Molecular Basis of Oligoubiquitin-DependentInternalization of Membrane Proteins inMammalian Cells

Herve Barriere1, Csilla Nemes1, DelphineLechardeur1, Mina Khan-Mohammad, KlausFruh2 and Gergely L. Lukacs1

1Program in Cell and Lung Biology, Hospital for SickChildren Research Institute and Department of LaboratoryMedicine and Pathobiology, University of Toronto, 555University Avenue, Toronto, Ontario, Canada, M5G 1X82Molecular Microbiology and Immunology, Oregon Healthand Science University, Vaccine and GeneTherapy Institute, 505 NW 185th Ave., Beaverton, OR,97006, USA*Corresponding author: G. L. Lukacs, glukacs@sickkids.ca

Ubiquitination induced down-regulation of cell surfaceproteins by internalization and lysosomal targetingplays a fundamental role in cell physiology and patho-genesis of diseases. The molecular basis of a singleubiquitin (Ub) as an autonomous endocytic signal, thewidely accepted mechanism, however, remains elusivein higher eukaryotes. Using Ub containing reporterproteins without signalling abilities, we present evidencethat only multiple Ub moieties, linked either covalentlyor assembled as oligomers with an intact interface forrecognition by Ub-interacting motifs (UIMs), are recog-nized by the endocytic machinery in vivo and associatewith a subset of Ub-binding clathrin adaptors in vitro.Genetic and pharmacological approaches show thatinternalization of plasma membrane proteins harbouringmultiple Ub moieties is clathrin-dependent, but caveolin-independent. Functional assays demonstrate the cargo-dependent involvement of eps15/15R and epsin, UIMcontaining clathrin adaptors, in the endocytosis ofmodel proteins, CD4 and the activated b2-adrenergicreceptor complex, containing polymeric or oligomericUb. These results provide a paradigm for the clathrin-mediated uptake of ubiquitinated membrane proteinsin mammalian cells, requiring the assembly of multipleUIM–Ub interactions to overcome the low affinity bind-ing of mono-Ub to UIM.

Key words: caveolin, CD4, clathrin, endocytosis, eps15,epsin, UIM, b2-adrenergic receptor, ubiquitin

Received 29 September 2005, revised and accepted forpublication 29 November 2005, published on-line 12January 2006

Post-translational attachment of ubiquitin (Ub) to polypep-

tides has a fundamental role in modulating the plasma

membrane protein composition. Polyubiquitination prevents

the delivery of non-native membrane proteins by signalling

their proteasome-dependent degradation at the endoplas-

mic reticulum (ER) or shunting them for vacuolar/lysoso-

mal proteolysis from post-ER compartments (1,2). While

ubiquitination serves as the primary endocytic signal for

the down-regulation of plasma membrane proteins in

yeast (3,4,5), a variety of short peptide motifs [e.g.

YXXF, where F is a bulky hydrophobic residue and (D/

E)XXXL(L/I)] have been established as internalization motifs

in mammalian cells (1). Recently, monoubiquitination also

emerged as a recognition signal in the retrieval of plasma

membrane receptors and transporters from the cell sur-

face in higher eukaryotes (3,4,8–10). The notion that

mono-Ub represents an autonomous endocytic signal in

mammalian cells was primarily based on the internalization

activity of fusion proteins, containing a Ub moiety and the

truncated epidermal growth factor (EGF), platelet-derived

growth factor (PDGF) and the IL-2 receptor (5,8,10).

Considering that these receptors have intrinsic homo-

and/or hetero-oligomerization propensity, as well as the

fused Ub moiety may have been subjected to cycles of

ubiquitination-deubiquitination at the plasma membrane/

endosomes (11), the presentation of oligomeric Ub

adduct(s) to the endocytic machinery cannot be ruled out.

Molecular recognition of Ub can be achieved by Ub-

receptors that contain Ub-binding module(s), such as Ub-

interacting motif (UIM), Ub associated, coupling of Ub to

endoplasmic reticulum associated degradation, Ub E2 var-

iant and Npl4 Zn finger domains (3,12). It is assumed that

decoding of mono-Ub as an endocytic signal is mediated

by modular adaptor molecules (e.g. epsin and eps15/

eps15R) (4,13,14), harbouring multiple UIMs and other

functional domains that trigger coat proteins assembly

and subsequent vesicular budding similar to cargo sorting

at the trans Golgi network and early endosomes (6,15,16).

Intriguingly, while the preferential association of poly Ub

chain to the conserved UIM of the S5a subunit of protea-

some correlates well with the proteasome-mediated

degradation of polyubiquitinated targets, the negligible

binding of mono-Ub to the UIMs of eps15 and epsin is at

variance with their presumed role in monoubiquitinated

cargo recruitment to clathrin-coated pits/vesicles (CCP/

CCV) (13,17–19).

Multiple internalization pathways have been invoked in the

uptake of ubiquitinated proteins from the cell surface.

While several plasma membrane proteins that undergo

oligo/polyubiquitination-induced down-regulation (e.g.

Traffic 2006; 7: 282–297# 2006 The Authors

Blackwell Munksgaard doi: 10.1111/j.1600-0854.2006.00384.x

282

CD4, major histocompatibility complex (MHC) class I,

b2-adrenergic receptor (b2-AR) and ENaC) are internalized

via the CCP and CCV, activated EGF receptor (EGFR) and

PDGF receptor (PDGFR) appear to be targeted for endo-

cytosis via caveolin- and clathrin-dependent mechanisms

(4,20–22). These observations, jointly, indicate that neither

the structural basis nor the molecular machinery respon-

sible for the recognition and internalization of ubiquitinated

plasma membrane proteins is fully understood in mamma-

lian cells (9,23).

To gain more insight into this process, we utilized model

proteins harbouring Ub as their primary endocytic signal.

Using a panel of CD4 chimeras, as well as other ubiquiti-

nated membrane proteins, in combination with functional,

biochemical and genetic techniques, we present data sug-

gesting that polymeric or oligomeric Ub is recognized as

an endocytic signal by a subset of UIM containing clathrin

adaptor molecules in higher eukaryotes.

Results

Chimeric membrane proteins to study the endocytic

activity of Ub

To examine the role of Ub as an endocytic signal, first we

used CD4, a monomeric type-I membrane protein, as a

reporter molecule. CD4 is targeted to the plasma mem-

brane by default and has been extensively used to deline-

ate sorting signals, and its truncated form lacks signalling

ability (1). Ub recognition by the endocytic sorting machin-

ery was optimized by creating linkers with various lengths

between the CD4 reporter molecule and the fused Ub

(Figure 1A). The cytoplasmic tail (43 amino acid residues)

Ii0

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IiT

53 kDa

Figure 1: Ubiquitin (Ub) fusion confers internalization activity to type-I and type-II plasma membrane proteins. (A) Schematic

representations of model proteins containing Ub fused to CD4 or invariant chain (Ii). The cytoplasmic tail of CD4T, CD4Tl and CD4 is drawn

to scale. The Ub linker (black line, 21 amino acid residues) and the additional spacer (orange line) are indicated and specified in the

Materials and Methods. To preserve the distance between Ub and the plasma membrane, we replaced the flexible linker by the coiled-

coiled tetramerization domain (CC) of the Kir6.2 channel (small circles) in CD4TCC-Ub. (B) Plasma membrane expression of CD4 chimeras.

The chimeras were visualized by indirect immunostaining and confocal fluorescence microscopy using anti-CD4 antibody in transiently

transfected, non-permeabilized COS-7 cells. Bar: 10 mm. (C) Immunoblot analysis of CD4Tl, CD4Tl-Ub, CD4Tl-UbAllR and CD4Tl-UbnGG

expression in transiently transfected COS-7 cells. Equal amounts of cell lysates were separated by SDS-PAGE and visualized by enhanced

chemiluminescence (ECL) assay with polyclonal anti-CD4 antibody. Actin was visualized as loading control. Black triangle, CD4Tl; grey

triangle, CD4Tl-Ub variants. (D) Ub fusion confers internalization signal to CD4 variants. Endocytosis rates of the indicated chimeras were

measured by the antibody-uptake assay in transiently transfected COS-7 cells at 37 �C for 5 min as described in Materials and Methods.

Internalization rates are expressed as percentage of CD4 antibody uptake in 5 min relative to the initial amount at the cell surface

(mean � SE, n ¼ 3–4). Experiments were performed in triplicates. Separate experiments verified that antibody uptake is linear for 5 min.

(E) Ub fusion confers internalization signal to the truncated IiT. Internalization rates of the indicated construct were measured by the anti-Ii

antibody uptake as described in Materials and Methods (mean � SEM, n ¼ 3–4).

Oligoubiquitin Recognition as Endocytic Signal

Traffic 2006; 7: 282–297 283

of CD4 was preserved in the CD4-Ub chimera, while it

was truncated at the membrane plane and replaced with a

flexible 20 or 40 amino acid linker in CD4T-Ub and CD4Tl-

Ub, respectively (Figure 1A) as described in Materials and

Methods. Immunostaining and immunoblotting demon-

strated the expression of CD4Tl, CD4T and CD4, as well

as their Ub fusions at plasma membrane and in the lysates

of COS-7 cells (Figure 1B,C and data not shown).

The endocytosis of chimeras was measured by anti-CD4

antibody uptake assays in transiently transfected COS-7

cells. After binding the anti-CD4 antibody at 4 �C, internal-ization was resumed at 37 �C for 3–5 min, and the amount

of cell surface-remaining antibody was quantified by radio-

active or fluorescence assay as described in Materials and

Methods. Ub fusion accelerated the internalization of CD4Tl

from 9.6 � 0.9 to 41.9 � 2.6% in 5 min, while it was less

effective upon fusion to the CD4 and CD4T, monitored by

anti-CD4 antibody uptake (Figure 1D). The endocytic rates

of CD4Tl-Ub were similar in CHO, HeLa and HEK293 cells

(data not shown). Inducing Ub unfolding by replacing Val26

with Gly (CD4Tl-UbV26G) abolished the internalization of the

chimera (Figure 1D), implying that the native conformation

of Ub is a prerequisite for its recognition by the endocytic

machinery, as observed in yeast (5).

Ub was also recognized as an endocytic signal in type II

membrane protein (24). Attachment of Ub to the truncated

invariant chain (IiT), a homo-trimeric type-II membrane

protein lacking its di-Leu-based endocytic signal, increased

the internalization rate of IiT (6.9 � 2.0%/5 min) by seven-

fold (IiT-Ub, 49.9 � 4.2%/5 min) (Figure 1E).

Mono-Ub is not recognized efficiently by the

endocytic machinery as an internalization signal

in mammalian cells

To assess whether a single Ub moiety constitutes an

autonomous internalization signal, we prevented Ub-

chain synthesis by replacing all the seven Lys residues

with Arg (UbAllR) or deleting the carboxy-terminal Gly

residues (UbnGG) in Ub (25). While CD4Tl-UbnGG

uptake was similar to the CD4Tl-Ub, the internalization of

both CD4Tl-UbAllR (10.0 � 2.1%/5 min) and IiT-UbAllR

(8.6 � 0.5%/5 min) was reduced to the level of the

respective reporter molecule (CD4Tl, 9.6 � 0.9%/5 min;

and IiT, 6.9 � 2.0%/5 min) (Figure 2A). Although the

steady-state expression of the CD4Tl and CD4Tl-UbAllR

was fourfold to fivefold higher, saturation of the internal-

ization machinery can account for their impeded uptake.

Reducing the cell surface density of CD4Tl by fourfold did

not influence its endocytosis rates (data not shown). The

slow signal-independent endocytosis of CD4Tl and CD4Tl-

UbAllR is likely mediated by bulk membrane flow (26) and

was comparable to that observed for CD4Tl in COS-7 and

CHO cells (Figure 2A,2H). These observations suggest

that monomeric Ub cannot be recognized as an autono-

mous endocytic signal in mammalian cells, a conclusion

that was reinforced by subsequent results.

Next, we tested whether reinsertion of a single Lys into the

UbAllR is sufficient to render endocytic activity to the CD4Tl-

UbAllR chimera. Reverting Arg48 into Lys residue comple-

tely restored the CD4Tl-UbAllR internalization rates to the

CD4Tl-Ub level, likely by providing an acceptor site for Ub

conjugation (Figure 2B). The endocytic activity was partially

recovered by Lys63, but not by Lys6 or Lys29 reinsertion

into UbAllR (Figure 2B). In line with the redundancy of the

branching site for Ub chain elongation, replacement of Lys48

with Arg in wild-type Ub caused only partial inhibition of the

CD4Tl-UbK48R internalization (approximately 37%), while

neither Lys63Arg, nor Lys29Arg mutations had an inhibitory

effect (Figure 2B). These observations suggest that Lys48 is

preferential, but not the exclusive acceptor site for the first

isopeptide bond formation in CD4Tl-UbAllR. Thus incorpor-

ation of UbK48R in fusion proteins does not preclude the

possibility of Ub conjugation. Tandem fusion of two Ubs

(CD4Tl-2Ub) or three Ubs (CD4Tl-3Ub) significantly acceler-

ated the initial uptake rates compared with CD4Tl-Ub, sup-

porting the notion that Ub multimerization facilitates cargo

uptake (Figure 2C). In the latter measurements, the inter-

nalization time was reduced from 5 to 3 min to assess the

rapid initial rates of endocytosis.

Biochemical evidence for the ubiquitination of CD4Tl-Ub

was obtained by determining the apparent molecular

mass and Ub conjugation of CD4Tl-Ub. Cell surface resi-

dent and endocytosed reporter molecules were selec-

tively immunoprecipitated following in vivo labelling the

cells with anti-CD4 antibody to preclude the isolation of

ubiquitinated adducts from the ER. Immunoprecipitates

were isolated by protein G sepharose and probed with

anti-CD4 antibody. While CD4Tl was represented by an

approximately 52 kDa polypeptide corresponding to its

predicted mass (Figure 2D, lane 3), CD4Tl-Ub appeared

as a doublet with molecular masses of approximately

58 kDa, representing CD4Tl-Ub and with MW of approxi-

mately 65–66 kDa, likely reflecting monoubiquitinated

adduct of CD4Tl-Ub (Figure 2D, lane 4). Both of the

mono- and di-Ub containing CD4Tl-Ub polypeptides were

recognized by anti-Ub antibody (Figure 2D, lower panel). In

addition, polyubiquitination of CD4Tl-Ub, but not CD4Tl or

CD4Tl-UbAllRnGG, could be observed, indicated by the

high molecular weight ladder formation in the precipitates

(Figure 2D, lane 4, lower panel and data not shown).

Polymeric Ub chain and multimers of mono-Ub serve

as endocytic signals

If mono-Ub cannot be recognized by the endocytic

machinery, non-covalently linked multimers of UbAllR

representing multiple monoubiquitination (8) or oligo-

merization of monoubiquitinated native cargo molecules

(27) may serve as an efficient internalization signal.

Tetramerization of CD4Tl-UbAllR was induced by inserting

the tetramerization coiled-coil (CC) domain of the Kir6.2

potassium channel between the membrane-spanning seg-

ment of CD4 and the UbAllR moiety (CD4TCC-UbAllR

Figure 1A) (28). The ability of the CC domain to induce

Barriere et al.

284 Traffic 2006; 7: 282–297

tetramerization of a soluble protein was confirmed by

the accumulation of higher order oligomers of GSTCC-

Ub (MW approximately 37 kDa) with an apparent mole-

cules mass =150 kDa determined by size-exclusion

chromatography (Figure 2E). Notably, the CC domain

insertion increased the internalization rates of CD4TCC-

UbAllR by fivefold (53.8 � 2.5%/5 min), while it had no

effect on the slow uptake of CD4TCC (6.7 � 1.6%/5 min,

B

D

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F G

H

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- wt ∆GG AllR wtIiT

AllR0

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CD4Tl - Ub - Ub97

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kDa1 2 3 4

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GSTCC-Ub

146GST-Ub

% Internalized in 5 min

CC

CC-Ub

UbAllR

CC-UbAllR

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CD

4T(l)

CD4TlCD4Tl-UbAllR

0 25 50

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control

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Oligoubiquitin Recognition as Endocytic Signal

Traffic 2006; 7: 282–297 285

Figure 2F). As a complementary approach to induce

CD4Tl-UbAllR oligomerization, antibody-mediated cross-

linking was applied. Cell surface bound anti-CD4 antibody

was crosslinked by secondary antibody at 4 �C, and inter-

nalization was measured by two different protocols. Non-

covalent crosslinking increased the internalization of

CD4Tl-UbAllR, but not CD4Tl, by fivefold at 37 �C(Figure 2G). These results not only rule out that conforma-

tional defect of UbAllR accounts for the defective endocy-

tosis of CD4Tl-UbAllR but, more importantly, demonstrate

that the internalization machinery can recognize Ub multi-

mers in covalently linked Ub chain, as well as in oligomeric

complexes.

If Ub-chain synthesis is required for CD4Tl-Ub endocyto-

sis, inactivation of the cellular ubiquitination machinery

should impede this process. Down-regulation of the

thermo-sensitive E1 Ub-activating enzyme was achieved

by exposing the transiently transfected ts20 cells to 40 �C(29). Inactivation of the E1 enzyme inhibited CD4Tl-Ub

endocytosis by 63%, compared with that measured in

E36 cells containing the wild-type E1 enzyme

(Figure 2H). The incomplete inhibition of endocytosis

may be explained by the residual activity of E2/E3 after

E1 inactivation. Considering that putative components of

the Ub-dependent internalization machinery are suscepti-

ble to monoubiquitination (e.g. eps15/15R and epsin)

(13,19,30), which may influence cargo recognition, we

also assessed the impact of E1 ablation on the activity of

the Ub-specific internalization machinery. To accomplish

this, we used the tetrameric CD4TCC-UbAllR as cargo,

because this chimera is not susceptible to ubiquitination

at the cell surface (Figure 2F). E1 down-regulation has

less-pronounced inhibition on the CD4TCC-UbAllR

endocytosis (43%) as compared with that on CD4Tl-Ub

uptake (63%, Figure 2H), lending credence to the hypoth-

esis that ubiquitination of the endocytic machinery and/or

the elevated cytoplasmic Ub concentration can influence

cargo recognition efficiency at the cell surface. Notably, E1

inactivation failed to alter the endocytosis of the CD4-

Lamp1 chimera and the transferrin receptor (TfR)

(Figure 2H). This suggests that recognition of the Tyr-

based endocytic motifs by the AP-2-dependent internaliza-

tion pathway is independent of the ubiquitination machin-

ery activity.

Clathrin-dependent endocytosis of ubiquitinated

chimeras

To unveil the identity of the internalization pathway of

ubiquitinated membrane proteins, we used Ub chimeras

without signalling ability. Established inhibitors of CCV

formation, such as hypertonicity, Kþ-depletion or cytoplas-

mic acidification abolished the Ub-dependent internaliza-

tion of the CD4Tl-Ub, similar to the dominant negative

dynamin1K44A overexpression (Figure 3A). The effect of

dynamin1K44A cannot be attributed to defective caveo-

some formation, because blocking caveosome-mediated

endocytosis by cholesterol depletion with filipin, nystatin

or mevinolin had no effect on CD4Tl-Ub uptake

(Figure 3B). These drugs efficiently reduced the cellular

cholesterol content, indicated by the impeded cholera

toxin B (CTB) uptake (Figure 3B), a cargo of caveolae-

mediated internalization (31). To validate these results

and rule out possible crosstalk of inhibitors, we deter-

mined CD4Tl-Ub endocytosis in clathrin heavy chain

(CHC) or caveolin1-depleted cells. Delivery of small inter-

fering RNA (siRNA) resulted in >80% loss of CHC and

caveolin1 content in HEK293 cells (Figure 3C) and caused

Figure 2: Monomeric ubiquitin (Ub) is not sufficient to signal internalization. (A) In-frame fusion of UbAllR is not recognized as an

internalization signal in type-I and -II plasma membrane proteins. Endocytosis rates of the indicated CD4 and invariant chain chimeras were

determined by the antibody uptake assay in transiently transfected COS-7 cells. (B) Oligo-ubiquitination is required for efficient internalization of the

CD4Tl-UbAllR chimera. Endocytosis rates of the indicated chimeras were determined as in panel (A) (means � SEM, n ¼ 3). For comparison,

dashed and solid lines indicate the internalization rates of CD4Tl-UbAllR and CD4Tl-Ub, respectively. The significance of change was calculated by

two-tailed unpaired t-test and indicated as follows: *, p ¼ 0.01 for R63K versus CD4Tl-UbAllR; **, p > 0.3 for R48K, K29R or K63Rmutants versus

CD4TlUb and ***, p < 0.01 for K48R mutant versus CD4Tl. (C) The internalization rates are proportional to the number of Ub moieties fused to

CD4Tl. Endocytosis of CD4Tl harbouring mono-, di- or tri-Ub moieties weremeasured in transiently transfected COS-7 cells. Antibody uptake rates

were calculated after 3 min of internalization. (D) Ubiquitination of CD4Tl-Ub. Cell surface resident and endocytosed CD4Tl and CD4Tl-Ub were

selectively labelled by incubating stably transfectedHEK293 cellswith CD4 antibody for 30 min at 37 �C. CD4-Ab complexeswere immunoisolated

on protein G. Imunoprecipitates and lysates were probed with anti-CD4 (top panel) and anti-Ub antibodies (bottom panel). CD4Tl-Ub and its

monoubiquitinated version are marked by black and grey arrow head, respectively. Asterisk denotes polyubiquitinated adducts of CD4Tl-Ub,

undetectable for CD4Tl-Ub. (E) Oligomerization tendency of the coil-coiled (CC) tetramerization domain containing GST-Ub. Bacterial extracts,

expressing GST-Ub or GSTCC-Ub were fractioned by size-exclusion chromatography. Fractions were analysed by dot-blotting using anti-GST Ab

and chemiluminescence (ECL). (F) The tetramerization domain (CC) restores the CD4Tl-UbAllR internalization activity. Endocytosis rates of

CD4TCC, CD4TCC-Ub, CD4TCC-UbAllR and CD4Tl-UbAllR were measured by antibody uptake in COS-7 cells (means � SE, n ¼ 3–4). (G)

Oligomerization of CD4Tl-UbAllR by antibody cross-linking induces internalization, measured in COS-7. Following primary and secondary antibody

binding (prot. 1) or primary, biotinylated secondary antibody and streptavidin-horseradish peroxydase (HRP) cross-linking (prot. 2) on ice, internaliza-

tion was resumed for 5 min at 37 �C. Data are expressed as percentage of uptake of the initial amount of antibody or streptavidin-HRP, measured

as described in Materials and Methods (means � SE, n ¼ 3). (H) Active ubiquitination machinery is required for efficient internalization of

ubiquitinated reportermolecules, but not for transferrin receptor (TfR) or the CD4-Lamp1 chimera. Internalization ratesweremeasured in transiently

transfected ts20 or E36 cells, after incubation at 40 �C for 2 h to down-regulate the E1 enzyme. CD4-Lamp1 and TfR uptakewas determined under

the same conditions using CD4 Ab and Tf-biotin, respectively, as detailed inMaterials andMethods. Inset: down-regulation of the thermo-sensitive

E1 enzyme was monitored by immunoblotting after 2 h of incubation at 40 �C with anti-E1 antibody and ECL.

Barriere et al.

286 Traffic 2006; 7: 282–297

0

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HE K293

Figure 3: Clathrin-dependent internalization of (Ub) ubiquitin chimeras. (A) Pharmacological inhibition of CD4Tl-Ub internalization.

Rates of CD4Tl-Ub endocytosis were measured by antibody uptake after Kþ depletion, exposure to hypertonic medium, cytoplasmic

acidification or following the transient over-expression of dominant negative (DN) dynamin1K44A (see Materials and Methods). (B) Effect

of cholesterol depletion on CD4Tl-Ub and CTB-HRP (horseradish peroxydase-conjugated cholera toxin B) internalization in HEK293 cells.

CTB-HRP (10 nM) was bound at 0 �C for 1 h and then internalized at 37 �C for 5 min. The amount of cell surface bound CTB-HRP was

quantified by the Amplex�Red assay. CD4Tl-Ub uptake was determined as described in Figure 1D. Cells were preincubated with nystatin

(25 mg/mL) and filipin (0.5 mg/mL) for 1 h at 37 �C. HEK293 cells were treated with 1 mM mevinolin for 24 h. Data are obtained from three

to four independent experiments. (C) Clathrin heavy chain (CHC) and caveolin1 (cav) depletion by small interfering RNA (siRNA). CHC and

caveolin1 expression was measured by immunoblotting after siRNA treatment of the indicated cells as described in Materials and

Methods. Equal amounts of protein were loaded. Invariant chain (IiT)-Ub was transiently cotransfected with the double stranded siRNA as

described in Materials and Methods. (D) Functional effects of CHC and caveolin1 depletion. Caveolin- and clathrin-dependent endocytosis

was monitored by CTB-HRP and transferrin-biotin (Tf-biotin) uptake in mock transfected cells as well as in cells treated with non-specific

(ns) or specific siRNA from 5 min at 37 �C as described in Materials and Methods. (E) CHC dependence of CD4Tl-Ub and IiT-Ub uptake.

Internalization rates of CD4 variants and IiT-Ub were measured (5 min, 37 �C) by the antibody uptake assay after CHC or caveolin1

depletion in HEK293 and HeLa cells, respectively. Means � SEM, n ¼ 3–4.

Oligoubiquitin Recognition as Endocytic Signal

Traffic 2006; 7: 282–297 287

near complete elimination of TfR and CTB uptake, respec-

tively (Figure 3D). Importantly, down-regulation of CHC,

but not caveolin1, suppressed CD4Tl-Ub endocytosis to

basal level (Figure 3E). Similar results were obtained with

the IiT-Ub chimera (Figure 3E).

Ubiquitinated reporter was excluded from detergent-resistant

membranes, because CD4Tl-Ub (and CD4Tl), but not CTB

binding to ganglioside 1 (GM1) was eliminated by TritonX-

100 extraction at 4 �C, visualized by fluorescencemicroscopy

(Figure 4A,B). Immunostaining confirmed that CD4Tl-Ub was

excluded from plasma membrane rafts, while it was partially

co-localized with the TfR (Figure 4C,D). These data, collec-

tively, reinforce the notion that ubiquitinated cargoes without

signalling ability are excluded from cholesterol-rich rafts and

preferentially targeted for clathrin-dependent endocytosis.

Biochemical interaction of clathrin adaptors with

ubiquitinated cargo

It has been postulated that eps15, eps15R and/or epsin

may play critical role in the recruitment of ubiquitinated

cargo into CCP and CCV because these adaptors (i)

harbour multiple UIMs (1); (ii) bind directly or indirectly to

CHC and AP-2 (16); (iii) have been localised to CCP and

CCV by electron microscopy (22,32) and (iv) their ortholo-

gues are implicated in the down-regulation of ubiquitinated

plasma membrane proteins in Drosophila melanogaster

(33) and yeast (14). Biochemical association of potential

clathrin adaptors, involved in the recognition of ubiquiti-

nated plasma membrane proteins, was examined next.

Binding of eps15, eps15R and epsin1 to GST-Ub fusion pro-

teins was measured by pull-down assays after incubating the

recombinant proteinswith HeLa cytosol. Although these adap-

tors were unable to bind to GST-Ub (and GST), significant

fraction of the lysate was associated with GST-2Ub and GST-

3Ub (Figure 5A). Misfolding of GST-Ub cannot account for

its inability to bind to the adaptor proteins, because Hrs

(34) and the E1 Ub-activating enzyme were bound to

mono-Ub (data not shown). In light of the dimerization

propensity of GST (Figure 2E), it is plausible to assume

that presentation of oligomeric Ub represents the efficient

binding partner for these adaptors by increasing their avid-

ity to Ub containing complex. This notion was further

supported by the inability of recombinant eps15 to bind

to mono-, di- or tri-Ub, but efficiently associate with oligo-

Ub, containing four or more Ub residues conjugated via

K63 or K48 (Figure 5B). These observations, together with

the preferential poly Ub binding of UIMs of eps15 and

epsin1 (13,18,19,35), are consistent with our functional

data, and the hypothesis that multivalent substrate – adap-

tor interactions are necessary to overcome the low affinity

binding of mono-Ub to various UIM domains (Kd approxi-

mately 0.1–1 mM) (17,36,37). The AP-2 association with

GST-2Ub and GST-3Ub is, presumable, indirect and

mediated by eps15/15R or epsin1 (Figure 5A), because

the AP-2 complex has eps15 and epsin1-binding sites,

but lacks Ub-binding domain (38).

Association of eps15 with the Ub chimera was demon-

strated by coimmunoprecipitation of eps15 with CD4Tl-

3Ub, but not with CD4Tl in transiently transfected COS-7

cells (Figure 5C and data not shown). We were unable to

detect eps15 binding to CD4Tl-Ub. Combination of rapid

deubiquitination, decreased binding affinity of adaptors to

and the low expression level of the oligo-ubiquitinated

CD4Tl-Ub may explain the lack of eps15 detection in

CD4Tl-Ub precipitates.

Functional relevance of eps15/15R and epsin1 in the

endocytosis of ubiquitinated cargo

The functional relevance of eps15, eps15R, epsin1 and

AP-2a in ubiquitinated cargo endocytosis was evaluated

after depletion of these adaptors by siRNA. Immunoblot

analysis revealed that the UIM adaptors were reduced by

73–76%, while AP-2a was depleted by approximately

84% in HEK293 cells (Figure 6A,B). While eps15,

eps15R and epsin1 depletion caused 73, 84 and 73%

inhibition of the Ub-dependent internalizaion of CD4Tl-

Ub, respectively (Figure 6C), TfR endocytosis was dimin-

ished by approximately 55% (Figure 6D). Inhibition of TfR

uptake is consistent with the role of these adaptors as

accessory proteins in CCV formation (39,40). However, a

more dramatic difference was observed in the sensitivity

to AP-2a depletion. While AP-2a siRNA treatment had only

marginally inhibited (approximately 11%) CD4Tl-Ub uptake

(Figure 6C), TfR endocytosis was attenuated by approxi-

mately 75% (Figure 6D). Notably, internalization of the

oligomeric CD4TCC-UbAllR was attenuated comparable

to that of CD4Tl-Ub in CHC, eps15 and epsin knockdown

cells (Figure 6E), implying that an overlapping set of UIM

containing endocytic adaptors can recognize cargo mole-

cules tagged with oligo-Ub chain(s) or oligomeric Ub.

These results also suggest that ubiquitinated plasma

membrane proteins may represent a diverse group of

ligands that can be recruited via clathrin-dependent, but

AP-2-independent internalization mechanism, similar to

the LDL-R family (16). Although GGA3, a Golgi/endosome

adaptor, contributes to the sorting of ubiquitinated cargo

at Golgi and endosomal compartments (15), GGA3

appears to have no role in the recognition of the Ub

chimeras at the plasma membrane in HEK293 cells.

The established biochemical interaction network of eps15/

15R, epsin and AP-2 adaptors suggest that ubiquitinated

cargo recruitment to CHC may proceed by eps15/eps15R

and AP-2, or epsin1 in cargo and cell type-dependent

manner (5,9,23,41) (Figure 7). In line with this hypothesis,

AP-2a and epsin depletion together inhibited CD4Tl-Ub

uptake by approximately 90%, while their individual ablation

reduced cargo uptake by approximately 11 and 73%, respec-

tively (Figure 6C). Furthermore, epsin1 and eps15 siRNA

combination inhibited CD4Tl-Ub internalization by approxi-

mately 90% (data not shown), suggesting that they have

partially overlapping function in ubiquitinated cargo uptake

(23,39). This phenomenon is reminiscent of the functional

co-operativity of the yeast eps15 and epsin orthologues

Barriere et al.

288 Traffic 2006; 7: 282–297

CTB CD4Tl-Ub

Con

trol

Ext

ract

edC

ontr

olE

xtra

cted

Overlay

CTB CD4Tl OverlayA

B

C

D

CD4Tl CTB

CD4Tl-Ub CTB

Overlay

Overlay

TfR

CD4Tl-Ub

CD4Tl Overlay

TfR Overlay

Figure 4: CD4Tl-ubiquitin (Ub) is excluded

from cholesterol-rich rafts. (A–B) Detergent

extractability of (A) CD4Tl and (B) CD4Tl-Ub.

COS-7 cells were transiently transfected with

CD4Tl or CD4Tl-Ub. Cells were stained with

cholera toxin B (CTB)-FITC, monoclonal anti-

CD4 and TRITC-conjugated anti-mouse antibo-

dies. Extraction of non-raft associated mem-

brane proteins was performed in 1% TritonX-

100 (0 �C, 15 min) before fixation. Non-treated

cells were used as controls. Optical sections of

representative cells were obtained by laser con-

focal fluorescence microscopy (LCFM) and pro-

jected images are shown. Note that the laser

light intensity was increased by 10-fold to visua-

lize the small amount of CD4 variants remaining

after TritonX-100 extraction. Bar: 10 mm. (C)

CD4Tl-Ub does not co-localize with raft micro-

domains, enriched in GM1. COS-7 cells were

transfected with CD4Tl or CD4Tl-Ub and

patched with mouse anti-CD4 antibody fol-

lowed by anti-mouse TRITC-conjugated anti-

body as described in Materials and Methods.

GM1 containing lipid rafts were labelled with

CTB-FITC. Boxed areas are enlarged and

depicted in the right column. (D) Partial co-loca-

lization of CD4Tl-Ub and transferrin receptor

(TfR). HEK293 cells were transiently trans-

fected with CD4Tl or CD4Tl-Ub. Cells, plated

on poly Lys-coated coverslips, were incubated

with sheep anti-CD4 and mouse anti-Tf antibo-

dies for 1 h at 12 �C, followed by incubation

with the relevant FITC- and TRITC-conjugated

secondary antibody under the same conditions

and imaged by LCFM.

Oligoubiquitin Recognition as Endocytic Signal

Traffic 2006; 7: 282–297 289

(Ede1p and Ent1/2p) (14). Complete restoration of the ubiqui-

tinated a-factor receptor endocytosis in the background of

ent1D/ent2D/ede1D strain could be only achieved upon com-

plementation with both plasmid-borne Ent1p and Ede1 (14).

Because epsin as well as eps15/15R are susceptible

to monoubiquitination and oligomerization in vivo

(13,19,30,42), multiple UIM and Ub interactions may

stabilize the Ub-adaptor network. While the precise

mechanism of this process remains to be discovered, it

is plausible to assume that cargo binding itself may alter

the avidity of UIM–Ub interactions. Inactivation of the E1

Ub-activating enzyme in ts20 cells may interfere with the

formation of the UIM-Ub adaptor network and therefore

contributes to the observed inhibition of ubiquitinated

cargo uptake (Figure 2H).

To demonstrate the functional relevance of UIM contain-

ing adaptors in the endocytosis of Ub chimeras, we

mutated the UIM-binding interface of Ub. These experi-

ments were performed on CD4TCC-UbAllR to avoid the

incorporation of wt Ub into the oligo-Ub chain of CD4Tl-

Ub. Based on structural studies and mutagenesis, the

interface of the UIM-Ub complexes is largely formed by

the hydrophobic residues of 44Ile, 8Leu and 70Val of Ub

(17,37). Replacement of 44Ile (UbAllR-1A) or 8Leu and

70Val (UbAllR-2A) with Ala inhibited CD4TCC-UbAllR

uptake by approximately 74% (Figure 8A), implying an

essential role of Ub–UIM interactions in ubiquitinated

cargo endocytosis. Notably, the Ile44Ala mutation similarly

prevented the antibody-induced uptake of oligomeric

CD4Tl-Ile44AlaUbAllR (data not shown), while it had a mod-

est effect on CD4Tl-Ub internalization (Figure 8A). The latter

observation is consistent with oligo-Ub chain formation in

vivo, incorporating wt Ub moieties readily recognizable by

the relevant endocytic adaptors. Combining the three point

mutations (UbAllR-3A) had no additive effect, suggesting

that residual binding of UIM adaptors and/or alternative

adaptors may be also involved in the internalization process

(Figure 8A).

The role of epsin1 and eps15/15R in clathrin-dependent

internalization of ubiquitinated CD4 and b2-AR

The physiological relevance of Ub-binding adaptors was

validated by establishing their role in the internalization of

ubiquitinated b2-AR/arrestin complex. Because clathrin-

95

51

197

11695

WB:α-CD4

WB:α-eps15

lysate IP: α-CD4

CD4Tl-3Ub– + +

kDa

CD4Tl+– ––

+––

IB: α-Ub200

513729

20

7

poly-Ub (K48)

poly-Ub (K63)

mono-Ub

–

–

–

–

–

–

– – –

–

–

–

–

–

–

–

–

–

+

+

+

+

+

+

+

+

+

GST GST-eps15

Ub

Ub2

Ub3

Ub4

Ub5

Ub6

Ub7

96

kDa

20011596

51

37

29

GSTGST-eps15

α-eps15R

Ponceau

α-AP2

α-GGA3

α-eps15

120100

100120

55

3829

201

A B

C

120

GST – Ub 2Ub 3Ub lys

100 α-epsin

kDa

100120

input

kDa

Figure 5: Biochemical interactions of endocytic adaptors with ubiquitin (Ub) and ubiquitinated cargo. (A) Endocytic adaptor

binding to Ub in vitro. Pull-down assays were performed with HeLa cell extracts and the indicated GST fusion protein containing none,

one, two, or three tandem Ub moieties. Bound polypeptides and cell lysates (Lys) were immunoblotted for eps15, eps15R, epsin1, AP-2aand GGA3. (B) Poly Ub-binding specificity of eps15. GST and GST-eps15 were purified on glutathione beads and visulized by SDS-PAGE

and SimplyBlue Safe Stain (lower panel). To measure Ub binding to eps15, purified GST and GST-eps15 was prebound to glutathione

beads and incubated with K48 or K63 poly Ub chains and bovine mono-Ub. Bound Ub was visualized by immunoblotting with the FK2 anti-

Ub antibody and chemiluminescence (upper panel). Note that tetra-Ub binding to eps15 was revealed by long exposure (not shown). (C)

CD4Tl-3Ub binds to eps15 in vivo. CD4Tl and CD4Tl-3Ub were immunoprecipitated from transiently transfected COS-7 cells. The lysates

and the precipitates were probed with CD4 and eps15-specific antibodies.

Barriere et al.

290 Traffic 2006; 7: 282–297

dependent internalization of agonist-stimulated b2-ARexclusively relies on arrestin ubiquitination by the Mdm2

E3 ligase (43), the endocytic adaptor sensitivity of constitu-

tive and agonist-stimulated b2-AR uptake was compared.

The constitutive internalization (14.5 � 3.5%/5 min) of b2-AR was not or modestly inhibited by eps15, eps15R and

epsin1 depletion in HEK293 cells expressing the Flag-

tagged receptor (Figure 8B). Isoproterenol-induced

– + – + – +

– + – + – +0

10

20

30

40

epsi

n

eps1

5

eps1

5R

GG

A 3

CH

C

AP 2α

AP

2α

epsin

eps15

eps15R

GGA 3

AP-2αepsin

AP-2α

AP-2α

epsinAP-2α

epsin

eps15

eps15R

GGA 3

AP-2α

GGA 3 CHC

eps15

A B

C D

E

eps15R epsin

actinsiRNA

actinsiRNA

0 25 50 75 100

% Inhibition of CD4TCC-UbAllRinternalization

eps15

epsin

CHC

0 25 50 75 100

0 25 50 75 100

% Inhibition of CD4Tl-Ubinternalization

% Inhibition of Tf internalization

Exp

ress

ion

(% o

f con

trol

)

Figure 6: Distinct subset of clathrin adaptors is required for endocytosis of ubiquitinated chimeras and the transferrin receptor.

(A) Down-regulation of endocytic proteins in HEK293 cells by small interfering RNA (siRNA). Specific and non-specific siRNA treatments

were performed as described in Materials and Methods. Equal amounts of cellular proteins were separated by SDS-PAGE and subjected

to immunoblotting. (B) Quantitative immunoblot analyses of the indicated siRNA efficiency in HEK293 cells. Densitometric analysis of

immunoblots is shown on panel (A). Protein expression levels were expressed relative to that measured in the presence of non-specific

(ns) siRNA. Means � SEM (n ¼ 3–5). (C) Inhibition of CD4Tl-ubiquitin (Ub) internalization in HEK293 cells after eps15, eps15R, epsin,

AP-2a, GGA3 and combination of epsin and or AP-2a siRNA treatment. Internalization rates were expressed as percentage of inhibition

relative to the Ub-dependent uptake of the CD4Tl-Ub, measured in the presence of ns siRNA (means � SE, n ¼ 3–5). (D) Effect of

adaptors depletion on transferrin receptor internalization. Inhibition of Tf-biotin uptake was determined as described in Materials and

Methods in siRNA-treated HEK293 cells. (E) Effect of adaptor and clathrin heavy chain depletion on the internalization efficiency of the

CD4TCC-UbAllR in HEK293 cells. Internalization was measured in siRNA treated cells as described in panel (C). Means � SEM (n ¼ 3).

Oligoubiquitin Recognition as Endocytic Signal

Traffic 2006; 7: 282–297 291

ubiquitination stimulated the internalization rate of the

b2-AR complex by threefold (42.5 � 2.5%/5 min, data

not shown) (44). The activated receptor endocytosis was

highly sensitive to eps15, eps15R and epsin depletion,

confirming their function as clathrin adaptors in polyubiqui-

tinated b2-AR complex uptake (Figure 8B).

As a second model to demonstrate the physiological rele-

vance of Ub-binding adaptors, we took advantage of the

Ub-dependent down-regulation of cell surface CD4 mole-

cules upon over-expression of viral membrane-anchored

E3 ligases (e.g. M153R) (45,46), or their human ortholo-

gues (e.g. March-IV) (47). Transient co-transfection of

March-IV, a Golgi-associated E3 enzyme (47), with CD4,

but not with CD4-lacking Lys residues in its cytoplasmic

tail, provoked approximately threefold-accelerated interna-

lization (Figure 8C). Co-expression of March-VIII, an E3

ligase, localized to the endosomal and lysosomal compart-

ments (47), had no effect on CD4Tl-Ub endocytosis.

Thus March-IV-mediated ubiquitination is necessary and

sufficient for rapid down-regulation of CD4 from the cell

surface (Figure 8C) (47). The constitutive and Ub-indepen-

dent internalization of CD4 was modestly sensitive to

eps15 and epsin depletion by siRNA (Figure 8D). In con-

trast, March-IV-induced CD4 endocytosis displayed similar

sensitivity to UIM containing adaptor knockdown as

observed for CD4Tl-Ub (Figure 6C). The internalization of

ubiquitinated CD4 was inhibited by 75–87% by eps15 and

epsin1 siRNA, while AP-2a depletion had only 50% block-

ing effect (Figure 8D). These observations with the

approximately 75% inhibition of CD4 internalization by

CHC siRNA (Figure 8D) provide supporting evidence for

the functional relevance of eps15/15R and epsin1 in the

down-regulation of oligo/polyubiquitinated plasma mem-

brane proteins via the clathrin-dependent pathway.

Discussion

Our results highlight three fundamental hallmarks of Ub as

an endocytic signal in mammalian cells. First, using RNA

interference in combination with internalization measure-

ments, we provide evidence for the involvement of the

UIM containing eps15/eps15R and epsin1 adaptors in the

recruitment of ubiquitinated cargo into CCV. The import-

ance of Ub recognition by UIM was demonstrated by

mutagenesis of the UIM-docking surface of the Ub moiety

(Figure 8A). These results, jointly with the observation that

AP-2a depletion caused negligible inhibition of the clathrin-

dependent CD4Tl-Ub uptake but approximately 75% block

of TfR endocytosis, strongly support the proposition that

eps15/15R and epsin1 are functional as clathrin-associated-

sorting proteins for ubiquitinated plasma membrane pro-

teins in higher eukaryotes (1,4,5,16,23).

Second, using morphological and genetic techniques, we

show that Ub attachment to type-I and -II membrane pro-

teins without signalling ability and endocytic motifs can

target reporter molecules for efficient internalization via a

clathrin-dependent and caveolin-independent pathway.

These observations are consistent with an earlier report,

demonstrating that overexpression of a Ub chimera com-

petes with the internalization of membrane proteins har-

bouring Tyr-based endocytic motifs (24). Our results are

also in agreement with data obtained by functional, bio-

chemical and morphological techniques, documenting that

polyubiquitinated CD4, MHC class I and b2-AR/arrestincomplex undergo clathrin-dependent internalization

(48,49). Notably, an alteration of EGFR internalization from

clathrin-dependent to caveolin-dependent has been

reported upon increasing EGF concentration (21). A hier-

archical interplay between Ub- and signalling-dependent-

sorting mechanisms may offer a plausible explanation for

this phenomenon. While the latter may target the activated

EGFR receptor to a detergent-resistant, cholesterol-rich

membrane (50), multiple-monoubiqutination of the receptor

would provide a direct link to the clathrin-dependent uptake

route via Ub-binding clathrin adaptors. The activation state

of the different signalling molecules and their interaction

with the signal-decoding machinery may define the ultimate

route(s) of EGFR endocytosis in a cell type specific manner.

The third characteristic of the Ub-dependent internalization

signal is that a single Ub moiety is insufficient to confer

endocytic activity to cell surface resident reporter

CHC

AP-2

epsin

eps15 eps15R

Ubiquitinated cargo

UIM

Ub

Figure 7: Schematic model for the recruitment of ubiquiti-

nated membrane proteins to clathrin-coated pits/vesicles

(CCP/CCV) based on previously established interactions and

the present work.

Barriere et al.

292 Traffic 2006; 7: 282–297

molecules and by inference to native cargos in mammalian

cells. CD4Tl-Ub internalization was inhibited by E1 inacti-

vation or by replacing the Ub moiety with UbAllR.

Meanwhile, defective endocytosis of CD4Tl-UbAllR was

rescued by the formation of polymeric Ub upon oligomer-

ization of the chimera, or by reintroducing a single Lys

residue into UbAllR. The oligoubiquitination propensity of

CD4Tl-Ub and IiT-Ub cannot be attributed to specific char-

acteristics of the chimeras per se, considering their similar

behaviour in the context of type-I and type-II membrane

proteins. Supporting the notion that oligoubiquitin is recog-

nized as an efficient internalization signal in mammalian

cells, modulator of immune recognition 1 an E3 Ub-ligase

encoded by the Kaposi’s sarcoma-associated herpesvirus

triggers the polyubiquitination and efficient endocytosis of

a Lys-less MHC I molecules, harbouring a single Cys resi-

due in its cytoplasmic tail as the Ub-acceptor site (51).

Finally, polyubiquitination of the TrkA receptor has been

recently reported to be indispensable for its ligand-induced

internalization (52).

Importantly, our results uncovered the structural diversity

of oligo-Ub recognition as an endocytic signal, represented

by its polymeric and oligomeric forms, in higher eukaryotic

cells. Induction of non-covalent oligomers of CD4Tl-UbAllR

by antibody cross-linking or inducing tetramerization

restored the endocytic activity of the reporter molecule

at least to that of CD4Tl-Ub (Figure 2F). These observa-

tions provide a mechanistic basis for the equally efficient

internalization of numerous oligomeric and monomeric

plasma membrane proteins that are subjected to mono-

ubiquitination, multiple-monoubiquitination and/or polyubi-

quitination [e.g. ROMK1 (27), ENaC (53), EGFR (10,54),

b2-AR/arrestin complex (44), CD4 (47) and MHC I, B7.2

(55)]. The low affinity binding of Ub to a variety of UIMs in

vitro (17,36,37,56) offers a possible explanation for the

inability of mono-Ub to be efficiently recognized as an

autonomous internalization signal in mammalian cells.

This inference is consistent with binding studies, demon-

strating that polymeric Ub, but not the monomeric Ub,

binds to the hetero-oligomeric complexes of eps15

0 10 20 30% Internalized

40 50 60

CD4CD4 Lys-less

March-IV

March-VIII

March-VIII

March-IV

-

-

CD4Tl-Ub1A

UbAllR

A B

C D

UbAllR-1A

UbAllR-2A

UbAllR-3A

0 25% Inhibition

50 75 0 25 50 75

0 25 50 75 100% Inhibition of CD4 internalization

+March-IV –March-IV

eps15epsin

AP-2αCHC

eps15

epsinAP-2α

CHC

CD

4TC

C-

% Inhibition of β2-AR internalization

100

eps15

eps15R

epsin

eps15eps15R

epsin

siRNA

CHCeps15 eps15R epsin

– – + – + – ++

+Isoproterenol

Non-stimulated

Figure 8: Adaptor requirement for efficient internalization of ubiquitinated CD4 and b2-adrenergic receptor (b2-AR). (A)

Recognition surface of ubiquitin (Ub) by endocytic adaptors. COS-7 cells transiently transfected with CD4TCC-UbAllR, CD4TCC-UbAllR-

1A (I44A), CD4TCC-UbAllR-2A (L8A, V70A), CD4TCC-UbAllR-3A (L8A, I44A and V70A) and CD4Tl-Ub1A (I44A). Endocytic rates of

chimeras were determined by antibody uptake (5 min, 37 �C) and expressed as percentage of CD4TCC-UbAllR and CD4Tl-Ub, respec-

tively. (B) Adaptor sensitivity of agonist-stimulated b2-AR internalization. Endocytosis of the Flag-b2-AR was measured in the absence or

presence of 10 mM isoproterenol in small interfering RNA (siRNA)-treated HEK293 cells (5 min, 37 �C) as described in Materials and

Methods. The lower panel illustrates the indicated siRNA efficiency on protein expression in a representative experiment, visualized by

immunoblotting. Non-specific siRNA was used as control. (C) March-IV-mediated ubiquitination accelerates CD4 internalization.

Internalization rates of CD4 and Lys-less CD4 were determined in the absence or presence of heterologously expressed March-IV

(membrane-associated RING-CH) or March-VIII by the antibody uptake assay (5 min, 37 �C) in HEK293 cells. (D) Clathrin heavy chain and

adaptor dependence of ubiquitinated CD4 internalization. HEK293 cells were treated with siRNA and after 48 h co-transfected with CD4

and March-IV or March-VIII. Forty-eight hours later, internalization of CD4 was measured by antibody uptake. Means � SEM, n ¼ 4.

Oligoubiquitin Recognition as Endocytic Signal

Traffic 2006; 7: 282–297 293

isolated from HeLa cell lysates (Figure 5A), or to purified,

recombinant eps15 (Figure 5B) and the UIMs of epsin and

eps15 (19,35). We propose that productive recognition

and uptake of ubiquitinated cargo necessitate polyvalent

Ub–UIM interactions to stabilize the low affinity mono-Ub-

UIM binding. This process could be facilitated by the

oligomerization of eps15/15R and epsin, increasing their

binding avidity to cargo molecules with polymeric Ub moi-

eties and/or by post-translational modifications of adaptors

(13). Association of ubiquitinated cargo may enhance the

affinity of the eps15/15R and epsin adaptors to oligo-Ub as

well as to each other in a co-operative manner. Due to

structural differences, certain Ub-binding motifs may have

preferential binding to oligo-Ub itself, as described for the

C-terminal UIM of the S5a subunit of the proteasome (57)

and the tandem UIMs in epsin and eps15 (19,35).

Uncovering the contribution of these mechanisms to ubi-

quitinated cargo internalization in vivo will require further

experimentations.

Materials and Methods

Cells and transfectionCOS-7, HEK293T, HeLa, CHO E36 cells were grown in Dulbecco’s

Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum in

thermostated cell culture incubator in 5% CO2 at 37 �C. CHO ts20 cells

were cultured at 32 �C and incubated for the indicated time at 40 �C to

down-regulate the E1 Ub-activating enzyme (29). HEK293 cells stably

expressing CD4Tl or CD4Tl-Ub were selected after transfection with the

pIRES2 expression plasmid (BD Biosciences Inc, Mississauga, ON,

Canada), encoding the respective polypeptide in the presence of 10 mg/mL puromycin. HEK293 cells stably expressing the N-terminally tagged

Flag-b2-AR (58) were kindly provided by Dr Mark von Zastrow (University

California San Francisco, San Francisco, CA, USA). Transient transfection

of COS-7 and HeLa cells was performed with FuGENETM 6 (Roche, Basel,

Switzerland) according to manufacture’s recommendation and analysed

after 48 h.

Plasmid constructionsTo generate the human CD4 chimeras, we fused Ub at the COOH terminus

of CD4, CD4Tl or CD4T via the flexible PRARDPGGGSGGGTGGGSGGG

linker. The linker-flanked Ub was fused in frame to the full-length CD4 in

CD4-Ub, and to the extracellular and transmembrane domain of CD4

(terminated at Phe418) either directly in CD4T-Ub, or via the

SRQEVEVQVEGGSGGGGSG spacer in CD4Tl-Ub (Figure 1A). Mutant Ubs

were generated by PCR mutagenesis, using appropriate templates and

cloned into the XmaI/ClaI sites of CD4Tl-Ub. The cDNA of wt Ub and the

Lys-less Ub (UbAllR) were kindly provided by Dr L. Hicke (North-western

University, Evanston, IL, USA). The cDNAs of R48KUbAllR and

R63KUbAllR were kind gifts of Dr Y. Yarden (The Weizmann Institute of

Science, Rehovot, Israel). The cDNA of UbK29R, UbK48R and UbK63R

were kind gifts of Dr R. Haguenauer-Tsapis (Institute Jacques Monod-

CNRS Universites Paris VI and Paris VII, Paris, France). R6KUbAllR,

R29KUbAllR, UbAllR-1 A (I44A), UbAllR-2 A (L8AV70A) and UbAllR-3 A

(L8AI44AV70A) were generated by overlapping PCR. The cDNA of CD4

containing only Arg residues in its cytoplasmic tail (Lys less) was kindly

provided by Dr U. Schubert (University of Erlangen-Nuremberg, Germany).

GST and CD4T containing the tetramerization CC domain of the potassium

channel Kir6.2 tail were generated by PCR, using the ZZ-pQE60-CC6.2-KKK

plasmid as template, kindly provided by Dr B. Schwappach (ZMBH,

Heidelberg, Germany). The tetramerization domain was attached to the

carboxyl terminus of CD4T (CD4TCC) and to the carboxyl terminus of GST

(GSTCC). Wt or mutant Ub was fused in frame to the CC domain (e.g.

GSTCC-Ub and CD4TCC-Ub). CD4Tl-2Ub and CD4Tl-3Ub were constructed

by subcloning the cDNA encoding the two or three tandem Ub from the

respective pGEX4T plasmids (see below). The dominant negative

dynamin1K44A was a kind gift of Dr S. Schmid (Scripps Institute, CA,

USA). Ii chimeras were engineered by PCR mutagenesis, using the full-

length Ii cDNA (Mammalian Gene Collection, clone BC018726) and sub-

cloned into the pcDNA3.1. The N-terminal 33 amino acid residues of Ii,

containing an internalization signal, were deleted by PCR (IiT) as described

(24). Wt Ub and UbAllR lacking 76GlyGly were fused in frame to the

N-terminus of IiT, yielding IiT-Ub and IiT-UbAllR. All constructs were ver-

ified by DNA sequencing. The construction of CD4-Lamp1 chimera was

described (59).

Recombinant protein purification and pull-down

assayGST fusions, containing one (GST-Ub), two (GST-2Ub) or three (GST-3Ub)

tandem Ub, were constructed by inserting the relevant Ub moieties into

pGEX-4T1. The cDNA for single and multiple Ub fusions were PCR ampli-

fied using pcDNA3.1-2xUb-PL-Bla and pcDNA3.1-3xUb-PL-Bla as tem-

plates (60), containing tandem Ub moieties lacking their C-terminal Gly

residues, kindly provided by Heather Clarke (Aurora Biosciences

Corporation, San Diego, CA, USA). GST-eps15 was obtained by subcloning

hEps15 from pHAT-Eps15, kindly provided by Dr H. Stenmark, into pGEX-

6P1. Protein expression was induced in HB101 cells with 0.3 mM IPTG for

3 h at 37 �C. Recombinant proteins were purified using glutathione-

Sepharose 4B and stored in 150 mM NaCl, 1 mM MgCl2 and 5% glycerol

at �80 �C. For GST and GST-Ub/2U/3Ub pull-down assays, 60 mg recom-

binant proteins were bound to glutathione-Sepharose 4B (150 min, 4 �C),washed three times (150 mM NaCl, 1 mM MgCl2, 0.5 mM EGTA, 20 mM

HEPES/NaOH, 5% glycerol, 0.1% TritonX-100 and pH 7.4) and incubated

with 8 mg HeLa cell lysate (150 min, 4 �C). The beads were washed on a

step-gradient consisting of 17% glycerol and 37% sucrose (0.4 mL), 17%

glycerol (0.2 mL) and 0.2% NP-40 in 150 mM NaCl, 20 mM Hepes/NaOH

pH 7.4, 1 mM MgCl2 (0.2 mL). Bound proteins were eluted with 20 mM

glutathione in 20 mM Tris/HCl, pH 7.4 (25 min, 22 �C).

In the case of GST-Eps15 pull-down, 20 mg purified recombinant proteins

were bound to glutathione beads (120 min at 4 �C), washed three times

(20 mM TRIS/HCl pH 7.4, 150 mM NaCl, 5% glycerol) and incubated with

mono-Ub (50 mg, Sigma, St Louis, MO, USA), polyUb K48-linked chain

(12 mg, Boston Biochem, Cambridge, MA, USA) and polyUb K63-linked

chain (6 mg, Boston Biochem) for 120 min at 4 �C. The beads were washed

as described above, and the bound proteins were eluted with 30 mM

glutathione in 100 mM TRIS/HCl, pH 7.4 (25 min at RT).

Western blotting and immunoprecipitationCD4 (H-370), epsin1 (R-20), eps15 (C-20) antibodies were from Santa Cruz

Biotechnology, Inc., Santa Cruz, CA, USA CHC (610499, clone 23), caveo-

lin1 (610059, clone pAb), GGA3 (612310, clone 8) and invariant chain Ii

(555612, clone LN2) antibodies were from BD Biosciences, Inc. Anti-E1

enzyme antibody was from Covance, Inc., Berkeley, CA, USA and OKT4

from ATCC, Manassas, VA. Eps15R (AP2160a) antibody was purchased

from ABGENT, Inc (San Diego, CA, USA). For immunoblotting, cells were

lysed in RIPA buffer (150 mM NaCl, 20 mM Tris-HCl, 1% (v/v) TritonX-100,

0.1% (w/v) SDS and 0.5% (w/v) sodium deoxycholate, pH 8.0) supplemen-

ted with protease inhibitors (10 mg/mL leupeptin, 10 mg/mL pepstatin and

1 mM PMSF). Polypeptides were separated by SDS-PAGE, transferred to

nitrocellulose membranes and visualized by enhanced chemiluminescence

Amersham Biosciences, Inc. (Quebec, Canada). Densitometric analyses

were performed with NIH IMAGE 1.62 software as described (59).

CD4 chimeras, confined to the cell surface and endosomes, were immuno-

isolated from transiently transfected COS-7 cells after internalization of

OKT4 anti-CD4 antibody (30 min, 37 �C). Excess antibody was then

removed, and cells were lysed in 150 mM NaCl, 10 mM Tris-Cl, 0.2% NP-

40 and pH 7.4, supplemented with 10 mg/mL pepstatin, 10 mg/mL leupep-

tin, 1 mM PMSF, 10 mM MG132 and 5 mM NEM. Immunocomplexes were

isolated on protein G Sepharose (Sigma). Immunopecipitation of CD4

variants from cell lysates was carried out similarly.

Barriere et al.

294 Traffic 2006; 7: 282–297

Recombinant GST-Ub and GSTCC-Ub production was induced with 0.3 mM

IPTG in HB101 cells. The bacteria were sonicated in 150 mM NaCl, 10 mM

Tris-Cl, 5 mM EGTA, 10 mg/mL leupeptin, 10 mg/mL pepstatin and 1 mM

PMSF. Insoluble materials were sedimented by ultracentrifugation

(110,000 � g, 30 min at 4 �C), and polypeptides were fractionated by

size-exclusion chromatography using a Superdex200 10/300G column

(Amersham Biosciences) with 100 mM Tris-Cl, 150 mM NaCl, 5% glycerol,

5 mM EGTA, 1 mM DTT and pH 7.5 as elution buffer. Fractions were

immunoblotted with the polyclonal anti-GST antibody (Amersham

Biosciences)

Cell surface density and internalization

measurementsThe cell surface density of CD4 chimeras was determined by mouse

monoclonal primary anti-CD4 (RPA-T4 or OKT4, 0 �C, 1 h) and 125I-labelled

goat anti-mouse secondary antibody (3 mCi/mL, Amersham Biosciences,

Inc. (Quebec, Canada), 0 �C, 1 h) binding in PBS supplemented with 1 mM

CaCl2, 1 mM MgCl2 (PBS/Ca2þMg2þ) and 0.5% BSA. Alternatively, the

primary antibody was detected by horseradish peroxydase (HRP)-conju-

gated goat anti-mouse secondary antibody (Amersham Biosciences)

using Amplex�Red (10-acetyl-3,7-dihydroxyphenoxazine, Molecular

Probes, Eugene, OR, USA) as substrate. The fluorescence was measured

by reading with POLARstar OPTIMA (BMG Labtech Inc., Offenburg,

Germany) plate-reader using filters 544 nm for excitation and 590 nm for

emission. Specific binding was calculated by subtracting the signal in the

presence of 10 mg/mL non-immune IgG (Santa Cruz Biotechnology Inc.)

and the respective secondary antibody. Non-specific binding was usually

5–10% of the specific signal.

Following the cell surface binding of anti-CD4 antibody (4 �C for 1 h),

internalization rates of CD4 and CD4 chimeras were calculated from the

loss of cell surface bound anti-CD4 antibody during 5 min internalization at

37 �C and expressed as percentage of radioactivity or fluorescence relative

to that detected initially. Comparable results were obtained with the radio-

active and the fluorescence technique (data not shown). To obtain the

initial uptake rates, we reduced the internalization time to 3 min for

CD4Tl-2Ub and CD4Tl-3Ub at 37 �C (Figure 2C). The internalization rates

of CD4 are not influenced by the valency of the antibody (61) and by the

variations of cell surface expression level of the chimera (see Result).

While the internalization deficient chimeras displayed a fourfold to fivefold

higher cell surface expression level than CD4Tl-Ub, insertion of the tetra-

merization domain decreased the relative cell surface density of the

CD4TCC-Ub and CD4TCC-UbAllR by 40% compared with CD4Tl-Ub.

Chimeras displaying the lowest cell surface expression (CD4Tl-2Ub and

CD4Tl-3Ub) yielded specific signal that was still twofold to threefold higher

than the background signal (data not shown). Antibody uptake assay was

used to determine the internalization of the Ii chimeras and the Flag-b2-ARwith anti-Ii and M2 anti-Flag antibodies, respectively. Tf uptake was mon-

itored after binding of 3 mg/mL Tf-biotin (Molecular Probes, T23363) to

serum-deprived cells in PBS/CM and 0.5% ovalbumin (0 �C, 1 h). The

amount of cell surface associated Tf-biotin before and after internalization

was determined by Amplex�Red, using 0.25 mg/mL streptavidin-HRP (1 h,

0 �C). Endocytosis of CTB was monitored at subsaturating concentration

(10 nM) of HRP-conjugated CTB (C-4672, Sigma) (data not shown).

Endocytosis rates were calculated as described for the CD4 chimeras.

Each internalization experiment was performed in at least triplicates.

CD4 cross-linking at the plasma membraneNon-covalent cross-linking of CD4Tl-UbAllR and CD4Tl was performed by

two protocols in COS-7 cells. Protocol 1 entails sequential binding of

primary anti-CD4 (OKT4, 0 �C, 1 h) and goat anti-mouse Ab (115-005-164,

Jackson ImmunoResearch Laboratory Inc., West Grove, PA, USA 0 �C,1 h). Internalization of CD4-antibody complex was detected by HRP-con-

jugated rabbit anti-goat tertiary antibody (0 �C, 1 h) (305-035-003, Jackson

ImmunoResearch Laboratory Inc.). In Protocol 2, cells were sequentially

incubated with anti-CD4 primary, biotinylated secondary antibody and

finally with streptavidin-HRP (0 �C, 1 h for each incubation). The cell sur-

face density of complexes was measured before and after 5 min of inter-

nalization at 37 �C by Amplex�Red.

Inhibition of clathrin- and caveolae-dependent

endocytosisThree different approaches were employed to inhibit CCV formation in

COS-7 cells as described previously (62). (i) Depletion of the cytosolic Kþ

content was achieved by hypotonic swelling of the cells in 50% diluted

medium, containing 1 mM ouabain (5 min, 37 �C) and then in Kþ-freemedium (100 mM NaCl, 50 mM Hepes, 1 mM MgCl2, 0.1 mM CaCl2,

10 mM glucose, pH 7.3) for 20 min at 37 �C. Internalization was measured

in Kþ-free medium. (ii) Cells were exposed to hypertonic DMEM supple-

mented with 0.3 M sucrose for 30 min at 4 �C, and internalization was

measured in the same medium. (iii) Acidification of the cytosol was accom-

plished with the NH4Cl prepulse method. Cells were incubated in DMEM

supplemented with 40 mM NH4Cl (5 min, 37 �C), transferred to Naþ-freemedium (140 mM N-methyl-D-glucamine-Cl, 10 mM Hepes, 1 mM MgCl2,

0.1 mM CaCl2, 10 mM glucose, pH 7.3). CD4Tl-Ub uptake was monitored in

Naþ-free medium. Cholesterol sequestration was achieved by incubating

HEK293 cells with 25 mg/mL nystatin or 0.5 mg/mL filipin for 1 h at 37 �C(21). Cholesterol synthesis was inhibited with 1 mM mevinolin for 24 h.

SiRNAsiRNA duplexes (Dharmacon Inc. Lafayette, CO, USA) were resuspended in 1X

siRNA Universal Buffer (Dharmacon) at 20 mM concentration. HEK293 cells

were transfected with oligofectamine (Invitrogen, Inc. Burlington, Canada) at

final concentration of 80 nM siRNA according to the manufacture’s recommen-

dation. Depletion of target proteins was optimized by repeated siRNA transfec-

tions with previously validated siRNA as indicated. CHC

(5’CCTGCGGTCTGGAGTCAAC) (63), AP-2a (5’-GAGGTGTGGTACCGAGTCA)

(63), caveolin1 (5’-TGTCTGGGGGCAAATACG) (64) and GGA3 (5’-

AAACGGCTTCCGCATCCTC) (65) knockdown was achieved by transfecting

the respective siRNA twice at 24 h intervals, and experiments were done

after 48 h of the second transfection. Eps15 (5’’-AAACGGAGCTACAGATTAT)

(66), eps15R (5’-GCACTTGGATCGAGATGAG) (66) and epsin1 (5’-

GGAAGACGCCGGAGTCATT) (66) siRNAs were transfected three times with

24 h intervals between each transfection, and experiments were performed

after 48 h of the last transfection. When two siRNAs were used together, the

concentration of individual siRNA was 40 nM. Controls included mock transfec-

tion in the absence of siRNA as well as using 80 nM non-specific (ns) siRNA.

MicroscopyImmunofluorescence localization of CD4 variants was performed using a

mouse anti-CD4 (RPA-T4) antibody (BD Biosciences) and FITC-conjugated

donkey anti-mouse antibodies. Co-localization of CD4Tl-Ub with GM1-

enrichedmembranemicrodomainswas achieved after patching CD4 variants

with OKT4 anti-CD4 antibody (1 h, 12 �C) and then with anti-mouse TRITC-

conjugated antibody (1 h, 12 �C). CTB-FITC (Sigma) was included with the

secondary antibody to visualize rafts. CD4 chimeras and TfRwere co-patched

using polyclonal sheep anti-CD4 and mouse monoclonal anti-TfR (OKT9)

antibody (1 h, 12 �C), followed by a second incubation with FITC-conjugated

anti-sheep and TRITC-conjugated anti-mouse secondary antibodies (1 h,

12 �C) before fixation. Membrane proteins excluded from rafts were

removed with 1% TritonX-100 containing PBS (20 min, 4 �C) prior to fixation.

Images were acquired with a Zeiss LSM510 laser confocal microscope

equipped with a Plan-Achromat 63X/NA 1.4 (Carl Zeiss Microimaging, Inc.,

Jena, Germany).

Statistical analysisExperiments were repeated at least three times. Data, means � SEM.

Significance was assessed by calculating two-tailed p-values at 95% con-

fidence level with unpaired t-test, using the PRISM software (GraphPad

Software, Inc. San Diego, CA, USA).

Acknowledgments

We are indebted to R. Haguenauer-Tsapis, L. Hicke, U. Schubert, S.

Schmid, B. Schwappach, Y. Yarden, H. Stenmark and M. von Zastrow for

generously providing valuable reagents for our studies. H.B was supported

Oligoubiquitin Recognition as Endocytic Signal

Traffic 2006; 7: 282–297 295

in part by Fellowships from the RESTRACOM, Hospital for Sick Children

and The Canadian Cystic Fibrosis Foundation (CCFF). This work was sup-

ported by grants to G.L. from the Canadian Institutes of Health Research

(CIHR), the Premier Research Excellence Award of the Ontario Ministry of

Energy and Education and CCFF. The instrumentation was supported in

part by a Block Term Grant from the Ontario Thoracic Society.

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