-clearing step using Ni-NTA resin
(Sigma) was also included to remove the haemoglobin. Samples were
clarified by centrifugation at 14,000 g for 10 minutes at 4 ˚C, and
immunoprecipitations were either performed overnight with the indicated
antibodies, and then Protein-A–sepharose for 2 hours the following day,
or, for TSG101, were performed using anti-HA-conjugated agarose
(Sigma) overnight. After washing three times in immunoprecipitation
buffer, SDS-PAGE sample buffer was added to the resin for analysis.
Following SDS-PAGE, the gels were dried and visualised by
phosphoimaging using a BAS5000 imager (Raytest; Surrey, UK).
AIDA software was used for quantification.
Competing interests
The authors declare no competing interests.
Author contributions
L.W., K.B. and F.S. contributed to the design of the project, and performed and
analysed experiments. L.T. contributed to the design of the project. P.W.
contributed to the design of the project and wrote the manuscript.
Funding
This study is supported by grants from the Medical Research Council UK [grant
number G0701140]; and Biotechnology and Biological Sciences Research
Council [grant number BB/K008773/1]. Deposited in PMC for immediate release.
Supplementary material
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.140673/-/DC1
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