Regulation of WASH-Dependent Actin
Polymerization and Protein Trafficking
Yi-Heng Hao,1,7Jennifer M. Doyle,1,7Saumya Ramanathan,1Timothy S. Gomez,6Da Jia,2Ming Xu,3Zhijian J. Chen,3,4
Daniel D. Billadeau,6Michael K. Rosen,2,4and Patrick Ryan Potts1,5,*
1Department of Physiology
2Department of Biophysics
3Department of Molecular Biology
4Howard Hughes Medical Institute
5Department of Pharmacology
UT Southwestern Dallas, TX 75390, USA
6Department of Immunology and Division of Oncology Research, Mayo Clinic, Rochester, MN 55905, USA
7These authors contributed equally
Endosomal protein trafficking is an essential cellular
process that is deregulated in several diseases and
targeted by pathogens. Here, we describe a role for
ubiquitination in this process. We find that the E3
RING ubiquitin ligase, MAGE-L2-TRIM27, localizes
to endosomes through interactions with the retromer
complex. Knockdown of MAGE-L2-TRIM27 or the
Ube2O E2 ubiquitin-conjugating enzyme signifi-
cantly impaired retromer-mediated transport. We
further demonstrate that MAGE-L2-TRIM27 ubiquitin
F-actin by the WASH regulatory complex, a known
regulator of retromer-mediated transport. Mecha-
nistic studies showed that MAGE-L2-TRIM27 facili-
tates K63-linked ubiquitination of WASH K220.
Significantly, disruption of WASH ubiquitination
impaired endosomal F-actin nucleation and retro-
mer-dependent transport. These findings provide
a cellular and molecular function for MAGE-L2-
TRIM27 in retrograde transport, including an unap-
preciated role of K63-linked ubiquitination and
identification of an activating signal of the WASH
Endosomal protein recycling pathways facilitate the transfer of
membrane proteins from early and late endosomes back to the
trans-Golgi network (TGN) or plasma membrane (Bonifacino
and Rojas, 2006). In doing so, these pathways generally function
to prevent lysosomal delivery and degradation of membrane
proteins. Endosome-to-Golgi transport, referred to as retro-
grade transport, is an important cellular process that facilitates
the recycling of a variety of proteins, including sorting receptors
(such as CI-M6PR), SNARE membrane fusion proteins, mem-
brane receptors, metabolite transporters, and several proteins
that undergo polarized localization/secretion (Bonifacino and
Rojas, 2006; Johannes and Popoff, 2008). Importantly, retro-
grade transport has been implicated in a number of different
human pathologies. Endosome-to-Golgi transport is essential
for cellular entry of pathogenic toxins, such as Shiga, cholera,
and ricin, as well as viral pathogens such as HIV (Brass et al.,
2008; Sandvig and van Deurs, 2005). Furthermore, components
of the retrograde transport pathway are downregulated in Alz-
heimer’s disease and upregulated in cancer (Scott et al., 2009;
crucial for different aspects of this process, including cargo
recognition, endosomal membrane budding, tubulation and
scission, and vesicle transport, tethering, and fusion at the
TGN (Bonifacino and Rojas, 2006; Cullen and Korswagen,
2012). One critical component is the retromer protein complex
that consists of VPS26, VPS29, and VPS35 and functions to
recognize retrograde cargo on endosomes (Bonifacino and Hur-
ley, 2008). Another essential factor in retrograde transport is
WASH. WASH is a member of the Wiskott-Aldrich syndrome
protein (WASP) family consisting of WASP/N-WASP, WAVE,
WHAMM, JMY, and WASH (Campellone and Welch, 2010).
Like other WASP family members, WASH contains a carboxy-
terminal VCA (verprolin homologous or WH2, central hydro-
phobic, and acidic) motif that binds to actin and the Arp2/3
complex to stimulate actin filament nucleation (Derivery et al.,
2009; Duleh and Welch, 2010; Jia et al., 2010; Linardopoulou
et al., 2007; Liu et al., 2009). Recent studies have demonstrated
complex termed the WASH regulatory complex (SHRC) and
functions downstream of the retromer complex to facilitate
endosome-to-Golgi transport (Derivery et al., 2009; Duleh and
Welch, 2010; Gomez and Billadeau, 2009; Linardopoulou et al.,
Cell 152, 1051–1064, February 28, 2013 ª2013 Elsevier Inc. 1051
2007). Importantly, retromer and SHRC have been implicated
in other endosomal recycling pathways, including endosome-
to-plasma membrane sorting of integrins (Zech et al., 2011).
SHRC consists of at least five core factors, CCDC53, FAM21,
SWIP, Strumpellin, and WASH (Derivery et al., 2009; Gomez
and Billadeau, 2009; Jia et al., 2010). The SHRC is recruited to
early endosomes by multivalent interactions between repeat
elements in FAM21 and the retromer subunit VPS35 (Harbour
et al., 2012; Jia et al., 2012). WASH is essential for Arp2/3-
induced F-actin accumulation on endosomes (Derivery et al.,
2009; Gomez et al., 2012). With WASH suppression, scission
of membrane tubules emanating from endosomes was sug-
gested to be impaired (Derivery et al., 2009; Gomez and Billa-
deau, 2009) and knockout of WASH in MEFs resulted in the
collapse of the early endosomal and lysosomal networks
(Gomez et al., 2012).
a variety of signaling events that converge on VCA motif activa-
tion/exposure (Padrick and Rosen, 2010). The VCA motifs of
tramolecular and intermolecular interactions (Chen et al., 2010;
Ismail et al., 2009; Jia et al., 2010; Kim et al., 2000; Miki et al.,
1998). Several signaling molecules have been identified to
promote the activation/exposure of WASP/N-WASP and WAVE
VCA motifs to allow timed and localized F-actin nucleation,
including small GTPases Cdc42 and Rac1, PIP2and PIP3phos-
pholipids, and phosphorylation by the Src, Abl, and Cdk family
of kinases (Eden et al., 2002; Ismail et al., 2009; Kim et al.,
2000; Lebensohn and Kirschner, 2009; Miki et al., 1998; Padrick
and Rosen, 2010). Contrary to WASP/N-WASP and WAVE,
the mechanisms regulating WASH activation have been more
elusive. The Rho GTPase has been genetically linked to activa-
tion of WASH in Drosophila (Liu et al., 2009) but is not sufficient
to directly activate human WASH in vitro (Jia et al., 2010).
Ubiquitination is a posttranslational modification in which a
small 76 amino acid protein is covalently attached to lysine resi-
dues in substrate proteins through a three step E1, E2, and E3
enzymatic cascade (Fang and Weissman, 2004). Ubiquitination
can have pleiotropic effects on its substrates depending on the
length and type of ubiquitin chains. K48-linked ubiquitin chains
typically target proteins for degradation by the 26S proteasome
(Bochtler et al., 1999). However, K63-linked ubiquitination typi-
protein-protein interactions, protein conformations, or targeting
proteins for lysosomal delivery (Sun and Chen, 2004). Recently,
we identified a class of proteins that bind to and enhance the
activity of E3 RING ubiquitin ligases (Doyle et al., 2010). These
ubiquitin ligase enhancers are known as melanoma antigen
(MAGE) genes and comprise a family of over 50 unique human
genes (Chomez et al., 2001). Although the biochemical function
of MAGE proteins in ubiquitination has been elucidated, the
cellular processes in which specific MAGE proteins act are
unclear. Here, we investigate the cellular function of MAGE-L2,
a paternally imprinted gene that is abundantly expressed in the
brain, maps to the Prader-Willi syndrome deletion locus, and is
implicated in normal circadian rhythm and the hypothalamic-
endocrine axis (Bischof et al., 2007; Boccaccio et al., 1999; Ko-
zlov et al., 2007; Tennese and Wevrick, 2011).
Identification of MAGE-L2 E3 RING Ubiquitin Ligase
Partner and Subcellular Localization
To determine the specific E3 RING ubiquitin ligase partner of
MAGE-L2 and gain insight into its cellular function, we exam-
ined MAGE-L2 binding partners by tandem affinity purification
(TAP) coupled to mass spectrometry. The E3 RING ubiquitin
ligase TRIM27 was identified as a major binding partner of
MAGE-L2 (Figure 1A). Binding of MAGE-L2 and TRIM27 was
confirmed by reciprocal coimmunoprecipitation experiments
(Figures 1B and S1C available online). Moreover, in vitro trans-
lated MAGE-L2 bound recombinant GST-TRIM27 but not GST
alone, indicating the interaction between the two proteins is
direct (Figure 1C). To confirm the association of MAGE-L2 and
TRIM27, we stably expressed GFP-MAGE-L2 and mCherry-
TRIM27 in U2OS cells and examined their colocalization by
live-cell microscopy. We found that the two proteins colocalized
in discrete cytoplasmic puncta (Figure 1D), as well as in a
smaller nuclear pool (data not shown). These findings suggest
that TRIM27 binds MAGE-L2 and that the MAGE-L2-TRIM27
ubiquitin ligase complex localizes to discrete cytoplasmic
MAGE-L2-TRIM27 Binds and Localizes to Retromer-
To determine the identity of the MAGE-L2-TRIM27 structures,
we reanalyzed our mass spectrometry data of MAGE-L2 inter-
acting proteins for clues. Interestingly, MAGE-L2 was identified
to interact with VPS35 and VPS26 (Figure 1A), two components
of the endosomal retromer complex. mCherry-TRIM27 colocal-
ized with GFP-tagged VPS35, VPS29, and VPS26, as well as
the retromer-associated SHRC proteins, WASH and FAM21
(Figure S1A). Furthermore, costaining for endogenous VPS35
and TRIM27 clearly identified TRIM27 cytoplasmic structures
as retromer-positive endosomes (Figure 1E). Unfortunately, we
were unable to observe endogenous MAGE-L2 due to the lack
of specific, high-quality antibodies (data not shown). More
detailed analysis of TRIM27 localization revealed its localization
ure 1F), a property shared with the retromer complex (Arighi
et al., 2004; Bonifacino and Hurley, 2008). These findings sug-
gest that MAGE-L2-TRIM27 localizes to the retromer-positive
subset of endosomes.
Next, we confirmed the interaction between MAGE-L2-
TRIM27 and the retromer complex initially observed by mass
spectrometry. MAGE-L2 and TRIM27 coimmunoprecipitated
with all three components of the retromer complex (Figures
S1B and S1C and data not shown) and MAGE-L2 directly bound
S1D, S1G, and S1H). In addition, MAGE-L2 interaction with
VPS35 did not impair VPS35 binding to the SHRC component
FAM21 (Figure S1I). Furthermore, MAGE-L2-VPS35 interaction
is functionally important because knockdown of VPS35 dramat-
ically inhibited MAGE-L2 and TRIM27 endosomal localization
(Figures S1E and S1F and data not shown). These findings sug-
gest that VPS35 recruits MAGE-L2-TRIM27 to retromer-positive
endosomes by binding to MAGE-L2.
1052 Cell 152, 1051–1064, February 28, 2013 ª2013 Elsevier Inc.
MAGE-L2-TRIM27 Is Required for Endosomal Protein
We next assessed whether MAGE-L2-TRIM27 participates in
endosome-to-Golgi transport. Two independent siRNAs tar-
geting MAGE-L2 or TRIM27 were identified that significantly
reduced their target protein’s levels but had no effects on the
levels of other known essential factors required for retrograde
transport (Figures S2A–S2C). Multiple siRNAs targeting MAGE-
L2 or TRIM27 resulted in impaired CI-M6PR and TGN46 traf-
ficking to a degree similar to VPS35-RNAi (Figures 2A–2C and
S2D). Importantly, the overall organization of the TGN was unaf-
fected in MAGE-L2- or TRIM27-RNAi cells (Figure S2E). The
steady-state defects in CI-M6PR localization were corroborated
by examining transport of a small pool of surface localized
CI-M6PR to the TGN in MAGE-L2- and TRIM27-RNAi cells.
MAGE-L2- or TRIM27-RNAi impaired transport of surface-
labeled CI-M6PR to the TGN (Figure 2D). Importantly, the dis-
persed steady-state or surface-labeled CI-M6PR in TRIM27- or
MAGE-L2-RNAi cells colocalized with the endosomal marker
EEA1 (Figures 2E and S2F). Furthermore, CI-M6PR protein
levels were reduced in MAGE-L2- and TRIM27-RNAi cells and
Cathepsin D processing and trafficking were impaired (Fig-
ure 2F). Finally, overexpression of MAGE-L2 in combination
with TRIM27 increased CI-M6PR endosomal retrieval kinetics
(Figure S2G). These results suggest that MAGE-L2-TRIM27 is
required for endosome-to-Golgi retrograde transport.
To extend our findings, we examined the requirement of
TRIM27 on the trafficking of two additional physiologically and
pathologically relevant substrates of the retromer and SHRC
complexes. First, knockdown of TRIM27 significantly impaired
trafficking of the retromer cargo cholera toxin subunit B (CTxB)
to the TGN (Figure 2G). However, the trafficking of the SHRC-
independent cargo Transferrin receptor to perinuclear recycling
leh and Welch, 2010; Gomez and Billadeau, 2009; Gomez et al.,
2012). In addition, the retromer and SHRC complexes have been
implicated in the recycling of endosomal proteins to the plasma
membrane, including integrins (Duleh and Welch, 2012; Zech
Overlay + DNA
TRIM27VPS35 Overlay + DNA
Rr = 0.7
Rr = 0.8
Figure 1. MAGE-L2 Binds TRIM27 and Localizes to Retromer-Positive Endosomes
(A) TAP-Vector or TAP-MAGE-L2 were isolated from HEK293 stable cell lines, separated by SDS-PAGE, Coomassie stained, and the identity of specific bands
was determined by LC-MS/MS. Asterisks indicate Usp7.
(B) The indicated proteins were expressed in cells for 48 hr, anti-Myc IP was performed, and proteins were detected by western blot (WB).
(C) Binding of Myc-MAGE-L2 to recombinant GST-TRIM27 or GST alone was determined by GST pull-down assays and anti-Myc immunoblotting.
(D) Stably expressed GFP-MAGE-L2 and mCherry-TRIM27 colocalize in specific cytoplasmic puncta.
(E) U2OS cells were stained for endogenous VPS35 (red), endogenous TRIM27 (green), and DNA (blue). XZ and YZ projection stacks are shown.
(F) Live-cell imaging of stably expressed mCherry-TRIM27 shows localization to tubule-like protrusions (yellow arrowheads) from endosomes.
Pearson’s correlation coefficients (Rr) are shown. Scale bars, 20 mm. See also Figure S1.
Cell 152, 1051–1064, February 28, 2013 ª2013 Elsevier Inc. 1053
siControl siTRIM27 #2
siMAGE-L2 #3 siVPS35
CI-M6PR + DNA
siControl siTRIM27 #2 siMAGE-L2 #3siVPS35
Overlay + DNA
siControl siTRIM27 #2siMAGE-L2 #3
siTRIM27 #1 siTRIM27 #2
siMAGE-L2 #1 siMAGE-L2 #3
Cells with Dispersed
Cells with Juxtanuclear
Internalized CI-M6PR (%)
TGN46 + DNA
0 2040 60
Cells with Perinuclear Localized
CTxB or Transferrin (%)
siControl - CTxB
siTRIM27 - CTxB
siControl - Tf
siTRIM27 - Tf
Integrin α5 + EEA1
86.8 ± 19.5
30.6 ± 4.6
56.1 ± 6.6
22.7 ± 6.7
Surface Integrin α5 (RFU)
Rr = 0.1Rr = 0.7Rr = 0.6
Figure 2. MAGE-L2-TRIM27 Is Required for Endosomal Protein Recycling
(A) Cells were treated with the indicated siRNAs for 72 hr and stained for CI-M6PR (green) and DNA (blue).
(B) Quantitation of cells shown in (A). Compact juxtanuclear or dispersed CI-M6PR was scored and the percentage of cells with dispersed CI-M6PR is shown.
(C) Cells were treated with the indicated siRNAs for 72 hr and stained for TGN46 (red) and DNA (blue).
(D) Cells were treated with the indicated siRNAs. Cell surface CI-M6PR was labeled with anti-CI-M6PR antibody for one hour before imaging the pool of
internalized cell surface-labeled CI-M6PR.
(E) Cells were treated with the indicated siRNAs and stained for CI-M6PR (green), EEA1 (red), and DNA (blue). XZ and YZ projection stacks and Pearson’s
correlation coefficients (Rr) are shown.
(F) Cells were treated withthe indicated siRNAs for 72hr and cell lysates and media were analyzed by immunoblotting.pCatD represents unprocessed pro-CatD,
iCatD indicates intermediate processed CatD, and mCatD represents fully matured CatD.
(G) Cells were treated with the indicated siRNAs for 72 hr, and transport of CTxB-488 or Tf-568 was determined at the indicated times.
(legend continued on next page)
1054 Cell 152, 1051–1064, February 28, 2013 ª2013 Elsevier Inc.
et al., 2011). Therefore, we examined whether MAGE-L2 and
TRIM27 function extends beyond endosome-to-Golgi trafficking
by examining their contribution to the recycling of integrin a5 to
the plasma membrane. Knockdown of MAGE-L2 or TRIM27
significantly decreased cell surface integrin a5 (Figure 2H) and
increased the intracellular pool of integrin a5 in EEA1-negative
endosomes (Figure 2I). Importantly, this decrease in cell surface
integrin a5 in MAGE-L2- or TRIM27-RNAi cells is functionally
significant because invasion of cells through matrigel, a process
regulated by integrins, is significantly impaired in MAGE-L2- or
TRIM27-RNAi cells (Figure 2J). These results suggest that
MAGE-L2-TRIM27 functions with the retromer and SHRC com-
plexes in both endosome-to-Golgi and endosome-to-plasma
membrane protein recycling.
Next, we determined whether the requirement for MAGE-L2-
TRIM27 in retrograde transport involved its ubiquitin ligase
activity by utilizing a TRIM27 RING mutant lacking E3 ubiquitin
ligase activity. Unlike wild-type TRIM27, expression of an RNAi
resistant TRIM27 RING mutant was unable to rescue retrograde
transport of CI-M6PR in TRIM27-RNAi cells (Figures 3A and
S3A). Importantly, the TRIM27 RING mutant localized similarly
to the wild-type protein (Figure S3B). These results suggest
that the ubiquitin ligase activity of MAGE-L2-TRIM27 is impor-
tant for proper retrograde transport.
Next, we examined which of the more than 35 different
E2 ubiquitin-conjugating enzymes functions with MAGE-L2-
TRIM27 to facilitate retrograde endosome-to-Golgi trafficking
of CI-M6PR. Of the 35 E2 enzymes examined, knockdown of
only Ube2O and Ube2L6 resulted in dramatic alteration of CI-
M6PR localization (Figures 3B and S3C, and data not shown).
Ube2O-RNAi showed similar penetrance as TRIM27-RNAi (Fig-
ures 3B and 3C), resulted in CI-M6PR dispersion to EEA1-
positive endosomes (Figure 3D), and reduced total CI-M6PR
(Figure 3E). However, Ube2L6-RNAi did not redistribute CI-
M6PR to EEA-positive endosomes (Figure 3D) or promote lyso-
somal degradation of CI-M6PR (Figure 3E) but rather disrupted
TGN organization (Figure S3D). Notably, Ube2O, but not
Ube2L6, was also identified in a yeast-2-hybrid screen for E2
enzymes interacting with TRIM27 (Markson et al., 2009). These
resultssuggest that the Ube2OE2 ubiquitin-conjugating enzyme
is the physiologically relevant E2 enzyme functioning with
MAGE-L2-TRIM27 in supporting retrograde transport.
Retrograde Transport Requires K63-Linked Ubiquitin
We next investigated whether retrograde transport was depen-
dent on ubiquitin, and if so, which type of polyubiquitin chain.
To do so, we utilized the previously developed system (Xu
et al., 2009) to inducibly knockdown ubiquitin by addition of
tetracycline and replace it with a ubiquitin variant that is unable
to produce a specific ubiquitin chain, namely ubiquitin in which
lysine 48 or lysine 63 has been mutated to arginine (K48R or
K63R, respectively; Figures S3E and S3F). We found that deple-
tion of ubiquitin from cells dramatically affected CI-M6PR
trafficking (Figures 3F and S3G). Addition of wild-type or K48R
ubiquitin completely rescued CI-M6PR localization (Figures 3F
and S3G), suggesting that ubiquitin is required for retrograde
trafficking, but K48-linked ubiquitin chains are not. In contrast,
depletion of K63-ubiquitin chains dramatically blocked CI-
M6PR TGN localization and relocalized it to EEA1-positive endo-
somes (Figures 3F and S3G and data not shown) but had no
significant effect on the overall organization of the TGN (Fig-
ure S3H). Similarly to the steady-state behavior, retrograde
transport of cell surface-labeled CI-M6PR to the TGN is also
dependent on K63-linked ubiquitination (Figures 3G and S3I).
Furthermore, depletion of K63-linked ubiquitin chains altered
trafficking of the lysosomal hydrolase Cathepsin D, resulting in
the reduction of mature Cathepsin D, accumulation of pro- and
intermediate-Cathepsin D, and the secretion of pro-Cathepsin
D into the cell culture media (Figure 3H). These results suggest
that K63-linked ubiquitination is important for proper retrograde
MAGE-L2-TRIM27, Ube2O, and K63-Linked Ubiquitin
Chains Are Required for Efficient Endosomal F-Actin
Next, we assessed the precise mechanism by which MAGE-L2-
TRIM27, Ube2O, and K63-linked ubiquitination promotes retro-
grade transport. Retrograde transport requires several ordered
steps to facilitate endosome-to-Golgi transport, including endo-
somal localization of the retromer complex and localization and
activation of the SHRC to facilitate endosomal F-actin accumu-
lation by the Arp2/3 complex (Cullen and Korswagen, 2012). We
interrogated several of these specific steps to determine when
ubiquitination may be important. The localization of the retromer
complex to CI-M6PR substrate-containing endosomes was un-
affected by depletion of TRIM27 or K63-linked ubiquitin chains
(Figures S4A and S4B). In addition, the SHRC was still recruited
normally to retromer-positive endosomes in TRIM27-RNAi cells
(Figure S4C). However, knockdown of MAGE-L2 or TRIM27 re-
sulted in reduced endosomal F-actin to a degree similar to
WASH-RNAi (Figures 4A and 4B). Similarly, knockdown of the
physiologically relevant Ube2O E2 enzyme, but not Ube2L6, re-
sulted in a reduced endosomal F-actin (Figures 4A and 4B).
Consistent with reduced endosomal F-actin, knockdown of
MAGE-L2, TRIM27, or Ube2O, but not Ube2L6, significantly
reduced the accumulation of the Arp2/3 complex subunit,
ARPC5, on SHRC-positive endosomes (Figures 4C and 4D).
Notably, there was no defect in total ARPC5 or F-actin levels
(Figure 4D and data not shown). Likewise, depletion of K63-
linked ubiquitin chains resulted in the specific reduction of
(H) Cells were treated with the indicated siRNAs for 72 hr and surface integrin a5 was determined by flow cytometry. Grey curve indicates control IgG staining.
(I) Cells were treated with the indicated siRNAs for 72 hr and immunostained with integrin a5 (green), EEA1 (red), and DNA (blue).
(J) Invasive potential of cells treated with the indicated siRNAs was assayed on matrigel-coated transwell invasion chambers. Invaded cells were stained with
crystal violet and quantitated.
All data are represented as the mean ±SD. Asterisk indicates p < 0.05. Scale bars, 20 mm. See also Figures S2 and S7.
Cell 152, 1051–1064, February 28, 2013 ª2013 Elsevier Inc. 1055
Ubiquitin E2 Enzymes
Mock #1 Mock #2
siUbe2R2 siUbe2R1 siUbe2D3 siUbe2D2 siUbe2D4siUbe2D1
siUbe2E3 siUbe2E2 siUbe2E1
Cell with Dispersed CI-M6PR (%)
Overlay + DNA
siControl siUbe2O siUbe2L6
Cells with Dispersed CI-M6PR (%)
CI-M6PR Intensity (RFU)
Cells with Dispersed CI-M6PR (%)A
– Tet+ Tet
Cells with Dispersed CI-M6PR (%)
Cells with Juxtanuclear
Internalized CI-M6PR (%)
Rr = 0.1Rr = 0.1Rr = 0.7
Figure 3. Ube2O E2 and K63-Ubiquitin Chains Are Required for Retrograde Transport
(A) Cells were treated with control or TRIM27 siRNAs for 24 hr before transfection of RNAi-resistant wild-type or RING mutant TRIM27. Forty-eight hours after
transfection, cells were stained for CI-M6PR. The percentage of transfected cells with dispersed CI-M6PR is shown.
(B) Cells were treated with siRNAs targeting the indicated E2 enzymes for 72 hr, stained with anti-CI-M6PR, imaged, and quantitated. Dotted line denotes cutoff
that reproducibly indicates CI-M6PR trafficking defect.
(C) Cells were treated with the indicated siRNAs for 72 hr, stained with CI-M6PR, and imaged. The percentage of cells with dispersed CI-M6PR is shown.
(D)Cellsweretreatedwiththeindicated siRNAsfor72hrand stainedforCI-M6PR(green),EEA1(red),andDNA(blue).XZandYZprojectionstacks andPearson’s
correlation coefficients (Rr) are shown.
(E) Cells were treated with the indicated siRNAs for 72 hr, stained for CI-M6PR, imaged, and CI-M6PR intensity was determined.
(F) Ubiquitin replacement cell lines were treated with or without tetracycline for 72–96 hr before steady-state CI-M6PR localization was determined by immu-
nostaining. The percentage of cells with dispersed CI-M6PR is shown.
(legend continued on next page)
1056 Cell 152, 1051–1064, February 28, 2013 ª2013 Elsevier Inc.
endosomal F-actin and Arp2/3 complex, without affecting total
F-actin, Arp2/3 complex, or SHRC levels (Figures 4E–4H, S3J,
and data not shown). Consistent with the previously described
role of SHRC and endosomal F-actin in membrane tubule
scission (Derivery et al., 2009; Gomez and Billadeau, 2009),
endosomal tubules (Figures S4D and S4E). These results sug-
gest that K63-linked ubiquitination by MAGE-L2-TRIM27 and
Ube2O is required for localization of the Arp2/3 complex to the
SHRC and generation of endosomal F-actin.
Uninhibited Endosomal Actin Assembly Rescues
Endosome-to-Golgi Transport Defects of TRIM27-RNAi
and K63-Ubiquitin Depleted Cells
We next examined whether the defective endosome-to-Golgi
retrograde trafficking in TRIM27-RNAi or shUb-Ub(K63R) cells
is due to decreased endosomal F-actin. First, we designed a
fusion protein in which endosomal F-actin accumulation could
be induced by an uninhibited WASH VCA motif. WASH VCA
motif was fused to the C-terminal domain of FAM21 (D356N),
which binds VPS35 for endosomal targeting (Figure 5A) (Gomez
and Billadeau, 2009; Jia et al., 2012). Upon expression in cells,
this fusion protein localized to retromer-positive endosomes
(Figure 5A) and increased F-actin accumulation on endo-
somes (Figures 5A and 5B). We next determined whether this
FAM21-WASH-VCA fusion could rescue retrograde transport
in TRIM27-RNAi cells. Indeed, this was the case for both
Cathepsin D trafficking (Figure 5C) and CI-M6PR TGN localiza-
tion (Figures 5D and S4F). Furthermore, the FAM21-WASH-
VCA fusion rescued CI-M6PR trafficking in cells deficient for
K63-linked ubiquitin chains (Figures 5E and S4G). Therefore,
the primary requirement for TRIM27 and K63-linked ubiquitin
chains in retrograde trafficking appears to be for accumulation
of endosomal F-actin.
Endosomal F-Actin Assembly
Generation of endosomal F-actin is facilitated by the SHRC,
which binds the Arp2/3 complex and actin through a conserved
VCA domain in WASH (Derivery et al., 2009; Gomez and Billa-
deau, 2009). However, the WASH VCA domain exists in an
autoinhibited state that must be relieved before nucleation of
F-actin by the Arp2/3 complex can be achieved (Jia et al.,
2010). Therefore, we speculated that MAGE-L2-TRIM27 may
facilitate WASH-VCA exposure, Arp2/3 complex and actin bind-
ing, and consequent F-actin nucleation on endosomes through
targeted ubiquitination of the SHRC. Based on the highly homol-
ogous WAVE regulatory complex, regulators of SHRC activation
are predicted to act on WASH itself or SWIP (Chen et al., 2010;
Jia et al., 2010). Although we were unable to detect any ubiquiti-
nation of endogenous SWIP (Figure S5A), endogenous WASH
was highly ubiquitinated (Figure 6A). WASH ubiquitination was
TRIM27-dependent, as knockdown of TRIM27 dramatically
reduced endogenous WASH ubiquitination (Figure 6B). Further-
more, WASH ubiquitination was K63 linked, as WASH was
unable to be polyubiquitinated by K63R ubiquitin (Figure 6C).
These findings suggest that TRIM27 is required for K63-linked
ubiquitination of WASH.
Next we investigated the importance of WASH ubiquitination.
To do so, we first determined the specific lysine in WASH that is
conjugated to K63-linked ubiquitin chains. A single lysine, K220,
in WASH had been identified in global proteomics studies to be
ubiquitinated (Kim et al., 2011). Significantly, K220 is a highly
conserved residue in species that express TRIM27 orthologs
(Figure S5D). This residue is located in a region of WASH that
is analogous to the ‘‘meander’’ region of WAVE, which is known
to make contacts necessary for intracomplex inhibition in the
WAVE regulatory complex (Chen et al., 2010). Therefore, we
examined whether WASH ubiquitination was dependent on
K220. Endogenous WASH was knocked down by RNAi and
YFP-tagged wild-type or K220R WASH was re-expressed to
insure integration of the re-expressed WASH into the SHRC.
Unlike YFP-WASH wild-type, YFP-WASH K220R failed to be
ubiquitinated (Figure 6C), suggesting that WASH ubiquitination
is dependent on K220. We next examined whether WASH
ubiquitination is important for retrograde transport. Using our
re-expression system, YFP-WASH K220R, unlike wild-type
YFP-WASH, could not support proper retrograde transport
resulting in CI-M6PR dispersion (Figures 6D and S5B) and
degradation (Figures 6E and S5B) and Cathepsin D secretion
(Figure 6F). These results suggest that K63-linked ubiquitination
We next examined whether WASH K220 ubiquitination may
act as a signal to relieve WASH autoinhibition and promote its
activity toward the Arp2/3 complex. As previously reported
(Derivery et al., 2009), knockdown of WASH resulted in reduced
endosomal Arp2/3 complex localization (Figures 6G and S5C).
Unlike wild-type YFP-WASH, re-expression of YFP-WASH
K220R was unable to rescue proper endosomal Arp2/3 complex
localization (Figures 6G and S5C). Importantly, YFP-WASH
K220R localized properly to endosomes indicating that it still
incorporated into the SHRC (Figure S5C). These results suggest
that ubiquitination of WASH K220 is required for WASH activa-
tion and Arp2/3 complex endosomal localization.
In Vitro Reconstitution of MAGE-L2-TRIM27 and K63-
Ubiquitin-Dependent SHRC Activity
Finally, we developed an in vitro reconstitution system to directly
test whether ubiquitination of WASH is required for its actin
assembling activity. To do so, we reconstituted WASH knock-
out MEFs (Gomez et al., 2012) with stable expression of
(G) Ubiquitin replacement cells were treated with or without tetracycline for 72–96 hr. Cell surface CI-M6PR was then labeled with anti-CI-M6PR antibody for one
hour and internalized cell surface-labeled CI-M6PR was imaged. CI-M6PR localization was determined and the percentage of cells showing juxtanuclear
internalized CI-M6PR is shown.
(H) Cells were treated with the indicated siRNAs for 72 hr before Cathepsin D processing and secretion was determined by immunoblotting. pCatD represents
unprocessed pro-CatD, iCatD indicates intermediate processed CatD, and mCatD represents fully matured CatD.
Data are represented as the mean ±SD. Asterisk indicates p < 0.05. Scale bars, 20 mm. See also Figure S3.
Cell 152, 1051–1064, February 28, 2013 ª2013 Elsevier Inc. 1057
siControl siTRIM27 #2siMAGE-L2 #3
Overlay + DNA
Overlay + DNA
ARPC5 Intensity (RFU)
Overlay + DNA
F-Actin Intensity on
VPS35 Endosomes (RFU)
siWASH siControl siTRIM27 #2siUbe2O
ARPC5 Intensity (RFU)
F-Actin Intensity on
WASH Endosomes (RFU)
Overlay + DNA
Figure 4. MAGE-L2-TRIM27, Ube2O, and K63-Linked Ubiquitination Are Required for Endosomal F-Actin Assembly
(A) Cells were treated with the indicated siRNAs for 72 hr before staining VPS35 (green), F-actin (red), and DNA (blue). XZ and YZ projection stacks are shown.
(B) F-actin intensity on VPS35-positive endosomes of cells described in (A).
(C) Cells were treated with the indicated siRNAs for 72 hr before staining ARPC5 (green), FAM21 (red), and DNA (blue). XZ and YZ projection stacks are shown.
(D) Cells treated as in (C) were imaged and endosomal (solid bars) or total (open bars) ARPC5 levels were determined.
(E) shUb-Ub(K63R) ubiquitin replacement cells were treated with or without tetracycline for 96 hr before staining for WASH (green), F-actin (red), and DNA (blue).
XZ and YZ projection stacks are shown.
(F) Cells described in (E) were imaged, and F-actin intensity on retromer-positive endosomes was quantitated and is shown.
(legend continued on next page)
1058 Cell 152, 1051–1064, February 28, 2013 ª2013 Elsevier Inc.
HA-GFP-WASH wild-type, DVCA, or K220R. The intact SHRC
was then purified from each of these cell lines by anti-HA chro-
matography to near homogeneity (Figures S6A and S6B). Under
these conditions, very little free WASH is present (Figure S6B).
The activity of the reconstituted SHRCs was first assayed by
examining their capacity to assemble F-actin on beads in cell
accumulation on beads in a manner dependent on the VCA motif
ofWASHand wasinhibited bycytochalasinD(Figure7A).Impor-
tantly, WASH knockout MEFs reconstituted with WASH K220R
mutant did not support actin assembly on beads (Figure 7A). In
addition, the activity of SHRC was dependent on MAGE-L2-
TRIM27, as SHRC isolated from MAGE-L2- or TRIM27-RNAi
cells was significantly less active (Figure 7B). Furthermore, we
examined the activity of purified SHRC in Arp2/3 complex-
dependent pyrene-actin assembly assays. SHRC reconstituted
(G) shUb-Ub(K63R) ubiquitin replacement cells were treated with or without tetracycline for 96 hr before staining with ARPC5 (green), FAM21 (red), and DNA
(blue). XZ and YZ projection stacks are shown.
(H) Cells treated as in (G) were imaged and endosomal (solid bars) or total (open bars) ARPC5 levels were determined.
Data are represented as the mean ±SD. Asterisk indicates p < 0.05. Scale bars, 20 mm. See also Figure S4.
Endosomal F-Actin Intensity (RFU)
357 1341316 468
Overlay + DNA
Cells with Dispersed CI-M6PR (%)
Cells with Dispersed CI-M6PR (%)
Figure 5. Uninhibited WASH-VCA Bypasses the Requirement for TRIM27 and K63-Linked Ubiquitin Chains in Retrograde Transport
(A) Schematic of uninhibited, endosomal localized WASH-VCA (top). Cells were transiently transfected for 48 hr before staining GFP-FAM21D356N-WASH-VCA
(green), F-actin (red), VPS35 (cyan), and DNA (blue). Scale bar, 20 mm. Pearson’s correlation coefficient (Rr) is shown.
(B) Cells were transfected with GFP-alone or GFP-FAM21D356N-WASH-VCA and VPS35-localized F-actin intensity was determined.
(C) Cells were treated with control or TRIM27 siRNAs for 24 hr before transfection with control YFP-WASH or constitutively active GFP-FAM21D356N-WASH-VCA.
Forty-eight hours later, the indicated proteins were examined by immunoblotting.
(D) Cells were treated with control or TRIM27 siRNAs for 24 hr before transfection with GFP alone or GFP-FAM21D356N-WASH-VCA. Forty-eight hours after
transfection, cells were immunostained for CI-M6PR and the percentage of transfected cells with dispersed CI-M6PR was determined.
(E) shUb-Ub(K63R) cells were treated with or without tetracycline and transfected with either GFP alone or GFP-FAM21D356N-WASH-VCA. Cells were immu-
nostained after 96 hr for CI-M6PR and the percentage of transfected cells with dispersed CI-M6PR was determined.
Data are represented as the mean ±SD. Asterisk indicates p < 0.05.
Cell 152, 1051–1064, February 28, 2013 ª2013 Elsevier Inc. 1059
– – Myc-Ubiquitin
Endosomal ArpC5 Intensity (RFU)
WT K220R shWASH+YFP-WASHWT WT
Endosomal CI-M6PR Intensity (RFU)4000
Cells with Dispersed CI-M6PR (%)
Figure 6. WASH K63-Linked Ubiquitination by TRIM27 Is Required for Endosomal F-Actin Nucleation and Retrograde Transport
(A and B)Cells weretreatedwith theindicated siRNAs for 24hrbefore transfection of theindicated vectors.Forty-eight hours after plasmid transfection, anti-Myc
IP was performed. Whole-cell lysates (WCL) or anti-Myc IP samples were immunoblotted for WASH and TRIM27.
(C) Cells were transfected with the indicated Myc-ubiquitin vectors and dual-knockdown/re-expression vectors to knockdown endogenous WASH and re-
immunoblotted for WASH and Myc-ubiquitin.
(D and E) Cells were transfected with the indicated dual-knockdown/re-expression vectors to knockdown endogenous WASH and re-express YFP-WASH
variants. Seventy-two hours after transfection cells were immunostained for CI-M6PR and dispersed CI-M6PR (D) and endosomal CI-M6PR abundance (E) was
determined in transfected cells.
(F) Cells were transfected with the indicated dual-knockdown/re-expression vectors as indicated. Seventy-two hours after transfection, Cathepsin D secretion
into the cell culture media was determined by immunoblotting. Two independent samples from each condition are shown.
(G) Cells described in (D) were immunostained for ARPC5 and the endosomal-localized pool of ARPC5 in transfected cells was quantitated.
Data are represented as the mean ±SD. Asterisk indicates p < 0.05. See also Figure S5.
1060 Cell 152, 1051–1064, February 28, 2013 ª2013 Elsevier Inc.
with wild-type, ubiquitinated WASH displayed increased activity
toward the Arp2/3 complex compared to SHRC reconstituted
with nonubiquitinated WASH K220R (Figure 7C). To further
examine the interplay of WASH ubiquitination and activity, we
treated purified wild-type SHRC with the K63-specific deubiqui-
tinating enzyme AMSH. This treatment deconjugated K63-
ubiquitin chains from WASH (Figure 7D) and significantly in-
hibited SHRC activity on beads (Figure 7E) and in pyrene-actin
assembly assays (Figure 7F). These results suggest that K63-
ubiquitination of WASH K220 by MAGE-L2-TRIM27 facilitates
the activation of SHRC in a reversible manner.
To determine if ubiquitination of WASH K220 may facilitate
SHRC activation by disrupting autoinhibitory contacts in the
meander region around WASH K220, we mutated WASH K220
to aspartic acid (K220D) to destabilize autoinhibitory contacts
in this region. Unlike inactive WASH K220R, WASH K220D is
active in vitro (Figures 7A and 7C) and facilitates endosome-
to-Golgi retrograde transport (Figures 6D–6F) and endosomal
Arp2/3 complex localization (Figure 6G) in cells. These results
suggest that destabilizing the WASH meander region around
the ubiquitination site can facilitate SHRC activity independent
MAGE proteins are a family of proteins that contain a conserved
domain known as the MAGE homology domain. Recently, we
showed that MAGE proteins function biochemically to bind to
and enhance the activity of E3 RING ubiquitin ligases (Doyle
et al., 2010). In this study we investigated the cellular function
of one specific MAGE protein, MAGE-L2. Proteomic analysis re-
vealed that MAGE-L2 specifically bound the TRIM27 E3 RING
ubiquitin ligase. TRIM27 belongs to a large family of E3 RING
ubiquitin ligases known as tripartite motif (TRIM) proteins.
TRIM27 was originally identified and named Ret finger protein
(RFP), due to its discovery as a gene that undergoes a transloca-
tion event with the Ret tyrosine kinase receptor in thyroid car-
cinomas (Saenko et al., 2003; Takahashi and Cooper, 1987).
Subsequent work has implicated it in several processes, includ-
ing transcriptional regulation, NFkB signaling, CD4+ T cell
homeostasis, and as an oncogene (Cai et al., 2011; Kru ¨tzfeldt
et al., 2012). It will be of particular interest in the future to deter-
mine if any of these diverse functions of TRIM27 are attributed to
its regulation of WASH and vesicular transport.
Cellular studies revealed that MAGE-L2 and TRIM27 colocal-
ized on cytoplasmic structures that were determined to be
retromer-positive endosomes. Of note, TRIM27 was previously
shown to localize to similar structures that were unidentified at
the time (Harbers et al., 2001). Furthermore, the localization of
TRIM27 was regulated by PKC, JNK, and RAS signaling path-
ways. To determine if any of these signaling pathways may con-
tribute to the upstream regulation of MAGE-L2-TRIM27 and
endosome-to-Golgi retrograde transport, we examined the
effects of short-term inhibition of the PKC, JNK, MEK, and
PI3K signaling pathways on the kinetics of surface CI-M6PR
trafficking to the TGN. Specific inhibition of the JNK signaling
pathway blocked CI-M6PR retrieval to the TGN (Figure S7).
Future studies into the mechanism by which JNK signaling
regulates endosomal protein recycling will be of particular
Our mechanistic studies uncovered that K63-linked ubiquiti-
nation of WASH K220 by MAGE-L2-TRIM27 is required for
endosomal F-actin nucleation and retrograde transport. WASH
K220 is predicted to exist in an analogous region on the SHRC
as the meander region on the known WAVE regulatory complex
structure. The meander region makes contacts with WAVE
itself and SRA1 (analogous to SWIP in SHRC) and is essential
for maintaining WAVE in an inactive state (Chen et al., 2010).
Importantly, this region is subjected to regulation by phosphory-
lation (Padrick and Rosen, 2010). Thus, the proposed meander
region on both the WAVE and WASH regulatory complexes
may be regulated by posttranslational modifications, phosphor-
ylation, and ubiquitination, respectively. In addition, WASH K220
is highly conserved in vertebrates where TRIM27 is found. How-
are not present (Boudinot et al., 2011). Thus, regulation of SHRC
by ubiquitination is likely highly conserved in vertebrates, but
additional forms of regulation are predicted in invertebrates.
Our results suggest that ubiquitination of WASH facilitates
activation by directly destabilizing autoinhibitory contacts in
the SHRC, thus allowing VCA exposure, Arp2/3 complex
binding, and subsequent F-actin assembly (Figure 7G). Con-
sistently, destabilizing these autoinhibitory contacts bypasses
the requirement for WASH ubiquitination. In the context of the
cell, other factors likely cooperate to further enhance activity,
perhaps by clustering the active SHRC. Another likely point
of regulation is in deactivating WASH after sufficient endo-
somal F-actin nucleation for retrograde trafficking. Indeed, we
find that ubiquitin-mediated activation of SHRC is reversible.
Future studies into relevant deubiquitinating enzymes will be of
Our findings also provide important insights into pathological
conditions associated with MAGE-L2, TRIM27, and retrograde
transport. Genes required for retrograde transport are fre-
quently amplified in melanomas, contribute to tumorigenesis,
and mediate trafficking of proteins required for tumor progres-
sion (Scott et al., 2009; Zech et al., 2011). Our findings extend
this work to show that the TRIM27 oncogene facilitates integrin
a5 recycling, an important event in tumorigenesis. Additionally,
retrograde transport has been implicated in Alzheimer’s disease,
where components of the pathway are downregulated and
mutated (Small, 2008). Similarly, MAGE-L2 has been reported
to be downregulated in the hippocampus of patients with incip-
ient Alzheimer’s disease (Blalock et al., 2004). Finally, retrograde
transport is an essential pathway in which many microbial toxins
trafficking of cholera toxin. Our results suggest a potential
strategy to combat these pathogens.
Cell Culture, Transfections, siRNAs, and Antibodies
Cells were cultured under standard conditions and transfected according
to manufacturer’s recommendation. Detailed descriptions of cell culture
conditions, transfection procedures, siRNA sequences, and antibodies are
described in the Extended Experimental Procedures.
Cell 152, 1051–1064, February 28, 2013 ª2013 Elsevier Inc. 1061
WT WT + CytoDΔVCA K220RK220D
Fluorescence Intensity (AU)
0 500 1000
Fluorescence Intensity (AU)
WT + Buffer
WT + AMSH
Figure 7. In Vitro Reconstitution of MAGE-L2-TRIM27 and K63-Ubiquitin-Dependent SHRC Activity
(A) Cell lysates from WASH knockout MEFs reconstituted with the indicated HA-GFP-WASH proteins were incubated with anti-HA agarose beads and F-actin
stained with phalloidin-568. WT+CytoD was treated with 10 mM cytochalasin D before addition of beads.
(B) WASH knockout MEFs reconstituted with wild-type HA-GFP-WASH were transfected with the indicated siRNAs for 96 hr and actin assembly was determined
as described in (A).
(C) The activity of purified SHRC from WASH knockout MEFs reconstituted with the indicated HA-GFP-WASH variants was examined by pyrene-actin assembly
(legend continued on next page)
1062 Cell 152, 1051–1064, February 28, 2013 ª2013 Elsevier Inc.
Tandem Affinity Purification and Mass Spectrometry
TAP was performed using 293/TAP-Vector or 293/TAP-MAGE-L2 stable cell
lines as described previously (Doyle et al., 2010) and in the Extended Experi-
Immunoprecipitation, Immunoblotting, and Cathepsin D Secretion
Immunoprecipitation and immunoblotting were performed as described previ-
ously (Potts and Yu, 2005). Cathepsin D secretion assay details are described
in the Extended Experimental Procedures.
Protein Purification and In Vitro Binding Assays
the Extended Experimental Procedures. In vitro binding assays were per-
formed as described previously (Doyle et al., 2010) and specified in the
Extended Experimental Procedures.
Immunofluorescence, Microscopy, and Quantitative Measurements
Immunofluorescence was performed essentially as described previously
(Potts and Yu, 2007) and in Extended Experimental Procedures. Retrograde
transport of cell surface CI-M6PR was performed as described previously
(Gomez and Billadeau, 2009) and detailed in the Extended Experimental
Actin Assembly Assays and Purification of SHRC
WASH knockout MEFs (Gomez et al., 2012) were reconstituted with HA-GFP-
tagged WASH and the resulting SHRC variants were purified as described
in the Extended Experimental Procedures. Bead-based and pyrene-actin
assembly assays were performed as described previously (Cory et al., 2003;
Jia et al., 2010) and detailed in the Extended Experimental Procedures.
Supplemental Information includes Extended Experimental Procedures and
seven figures and can be found with this article online at http://dx.doi.org/
We thank Mitsuaki Tabuchi (Kawasaki Medical School, Japan) for providing
the retromer construct. We also thank Potts lab members for helpful discus-
sions and critical reading of the manuscript. This work was supported by the
Cancer Prevention and Research Initiative of Texas R1117 (P.R.P.), Depart-
ment of Defense CA110261 (P.R.P.), Howard Hughes Medical Institute
(M.K.R.), Mayo Foundation (D.D.B.), Michael L. Rosenberg Scholar in Medical
Research fund (P.R.P.), NIH R01-AI065474 (D.D.B.), NIH R01-GM063692
(Z.J.C.), NIH R01-GM56322 (M.K.R.), Sara and Frank McKnight Fellowship
(P.R.P.), and Welch Foundation I–1544 (M.K.R.) and I-1389 (Z.J.C.).
Received: July 25, 2012
Revised: November 29, 2012
Accepted: January 24, 2013
Published: February 28, 2013
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