Control of the Intracellular Pathway of CD1e

# 2008 The Authors

Journal compilation # 2008 Blackwell Publishing Ltd

doi: 10.1111/j.1600-0854.2008.00707.xTraffic 2008; 9: 431–445Blackwell Munksgaard

Control of the Intracellular Pathway of CD1e

Blandine Maıtre1,2, Catherine Angenieux1,2,

Jean Salamero3,4, Daniel Hanau1,2,

Dominique Fricker1,2, Francxois Signorino1,2,

Fabienne Proamer1,2, Jean-Pierre Cazenave2,5,

Bruno Goud4, Sylvie Tourne1,2 and

Henri de la Salle1,2,*

1INSERM, U725, Etablissement Francxais duSang-Alsace, Strasbourg 67065, France2Universite Louis-Pasteur, Strasbourg 67000, France3Cell and Tissue Imaging-RIO, CNRS, UMR144,Institut Curie, Paris 75005, France4Molecular Mechanisms of Intracellular Transport,CNRS, UMR144, Institut Curie, Paris 75005, France5INSERM, U311, Etablissement Francxais duSang-Alsace, Strasbourg, 67065 France*Corresponding author: Henri de la Salle,[email protected]

CD1e is a membrane-associated protein predominantly

detected in the Golgi compartments of immature human

dendritic cells. Without transiting through the plasma

membrane, it is targeted to lysosomes (Ls) where it

remains as a cleaved and soluble form and participates

in the processing of glycolipidic antigens. The role of

the cytoplasmic tail of CD1e in the control of its intracel-

lular pathway was studied. Experiments with chimeric

molecules demonstrated that the cytoplasmic domain

determines a cellular pathway that conditions the endo-

somal cleavage of these molecules. Other experiments

showed that the C-terminal half of the cytoplasmic tail

mediates the accumulation of CD1e in Golgi compart-

ments. The cytoplasmic domain of CD1e undergoes

monoubiquitinations, and its ubiquitination profile is

maintained when its N- or C-terminal half is deleted.

Replacement of the eight cytoplasmic lysines by argi-

nines results in a marked accumulation of CD1e in trans

Golgi network 461 compartments, its expression on the

plasma membrane and a moderate slowing of its trans-

port to Ls. Fusion of this mutated form with ubiquitin

abolishes the accumulation of CD1e molecules in the

Golgi compartments and restores the kinetics of their

transport to Ls. Thus, ubiquitination of CD1e appears to

trigger its exit fromGolgi compartments and its transport

to endosomes. This ubiquitin-dependent pathway may

explain several features of the very particular intracellular

traffic of CD1e in dendritic cells compared with other CD1

molecules.

Key words: CD1, endosome, Golgi, ubiquitin

Received 14 June 2007, revised and accepted for publica-

tion 11 January 2008, uncorrected manuscript published

online 15 January 2008, published online 13 February

2008

Among the five human CD1 genes, four of them, CD1A, B,

C and D, encode cell surface-expressed glycoproteins that

have the unique capacity to bind lipidic antigens and present

them to T cells (1,2). The different antigen-presenting prop-

erties of thesemolecules can be explained by the structures

of their antigen-binding sites and by their cellular pathways.

CD1a, b, c and d are first expressed on the plasma mem-

brane of monocyte-derived dendritic cells (DCs) and then

internalized into the endocytic system, each of them follow-

ing its own cellular pathway. In immature Langerhans cells,

CD1a reaches sorting early endosomes (EEs) and then

recycling EEs where it is retained before returning to the

cell surface (3). In immature DCs (iDCs), CD1b is targeted to

the peripheral membranes of HLA-DRþ multilaminar lyso-

somes (Ls) (4) where it stays transiently and then returns to

the plasma membrane. CD1c is found mainly on the cell

surface, while the remaining fraction is localized in EEs and

late endosomal compartments [late endosomes (LEs)/Ls]

(5,6), as is human CD1d (7). Thematuration of DCs results in

a rapid relocalization of HLA class II molecules to the plasma

membrane and loss of the internal membranes of the Ls,

also called ‘mature DC lysosomes’ (MDLs). In contrast, the

cellular distribution of CD1a–c molecules is not modified by

the maturation of monocyte-derived DCs (8). In particular,

CD1b and c are still observed in the MDLs (4). The inter-

nalization and/or targeting of human CD1b and c and mouse

CD1d to endosomes depends, at least in part, on the

presence in their cytoplasmic tails of tyrosine motifs, which

bind the adaptor protein (AP)-2 or AP-3 molecules (7,9–11).

AP-2 is involved in internalization from the plasmamembrane

into EEs, while AP-3 controls transport from EEs to LEs.

CD1e displays very particular properties in terms of its

cellular distribution (12,13). In the steady state, it is mainly

located in the Golgi apparatus and trans Golgi network

(TGN) of iDCs, while a minor part can be found in EEs, LEs

and HLA-DRþ or CD1bþ Ls. The induction of DCmaturation

leads to the rapid mobilization of TGN-localized CD1e

molecules to the endosomal network, with the result that

only lysosomal CD1e can be detected a few hours later.

Under no circumstances does one observe any transit of

CD1e through the plasma membrane. In endosomes, the

luminal part of CD1e is cleaved at the junction to the

transmembrane domain (12) and hence endosomal CD1e

is in a soluble form. The soluble lysosomal CD1e molecules

are biologically active and have been shown to facilitate the

processing of a mycobacterial glycolipid by lysosomal

a-mannosidase. The involvement of CD1e allows the gen-

eration of an antigen, which is loaded onto CD1b molecules

transiting in Ls before they return to the plasma membrane

to present it to antigen-specific T cells (14). The particular

and physiologically important cellular behavior of CD1e thus

appears to optimize its biological function in defense against

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microbial infections and raises the question of how its

cellular pathway is controlled. The cytoplasmic domain of

CD1e contains 53 or 61 amino acids depending on an

alternative splicing, which is much longer than the corres-

ponding domains of other human CD1 molecules (6–12

amino acids). It does not contain typical Golgi- or lysosomal-

targeting motifs and, unlike in CD1b, c and d molecules, no

tyrosine-based endosomal-addressing motif is present

(7,15). Although di-leucine-like pairs are present in CD1e,

they are not associatedwith the acidic amino acids generally

found in such motifs, especially the Golgi-localized g ear-

containing ARF binding adaptor protein (GGA)-dependent

motifs that might be expected to be used for direct transport

from the TGN to EEs (13,16). The aim of this study was

therefore to clarify the mechanisms controlling the distribu-

tion of CD1e using a model of transfected M10 melanoma

cells, which has been previously validated (12,13).

Results

The generation of soluble lysosomal CD1e is

controlled by the cytoplasmic tail

CD1e and CD1b colocalize in the same late endosomal

compartments (13,14), but only CD1e is cleaved (12,17).

This suggests that either CD1b does not bear an endosomal-

cleavage site, or CD1b andmembrane-associated CD1e do

not localize on, or transit through, the same membrane

structures. To discriminate between these alternatives,

we expressed two CD1e–CD1b chimeras in M10 cells

(Figure 1A). The first, CD1ebb, contained the a1 and

a2 domains and a few amino acids of the a3 domain of

CD1e, the remaining amino acids being derived from

CD1b. The second, CD1ebe, was derived from CD1ebb

by replacing the cytoplasmic tail of CD1b by that of CD1e.

Like CD1b, CD1ebb was expressed on the plasma mem-

brane (Figure 2A). We confirmed that as previously re-

ported (14), this chimera was present in CD63þ LEs/Ls

(Figure 2B), as is CD1b (9). CD1ebe, like CD1e, was not

detected on the plasma membrane of transfected cells but

was found in CD63þ vesicles (Figures 2A,B). Interestingly,

a biochemical analysis revealed that CD1ebb was ineffi-

ciently cleaved (14) (Figure 2C), even after 8 h of chase

(data not shown). On the other hand, CD1ebe underwent

a biochemical maturation similar to that of CD1e, being

cleaved in bafilomycin-sensitive compartments. In addi-

tion, after 4 h of chase in the presence of bafilomycin,

CD1ebe coimmunoprecipitated with a 27-kD protein called

p27, as previously observed for CD1e (12,13).

We also expressed in M10 cells another chimera in which

only the cytoplasmic tail of CD1e was replaced by that of

CD1b (Figure 1A). This chimera (CD1eeb) displayed the

same plasma membrane and intracellular distribution as

CD1b (data not shown). Cleavage of CD1eeb still occurred

but was also strongly retarded (Figure 2D), suggesting

that structural differences between CD1e and CD1b, in the

a3 and/or transmembrane domains, result in sensitivity

to endosomal cleavage of CD1e but not CD1b.

Altogether, our results demonstrate that the lack of

cleavage of CD1b in Ls is not because of the absence of

a cleavage site in its a3 domain. They strongly suggest that

CD1e and CD1b molecules are targeted to Ls through two

different cellular pathways, transiting through different

membrane environments. In addition, the data show that

the cellular pathways of CD1e and CD1b are controlled by

their respective cytoplasmic tails.

The cytoplasmic tail mediates the TGN localization

and intracellular retention of CD1e molecules

To define the targeting functions of the cytoplasmic

domain of CD1e, we generated three deletion mutants

Figure 1: Structures of CD1e–CD1b chimeras and of the mutated forms of CD1e. A) Schematic drawing of the structures of CD1e,

CD1b and the CD1e–CD1b chimeras. The cDNA blocks encoding the different a domains of CD1e and CD1b are aligned, and the positions

of restriction sites used to derive the different chimeric forms are shown (see Materials and Methods). Cyt, cytoplasmic domain;

TM, transmembrane segment. B) Amino acid sequences of the native and mutated cytoplasmic domains of CD1e.

432 Traffic 2008; 9: 431–445

Maıtre et al.

(Figure 1B). The N- or C-terminal half of the cytoplasmic

domain was deleted (CD1eDN and CD1eDC), or the

complete cytoplasmic tail was replaced by an artificial

11-amino acid sequence (CD1eDCyt).

Stably transfected M10 cells expressing these deletion

mutants were isolated and compared with M10 cells

expressing complete CD1e. The cell surface and total ex-

pression of CD1e molecules in these cells were quantified

Figure 2: Role of the cytoplasmic

domain of CD1e in the generation

of its soluble form. The CD1ebb and

CD1ebe chimeras were studied in

stably transfected M10 cells. A) The

expression of CD1e chimeras on

the plasma membrane and its total

expression were quantified by flow

cytometry after staining, respect-

ively, intact cells and fixed, permea-

bilized cells with the mAb 20.6 or

control IgG, followed by counter-

staining with PE-conjugated goat

anti-mouse IgG (unfilled and filled

histograms, respectively). B) Accu-

mulation of the chimeras in LEs/Ls

was confirmed by confocal IF micro-

scopy of fixed, permeabilized cells

stained with anti-CD1e and anti-CD63

mAbs. Scale bars: 10 mm. The bio-

chemical maturation of CD1ebb and

CD1ebe (C)and CD1eeb (D) was ana-

lyzed by pulse–chase labeling in the

presence or absence of bafilomycin

(Baf), followed by immunoadsorption

on the mAb 20.6. Immunopurified

proteins were treated with PNGase

F (F) or not (NT), separated by SDS–

PAGE and revealed by autoradiogra-

phy. Membrane-associated, soluble

molecules and p27 are indicated by

m, s and p, respectively.

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Ubiquitination of CD1e

by flow cytometry, and the ratio of the specific mean

fluorescence intensity (MFI) of cell surface CD1e to the

specific MFI of total CD1e was calculated in order to obtain

a semiquantitative measure of the intracellular retention of

the different forms of CD1e (see Materials and Methods)

(Figure 3). Complete CD1e and the deletion mutant

CD1eDN were barely detected on the plasma membrane

(less than 1 and 4% of the total CD1e, respectively). When

the C-terminal half of the cytoplasmic tail was deleted

(CD1eDC), 40% of the total CD1e was present on the cell

membrane. Finally, replacement of the whole cytoplasmic

tail by an artificial sequence (CD1eDCyt) allowed more

than 60% of CD1e molecules to be expressed on the

plasma membrane in the steady state.

Because wild-type CD1e accumulates in the TGN in trans-

fected cells, we next investigated the colocalization of the

different forms of CD1e with TGN46 (Figure 4). Deletion of

the N-terminal half of the cytoplasmic tail (CD1eDN) resultedin a pronounced accumulation of CD1e molecules in the

TGN. In contrast, when the C-terminal half of the cytoplas-

mic domain was deleted (CD1eDC) or the cytoplasmic tail

was replaced by an artificial sequence (CD1eDCyt), a carefulanalysis of the micrographs showed that CD1e and TGN46

labeling were in general juxtaposed but not superimposed.

These CD1e molecules thus appeared to be only marginally

present in, if not absent from, the TGN.

This analysis revealed a major contribution of the

C-terminal half of the cytoplasmic domain to the intracel-

lular retention of CD1e and its accumulation in the TGN.

The cytoplasmic tail facilitates the generation of

soluble CD1e in late endosomal compartments

Despite differences in the accumulation of the mutated

forms of CD1e in the TGN, confocal microscopy revealed

that all were present in CD63þ late endosomal compart-

ments (Figure 5A). As it is in these compartments that the

soluble form of CD1e is generated, we looked at the

effects of the deletions on the biochemical maturation

of CD1e molecules. Pulse–chase labeling of transfected

cells with [35S] methionine and cysteine was performed

in the presence or absence of bafilomycin, which inhibits

the endosomal adenosine triphosphatase responsible for

acidification of LEs and consequently cleavage of CD1e in

LEs/Ls (12). CD1e molecules were immunoprecipitated

with the monoclonal antibody (mAb) 20.6, and their

glycosylation was assayed by digestion with endoglyco-

sidase H (endo H) or peptide N-glycosidase F. To compare

the kinetics of cleavage of the different CD1e molecules,

the lanes of the autoradiograms corresponding to mol-

ecules immunoprecipitated by mAb 20.6 after 4 h of chase

and treated with endoglycosidase F (endo F) were scanned

and the relative intensities of the bands corresponding to

the different soluble forms were measured (see Materials

and Methods).

All the natural and mutated forms of CD1e behaved

qualitatively in accordance with the immunofluorescence

(IF) analysis. In other words, all forms were cleaved

(Figure 5B), bafilomycin inhibited the cleavage of all CD1e

mutants and a 27-kD protein coimmunoprecipitated with

CD1e molecules in bafilomycin-treated cells (Figure 5B).

However, significant differences were observed in the

kinetics of their cleavage (Figure 5C). After 4 h of chase,

80% of neosynthesized wild-type CD1e molecules were

cleaved compared with only approximately 50% of the

deletion mutants CD1eDN and CD1eDC and 15% of

CD1eDCyt molecules.

Altogether, these results emphasized the role of the

cytoplasmic tail of CD1e in the generation of soluble

CD1e molecules.

Figure 3: The cytoplasmic tail mediates the intracellular retention of CD1e molecules. Transfected M10 cells stably expressing the

different molecules were isolated. The expression of CD1e on the plasma membrane and its total expression were quantified by flow

cytometry after staining respectively intact cells and fixed, permeabilized cells with the mAb 20.6 (unfilled histograms). Cells were stained

using a control isotype-matched mAb (filled histograms). The proportion of plasma membrane-expressed CD1e with respect to the total

CD1e was calculated as described in the Materials and Methods.

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The cytoplasmic domain of CD1e is ubiquitinated

The differences in the processing of CD1ebb and CD1ebe

strongly suggest that CD1e gains access to the internal

membranes of multivesicular endosomes (MVEs). In fact,

immunoelectron microscopy showed that CD1e can be

detected on the internal vesiclesof LEs (Figure 6A) andwithin

multilaminar Ls in transfected M10 cells, similarly as in DCs

(13). Because the incorporation of proteins into the internal

vesicles of LEs can be mediated by an ubiquitin-dependent

pathway (18), we examined whether CD1e is ubiquitinated.

Figure 4: TGN colocalization of

deletion mutants of CD1e. Stably

transfected cells were fixed, per-

meabilized and stained with poly-

clonal goat anti-TGN46 Abs, revealed

with Cy5-conjugated donkey anti-goat

IgG and the anti-CD1e mAb 20.6,

revealed with Cy3-conjugated donkey

anti-mouse IgG. The cells were ana-

lyzed by confocal microscopy. Scale

bars: 10 mm.

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Figure 5: Transport of mutated forms of CD1e to CD631 Ls. A) Stably transfected cells were fixed, permeabilized and stained with the

anti-CD1e mAb 20.6, which was revealed with Cy3-conjugated donkey anti-mouse IgG. After blocking with non-immune mouse serum,

cells were incubated with biotinylated H5C6, revealed by A488-conjugated streptavidin. Cells were analyzed by confocal microscopy. Scale

bars: 10 mm. B) Stably transfected cell lines were pulse labeled and chased for 2 or 4 h in the absence of bafilomycin or for 4 h in the

presence of bafilomycin (þBAF). CD1e molecules were immunoprecipitated with the mAb 20.6, treated with endo H (H) or PNGase (F) or

not (NT), separated by SDS–PAGE and analyzed by autofluorography. The molecular species are membrane-associated CD1e (m), soluble

CD1e (s) and p27 (p). C) The percentage of cleaved forms with respect to the total CD1e after 4 h of chase in the absence of bafilomycin

was calculated by densitometry scanning of the autofluorograms from two independent experiments as described in the Materials and

Methods. Figure 5 continued on next page.

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Transfected M10 cells expressing CD1e were treated or

not with bafilomycin for 5 h. Bafilomycin inhibits the

maturation of EEs into MVEs (19) or MVEs into LEs

(20,21). After treatment, the cells were lysed, and CD1e

molecules were immunoprecipitated with the mAb 20.6.

Proteins were separated by SDS–PAGE, and ubiquitinated

proteins were identified by immunochemistry (Figure 6B).

The Western blots revealed ubiquitinated CD1e in un-

treated cells and in greater amounts after treatment with

bafilomycin. In some experiments, a non-specific protein

was revealed under all conditions, including in the control

with an irrelevant immunoglobulin G (IgG) (Figure 6B).

Therefore, essentially two CD1e species could be speci-

fically identified, and these had electrophoretic mobilities

compatible with the coupling of one or two ubiquitins.

The mutant forms CD1eDC and CD1eDN, which both

include four cytoplasmic lysines, were also ubiquitinated

(Figure 6C). The electrophoretic mobilities of the ubiquiti-

nated deletion mutants were, as expected, faster than

that of ubiquitinated complete CD1e. Moreover, the dif-

ferences in electrophoretic mobility confirmed that these

ubiquitinated species were actually CD1e molecules and

not unrelated ubiquitinated proteins coimmunoprecipitat-

ing with CD1e. Bafilomycin treatment again increased the

number of immunoprecipitated ubiquitinated molecules.

CD1eDCyt, which has no lysines in its cytoplasmic domain,

was not ubiquitinated, and control Western blots using an

anti-beta 2 microglobulin (b2m) antibody confirmed that

this mutant was equally efficiently immunoprecipitated

(data not shown).

Ubiquitination may represent a signal for the exit

of CD1e molecules from the TGN and their transport

to endosomal compartments

To further investigate the role of ubiquitination in the

transport of CD1e, we generated a cell line expressing

a mutant in which all eight cytoplasmic lysines had been

replaced by arginines (CD1e/8K-R) and another one where

this mutated form was fused with ubiquitin, generating

a constitutively monoubiquitinated molecule (CD1e/8K-

R-Ubi) (Figure 1A). CD1e/8K-R and CD1e/8K-R-Ubi mol-

ecules were expressed on the plasmamembrane in similar

proportions (25 and 30% of total CD1e, respectively)

Figure 5: Continued from previous page.

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(Figure 7A). Remarkably, CD1e/8K-R strongly colocalized

with TGN46, while CD1e/8K-R-Ubi was poorly detected

in TGN46þ compartments in most of the cells (Figure 7B).

In less than 20% of the cells, a partial colocalization with

TGN46 was observed, but it was not as marked as for

CD1e/8K-R (data not shown). Both forms were detected

in CD63þ compartments (Figure 7C).

The distributions of the CD1e/8K-R and CD1e/8K-R-Ubi

mutants in CD63þ LEs/Ls were compared with that of

native CD1e by analysis of wide-field deconvolved micro-

graphs (Figure S1) followed by quantification (Figure 8A).

The percentage of CD1eþ endosomes among CD63þ

endosomes, given by the ratio of the number of double

positive to CD63þ structures, was higher for CD1e (34%)

than that for CD1e/8K-R-Ubi (26%) and lower for CD1e/8K-

R (20%). The higher proportion of CD1eþ compartments

among CD63þ structures for wild-type CD1e suggests that

the physiological ubiquitination of the cytoplasmic tail of

CD1e directs the latter more efficiently to late endosomal

compartments. On the other hand, the percentage of

CD63þ compartments among CD1eþ structures was

similar for CD1e and CD1e/8K-R-Ubi (48%) and relatively

low for CD1e/8K-R (less than 14%), reflecting the apparent

strong localization of CD1e/8K-R in Golgi compartments.

This colocalization was deduced from the overlap of

structures labeled with the mAb 20.6 or anti-TGN46 Abs,

which was quite pronounced for CD1e/8K-R compared

with CD1e and CD1e/8K-R-Ubi (Figures 4 and 7B).

However, especially for CD1e and CD1e/8K-R-Ubi, a high

density of single-positive structures was superimposed in

the TGN46þ area. These structures displayed different

shapes and thus were not identical, so their superimposi-

tion does not mean true colocalization. Consequently, all

attempts to quantify the colocalization of CD1eþ and

TGN46þ compartments led to widely divergent results

between individual cells (high standard deviation), making

these estimations unreliable.

Figure 6: CD1e in transfected cells

transits through multivesicular

compartments and is ubiquitinated.

A) Cellular distribution of CD1e in M10

cells. Cryosections of transfected cells

expressing CD1e were stained with

the mAb 20.6 and analyzed by electron

microscopy. CD1e was detected in

Golgi compartments (Go), EE and MVE

and Ls (L). B and C) Ubiquitination of

CD1e in transfected cells. B) Trans-

fected M10 cells expressing CD1e

were left untreated (�) or incubated

with bafilomycin (BAF) for 5 h. CD1e

molecules were immunoprecipitated

with the mAb 20.6 and analyzed

by Western blotting using an HRP-

conjugated anti-ubiquitin mAb with

an irrelevant mAb (Ig) as a negative

control. Arrows indicate ubiquitinated

forms having a molecular mass corres-

ponding to one (1) or two (2) mono-

ubiquitinations. C) Untransfected cells

(M10) and cells expressing different

CD1e deletion mutants were treated

(þ) or not (�) with bafilomycin (BAF),

after which CD1e molecules were

analyzed as described in (B).

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Maıtre et al.

Figure 7: Role of the cytoplasmic lysines

and their ubiquitination in the intracellular

distribution and biochemical maturation of

CD1e. A) Relative plasma membrane expres-

sion of CD1e/8K-R and CD1e/8K-R-Ubi mol-

ecules. Viable and fixed, permeabilized stably

transfected cells were labeled with the mAb

20.6 and analyzed by flow cytometry. Colocali-

zation of CD1e/8K-R and CD1e/8K-R-Ubi with

TGN46 (B) or CD63 (C). Fixed and permeabilized

cells were stained as described in Figures 4 and

5A and analyzed by confocal microscopy.

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The cellular distributions of CD1e, CD1e/8K-R and

CD1e/8K-R-Ubi were also compared by immunolabeling

of cryosections of transfected cells. CD1e molecules were

detected by staining with the mAb 20.6, followed by

counterstaining with protein A-conjugated gold particles

and electron microscopy. The numbers of gold particles in

the endoplasmic reticulum, Golgi compartments, small

vesicles, tubules, EEs, MVEs and multilaminar (ML)/MVEs

and on the plasma membrane were counted (Figure S2

and Figure 8B). In the case of CD1e/8K-R, greater numbers

of particles were found in the Golgi and TGN compart-

ments, in agreement with the confocal microscopy obser-

vations. Larger numbers of small vesicles were stained in

cells expressing CD1e/8K-R or CD1e/8K-R-Ubi. Because

these two forms are well expressed on the plasma

membrane (Figure 7A), the small vesicles might represent

secretory structures. We also looked at the distribution of

gold particles within the hybrid ML/MVEs between the

outer limiting membranes, the internal multilaminar struc-

tures and the internal vesicles but did not find any

significant differences (data not shown).

Biochemical experiments revealed that soluble lysosomal

CD1emoleculeswere generated at a slower rate in the case

of CD1e/8K-R but at a normal one in the case of CD1e/8K-R-

Ubi (Figure 9A). Approximately 40% of CD1e/8K-R molecu-

les were cleaved after 4 h of chase compared with 80%

for wild-type CD1e or CD1e/8K-R-Ubi chimera. The ubiqui-

tination profiles of these two forms were analyzed by

Western blotting (Figure 9B). As expected, CD1e/8K-R

was not ubiquitinated, while some CD1e/8K-R-Ubi mol-

ecules remained monoubiquitinated and the others be-

coming polyubiquitinated but not diubiquitinated. Larger

amounts of monoubiquitinated and polyubiquitinated

CD1e/8K-R-Ubi were found in cells treatedwith bafilomycin.

In summary, an absence of lysines from the cytoplasmic

tail resulted in the accumulation of CD1e molecules in the

TGN at a level as strong as, if not stronger than, for wild-

type CD1e. This accumulation was abolished by fusion

with ubiquitin. While deletion and substitution mutants

were more slowly converted into soluble lysosomal CD1e,

constitutively ubiquitinated and natural CD1e molecules

were cleaved at the same rate. Electron microscopy of

immunolabeled cryosections confirmed the presence of

the different forms of CD1e in the endocytic network.

However, an analysis of IF micrographs revealed quantita-

tive differences in the colocalization of CD1e/8K-R and

CD1e/8K-R-Ubi with the lysosomal protein CD63, reflect-

ing differences in the kinetics of the generation of soluble

lysosomal CD1e molecules. Overall, these data strongly

suggest that ubiquitination of CD1e facilitates its exit from

the TGN, its transport to Ls and/or the generation of

soluble CD1e molecules.

CD1e is ubiquitinated in DCs

Finally, we looked at the ubiquitination of CD1e in DCs.

In iDCs, CD1e is mainly detected in the TGN, but incuba-

tion of these cells for 5 h with lipopolysaccharide (LPS)

results in its relocalization to CD63þ Ls (13). CD1e mol-

ecules accumulate in endosomal compartments when

iDCs are treated with bafilomycin (13), and we checked

that bafilomycin did not affect the relocalization of CD1e to

CD63þ compartments induced by LPS (Figure S3).

Immature DCs were incubated for 5 h in the presence or

absence of bafilomycin and/or LPS, and CD1e molecules

were immunoprecipitated and analyzed by Western blot-

ting (Figure 10). The profile of ubiquitinated CD1e in iDCs

was identical to that in transfected M10 cells. Two major

species having a molecular mass corresponding to mono-

ubiquitinated or diubiquitinated CD1e were detected.

Figure 8: Quantification of the cellular localization of CD1e

and mutant forms. A) Localization of CD1e, CD1e/8K-R and

CD1e/8K-R-Ubi (CD1e-Ubi) in LEs/Ls. Fixed and permeabilized

cells were stained with the mAbs 20.6 (anti-CD1e) and H5C6 (anti-

CD63) and analyzed by IF microscopy. Wide-field deconvolved

images (Figure S1) of at least 14 cells from the same samples

were analyzed (see Materials and Methods). In each cell, the

colocalization was quantified by determining the ratio of CD1e–

CD63 double-positive (DP) structures to CD1e or CD63 single-

positive structures (CD1e and CD63, respectively). The standard

error deviation is shown. B) Quantification of gold particles in

immunolabeled cryosections. Gold particles were counted on

15 different cryosections of complete cells taking into account

their location in the different cellular compartments. A total of 852,

866 and 1118 particles were counted for CD1e, CD1e/8K-R and

CD1e/8K-R-Ubi, respectively. S, surface; Tub, tubular structures;

Ves, small vesicles.

440 Traffic 2008; 9: 431–445

Maıtre et al.

When iDCs were treated with LPS alone, no ubiquitinated

CD1e molecules were detected, consistent with their

relocalization to lysosomal compartments and conversion

into a soluble form (13). Conversely, ubiquitinated CD1e

accumulated in iDCs treated with LPS and bafilomycin.

Thus, in LPS-treated iDCs as in M10 cells, ubiquitination of

CD1e molecules appeared to accompany their exit from

the TGN.

Discussion

To investigate the control of the cellular transport of CD1e

and the generation of soluble lysosomal CD1e molecules,

CD1e/CD1b chimeras and deletion and substitution mu-

tants of the cytoplasmic tail of CD1e were expressed in

the M10 melanoma cell line. These experiments provided

strong evidence that the cytoplasmic tails of CD1e and

CD1b control transport of the molecules by different

cellular pathways to specialized late endosomal subdo-

mains where they can be cleaved into a soluble form. On

the other hand, slow but significant cleavage of CD1eeb

chimera (Figure 2D) indicates that the cleavage of CD1e is

only facilitated by its membrane environment. One should

note that CD1b is targeted to lysosomal compartments in

an AP-3-dependent manner (9). It is mainly detected on the

outer membranes of LEs/Ls and rarely on the internal

membranes of MVEs (8). In contrast, the cytoplasmic tail

of CD1e does not include an AP3-dependent signal.

Consistent with this property, the accumulation of CD1e

in CD63þ LEs/Ls and its kinetics of cleavage are identical in

normal and AP3-deficient transfected human fibroblasts

(22). Contrary to CD1b molecules, CD1e molecules are

detected on the internal membranes of MVEs.

The N-terminal and C-terminal halves of the cytoplasmic

tail were both found to facilitate the generation of

the soluble form of CD1e. Thus, deletion of the N- or

C-terminal half of the cytoplasmic domain moderately

slowed generation of the cleaved form, while replacement

of the whole cytoplasmic tail by an artificial sequence

(CD1eDCyt) drastically retarded the bafilomycin-sensitive

generation of soluble CD1e.

In view of the fact that CD1e molecules were immuno-

detected on the internal membranes of MVEs, our

Figure 9: Ubiquitination positively

regulates the generation of lyso-

somal-soluble CD1e. A) Biochemical

maturation of CD1e/8K-R and CD1e/

8K-R-Ubi. The maturation of CD1e mol-

ecules was analyzed as outlined in Fig-

ure 5B. B) Ubiquitination of CD1e/8K-R

and CD1e/8K-R-Ubi. Transfected cells

were treated or not for 5 h with bafilo-

mycin (BAF) and lysed. CD1e molecules

were immunoprecipitated with the mAb

20.6 and analyzed by Western blotting

using an HRP-conjugated anti-ubiquitin

or anti-b2m mAb revealed by chemi-

luminescence at the indicated times.

Overexposure of the anti-ubiquitin blots

did not reveal trace amounts of ubiqui-

tinated CD1e. F, PNGase; H, endo H.

Traffic 2008; 9: 431–445 441


findings pointed to an ubiquitin-facilitated transport

through these vesicles. The profile of CD1e ubiquitination

revealed two major species, corresponding to forms

bearing one or two ubiquitins, for not only complete

CD1e but also mutants deleted of either half of the

cytoplasmic domain. This would suggest that, in terms

of function, the ubiquitination profile (monoubiquitination

or diubiquitination) is more important than the ubiquitina-

tion of specific cytoplasmic lysines. Replacement of the

cytoplasmic lysines by arginines (CD1e/8K-R) resulted in

stronger colocalization with TGN46 and expression of

more than 20% of the molecules on the plasma mem-

brane in the steady state. The cleavage of CD1e/8K-R in

LEs/Ls was slower than that of CD1e but similar to that of

CD1eDN or CD1eDC. Hence ubiquitin-independent cellu-

lar mechanisms, which remain to be characterized, are

also involved in the transport of CD1e to Ls. Such

ubiquitin-independent lysosomal transport is not particu-

lar to CD1e and has been described for the EGF receptor

(23). In contrast, fusion of CD1e/8K-R with ubiquitin

(CD1e/8K-R-Ubi) led to nearly complete absence of the

fusion molecule from the TGN at the steady state and

restored normal kinetics of its cleavage in Ls. On the

other hand, CD1e/8K-R and CD1e/8K-R-Ubi were ex-

pressed similarly on the plasma membrane. Given the

known role of ubiquitin in the direct transport of proteins

from the TGN to endosomes (24), our observations

strongly suggest that CD1e molecules leave the TGN

after their ubiquitination. Alternatively, non-ubiquitinated

CD1e molecules may continuously recycle between the

TGN and endosomes. After ubiquitination, CD1e mole-

cules leave this recycling pathway and are incorporated

into MVEs, allowing the generation of soluble lysosomal

CD1e.

Because the deletion mutant CD1eDC was monoubiqui-

tinated and diubiquitinated, but well detected on the

plasma membrane while CD1e/8K-R-Ubi was also ex-

pressed on the cell surface, the absence of detectable

native CD1e on the plasma membrane indicates that non-

ubiquitinated lysines fulfill an indispensable function or that

the right ubiquitination profile of the cytoplasmic tail

conditions the efficient recruitment of adaptors mediating

direct transport to the endosomal network.

While our initial conducting idea was that the transport of

CD1e through MVEs is driven by ubiquitination of its

cytoplasmic tail, electron microscopy of immunolabeled

cryosections failed to reveal any significant differences in

the distribution of CD1e, CD1e/8K-R and CD1e/8K-R-Ubi

between the internal and the peripheral membranes of

MVEs and ML/MVEs. Thus, CD1e appears to behave

differently from major histocompatibility complex (MHC)

class II molecules whose targeting to the internal mem-

branes of MVEs would seem to be more dependent on the

ubiquitination of the cytoplasmic tail of the a chain (25,26).

Ubiquitination of CD1e was observed not only in trans-

fected cells but also in iDCs, which contained a pool of

ubiquitinated CD1e. Addition of bafilomycin during LPS

treatment, which does not prevent exit of CD1e molecules

from the TGN or their accumulation in endosomal compart-

ments (13), increased the quantity of diubiquitinated CD1e.

Some polyubiquitinated molecules were also observed, in

agreement with recent findings suggesting that polyubi-

quitination is necessary for endosomal targeting of the

EGF receptor (23). In the absence of bafilomycin, treat-

ment for 4 h with LPS results in almost complete relocal-

ization of CD1e molecules from the TGN to Ls (13). We

found that under these conditions, ubiquitinated CD1ewas

no longer detectable. It is thus tempting to speculate that

ubiquitination of CD1e molecules is increased during the

first hours of treatment with LPS, inducing their transport

from the TGN to the endosomal network or their escape

from the TGN to endosomes and further incorporation into

the MVE pathway. Similarly, ubiquitination of mature MHC

class II proteins in immature mouse DCs facilitates their

internalization into and retention in MVEs and conse-

quently regulates the cell surface expression of antigen-

presenting MHC class II molecules (25,26). Induction of

the maturation of these cells leads to the disappearance of

ubiquitinated MHC class II molecules fromMVEs and their

stabilization on the plasma membrane. CD1e therefore

represents a second system of molecules involved in

antigen presentation whose ubiquitin-dependent cellular

transport in DCs appears to be regulated by maturation.

Among the other human CD1 proteins, only CD1d con-

tains a cytoplasmic lysine. Ubiquitination of this lysine

has not been observed in normal cells but has been re-

ported after infection with Kaposi-associated herpesvirus.

Thus, the viral modulator of immune response 2 (MIR2)

was found to mediate the polyubiquitination of plasma

Figure 10: Ubiquitinated CD1e molecules are present in iDCs

and rapidly disappear upon induction of maturation by LPS.

Monocyte-derived DCs (lanes 1–4) were treated (þ) or not (�)

with LPS in the presence or absence of bafilomycin (BAF) and

processed as described in Figure 6B. Lane 0, negative control,

immunoprecipitate from untreated M10 cells. CD1e was im-

munoprecipitated with the mAb 20.6 and analyzed by Western

blotting using an HRP-conjugated anti-ubiquitin mAb. Arrows

indicate ubiquitinated forms having a molecular mass correspond-

ing to one (1) or two (2) monoubiquitinations.

442 Traffic 2008; 9: 431–445

Maıtre et al.

membrane-associated human CD1d, thereby increasing its

internalization but not its half-life (27). In cells infected with

Kaposi-associated herpesvirus, plasma membrane HLA

class I molecules were polyubiquitinated under control of

the K3 gene product and then internalized into Ls to be

degraded (28). Our results reveal another role of the

ubiquitination of HLA class I and class I-like molecules:

while ubiquitination of these molecules may be used by

viruses to escape from T-cell responses by inducing the

degradation of these molecules in Ls (28) or their retention

without degradation in LEs/Ls (27), ubiquitination of CD1e is

a physiological process that contributes to the cellular

mechanisms controlling the transport of this molecule from

the TGN to Ls and its release in a stable soluble form

necessary for the processing of glycolipidic antigens.

We previously reported that a 27-kD protein coimmuno-

precipitated with CD1e from extracts of DCs and

transfected cells treated with bafilomycin. This coimmuno-

precipitation was also observed for all the chimeric and

mutant forms analyzed except CD1ebb, which was the

only form found to be inefficiently cleaved. The identifica-

tion of this protein will be necessary in order to determine

whether it is truly associated with the transport and/or

endosomal cleavage of CD1e.

In summary, we have demonstrated that the traffic of

CD1e is controlled by signals present in the cytoplasmic

domain, which mediate the accumulation of CD1e mol-

ecules in the TGN, prevent their cell surface expression

and facilitate their targeting to LEs/Ls. While the lysines of

the cytoplasmic tail clearly play a role in the intracellular

distribution of CD1e, partly through ubiquitination, other

targeting mechanisms remain to be clarified. The traffic of

CD1e appears to be directed in a way that favors its

cleavage, whereas other CD1 molecules like CD1b are

excluded from this pathway but reach the same compart-

ments. The different pathways followed by CD1e and

CD1b to reach the same Ls make possible the function

of soluble CD1e, which is to facilitate the processing of

CD1b-restricted antigenic glycolipids, while membrane-

associated CD1e is not functional (14).

Materials and Methods

Cell lines and culture mediaThe M10 melanoma cell line (29) was grown in RPMI-1640 supplemented

with 10% fetal calf serum (Invitrogen). M10-CD1e cells, previously

described (12), express the CD1e isoform, characterized by its long

cytoplasmic tail (12). Cells were transfected with Fugene reagent (Roche

Biosciences), and stably transfected cells were selected with G418

(500 mg/mL; Invitrogen). DCs were differentiated from elutriated monocytes

in the presence of granulocyte–macrophage colony-stimulating factor and

interleukin-4 as previously described (13).

Antibodies and reagentsPolyclonal anti-TGN46 antibodies from sheep were purchased from Sero-

tec. The anti-CD1e mAb 20.6 recognizes the luminal domain of human

CD1e (12). The horseradish peroxidase (HRP)-conjugated anti-b2m Abs

were purchased from DakoCytomation. Secondary reagents were the

following: phycoerythrin (PE)-conjugated F(ab0)2 goat anti-mouse IgG

(DakoCytomation), Cy5-conjugated F(ab0)2 donkey anti-sheep IgG and

Cy3-conjugated F(ab0)2 donkey anti-mouse IgG (Jackson Immunoresearch),

A488-conjugated streptavidin and Cy5-conjugated goat anti-mouse IgG

(Molecular Probes) and control non-immune IgG1 and IgG2a (Beckman-

Coulter). The purified anti-CD63 mAb H5C6 (kindly provided by F. Lanza)

was biotinylated using a labeling kit from Molecular Probes. Bafilomycin

was purchased from LC Laboratories.

Genetic constructionsThe complementary DNA (cDNA) encoding the long isoform of CD1e (12)

was used for all mutagenesis experiments. To generate CD1e molecules

deleted of the N- or C-terminal half of the cytoplasmic domain, we took

advantage of the presence of an EcoRI restriction site in the middle of the

cytoplasmic domain and a HincII site at the junction of the transmembrane

and cytoplasmic domains (Figure 1). Deletion of the C-terminal half was

performed by introducing a stop codon immediately after the EcoRI

restriction site. The cDNA of CD1e was digested with EcoRI, filled in with

T4-DNA polymerase and then ligated to itself (CD1eDC). To delete the

N-terminal half, the cDNA was digested with HincII and EcoRI, treated with

T4-DNA polymerase and ligated to itself (CD1eDN). To obtain a CD1e

molecule with a cytoplasmic domain composed of an artificial sequence of

only 11 amino acids, the HincII site of the cDNA was ligated to the Eco47III

site of pEGFP-N3, after which the resulting plasmid was digested with

EcoRI, treated with T4-DNA polymerase and ligated to itself (CD1eDCyt). Inone mutant, we used a synthetic DNA sequence (Epoch Biolabs) encoding

a CD1e cytoplasmic tail where all the lysines had been replaced by arginines

(CD1e/8K-R). The C-terminal end of CD1e/8K-R was then fused with the

N-terminal end of human ubiquitin (CD1e/8K-R-Ubi).

A fusion mutant of CD1e and CD1b was generated by splicing their

respective cDNAs at the level of a PstI restriction site present in the coding

sequence of their a3 domains (CD1ebb) (Figure 1B). Then, the cytoplasmic

domain of CD1b in CD1ebb was replaced by that of CD1e (CD1ebe). Finally,

the cytoplasmic tail of CD1e was substituted by that of CD1b (CD1eeb).

All mutations were checked by DNA sequencing.

Flow cytometry and immunostainingTo quantify the cell surface expression of the different forms of CD1e, live

transfected cells were incubated with the mAb 20.6 and counterstained with

PE-conjugated goat anti-mouse IgG. Quantification of the total cellular CD1e

was performed using cells fixed in 3% paraformaldehyde, permeabilized in

0.05% saponin and stained with the mAb 20.6 as previously described (13).

Controls included staining with an isotype-matched irrelevant antibody.

The labeled cells were then analyzed with a FACScan cytometer (Becton

Dickinson). The proportion of cell surface-expressed CD1e molecules

was calculated using the formula (MFI of cells stained on the plasma mem-

brane with the mAb 20.6 � MFI of cells stained on the plasma membrane

with control IgG)/(MFI of fixed, permeabilized cells stained with the mAb

20.6 � MFI of fixed, permeabilized cells stained with control IgG).

Immunofluorescence microscopy of fixed, permeabilized adherent cells

and confocal laser scanning microscopy (Leica SP5 AOBS confocal

microscope; Leica Microsystems) were performed as previously described

(12). Briefly, adherent cells were fixed, permeabilized and labeled with the

mAb 20.6, counterstained with Cy3-conjugated anti-mouse Abs and

blocked with non-immune mouse serum. The cells were then incubated

with A488-conjugated H5C6 or biotinylated L243 and counterstained with

Cy5-conjugated streptavidin. Alternatively, the cells were labeled first with

polyclonal anti-TGN46 Abs, revealed with Cy5-conjugated donkey anti-

sheep IgG, and then with the mAb 20.6, revealed with Cy3-conjugated

donkey anti-mouse IgG.

Quantitative IF confocal microscopyCells were fixed, permeabilized and labeled with the anti-CD1e mAb 20.6

and either anti-TGN46 or anti-CD63 Abs and analyzed by three-dimensional

Traffic 2008; 9: 431–445 443


deconvolution microscopy (13). The percentage of colocalization of double

labels was calculated on deconvolved images with the dedicated METAMORPH

module (Universal Imaging Corp.). Thresholds were automatically deter-

mined after data processing by wavelet transforms (13,30), a process

allowing the separation of small and weak structures from large and

brighter structures (31). Quantitative analyses of coincident structures

were performed in all regions of the cells, except in the Golgi area when

one of the two signals was saturating.

Metabolic labeling, immunoprecipitation and

Western blot analysesPulse–chase labeling experiments were performed as described in previous

work (12). Confluent 75-cm2 flasks of transfected cells were labeled for

30min in 3mLofmethionine and cysteine-free RPMI-1640 containing 250mCi

of [35S] methionine and cysteine (Promix; GE Healthcare). The cells were

thenwashed and chased in 20mL of completemedium.When indicated, the

cells were labeled and chased in the presence of 0.1 mM bafilomycin. After

chase, the cells were lysed in 0.5mL of lysis buffer (20mM Tris pH 8, 150mM

NaCl, 5 mM ethylenediaminetetraacetic acid and 1% Triton-X-100) containing

a protease inhibitor cocktail (Complete�; Roche Biosciences) for 20 min on

ice. Lysates were centrifuged at 20 000 � g for 10 min, and supernatants

were precleared with 50 mL of protein A–Sepharose (GE Healthcare) for 2 h

before incubation with protein A–Sepharose and 5 mg of anti-CD1e or anti-

b2m mAb for 2 h. After extensive washing, the immunoprecipitates were

treated or not with endo H or endo F (Biolabs). Samples were separated on

12.5% SDS–PAGE) under reducing conditions. The gels were treated with

Amplify� (GE Healthcare) and exposed for autofluorography.

Autofluorograms were scanned to determine the rates of formation of

soluble endosomal CD1e. The lanes corresponding to samples treated with

endo F after 4 h of chase were analyzed using IMAGE QUANT TL software (GE

Healthcare), giving the ratio (R) of membrane-associated CD1e to soluble

CD1e. The percentage of cleaved forms with respect to the total CD1e was

calculated using the formula 100/(1 þ R).

To analyze the ubiquitination of CD1e, DCs or transfected M10 cells were

treated with bafilomycin (0.1 mM) for 5 h. The cells were then washed and

recovered with Versene (Invitrogen). After centrifugation, the cells were

lysed in cold lysis buffer supplemented with Complete and 20 mM

n-ethylmaleimide. CD1e molecules were immunoprecipitated, denatured in

Laemmli buffer, separated by SDS–PAGE and transferred to polyvinylidene

difluoride (PVDF) membranes. The membranes were saturated with 5% w/v

BSA in 20 mM Tris pH 7.6 containing 150 mM NaCl and 0.1% Tween-20.

Ubiquitinated proteins were revealed with a HRP-conjugated anti-ubiquitin

mAb (P4D1; Santa Cruz) and SuperSignal West Pico (transfected cells) or

SuperSignal West Femto (DCs) (Perbio Science).

Electron microscopyTransfected cells were fixed and cryosections were stained as previously

described (13) using the purified anti-CD1e mAb 20.6 (4–8 mg/mL) revealed

with protein A conjugated to 10-nm gold particles.

Acknowledgments

The authors are especially grateful to J. Mulvihil for excellent editorial

assistance and H. Bausinger for expert technical assistance. This work was

supported by Institut National de la Sante et de la Recherche Medical,

Etablissement Francxais du Sang-Alsace and Agence Nationale de la

Recherche (ANR-05-MIIM-006). B. M. was the recipient of a grant from

Association de Recherche et de Developpement en Medecine et Sante

Publique. We thank the members of the RIO – Cell and Tissue Imaging

Facility of UMR 144 Centre National de la Recherche Scientifique – Institut

Curie for their help with imaging approaches and the Region Ile de France

(SESAME program) for its support.

Supplementary Materials

Figure S1: Colocalization of CD63 with CD1e, CD1e/8K-R or CD1e/8K-

R-Ubi. Transfected cells were fixed, permeabilized and stained with the

mAb 20.6, which was revealed with Cy3-conjugated anti-mouse Abs. After

incubation with non-immune mouse serum, the cells were counterstained

with A488-conjugated H5C6. Two representative wild field deconvolved

images are shown for each form. Scale bars: 10 mm.

Figure S2: Immunodetection of CD1e by immunocryoelectron

microscopy. Cryosections of fixed transfected M10 cells expressing

CD1e, CD1e/8K-R and CD1e/8K-R-Ubi were labeled with the mAb 20.6.

The distribution of the three types of CD1e molecules is shown in three

wide-field images. In addition, representative views of labeling of Golgi

compartments in cells expressing CD1e/8K-R and of endosomes in cells

expressing different CD1e forms are shown.

Figure S3: Bafilomycin does not inhibit the LPS-induced relocalization

of CD1e molecules in iDCs. Immature DCswere treated or notwith 0.1mM

bafilomycin in presence or absence of 100 ng/mL LPS for 5 h. Then CD1e,

CD63 and TGN46 molecules were stained using the mAbs 20.6 and H5C6

and polyclonal anti-TGN46 Abs and then analyzed by confocal microscopy

as described in Materials and Methods. In iDCs, CD1e colocalizes with

TGN46. When cells are treated with LPS, colocalization of CD1e with

TGN46 decreases. Treatment with bafilomycin does not significantly alter

this distribution. In iDCs, CD1e poorly colocalizes with CD63, while

treatment with bafilomycin increases this colocalization. When cells are

treated with LPS, colocalization with CD63 is increased compared with

iDCs, while coincubation with bafilomycin results in a dramatic increase of

the fluorescence signal in CD63þ compartments. Scale bars: 10 mm.

Supplemental materials are available as part of the online article at http://

www.blackwell-synergy.com

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