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Biochem. J. (2012) 442, 209–220 (Printed in Great Britain) doi:10.1042/BJ20111761 209
Recruitment of the endosomal WASH complex is mediated by the extended
‘tail’ of Fam21 binding to the retromer protein Vps35
Michael E. HARBOUR1, Sophia Y. BREUSEGEM1and Matthew N. J. SEAMAN2
Cambridge Institute for Medical Research (CIMR)/Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Cambridge CB2 0XY, U.K.
The retromer complex is a conserved endosomal protein sorting
complex that sorts membrane proteins into nascent endosomal
tubules. The recognition of membrane proteins is mediated
by the cargo-selective retromer complex, a stable trimer of
the Vps35 (vacuolar protein sorting 35), Vps29 and Vps26
proteins. We have recently reported that the cargo-selective
retromer complex associates with the WASH (Wiskott–Aldrich
syndrome homologue) complex, a multimeric protein complex
that regulates tubule dynamics at endosomes. In the present
study, we show that the retromer–WASH complex interaction
occurs through the long unstructured ‘tail’ domain of the WASH
complex–Fam21 protein binding to Vps35, an interaction that
is necessary and sufficient to target the WASH complex to
endosomes. The Fam21-tail also binds to FKBP15 (FK506-
binding protein 15), a protein associated with ulcerative colitis, to
mediate the membrane association of FKBP15. Elevated Fam21-
tail expression inhibits the association of the WASH complex
with retromer, resulting in increased cytoplasmic WASH complex.
Additionally, overexpression of the Fam21-tail results in cell-
spreading defects, implicating the activity of the WASH complex
in regulating the mobilization of membrane into the endosome-
to-cell surface pathway.
Key words: endosome, FK506-binding protein 15 (FKBP15),
retromer, sorting, tubule, Wiskott–Aldrich syndrome homologue
(WASH) complex.
INTRODUCTION
Endosomes operate as major sorting stations that receive proteins
and lipids from the cell surface via endocytosis as well as
from the biosynthetic pathway [1]. Additionally, membrane flux
through the endocytic system plays a key role in the regulation
of cell size and shape [2–4]. The fidelity of endosomal protein
sorting is therefore of profound physiological importance and
there are numerous examples of pathologies resulting from a
failure of correct protein sorting, including the Niemann–Pick
disease [5], Charcot–Marie–Tooth disease and HSP (hereditary
spastic paraplegia) [6,7]. There is also much evidence to suggest
a causal link between the endosomal protein sorting of amyloid
precursor protein and the mechanisms that govern production of
the neurotoxic amyloid β-peptide that is a key pathological event
in Alzheimer’s disease [8].
Endosomal protein sorting occurs concomitantly with increas-
ing endosomal acidity. The influx of protons into endosomes
is mediated by the V-ATPase (vacuolar ATPase) and regulates
the association of some ligands (e.g. lysosomal hydrolases)
with their respective receptor [9,10]. One of the principal
components of endosomal protein sorting is the retromer complex.
Retromer is an evolutionarily conserved protein complex that
is required for the localization of several membrane proteins
of physiological importance, including the CIMPR (cation-
independent mannose 6-phosphate receptor) [11,12], the Vps10
(Vps is vacuolar protein sorting)-family sorting receptors
sortilin and SorL1 [13,14] and the divalent cation transporter
DMT1 [15].
The retromer complex recognizes cargo proteins through a
trimeric complex comprising Vps35, Vps29 and Vps26 [16,17]
that is recruited to the endosomal membrane by the small GTPase
Rab7 [18,19]. The Vps35 protein is believed to play a key role in
cargo recognition [20], although the structural similarity between
Vps26 and the arrestin family hints that Vps26 may also act
in cargo recognition [21,22]. In addition to the cargo-recognition
complex, retromer-mediated endosome-to-Golgi retrieval also
utilizes the activity of members of the Snx (sorting nexin)-BAR
(Bin/amphiphysin/Rvs) subfamily of Snxs, Snx1 and Snx2, in
complex with Snx5 and Snx6 [23]. The Snx-BAR sorting nexins
mediate the tubulation of endosomal membranes through their
C-terminal BAR domains [24].
The cargo-selective retromer complex associates with a
number of accessory proteins, including the Rab GTPase-
activating protein, TBC1D5 [TBC (Tre-2/Bub2/Cdc16) domain
family, member 5], the WASH (Wiskott–Aldrich syndrome
homologue) complex and FKBP15 (FK506-binding protein 15)
[19,25], a protein implicated in both ulcerative colitis and growth
cone collapse in neurons [26,27]. The WASH complex is a protein
complex containing WASH1, a member of the WASP (Wiskott–
Aldrich syndrome protein)/WAVE (WASP verprolin homologous)
family of actin nucleating promoting factors, KIAA1033 (also
known as SWIP), Strumpellin and Fam21. Loss of function of the
WASH complex results in dysregulation of endosomal tubules,
and since the Strumpellin protein is mutated in HSP, the activity of
the WASH complex has been of interest to researchers studying
the underlying pathology of HSP and related neuropathies
[25,28,29]. Although the precise function of the WASH complex
in endosomal protein sorting is yet to be determined, recent
studies in Dictyostelium discoideum have revealed a role for the
WASH complex in regulating the localization of V-ATPase [30].
Initial reports of the WASH complex association with
endosomes [28,29] contained a number of interesting
discrepancies. For example, the work by Gomez and Billadeau
Abbreviations used: BAR, Bin/amphiphysin/Rvs; CAPZ, capping protein (actin filament) muscle Z-line; CCDC, coiled-coil-domain-containing; CIMPR,
cation-independent mannose 6-phosphate receptor; FKBP15, FK506-binding protein 15; GFP, green fluorescent protein; GST, glutathione transferase; HSP,
hereditary spastic paraplegia; KD, knockdown; siRNA, small interfering RNA; Snx, sorting nexin; V-ATPase, vacuolar ATPase; Vps, vacuolar protein sorting;
WASH, Wiskott–Aldrich syndrome homologue; WASP, Wiskott–Aldrich syndrome protein; WAVE, WASP verprolin homologous; Y2H, yeast two-hybrid.
1These authors contributed equally to this work.
2To whom correspondence should be addressed (email mnjs100@cam.ac.uk).
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210 M. E. Harbour, S. Y. Breusegem and M. N. J. Seaman
[28] did not detect Strumpellin or KIAA1033 in association
with Fam21 and WASH1, whereas the study by Derivery et al.
[29] struggled to detect Fam21 that, when observed, was often
proteolysed and partially degraded. This could indicate that
the WASH complex is not a stable single entity but rather a
looser association of the constituent proteins. Studies conducted
by our laboratory showed that the WASH complex comprising
Strumpellin, KIAA1033, Fam21 and WASH1 bound to retromer
as a single unit. Questions remain, however, as to the nature and
assembly of the endosomal WASH complex [25].
Structural similarities between the endosomal WASH complex
and the analogous plasma membrane-localized WAVE
complex have been recently highlighted [31]. An important
difference between the WASH and WAVE complexes, however,
is the large unstructured ‘tail’ domain of the Fam21 protein. This
clearly distinguishes Fam21 from its WAVE complex counterpart,
Abi. Although Abi is ∼370 amino acids in size, Fam21 is a
much larger protein of ∼1300 amino acids containing a predicted
globular ‘head’ domain (∼200 amino acids) at its N-terminus
with a long (∼1100 amino acids) unstructured ‘tail’ that contains
a binding site for the actin capping protein [32].
In the present study, we show that Vps35 directly binds
the unstructured tail of Fam21. We show that the interaction
between the Fam21-tail and Vps35 is sufficient to target the
Fam21-tail to endosomes, consistent with the requirement for
the cargo-selective retromer complex to mediate the endosomal
recruitment of the WASH complex. Additionally, we report that
the Fam21-tail can bind to the FKBP15 protein, mediating its
endosomal localization. Overexpression of the Fam21-tail results
in displacement of FKBP15 from the endosome membrane and
competes with the endogenous WASH complex for binding to
retromer, resulting in cell spreading defects, thereby implicating
the activity of the WASH complex in regulating cell shape.
EXPERIMENTAL
Antibodies, general reagents and biochemicals
Unless otherwise stated, chemicals and reagents were purchased
from Sigma. 125I-labelled Protein A used in Western blotting
was obtained from PerkinElmer. Restriction enzymes and other
molecular biology reagents were purchased from New England
Biolabs. siRNA (small interfering RNA) oligonucleotides were
purchased from Dharmacon. Oligofectamine and Optimem used
in siRNA transfections were purchased from Invitrogen.
The anti-Fam21 sera (used for immunofluorescence) and
the anti-Strumpellin sera were both obtained from Santa
Cruz Biotechnology, the anti-FKBP15 serum used for
immunofluorescence was purchased from Abcam and the
anti-FKBP15 antibody used for Western blotting was generated
against a GST (glutathione transferase)–FKBP15 fusion protein
as described previously [25]. Anti-WASH1 antibody was
purchased from Sigma or Millipore. Anti-Fam21 serum used
for Western blotting was produced using a commercial service
(Charles River Laboratories; Margate, Kent, U.K.) using a GST–
Fam21-head domain fusion protein to immunize a rabbit. The
antiserum was affinity purified using GST–Fam21-head domain
coupled with CNBr-activated Sepharose (Amersham Pharmacia).
Monoclonal anti-Snx1 and anti-EEA1 (early endosome antigen
1) were purchased from BD Biosciences. Monoclonal anti-
GFP (green fluorescent protein) antibody was purchased from
Invitrogen, as were fluorescently labelled secondary antibodies
used in immunofluorescence. Polyclonal antisera against GFP,
Vps26, Vps35 and Snx1 have been described previously
[12,19].
Cell culture and production of stably transfected cell lines
HeLa M cells [33] were used throughout the present study
and are referred to simply as HeLa cells. Cells were cultured
in Dulbecco’s modified Eagle’s medium with the addition of
100 units/ml penicillin and 100 μg/ml streptomycin, 10 %(v/v)
fetal calf serum and 0.5 mg/ml G418 as necessary. The cells
stably expressing Vps29–GFP, GFP–Vps35 and Hrs1–GFP have
been described previously [19,25]. Cells stably expressing GFP–
Snx3, GFP–Snx1 and GFP–Fam21 were generated by transfecting
HeLa cells with the appropriate GFP-tagged construct in the
pIRESneo2 vector (Clontech) using Effectene (Qiagen) according
to the manufacturer’s instructions. Transfectants were selected
using G418 (Gibco) at 0.5 mg/ml and colonies that were G418
resistant were screened for expression of the respective construct
by fluorescence or immunofluorescence microscopy.
Production of the GFP-tagged constructs
In the present study, constructs to express GFP–Snx3, GFP–Snx1
and GFP–Fam21-head or -tail were generated. Other GFP-tagged
constructs have been described previously [25]. An IMAGE
consortium [Integrated Molecular Analysis of Genomes and their
Expression consortium (at St. Louis, MO, U.S.A., and at the
Human Genome Mapping Project, Hinxton Hall, Cambridge,
U.K.)] EST (expressed sequence tag) containing full-length
human Snx3 was obtained from GeneService and was used as
a template for a Pfu-mediated PCR reaction to amplify Snx3 and
add BamHI and SalI sites to the 5-and3
-ends respectively. The
PCR product was cloned first using pCRblunt (Invitrogen) and
then sequenced to confirm the identity and fidelity of the PCR
reaction product. The Snx3 ORF (open reading frame) was then
subcloned into pEGFP-C1 at the BglII and SalI sites. The GFP–
Snx3 construct was excised from pEGFP-C1 by digestion with
NheI–BamHI and then cloned into pIRESneo2 that had also been
digested with NheI and BamHI.
The Fam21–GFP construct was described previously [25]. The
Fam21-head and -tail regions were amplified by Pfu-mediated
PCR incorporating BamHI and SalI sites at the 5-and3
-
ends respectively and then cloned into pCRblunt. Following
sequencing, the fragments were subcloned into pEGFP-C1 by
digestion with BglII and SalI and then subsequently subcloned
into pIRESneo2 by excision with NheI and BamHI.
The GFP–Snx1 construct was generated by subcloning the
murine Snx1 coding region (except the start methionine) from
pGEX [12] into pEGFP C1. The GFP–Snx1 construct was then
subcloned into pIRESneo2.
Y2H (yeast two-hybrid) interaction assays
These were performed essentially as described previously [25].
In some instances, constructs were generated by subcloning from
pEGFP-C1 into versions of the pGBT9 (‘bait’) and pGAD424
(‘prey’) vectors that had been modified so that the multi-cloning
sites of the two vectors were the same reading frame as pEGFP-
C1. All constructs used in the Y2H assays were sequenced to
confirm that an in-frame fusion was made.
Automated microscopy and cell spreading assay
Flasks of HeLa cells or HeLa cells expressing GFP-tagged
Vps35 or Fam21-tail were trypsinized and seeded on to 24-well
plates (Greiner) at comparable density, using up to four wells
per cell line. After 75, 150, 250 or 480 min incubation in a
37 ◦C humidified incubator, cells were fixed at room temperature
(20 ◦C) with 4 %(w/v) paraformaldehyde, permeabilized with
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The interactions of the Fam21-tail 211
0.1 %Triton X-100 and stained with Hoechst 33258 and Alexa
Fluor®594-phalloidin. Cell spreading was measured using a
Thermo Fisher Cellomics Arrayscan automated microscope,
using its cell spreading assay protocol and a ×10 objective lens.
For each experiment, 1000 single cells were measured in four
replicate wells at each time point for the cells expressing GFP-
tagged constructs; for the HeLa cells, 800 cells were measured
in duplicate wells. The average single-cell perimeter per well
was obtained from Cellomics vHSC software, whereas further
averaging and normalization was performed using Origin 8.1
(OriginLab).
Microscopy and bafilomycin treatment
Immunofluorescence microscopy was performed essentially
as described previously [25]. For the bafilomycin treatment
experiments described in the present study, a 200×stock solution
of bafilomycin was made by dissolving 10 μgin800 μlofDMSO.
This was divided into aliquots and stored at −20 ◦C. A 1000×
stock solution of chloroquine was made up in water and stored
at −80 ◦C.
HeLa cells were seeded on to coverslips 24 h prior to the
experiment. At 1 h after replacing the medium with fresh medium,
the cells were incubated with 100 nM bafilomycin or 100 μM
chloroquine for 4 h at 37 ◦C. Control cells were treated with
just DMSO. At the end of the incubation period, the cells were
briefly washed with PBS and then fixed for 10 min at room
temperature with 4 %paraformaldehyde. The cells were then
permeabilized with 0.1 %Triton X-100 in PBS and then
blocked with 3 %BSA in PBS prior to incubation with primary
and secondary antibodies. After mounting the coverslips using
ProLong Anti-fade Gold (Invitrogen), the cells were viewed
and imaged using a Zeiss Axioplan epifluorescence microscope
operating with a Hamamatsu C10600 CCD camera (charge-
coupled-device camera). A total of 100 cells were blind scored
for Snx1 tubules in each of three independent experiments.
SDS/PAGE and Western blotting
SDS/PAGE was performed as described previously [12]. Western
blotting employed 125I-labelled Protein A for detection and was
performed as described previously [12]. Signals were obtained by
a phosphoimager (using a GE Typhoon Imager) or by exposure
to X-ray film.
Native immunoprecipitation and MS
Native immunoprecipitations were initially performed (in
Figures 2B and 3A) as described previously [24] using
the PBS +1%Triton X-100 lysis buffer. Subsequently (for
Figures 3B–3D) the buffer was changed to 20 mM Hepes/KOH
(pH 7.0), 50 mM potassium acetate, 1 mM EDTA, 200 mM
sorbitol and 0.1 %Triton X-100, but the methodology was
identical.
MS analysis of gel bands was performed as described
previously [19].
Cell fractionation assay
The assay to separate membrane-associated (pelletable – P)
proteins from cytosolic (soluble – S) proteins was performed as
described previously [19].
RESULTS
The interactions of Vps35 and Fam21
The Vps35, Vps29 and Vps26 proteins assemble to form the
cargo-selective retromer complex that is depicted schematically
in Figure 1(A). To better understand the interactions of Vps35,
we performed a systematic Y2H-based assay for protein–protein
interactions. In Figure 1(B), we show that full-length Vps35
binds to Vps26, Vps29, Fam21 and Snx3. The interactions with
Vps26 and Vps29 act as positive controls in the assay. The
interaction with Fam21 is consistent with our previously published
results [25]. The Vps35–Snx3 interaction is novel, although an
association between Snx3 and retromer has been demonstrated
by native immunoprecipitation [34,35].
Truncation of Vps35 so that the Vps29-binding domain is
separated from the rest of the protein indicated that Snx3 binds to
Vps35 at a site distinct from that of Vps29, but Fam21 interacts
only with full-length Vps35. Additional truncation of Vps35 to
define the Snx3 and Fam21 interaction sites was not possible
due to markedly increased auto-activation of the truncated Vps35
constructs (results not shown). As the Vps35–Snx3 interaction
and also the Vps35–Fam21 interaction were only observed when
Vps35 was expressed from the pGBT9 ‘bait’ plasmid, further
investigation of the interactions of Vps35 using the Y2H system
was not possible.
In addition to interacting with Snx3, Vps35 also binds to the
WASH complex protein Fam21. As stated previously, Fam21
comprises a predicted globular ‘head’ domain of ∼200 amino
acids and a large (∼1100 amino acids) unstructured ‘tail’. We
therefore set out to investigate the interactions of the head and tail
of Fam21 and determine whether the Vps35–Fam21 interaction
occurs via one domain or another. Using the Y2H system we tested
the Fam21 head and tail against the retromer proteins and several
retromer-interacting proteins. In Figure 1(C), full-length Fam21
protein demonstrates a robust interaction with KIAA1033/SWIP.
The Fam21–KIAA1033 interaction is mediated exclusively
through the Fam21-head domain, as no interaction was observed
for the Fam21-tail with KIAA1033/SWIP. The Fam21-head
domain is also able to bind to WASH1 and can weakly interact
with both Snx1 and Snx2.
The Fam21-tail interacts with Strumpellin, FKBP15 and the
actin capping protein CAPZa {CAPZ [capping protein (actin
filament) muscle Z-line], α}, interactions that are not observable
for full-length Fam21. By conducting the Y2H assay with the
Fam21 constructs expressed from the pGAD424 ‘prey’ vector,
auto-activation effects could be avoided, and in this configuration
we observe a robust interaction between Fam21 and Vps35 that
appears to be primarily mediated through the tail of Fam21,
whereas the head-domain of Fam21 demonstrates an interaction
with KIAA1033/SWIP (see Figure 1D).
Although we focused primarily on the interactions of Fam21
and Vps35, we also examined the interactions of WASH1,
FKBP15 and Strumpellin. In Supplementary Figure S1 (at
http://www.BiochemJ.org/bj/442/bj4420209add.htm) we show
that WASH1 interacts strongly with KIAA1033/SWIP and also
with Vps35. FKBP15 interacts strongly with itself and also with a
number of other proteins. Conversely, Strumpellin demonstrated
an interaction with KIAA1033/SWIP only.
Localization and
in vivo
interactions of the Fam21-tail
We next investigated whether the Fam21-head or -tail domain
mediate the endosomal localization of the Fam21 protein. GFP-
tagged Fam21-head or -tail constructs were generated and
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212 M. E. Harbour, S. Y. Breusegem and M. N. J. Seaman
Figure 1 Interactions of Vps35 and Fam21
(A) A simple schematic diagram showing the Vps35 protein and the approximate regions where Vps26 and Vps29 bind. The association of Vps26 with Vps35 requires the PRLYL motif at residue
∼100 in Vps35 [45]. (B) Vps35 in the Y2H ‘bait’ vector (pGBT9) was tested for interactions with a number of retromer and retromer-associated proteins. Interactions were observed for Vps26,
Vps29, Fam21 and Snx3. Truncation of Vps35 abolished the interaction with Fam21 but interactions with Vps26A and Vps29 were retained. (Cand D) Full-length Fam21 or the head or tail domains
were expressed in either the bait vector (pGBT9; C) or prey vector (pGAD424; D) and tested for interactions with retromer and retromer-associated proteins. Expression of the head or tail
domains separately resulted in strong auto-activating activity for the head and weaker auto-activating activity for the tail. In the case of the Fam21-head domain, it was necessary to conduct the
Y2H assays in the presence of 3-amino-1,2,4-triazole (3-AT) to reduce the growth from auto-activation. The Vps35–Fam21 interaction is mediated through the tail of Fam21, whereas the head is
responsible for binding to KIAA1033/SWIP.
transiently transfected into HeLa cells. In Figure 2(A), we show
that the GFP–Fam21-tail construct is sufficient for endosomal
localization and co-localizes with Vps26, similar to the full-length
Fam21–GFP construct. The GFP–Fam21-head domain appears to
be cytoplasmic.
To further investigate the interactions of the Fam21 protein,
we sought to generate a cell line stably expressing Fam21–GFP.
After repeated unsuccessful attempts, a GFP-positive cell line was
obtained in which ∼50 %of the cells expressed a GFP-tagged
protein. This cell line was used in a native immunoprecipitation
experiment applying methodology we have used recently [25].
In Figure 2(B), the proteins precipitated with anti-GFP from
cells expressing either Fam21–GFP or Vps29–GFP were analysed
by SDS/PAGE, MS and Western blotting. Vps29–GFP co-
immunoprecipitated other retromer proteins and also members
of the WASH complex. The Fam21–GFP protein was observed to
be much smaller than expected (the expected size on SDS/PAGE
is ∼220 kDa) and relatively few tryptic peptides from Fam21
were identified by MS compared with endogenous Fam21 in the
Vps29–GFP sample (see Figure 2C). The truncated Fam21 did,
however, associate with FKBP15. The interaction between Fam21
and FKBP15 was confirmed by Western blotting of samples
similar to those analysed by SDS/PAGE. In the experiments
presented below, the cells expressing the truncated Fam21–GFP
are designated Fam21–GFP(T).
The total absence of peptides from the N-terminal half of Fam21
in the sample from the Fam21–GFP(T) cells is inconsistent with
some form of post-lysis proteolysis. Also, as several attempts at
generating a cell line stably expressing the Fam21–GFP construct
were required before the Fam21–GFP(T) cell line was obtained,
we surmised that the truncation of the Fam21–GFP construct
occurred stochastically at the time the construct integrated
into the HeLa cell DNA. Attempts to define the N-terminus of
the truncated Fam21–GFP protein by Edman degradation were
unsuccessful due to the N-terminus being blocked. It is, however,
possible to assign the likely start methionine residue with some
confidence as there are relatively few methionine residues in the
Fam21 protein and the only methionine residue that could be
employed as a start methionine residue to produce the truncated
protein detected is at position 801 and is shown in Figure 2(C).
As the use of full-length Fam21–GFP to generate a stably
transfected cell line was problematic, we next used the GFP–
Fam21-tail and GFP–Fam21-head constructs to generate stably
expressing cell lines. We were repeatedly unsuccessful in
producing a GFP–Fam21-head-expressing cell line, but we did
obtain several different cell lines expressing the GFP–Fam21-tail
construct. These cell lines, designated GFP–Fam21-tail(A–D),
were used in native immunoprecipitation/MS experiments along
with positive and negative control cell lines.
Attempts to demonstrate an in vivo interaction between the
GFP–Fam21-tail and retromer were initially unreliable when
we used our standard lysis buffer comprising PBS and 1%
Triton X-100, although the GFP–Fam21-tail construct was able to
reliably co-immunoprecipitate the actin capping proteins CAPZa
and CAPZb (CAPZ, β) (see Figure 3A). We therefore switched
to using a Hepes/potassium acetate buffer with 0.1%Triton
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The interactions of the Fam21-tail 213
Figure 2 The tail of Fam21 is sufficient for targeting to endosomes
(A) HeLa cells were transiently transfected with GFP-tagged constructs of full-length Fam21, or just the head or tail. Both the full-length and the GFP-tail construct targeted to endosomes
and co-localized with Vps26 (indicated with arrowheads). Scale bar, 20 μm. (B) A truncated Fam21–GFP construct co-immunoprecipitates FKBP15. HeLa cells expressing either Vps29–GFP
or Fam21–GFP (a truncated construct) were lysed and treated with anti-GFP. The immunoprecipitations were analysed by SDS/PAGE, MS and Western blotting. FKBP15 was detected in both
immunoprecipitations but more strongly in the immunoprecipitation from Fam21–GFP expressing cells. (C) A schematic diagram showing where the Fam21–GFP construct has been truncated
post-transfection and where peptides detected by MS originate from.
X-100. The results of the large-scale native immunoprecipitation
experiments are shown in Figures 3(A) and 3(B). The GFP–
Fam21-tail cell lines A–C all express the full-length Fam21-tail
at varying levels. The GFP–Fam21-tail(D) cell line, however,
expressed a truncated form that constituted approximately
one-fifth of the Fam21-tail (see Supplementary Figure S2 at
http://www.BiochemJ.org/bj/442/bj4420209add.htm).
Using the Hepes-based lysis buffer, we detected retromer,
FKBP15 and the actin-capping proteins CAPZa and CAPZb in the
immunoprecipitations from cells expressing the full-length GFP–
Fam21-tail. Additionally, two proteins of unknown function,
CCDC22 (coiled-coil-domain-containing 22) and CCDC93, were
detected associated with GFP–Fam21-tail. These proteins were
largely absent in the lane from the GFP–Fam21-tail(D) cell line.
Interactions detected by MS were confirmed by Western blotting
(see Figure 3C).
The increased expression of the Fam21-tail is able to compete
with the endogenous WASH complex for retromer binding.
As shown in Figure 3(D), Vps26 co-immunoprecipitates the
WASH complex from HeLa cells and other cell lines that do not
express the full-length Fam21-tail, but the presence of Strumpellin
and endogenous Fam21 in the immunoprecipitations from the
GFP–Fam21-tail(A–C) cell lines was much reduced, particularly
when compared with the levels of Vps26 and Vps35 in those
immunoprecipitations. Protein levels in the lysates of the samples
shown in Figures 3(C) and 3(D) were examined by Western
blotting and are shown in Figure 3(E).
The reduction in the association of endogenous WASH complex
with retromer in the GFP–Fam21-tail expressing cells is not due
to effects on the WASH complex assembly as incubation of
lysates with anti-WASH sera was able to recover very similar
levels of Strumpellin and Fam21 (Figure 3F). This is consistent
with the Fam21-head domain mediating the interaction with
KIAA1033/SWIP and assembling into the WASH complex,
whereas the tail binds to retromer (through Vps35), FKBP15 and
the actin-capping proteins.
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214 M. E. Harbour, S. Y. Breusegem and M. N. J. Seaman
Figure 3
In vivo
interactions of the Fam21-tail
(A) HeLa cells stably transfected with various GFP constructs were lysed in PBS +1% Triton
X-100 (as we have used previously [25]) and the GFP-tagged proteins were immunoprecipitated
using anti-GFP. After SDS/PAGE, bands were excised and the proteins were identified using
MS. The actin-capping proteins CAPZa and CAPZb are readily detected in the lanes containing
full-length Fam21-tail or the truncated form characterized in Figure 2(B). (B) The same as in
(A), but the lysis buffer was 20 mM Hepes/KOH (pH 7.0), 50 mM potassium acetate, 1mM
EDTA, 200 mM sorbitol and 0.1% Triton X-100. The negative control was untransfected
HeLa cells. Retromer proteins are readily detected in the GFP–Fam21-tail(A-C) lanes (i.e.
full-length Fam21-tail). Two proteins of unknown function, CCDC22 and CCDC93, are also
detected in the Fam21-tail lanes. (C) Similar samples to those shown in (B) were analysed by
Western blotting. (D) Similar lysates to those in (B)and(C) were treated with anti-Vps26 to
immunoprecipitate retromer. The immunoprecipitations were analysed by Western blotting and
showed that increased Fam21-tail inhibits the association of endogenous WASH complex with
The cargo-selective retromer complex mediates the localization
of Fam21
The tail of Fam21 can bind Vps35 and is sufficient to target to
endosomes (see Figures 2 and 3). As shown in Supplementary
Figure S3(A) (at http://www.BiochemJ.org/bj/442/bj4420209add.
htm), in the GFP–Fam21-tail(B) cell line we observe that the
GFP–Fam21-tail is localized to Vps26-positive endosomes. The
GFP–Fam21-tail(A) and (C) cell lines demonstrated a similar
degree of co-localization (results not shown), but the GFP–
Fam21-tail(D) cell line that expresses the truncated Fam21-tail
did not exhibit any co-localization with retromer proteins and
the construct was instead cytoplasmic and also localized to the
nucleus (Supplementary Figure S3B).
Although the tail of Fam21 is sufficient to target to endosomes,
the Fam21-tail retains a requirement for retromer to be membrane-
associated. In Supplementary Figures S3(C) and S3(D), siRNA
KD (knockdown) of Vps26 or Vps35 resulted in no membrane-
associated GFP–Fam21-tail. The siRNA KD of FKBP15,
Strumpellin or endogenous Fam21, on the other hand, did not
cause the GFP–Fam21-tail protein to become cytosolic.
Fam21 mediates the localization of FKBP15
The tail of Fam21 binds the FKBP15 protein. We would therefore
predict that overexpression of the Fam21-tail will displace
FKBP15 from the membrane. In Figure 4(A), cells expressing
GFP–Fam21-tail (cell line B) were mixed with untransfected
HeLa cells and seeded on to coverslips. In cells expressing
GFP–Fam21-tail, the punctate localization of FKBP15 is much
reduced and the protein appears more cytosolic. The localization
of WASH1 was also affected, although to a lesser degree, whereas
the Vps26 localization did not appear to be affected.
Using a simple fractionation assay to separate membranes
from the cytosol, we observe a clear shift of FKBP15 from the
membrane fraction (P) to the cytosolic fraction (S) in the cell
lines expressing GFP–Fam21-tail (see Figures 4B and 4C), but
other cell lines expressing GFP-tagged proteins (e.g. GFP–Snx1)
do not elicit the same effect. Additionally, as the Fam21-tail can
bind to retromer through Vps35, it is able to compete with the
endogenous WASH complex for retromer binding and results in
a partial shift of the WASH complex into the cytosolic fraction as
more Strumpellin was detected in the soluble cytosolic fraction
in the cell lines expressing the full-length GFP–Fam21-tail (see
Figures 4B and 4D).
Although the overexpression of the Fam21-tail causes more
FKBP15 and WASH complex to become cytosolic, the effect
is not as pronounced as observed after siRNA KD of the
Vps26 protein. In Figures 4(E)–4(G), the amount of membrane-
associated FKBP15 and WASH complex (Strumpellin) was
quantitatively determined for the KDs of Vps26, FKBP15
or Fam21. The siRNA KD of either retromer (Vps26) or
Fam21 results in a shift of FKBP15 into the cytosolic fraction.
Additionally, as shown in Supplementary Figure S4 (http://www.
BiochemJ.org/bj/442/bj4420209add.htm), cells treated with
siRNA to KD the expression of Vps26, FKBP15 and Fam21
were fixed and labelled with antibodies against Vps26, FKBP15
retromer. (E) Lysates similar to those for (C)and(D) were analysed by Western blotting. The
levels of some proteins (e.g. FKBP15) vary somewhat between samples but differences in the
levels do not account for the levels detected in (C)and(D). (F) Lysates similar to those in
(A) were treated with antisera against WASH1. The expression of the GFP–Fam21-tail does not
affect the assembly of the WASH complex.
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The interactions of the Fam21-tail 215
Figure 4 The Fam21-tail mediates the membrane association of FKBP15
(A) The GFP–Fam21-tail(B) cells were mixed with untransfected HeLa cells and seeded on to coverslips. After 24 h, the cells were fixed and labelled with antibodies against GFP and either anti-Vps26,
anti-FKBP15 or anti-WASH1. Overexpression of the Fam21-tail does not affect the localization of Vps26 but localization of both FKBP15 and WASH1 is affected, appearing less punctate and more
diffuse in the GFP–Fam21-tail(B) cells. Scale bar, 20 μm. (B) Cells were fractionated into membrane (pelletable – P) and cytosolic (soluble – S) fractions and the samples were subjected to
SDS/PAGE and analysed by Western blotting. FKBP15 and (to a lesser extent) Strumpellin are shifted into the cytosolic fraction in both cell lines expressing the GFP–Fam21-tail construct. (Cand
D) Results from triplicate experiments were quantified and the level of membrane-associated FKBP15 (C) and Strumpellin (D) are shown. (E) Cells treated with various siRNAs were fractionated into
membrane (pelletable – P) and cytosolic (soluble – S) fractions and the samples were subjected to SDS/PAGE and analysed by Western blotting. FKBP15 becomes cytosolic after Vps26 or Fam21
KD. (Fand G) Results from triplicate experiments were quantified and the level of membrane-associated FKBP15 (F) and Strumpellin (G) are shown.
and Fam21 and co-stained with anti-Snx1 antibodies. Labelling of
endogenous FKBP15 and Fam21, although not especially strong,
showed co-localization with Snx1. The loss of Vps26 expression
results in both FKBP15 and Fam21 becoming cytosolic. KD of
FKBP15 does not, however, affect Vps26 or Fam21 localization,
but KD of Fam21 does abolish the localization of FKBP15.
Therefore FKBP15 localization to the endosome requires Fam21,
consistent with the interaction observed between the tail of Fam21
and FKBP15.
Inhibition of the V-ATPase also displaces FKBP15
Recent studies in Dictyostelium have reported that the WASH
complex regulates the luminal pH of endo-/lyso-somes by
mediating the sorting of the V-ATPase from lysosomes [30]. We
therefore asked whether the activity of the V-ATPase can influence
the functioning of the WASH complex using bafilomycin to
inhibit the V-ATPase. Following treatment with bafilomycin, we
observed that cells exhibited markedly less endosomally localized
FKBP15. Comparison of the labelling shown in Figures 5(A)
and 5(B) reveals that cells treated with bafilomycin have little
punctate FKBP15. Treatment with chloroquine, a weak base that
neutralizes endosomal and lysosomal pH, does not produce the
same effect (Figure 5C), indicating that the loss of FKBP15
localization is the result of inhibition of the V-ATPase and not
the result of perturbation of the pH gradient across the endosomal
membrane. We quantified the effect of bafilomycin on the level of
membrane-associated FKBP15 and observed a pronounced shift
(∼2-fold) of FKBP15 into the cytosolic fraction after treatment
with bafilomycin (see Figure 5D).
Interestingly, we also observed that cells treated with
bafilomycin exhibited almost no Snx1-positive tubules (see
Supplementary Figure S5 at http://www.BiochemJ.org/bj/442/
bj4420209add.htm), although, unlike FKBP15, Snx1 remained
associated with punctate endosomal structures positive for the
retromer protein Vps26.
Dominant-negative effects of the Fam21-tail on cell spreading
Over the course of these studies, we noticed that the cell lines
stably expressing the full-length GFP-tagged Fam21-tail were
slow to spread after trypsinization and seeding on to tissue culture-
treated plastic. Using an automated microscope, we therefore
examined the cell spreading of the four GFP–Fam21-tail cell lines
(A–D) along with cells expressing GFP–Vps35 and untransfected
HeLa cells. As shown in Figure 6, the GFP–Fam21-tail cell lines
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216 M. E. Harbour, S. Y. Breusegem and M. N. J. Seaman
Figure 5 Inhibition of V-ATPase redistributes FKBP15 into the cytoplasm
(A–C) HeLa cells were treated with just DMSO (A), 100 nM bafilomycin (B) or 100 μM chloroquine (C) for 4 h. After fixation the cells were labelled with antibodies against Snx1 and either anti-Vps26,
anti-FKBP15 or anti-Fam21. Incubation with bafilomycin results in FKBP15 redistributing to the cytoplasm, but chloroquine treatment does not. Scalebar,20μm. (D) Control or bafilomycin-treated
cells were fractionated into membrane (pelletable – P) and cytosolic (soluble – S) fractions and the samples were subjected to SDS/PAGE and analysed by Western blotting. FKBP15 becomes
cytosolic after incubation with bafilomycin. Results from triplicate blots were quantified and graphed.
A–C all exhibit slower cell spreading when compared with GFP–
Vps35, GFP–Fam21-tail(D) and also untransfected HeLa cells. As
the GFP–Fam21-tail(D) cell line differs from the GFP–Fam21-
tail(A–C) cell lines only by virtue of expressing a truncated form
of the Fam21-tail, the lack of cell spreading defect in this cell line
suggests that the cell spreading defect in the GFP–Fam21-tail(A–
C) cell lines is the result of the full-length constructs titrating a
protein (or proteins) into inactive complexes.
DISCUSSION
In the present study, we have investigated the interactions of the
retromer protein Vps35 and the WASH complex protein Fam21.
The Vps35 protein forms part of the cargo-selective retromer
subcomplex and, as such, associates with Vps26 and Vps29 via
its N- and C-terminal regions respectively [36]. Vps35 has been
directly implicated in cargo selection [20], and it is therefore
important to elucidate how the interactions of Vps35 contribute
to endosomal protein sorting.
Determination of the distinct interactions of the Fam21-head
and -tail domains
In addition to identifying a novel Vps35–Snx3 interaction, we also
show that Vps35 binds to the unstructured ‘tail’ of the Fam21
protein of the WASH complex (see Figure 1B). These novel
interactions were observed using the Y2H system. This proved
a useful tool for examining a large number of binary interactions
of retromer and associated proteins. In some instances, however,
further examination of the interactions using the Y2H system were
curtailed due to the auto-activating effects of particular constructs.
Dissecting Fam21 into its head- and tail-domains revealed
interactions that were not observed for full-length Fam21.
For example, the interactions between the tail of Fam21 and
the FKBP15 and Strumpellin proteins were detected when
no interactions were observed for full-length Fam21 (see
Figures 1C and 1D). Similarly, the Fam21-head domain bound to
WASH1 and the Snx1 and Snx2 proteins when no such interaction
was observed for the full-length Fam21 protein. It is possible that
the head of Fam21 interacts with a region of the tail and
that this interaction prevents full-length Fam21 from interacting
with FKBP15 and Strumpellin in the Y2H experiments. A Fam21-
head–tail interaction could also explain why the head and tail
domains were individually auto-activating but full-length Fam21
did not auto-activate.
Attempts to generate a cell line expressing full-length GFP-
tagged Fam21 were unsuccessful, although a cell line expressing
a truncated version of the Fam21–GFP construct was eventually
obtained that revealed an interaction between Fam21 and FKBP15
(see Figure 2B). This interaction had been predicted from the
Y2H results and therefore provides a useful in vivo confirmation
of the validity of the Y2H results. Although the function of
FKBP15 is yet to be determined, it is a protein of interest
to researchers studying ulcerative colitis and has also been
implicated in neuronal cell function [26,27].
It seems likely that overexpression of the ‘head’ of Fam21
may be toxic, as no cell line stably expressing the GFP–Fam21-
head construct could be obtained. The reason for the apparent
cytotoxicity of the Fam21-head domain is currently unknown and
merits further investigation.
Vps35 is central to the endosomal recruitment of Fam21
When cell lines expressing GFP–Fam21-tail were generated, the
tail of Fam21 was found to interact with a number of proteins
in addition to FKBP15. The actin-capping proteins CAPZa and
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The interactions of the Fam21-tail 217
Figure 6 Overexpression of the Fam21-tail causes cell spreading defects
(A) Average single cell perimeter of HeLa cells and HeLa cells expressing GFP–Vps35 or
GFP–Fam21-tail(A–D) measured at four time points after seeding and normalized to the first time
point (75 min post-seeding). Cells expressing full-length GFP–Fam21-tail(A–C) show markedly
slower cell spreading kinetics compared with control HeLa cells or HeLa cells expressing
GFP–Vps35. This experiment was repeated three times; a representative experiment is shown. (B)
Representative images of the phalloidin (F-actin) stain at three time points post-seeding for HeLa
control cells, HeLa cells expressing GFP–Vps35 and HeLa cells expressing GFP–Fam21-tail(B).
Scale bar, 300 μm. Note the greater proportion of rounder, smaller cells at 8 h for the cells
expressing GFP–Fam21-tail(B). For the cell perimeter analysis in (A), only single cells in the
images are analysed.
CAPZb were readily detected in native immunoprecipitations of
the full-length GFP–Fam21-tail construct, as were the retromer
proteins Vps35, Vps29 and Vps26. Two proteins of unknown
function, CCDC22 and CCDC93, were also detected and, in the
case of CCDC22, confirmed by Western blotting. As CCDC22
was detected only in the GFP–Fam21-tail immunoprecipitations
and not when retromer components were immunoprecipitated,
the CCDC22 protein (and possibly CCDC93) may associate with
Fam21-tail only when the Fam21-tail is not bound to retromer
(see Figures 3A–3C).
The tail of Fam21 is sufficient for recruitment to the endosomal
membrane, a process that requires the retromer cargo-selective
complex (see Supplementary Figure S3). In vivo, an interaction
between Vps35 and the WASH1 protein (revealed in the Y2H ex-
periment) may also contribute to the membrane association of the
WASH complex. Indeed, the inability to co-immunoprecipitate
retromer with the GFP–Fam21-tail under conditions (namely
PBS +1%Triton X-100) in which GFP–Vps35 or Vps29–GFP
are able to co-immunoprecipitate endogenous WASH complex
may be explained by the absence of the contribution of the Vps35–
WASH1 interaction when native immunoprecipitations of GFP–
Fam21-tail constructs were initially performed. The loss of the
Vps35–WASH1 interaction therefore necessitated the use of
the lower stringency Hepes/potassium acetate buffer that was able
to retain the retromer–Fam21-tail interaction. Alternatively, the
Vps35–WASH1 interaction might be involved in regulating
the activity of the WASH1 protein. In either case, the central
role that Vps35 plays in mediating the recruitment of the WASH
complex could explain why loss of Vps35 function in Drosophila
results in extensive actin dysregulation along with defects in
endocytosis [37].
The elevated levels of Fam21-tail in the cell lines expressing
the GFP–Fam21-tail construct disrupted the association of
endogenous WASH complex with retromer, resulting in an
increased cytoplasmic localization of WASH1 and Strumpellin
(see Figures 3D and 4A–4D). This observation significantly
extends our original finding that retromer mediates recruitment
of the WASH complex, which was based on phenotypes observed
after siRNA KD of retromer proteins [25]. Additionally, because
no direct interaction has been observed between WASH1 and the
Fam21-tail domain, these results suggest that the WASH complex
is more likely to be a stable entity in which the Strumpellin,
KIAA1033, WASH1 and Fam21 proteins are recruited to the
endosome as a single unit.
As the overexpression of the Fam21-tail was also able to
induce the displacement of FKBP15 from the membrane, we
suggest that the interaction between Fam21 and FKBP15 may
be the driving force in mediating the membrane association
of FKBP15, a hypothesis supported by the loss of FKBP15
membrane association after Fam21 KD (see Figure 4).
A mechanistic link between endosomal acidification and protein
sorting
Studies in D. discoideum have shown that the WASH complex
is required to mediate the trafficking of the V-ATPase [30].
Results presented in the present paper demonstrate that FKBP15
requires Fam21 for its membrane association (see Figure 4).
Intriguingly, we observed that inhibition of V-ATPase activity
by bafilomycin also markedly reduces the membrane association
of FKBP15, an effect similar to that observed when the Fam21-
tail is overexpressed (see Figures 4 and 5). The coupling of the
V-ATPase activity to the association of FKBP15 with endosomes
(and thereby the WASH complex) could provide a mechanism
to ensure that FKBP15 is only recruited to the membrane when
the V-ATPase is active and the endosome has achieved a measure
of maturity. At this time, however, the precise role of FKBP15
in regulating WASH complex function, or other aspects of
endosomal protein sorting, remains to be determined.
In addition to the displacement of FKBP15 from the membrane
after bafilomycin treatment, we also observed an almost total
loss of Snx1 tubules (see Supplementary Figure S5). We do not
believe, however, that loss of endosomally localized FKBP15
leads to a defect in Snx1-tubule formation as we have not observed
any effect on Snx1-tubules or endosome-to-Golgi retrieval
after FKBP15 KD [25]. A block in Snx1-tubule formation after
inhibition of the V-ATPase is potentially highly significant as it
may provide a regulatory mechanism for ensuring that Snx1-
tubule formation does not occur too early in the endocytic
pathway. This observation may also explain the apparent defect in
endosome-to-Golgi retrieval after pharmacological abolishment
of endosomal acidity [38].
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218 M. E. Harbour, S. Y. Breusegem and M. N. J. Seaman
Figure 7 Schematic diagrams of the WASH complex and its role in endosomal protein sorting
(A) Schematic diagram of the WASH and WAVE complexes showing similar overall architecture (based on Jia et al. [31] and the results of the present study). A key difference is the very long
Fam21-tail domain that is not present in the orthologous WAVE protein, Abi. (B) Schematic diagram summarizing the different Fam21 constructs analysed in the present study and the interactions
detected for the respective constructs. (C) A model for the action of the WASH complex and retromer in regulating endosomal protein sorting. Recruitment of the WASH complex by retromer facilitates
WASH complex-mediated sorting of the V-ATPase and other membrane proteins.
Assembly and physiological role of the WASH complex
The dominant-negative effect(s) resulting from the overexpression
of the Fam21-tail did not extend to assembly of the WASH
complex as there was no apparent defect in the ability of WASH1
to co-immunoprecipitate the Strumpellin or endogenous Fam21
proteins, indicating that the WASH complex was intact and that
the assembly of Fam21 into the WASH complex is most likely to
be mediated by the interaction of the Fam21-head domain with
the KIAA1033/SWIP protein (see Figures 1C, 1D and 3F). The
results presented here are therefore complementary to the in vitro
structural studies of WASH complex assembly [31] and confirm
that the assembly of the WASH complex in vivo is similar to
the assembly of the WAVE complex. A schematic diagram of the
WASH complex and the analogous WAVE complex is shown in
Figure 7(A) and the interactions of the various Fam21 constructs
are summarized in Figure 7(B).
Although we did not observe any profound changes in the
localization of several membrane proteins [e.g. the transferrin
receptor, the CIMPR and LAMP-1 (lysosome-associated mem-
brane protein-1)] in cells expressing the GFP–Fam21-tail (results
not shown), we did observe a significant cell spreading defect
in all three cell lines expressing the full-length GFP–Fam21-tail.
This phenotype was not observed in cells expressing the truncated
GFP–Fam21-tail or GFP–Vps35 and therefore is likely to be due
to dominant-negative effects exerted by the full-length tail (see
Figure 6). Cell spreading following loss of adherence requires
the mobilization of endocytosed membrane to expand the area
of the plasma membrane [2-4]. Interestingly, the genes encoding
Fam21 and WASH1 homologues in the amoeba Naegleria gruberi
(annotated as AM46 and AM5 respectively) have been implicated
in amoeboid locomotion due to their absence in related organisms
incapable of amoeboid motility [39]. Amoeboid motility is
a process that is conceptually similar to the spreading of a
trypsinized HeLa cell that is newly adhered to the substratum.
It is also noteworthy that the two proteins of unknown function,
CCDC22 and CCDC93, that interact with the Fam21-tail, could
be involved in this pathway. The Drosophila melanogaster
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The interactions of the Fam21-tail 219
homologue of CCDC93 has been reported to interact with exo70
[40], a protein of the exocyst complex. CCDC22 has been shown
to bind to Copine 1 and 4, calcium-binding proteins that have
been implicated in membrane trafficking [41,42]. Additionally,
mutations in CCDC22 have recently been identified as causal in
X-linked inherited learning disability [43].
We have previously demonstrated that loss of WASH-complex
function does not profoundly affect endosome-to-Golgi retrieval
of a well-characterized retromer cargo protein, namely the CIMPR
[25]. We therefore suggest that the function of the WASH
complex is more acutely required for membrane trafficking from
endosomes to the cell surface, a pathway that is necessary for
cell spreading [2-4] and recycling of membrane proteins such as
the β-adrenergic receptor [44]. The retromer complex mediates
the endosomal recruitment of the WASH complex through Vps35
binding to the Fam21-tail domain and thereby contributes to the
efficiency of protein sorting into the endosome-to-cell surface
pathway (see Figure 7C).
AUTHOR CONTRIBUTION
Michael Harbour performed the Y2H analysis, generated constructs and cell lines
and performed preliminary native immunoprecipitation experiments. Sophia Breusegem
performed the quantitative cell spreading assay and Snx1-tubule analysis (in the
Supplementary online data). Matthew Seaman performed the immunofluorescence
experiments, cell fractionation assays and native immunoprecipitations and wrote the
manuscript.
ACKNOWLEDGEMENTS
We are grateful to Kamburapola Jayawardena (a.k.a. Jay – ‘the mass-spec-guy’) for MS
identification of gel bands.
FUNDING
This work was funded by the Medical Research Council through a Senior Fellowship
Award [grant number G0701444 (to M.N.J.S.)] and an additional research grant [grant
number G0700750]. Funding for MS in the CIMR was provided by the Wellcome Trust
through a Strategic Award [grant number 079895/Z/06].
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Received 3 October 2011/9 November 2011; accepted 9 November 2011
Published as BJ Immediate Publication 9 November 2011, doi:10.1042/BJ20111761
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Biochem. J. (2012) 442, 209–220 (Printed in Great Britain) doi:10.1042/BJ20111761
SUPPLEMENTARY ONLINE DATA
Recruitment of the endosomal WASH complex is mediated by the extended
‘tail’ of Fam21 binding to the retromer protein Vps35
Michael E. HARBOUR1, Sophia Y. BREUSEGEM1and Matthew N. J. SEAMAN2
Cambridge Institute for Medical Research (CIMR)/Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Cambridge CB2 0XY, U.K.
Figure S1 The interactions of WASH1, FKBP15 and Strumpellin were analysed using the Y2H system
WASH1 demonstrated interactions with KIAA1033 and VPS35. FKBP15 interacted most strongly with itself and also with Fam21. Strumpellin only interacted with KIAA1033.
1These authors contributed equally to this work.
2To whom correspondence should be addressed (email mnjs100@cam.ac.uk).
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M. E. Harbour, S. Y. Breusegem and M. N. J. Seaman
Figure S2 GFP–Fam21-tail constructs
(A) Untransfected HeLa cells or cells expressing GFP-tagged Fam21 constructs were lysed (using PBS +1 % Triton X-100 buffer) and treated with anti-GFP antibody to precipitate (IP) the respective
GFP-tagged protein. The precipitated proteins were analysed by SDS/PAGE and MS. Gel bands marked with a circle were excised and subjected to MALDI–TOF MS (matrix-assisted laser-desorption
ionization–time-of-flight MS) as described previously [1]. The red-filled circles denote bands corresponding to the GFP-tagged Fam21 construct. The protein(s) identified from each band are
shown below the gel. Actin-capping proteins (CAPZa and CAPZb) are readily identifiable in samples that contain C-terminal regions of the Fam21-tail. (B) Peptides identified in gel bands of the
GFP-tagged Fam21 constructs are highlighted (in red boxes) on the Fam21 sequence. The darker blue shaded area is the Fam21-head domain. The light blue shaded area denotes the actin-capping
protein-binding region [2]. The region underlined in the GFP-Fam21(D) data is the region where the construct is likely to be truncated on the basis of the peptides detected in this sample and the
full-length GFP–Fam21-tail(A) sample. The Fam21-tail(D) construct is truncated prior to the actin-capping protein binding region and therefore does not interact with CAPZa or CAPZb.
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The interactions of the Fam21-tail
Figure S3 The tail of Fam21 co-localizes with retromer and requires retromer for its membrane association
(A) The GFP–Fam21-tail(B) cell line was fixed and labelled with anti-GFP and anti-Vps26 antibodies. Scale bar, 20 μm. The regions of the micrograph contained in the boxes is shown as merged
images in the right-hand panels. (B) The GFP–Fam21-tail(D) cell line that expresses a truncated tail does not co-localize with retromer. Scale bar, 20 μm. (C) The GFP–Fam21-tail(B) cell line
was treated with siRNA to KD expression of Vps26 or Vps35. Loss of the cargo-selective retromer complex results in the GFP–Fam21-tail construct becoming cytosolic, but siRNA KD of FKBP15,
Strumpellin or endogenous Fam21 (shown in D) does not affect the localization of the GFP–Fam21-tail(B) construct. Coincident labelling of GFP–Fam21-tail and Snx1 is indicated by arrowheads.
Scale bar, 20 μm. (E) Efficacy of the siRNA KDs. Lysates from HeLa cells treated with siRNA to KD expression of retromer, FKBP15 or WASH complex proteins were subjected to SDS/PAGE and
analysed by Western blotting. The siRNA efficiently silences expression of its respective target, and in some cases, loss of one protein (e.g. Vps26) leads to instability and loss of another (e.g. Vps35).
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Figure S4 Fam21 is required for the membrane association of FKBP15
(A) HeLa cells were treated with siRNA to KD Vps26, FKBP15 or Fam21. After fixation, the cells were labelled with antibodies against Snx1 and either Vps26, FKBP15 or Fam21. The punctate
localization of FKBP15 and Fam21 was lost after Vps26 KD, and FKBP15 localization was abolished by Fam21 KD, but FKBP15 KD did not markedly affect Fam21 localization. Scale bar, 20 μm.
Figure S5 Absence of Snx1 tubules after bafilomycin treatment
HeLa cells were treated with DMSO, 100 nM bafilomycin or 100 μM chloroquine for 4 h, fixed and labelled with anti-Snx1 and anti-VPS26 antibodies. Scale bar, 20 μm. (B) Cells were treated as in
(A) and 100 cells were blind scored in each of three independent experiments. Results are means +
−S.D.
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Received 3 October 2011/9 November 2011; accepted 9 November 2011
Published as BJ Immediate Publication 9 November 2011, doi:10.1042/BJ20111761
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