Báo cáo khoa học: The stop transfer sequence of the human UDPglucuronosyltransferase 1A determines localization to the endoplasmic reticulum by both static retention and retrieval mechanisms docx

The stop transfer sequence of the human UDP-
glucuronosyltransferase 1A determines localization to the
endoplasmic reticulum by both static retention and
retrieval mechanisms
Lydia Barre
´
, Jacques Magdalou, Patrick Netter, Sylvie Fournel-Gigleux and Mohamed Ouzzine
UMR 7561 CNRS-Universite
´
Henri Poincare
´
Nancy I, France
Biosynthesis of integral membrane proteins involves sev-
eral events such as targeting to the endoplasmic reti-
culum (ER), translocation of certain domains into the
ER lumen and integration of transmembrane domains
(TMD) into the lipid bilayer. These proteins are then
maintained in the ER by two modes, static retention or
dynamic retention by continuous retrieval of the escaped
proteins from the post-ER compartments. Retrieval sig-
nal sequences have been identified in both soluble [1]
and transmembrane [2] ER resident proteins. For
soluble ER resident proteins, a C-terminal tetrapeptide
KDEL in mammals and a closely related sequence
HDEL in yeast were shown to serve as specific ER
retention signals. The mechanism is based on the KDEL
receptor, which binds the escaped proteins in the Golgi
complex and returns them back to the ER [3]. For trans-
membrane type I ER resident proteins, a retrieval signal
KXKXX has been identified in the cytosolic tail (CT)
allowing for retrieval from the Golgi to the ER in a

coatomer-dependent manner [4,5]. Recently, a new ER
Keywords
endoplasmic reticulum retention; membrane
protein; stop transfer sequence;
transmembrane domain; UDP-
glucuronosyltransferase
Correspondence
M. Ouzzine, UMR CNRS 7561-Universite
´
Henri Poincare
´
Nancy 1, Faculte
´
de
Me
´
decine, BP 184, 54505 Vandœuvre-le
`
s-
Nancy, France
Fax: +33 3 83 68 39 59
Tel: +33 3 83 68 39 72
E-mail:
(Received 12 October 2004, revised 16
December 2004, accepted 24 December
2004)
doi:10.1111/j.1742-4658.2005.04548.x
Human UDP-glucuronosyltransferase 1A (UGT1A) isoforms are endoplas-
mic reticulum (ER)-resident type I membrane proteins responsible for the
detoxification of a broad range of toxic phenolic compounds. These pro-

teins contain a C-terminal stop transfer sequence with a transmembrane
domain (TMD), which anchors the protein into the membrane, followed
by a short cytosolic tail (CT). Here, we investigated the mechanism of ER
residency of UGT1A mediated by the stop transfer sequence by analysing
the subcellular localization and sensitivity to endoglycosidases of chimeric
proteins formed by fusion of UGT1A stop transfer sequence (TMD ⁄ CT)
with the ectodomain of the plasma membrane CD4 reporter protein. We
showed that the stop transfer sequence, when attached to C-terminus of
the CD4 ectodomain was able to prevent it from being transported to the
cell surface. The protein was retained in the ER indicating that this
sequence functions as an ER localization signal. Furthermore, we demon-
strated that ER localization conferred by the stop transfer sequence was
mediated in part by the KSKTH retrieval signal located on the CT. Inter-
estingly, our data indicated that UGT1A TMD alone was sufficient to
retain the protein in ER without recycling from Golgi compartment, and
brought evidence that organelle localization conferred by UGT1A TMD
was determined by the length of its hydrophobic core. We conclude that
both retrieval mechanism and static retention mediated by the stop transfer
sequence contribute to ER residency of UGT1A proteins.
Abbreviations
CT, cytosolic tail; Endo H, endoglycosidase H; ER, endoplasmic reticulum; FITC, fluoresceine isothiocyanate; PGNase F, peptide-N-
glycosidase F; TMD, transmembrane domain; UGT, UDP-glucuronosyltransferase.
FEBS Journal 272 (2005) 1063–1071 ª 2005 FEBS 1063
retention ⁄ retrieval motif CVLF has been described for a
splice variant SV1 of the voltage- and Ca
2+
-activated
K
+
channel alpha-subunit preventing plasma mem-

brane expression [6].
In the absence of positive transport signals, the
localization of a protein in the ER may result from
the properties of the TMD and its interaction with
the membranes. It has been demonstrated that TMD
of the yeast Sec12p and UBC6 (ubiquitin-conjugating
enzyme 6) and of the rabbit cytochrome b5 plays a
determinant role in the ER localization [7–9]. In
addition, TMD ER retention was shown to be static
in the case of UBC6 and cytochrome b5 and retrie-
val in the case of Sec12p [10]. Therefore, it has been
suggested that TMDs with centrally placed polar resi-
dues [10] can interact with Rer1p, which allows ER
retrieval from the cis-Golgi in COPI vesicles. Short
TMD (< 17 residues) with hydrophilic residues may
also promote ER targeting possibly by a Rer1p-inde-
pendent pathway [11]. Previous work suggested that
sorting of Golgi and plasma membrane proteins
depends on the length of the hydrophobic segment
of their TMD [12]. This is also true for ER mem-
brane proteins such as cytochrome b5 and UBC6 in
which lengthening of the TMD resulted in escape from
the ER and arrival at the plasma membrane [8,9].
Human UDP-glucuronosyltransferase 1As (UGT1A,
EC 2.4.1.17) are members of UGT superfamily that
plays a key role in the inactivation and elimination
of a broad range of toxic phenolic compounds by
conjugation to glucuronic acid, from the donor
cosubstrate UDP–glucuronic acid [13,14]. Members
of UGT1A are all encoded by a complex UGT1

gene locus consisting of 16 exons. The isoforms are
generated by alternative splicing of exon 1 to the
four common exons 2–5 resulting in isoforms with
an identical C-terminal half of the protein [15] and a
unique N-terminal end. UGT1A proteins are predic-
ted to be type I membrane proteins of the ER with
a glycosylated lumenal domain. It has also been
reported that UGT2B7 and UGT1A6 were expressed
in nuclear membrane [16]. The proteins contain a
stop transfer sequence at the C-terminus consisting
of a TMD of 17 residues followed by a short CT of
25 residues containing a KXKXX ER retrieval sig-
nal. In this study, we investigated the role of the
UGT1A stop transfer sequence (TMD ⁄ CT) in ER
residency. We showed, using a CD4 plasma mem-
brane protein as a reporter, that the UGT1A stop
transfer sequence acts as an ER retention signal. We
demonstrated that ER residency is determined by
both the retrieval mechanism mediated by the
KSKTH motif at the C-terminus of the CT and by
a static retention mediated by the hydrophobic
domain of the TMD. Furthermore, we showed that
the major determinant accounting for ER residency
conferred by the TMD is related to the length of its
hydrophobic core.
Results
The stop transfer sequence of UGT1A functions
as an ER targeting and retention signal in
mammalian cells
In order to analyse the ER retention capacity of the

TMD ⁄ CT domain, a chimera between the ectodomain
of plasma membrane CD4 glycoprotein (CD4 deleted
from the C-terminal anchoring domain) and the
TMD ⁄ CT of UGT1A was stably expressed in HeLa
cells (Fig. 1). The CD4 protein contains two N-linked
glycosylation sites and therefore glycosylation can be
used as a marker for subcellular localization. Indeed,
resistance to digestion with endoglycosidase H
(Endo H) indicated that glycoproteins have moved
from the ER compartment to at least the medial Golgi
apparatus and trans-Golgi apparatus, where complex
sugars are added. Analysis of CD4–TMD ⁄ CT pro-
tein on SDS ⁄ PAGE gave a single band of 44 kDa,
Fig. 1. Schematic representation of parental UGT1A6 (a member of
the UGT1A family), CD4 and chimeric proteins. CD4–TMD ⁄ CT,
ectodomain of CD4 fused to native stop transfer sequence of
UGT1A. CD4–TMD ⁄ CT
ser
, same as CD4–TMD ⁄ CT except that dily-
sine of KXKXX motif was mutated to serine residues.
CD4–TMD ⁄ CT
myc
, CD4–TMD ⁄ CT extended by myc-tag epitope at
the C-terminus. CD4–TMD, ectodomain of CD4 fused to the TMD
of UGT1A. CD4–TMD
21
and CD4–TMD
26
, same as CD4–TMD
except that the length of the TMD was extended by four and nine

hydrophobic Ala ⁄ Leu residues, respectively.
Retention of human UGT1A in endoplasmic reticulum L. Barre
´
et al.
1064 FEBS Journal 272 (2005) 1063–1071 ª 2005 FEBS
which was converted, after peptide-N-glycosidase F
(PGNase F) treatment, to a band  4 kDa smaller,
corresponding to the expected size of the unglycosyla-
ted fusion protein (Fig. 2A). Interestingly, a similar
band was generated upon treatment with Endo H indi-
cating that the protein was also sensitive to Endo H
digestion (Fig. 2A), thereby demonstrating that CD4–
TMD ⁄ CT was retained in the ER of HeLa cells. To
ascertain the intracellular localization of the CD4–
TMD ⁄ CT chimeric protein, immunofluorescence analy-
ses were carried out using monoclonal anti-CD4 sera.
Cells expressing recombinant full-length CD4 were
used as a control for cell surface expression (Fig. 2B).
Cells expressing CD4–TMD ⁄ CT were not labelled in
the absence of Triton X-100 permeabilization (Fig. 2B)
suggesting that CD4–TMD ⁄ CT protein was not
exposed to the cell surface but was retained in an
intracellular compartment. Interestingly, analysis of
Triton X-100-permeabilized cells showed a reticular
staining pattern characteristic of the ER localization of
CD4–TMD ⁄ CT protein. This location was confirmed
by the colocalization of the chimeric protein with the
ER marker protein, calnexin (Fig. 2B). Together, these
data showed that the UGT1A TMD ⁄ CT domain was
able to retain the CD4 plasma membrane protein in

the ER and therefore functions as an ER localization
signal.
The dilysine motif on the cytoplasmic tail
of UGT1A participates in ER retention
In order to investigate whether ER retention was medi-
ated by the dilysine KSKTH signal located in the CT
of the stop transfer sequence, we constructed two
mutant proteins: CD4–TMD ⁄ CT
ser
in which the
lysines of the KSKTH motif were mutated to serine
residues, and CD4–TMD ⁄ CT
myc
in which the length
of the cytoplasmic tail was extended by adding a myc-
epitope tag at its C-terminus so that the dilysine resi-
dues at critical positions )3 and )5 were positioned at
)14 and )16 (Fig. 1). The mutants were stably
expressed in HeLa cells and their sensitivity to endo-
glycosidase treatment was analysed. In contrast to
CD4–TMD ⁄ CT, Endo H treatment of CD4–TMD ⁄
CT
ser
protein resulted in a band with a molecular mass
similar to that of the nontreated polypeptide as well as
a band of 4 kDa smaller corresponding to the nongly-
cosylated form (Fig. 3A). The high molecular mass
band was sensitive to PGNase F but resistant to
Endo H (Fig. 3A) indicating that this polypeptide con-
tained complex-type oligosaccharides. This implies that

CD4–TMD ⁄ CT
ser
protein leaked from the ER into the
latter compartment in the secretory pathway. A similar
behaviour was observed in the case of CD4–
TMD ⁄ CT
myc
(data not shown). Immunofluorescence
A
B
Fig. 2. The TMD and CT of the UGT1A stop
transfer sequence determine subcellular
localization. (A) Sensitivity of CD4–TMD ⁄ CT
chimeric proteins to Endo H and PGNase F
treatment. CD4–TMD ⁄ CT construct has
been stably expressed in HeLa cells and
microsomal membranes of recombinant
cells were prepared as described in Experi-
mental procedures. Microsomal proteins
were subjected, or not, to Endo H and
PGNase F digestion and chimeric proteins
were then analysed by SDS ⁄ PAGE and
detected by Western blot analysis using
anti-CD4 sera. Nontransfected HeLa cells
were used as controls. (B) Cells expressing
native (CD4) and CD4–TMD ⁄ CT chimeric
protein were analysed by immunofluore-
scence microscopy. Cells were fixed with
paraformaldehyde, permeabilized or not (to
monitor cell surface expression) with Tri-

ton X-100, and immunostained with anti-
CD4–FITC conjugated sera. Cells expressing
CD4–TMD ⁄ CT were also stained for
calnexin as ER marker using rhodamine-con-
jugated secondary antibodies. Merge corres-
ponds to colocalization (yellow) of the
chimeric protein with the ER marker.
L. Barre
´
et al. Retention of human UGT1A in endoplasmic reticulum
FEBS Journal 272 (2005) 1063–1071 ª 2005 FEBS 1065
analysis of cells expressing CD4–TMD ⁄ CT
ser
showed
that the protein was detected in ER, Golgi and plasma
membrane compartments (Fig. 3B). In agreement, the
staining pattern of the chimeric protein overlapped
with that of calnexin, as well as with that of the Golgi
marker protein, GM130 (Fig. 3B). Cell-surface expres-
sion of the protein was confirmed by immunofluores-
cence staining of cells expressing CD4–TMD ⁄ CT
ser
in
the absence of detergent (Fig. 3B). Together, these
results indicated that although the dilysine KSKTH
motif of the TMD ⁄ CT domain plays a role in ER
retention, other determinants preventing escape of
CD4–TMD ⁄ CT
ser
from this organelle may exist.

The TMD of UGT1A is sufficient for ER retention
In order to investigate the role of the TMD of
UGT1A in ER retention, a CD4–TMD chimeric pro-
tein (CD4 ectodomain fused to the TMD of UGT1A)
was stably expressed in HeLa cells (Fig. 1). As des-
cribed above, glycosylation was used as a marker to
determine whether this protein without CT was
retained in the ER or moved forward to the distal
organelles in the secretory pathway. Endoglycosidase
analysis showed that CD4–TMD protein was Endo H
sensitive, as digestion with the endoglycosidase pro-
duced a single polypeptide, whose mass was reduced
by 4 kDa (Fig. 4A). Similar results were obtained after
PGNase F treatment (Fig. 4A). These data suggested
that CD4–TMD was retained in the ER. In agreement,
immunofluorescence studies showed that CD4–TMD
presented a typical reticular staining pattern and
colocalized with the ER marker protein, calnexin
(Fig. 4B). Taken together, these data indicated that the
TMD domain of UGT1A was sufficient to retain the
ectodomain of CD4 protein in the ER. Because
the carbohydrate moieties of proteins that are trans-
ported to the Golgi become resistant to Endo H, these
results also indicated that CD4–TMD was retained
in the ER without recycling from post-Golgi com-
partment.
TMD length determines the subcellular
localization
It has been proposed that the length of the TMD of
Golgi and plasma membrane proteins was in part

responsible for their subcellular localization. To
address whether the length of UGT1A TMD plays a
role in ER residency, its hydrophobic segment was
increased by four or nine amino acids (LALA or
LALALALAL), to a total of 21 (TMD
21
)or26
(TMD
26
) transmembrane residues, respectively
(Fig. 1). CD4–TMD
21
and CD4–TMD
26
proteins were
stably expressed in HeLa cells and then analysed by
endoglycosidase treatment. In contrast to CD4–TMD,
A
B
Fig. 3. The KSKTH dilysine motif on the
cytosolic tail of the UGT1A6 stop transfer
sequence acts as ER retention signal. The
construct expressing CD4–TMD ⁄ CT
ser
(same as CD4–TMD ⁄ CT except that dilysine
of the KSKTH motif was mutated to serine
residues) was stably expressed in HeLa
cells. (A) Microsomal membrane proteins of
the recombinant cells were or were not
subjected to Endo H and PGNase F diges-

tion, and analysed by Western blot using
anti-CD4 sera. Nontransfected HeLa cells
were used as controls. (B) Cells expressing
CD4–TMD ⁄ CT
ser
protein were Triton X-100
permeabilized, or not, and analysed by
immunofluorescence microscopy using anti-
CD4–FITC conjugated sera. Cells expressing
CD4–TMD ⁄ CT
ser
were also stained for cal-
nexin and for GM130 as ER and Golgi
marker, respectively, using rhodamine-conju-
gated secondary antibodies. Merge corres-
ponds to colocalization (yellow) of the
chimeric protein with each subcompartment
marker.
Retention of human UGT1A in endoplasmic reticulum L. Barre
´
et al.
1066 FEBS Journal 272 (2005) 1063–1071 ª 2005 FEBS
CD4–TMD
26
was partially sensitive as about half of
the polypeptides acquired resistance to Endo H
(Fig. 5A). However, these polypeptides were sensitive
to PGNase F (Fig. 5A). Taken together, these data
suggested that the CD4–TMD
26

protein leaked from
the ER and moved forward in the secretory pathway.
Similar results were obtained for CD4–TMD
21
protein
(data not shown).
Immunofluorescence analysis of cells expressing
CD4–TMD
26
, where the TMD segment was extended,
revealed the protein in the absence of detergent treat-
ment (Fig. 5B) indicating cell surface expression of
the chimera. CD4–TMD
26
was also located in the ER
and the perinuclear region corresponding to Golgi
complex, as shown by immunofluorescence analysis
(Fig. 5B). Indeed, the CD4–TMD
26
staining pattern
overlapped with the ER and Golgi markers, calnexin
and GM130, respectively (Fig. 5B). In the case of
CD4–TMD
21
, staining was observed in both the ER
and the perinuclear region (data not shown). These
findings suggest that the proteins may be sorted within
the secretory pathway based on interactions between
their TMDs and the surrounding lipid bilayer.
Discussion

Human UGTs are transmembrane type I glycoproteins
with an N-terminal cleavable signal peptide and a
C-terminal stop transfer sequence. Our laboratory has
been deeply involved in the identification of protein
domains that are determinant for membrane assembly
in the ER. We previously showed that deletion of the
signal peptide alone or in combination with that of the
TMD did not prevent membrane targeting and
insertion of the enzyme. These findings resulted in the
identification of an internal signal sequence localized
between residues 140 and 240 and led us to suggest
that the membrane assembly of UGT1A6 may involve
several topogenic elements [17]. This prompted us to
investigate the topogenic role of the stop transfer
sequence, which comprises a TMD followed by a short
cytosolic tail with the common KXKXX ER
retrieval ⁄ retention signal.
It is widely accepted that there are two mecha-
nisms for the localization of ER resident proteins;
one is the dynamic retrieval mechanism from post-
ER compartments, and the other is the static retent-
ion mechanism that prevents exit from the ER. In
type I membrane proteins such as UGTs, the retrie-
val signal has been defined as two lysine residues at
positions )3 and )5 from the C-terminus exposed
on the cytosolic side of the ER membrane [18]. We
report in this study that disruption of the dilysine
motif KSKTH of UGT1A by mutation of lysine to
serine residues or by extending the length of the
cytoplasmic tail to relocate the dilysine from the crit-

ical positions )3 and )5 to positions )14 and )16
affected the ER localization of the recombinant
CD4–TMD ⁄ CT protein, as evidenced by resistance
to Endo H treatment. However, a portion of the chi-
meric proteins did not acquire Golgi-specific carbo-
hydrate modifications. These results suggested that
part of the CD4–TMD ⁄ CT chimeric proteins escaped
from the ER compartment and moved forward to the
distal organelles in the secretory pathway, whereas
the other part was retained in the ER. This result
A
B
Fig. 4. TMD of UGT1A stop transfer
sequence determines ER retention.
(A) Microsomal membranes from cells
expressing CD4–TMD protein were treated,
or not, with Endo H and PGNase F, and
analysed by Western blot using anti-CD4
sera. (B) Cells expressing CD4–TMD were
analysed by immunofluorescence
microscopy using anti-CD4–FITC conjugated
sera. Cells were also stained for calnexin as
ER marker using rhodamine-conjugated
secondary antibodies. Merge corresponds to
colocalization (yellow) of the chimeric
protein with the ER marker.
L. Barre
´
et al. Retention of human UGT1A in endoplasmic reticulum
FEBS Journal 272 (2005) 1063–1071 ª 2005 FEBS 1067

indicates that the retrieval mechanism is not suffi-
cient to ensure ER residency, suggesting that an
additional mechanism may be involved.
TMDs of membrane proteins have often been shown
to contain important information for localization in
the ER [19]. We found that the TMD of UGT1A pro-
teins appended to the C-terminus of the ectodomain of
CD4 plasma membrane glycoprotein was able to retain
the chimeric protein in the ER as indicated by endo-
glycosidase treatments which showed that CD4 ⁄ TMD
was sensitive to Endo H, which removes the high-
mannose oligosaccharides that are found in the ER,
and by immunofluorescence analysis. These data sug-
gest that the TMD contains sufficient information for
ER retention probably acting via a static mechanism.
In the same manner, it has been demonstrated that the
TMD of transmembrane proteins cause ER localiza-
tion of a yeast type VI transmembrane protein UBC6
[9] and a rat type II membrane protein cytochrome b5
[20] by static retention. Further experiments showed
that extension of the TMD of UGT1A appended to
the CD4 ectodomain resulted in a chimeric protein
that was partially resistant to Endo H treatment, but
sensitive to PGNase F, indicating that complex glyco-
sylation occurred. This observation suggested that the
protein effectively moved through the medial-Golgi
compartment. In agreement, immunofluorescence
localization studies confirmed that lengthening the
TMD resulted in Golgi and cell-surface expression of
CD4–TMD chimera. The concept that TMD length

determines distribution between the Golgi and plasma
membrane was initially reported for both Golgi and
plasma membrane proteins [12]. Membrane thickness
(determined partly by cholesterol content) may help
segregate proteins with TMD of different lengths.
Recently, it has been suggested that differential target-
ing of IP3R in different cell types may depend on vari-
ations in lipid composition rather than the presence
of specific protein-sorting signals [21]. Our results,
together with these studies, are consistent with the idea
that a short membrane anchor may provide a mechan-
ism for the exclusion of ER-membrane proteins from
transport down the secretory pathway. Altogether,
these experiments suggest that the UGT1A stop trans-
fer sequence maintains ER residency by a combination
of both static and dynamic retrieval. In agreement, it
has been shown that both retention and retrieval
mechanisms operate to keep protein such as cyto-
chrome b5 in the ER compartment [8].
In conclusion, ER residency conferred by the
UGT1A stop transfer sequence involves at least two
determinants, the TMD probably acting by static ER
retention and the KSKTH for retrieval of escaped pro-
teins from the post-ER compartment.
A
B
Fig. 5. The length of the TMD of UGT1A
stop transfer sequence determines the
subcellular localization. (A) Microsomal
membranes from cells expressing

CD4–TMD ⁄ CT
26
were treated, or not, with
Endo H and PGNase F, and analysed by
Western blot using anti-CD4 sera. (B) Cells
expressing CD4–TMD ⁄ CT
ser
were analysed
by immunofluorescence microscopy using
anti-CD4–FITC conjugated sera. Cells were
also stained for calnexin and for GM130 as
ER and Golgi markers, respectively, using
rhodamine-conjugated secondary antibodies.
Merge corresponds to colocalization (yellow)
of the chimeric protein with each sub-
compartment marker.
Retention of human UGT1A in endoplasmic reticulum L. Barre
´
et al.
1068 FEBS Journal 272 (2005) 1063–1071 ª 2005 FEBS
Experimental procedures
Chemicals were from Merck (Darmstadt, Germany) or
Sigma (St. Louis, MO, USA). Vent DNA polymerase,
restriction enzymes, Endo H, PGNase F and Phototope
Ò
-
HRP Western detection system were from New England
Biolabs (Beverly, MA, USA). Escherichia coli JM109 was
from Promega (Madison, WI, USA). ExGen 500 transfec-
tion reagent was from Euromedex (Souffelweyersheim,

France). Dulbecco’s modified Eagle’s medium was from
Life Technologies (Rockville, MD, USA). Polyclonal
anti-CD4 antibodies were purchased from Santa Cruz Bio-
technology (Santa Cruz, CA, USA). Monoclonal anti-
CD4–FITC conjugated sera were from Sigma. Monoclonal
anti-GM130 Golgi protein and anti-calnexin ER protein
sera were from BD Transduction Laboratories (Lexington,
KY, USA) and Affinity Bioreagents (Golden Co, USA),
respectively.
Plasmid constructions
To generate the pCD4–TMD ⁄ CT vector expressing CD4
ectodomain sequence (cell surface-expressed CD4 polypep-
tide) in fusion with the C-terminal 43 amino acids of
human UGT1A stop transfer sequence, TMD ⁄ CT coding
sequence was amplified by PCR using UGT1A6 cDNA [13]
(a member of UGT1A family) as template and two primers,
a5¢ primer 5¢-GGATCCGTGATTGGTTTCCTCTTG-3¢
containing a BamHI site and UGT1A6 nucleotides 1467–
1482 and a 3¢ primer 5¢-CTCGAGTCAATGGGTCTTG
GATTTGTG-3¢ encoding for the last six residues of human
UGT1A6 (1593–1576) followed by a stop codon and XhoI
site. The PCR product was cut with BamHI and XhoI and
cloned into BamHI and XhoI sites of pTM1 expression vec-
tor in frame with CD4 ectodomain (a gift from Dr J. Dub-
uisson, IBL ⁄ Institut Pasteur, Lille, France) to generate
pCD4–TMD ⁄ CT vector (Fig. 1).
Vector expressing the CD4–TMD was constructed by
PCR using a sense primer as above and an oligonucleotide
corresponding to nucleotides 1498–1515 of UGT1A6 fol-
lowed by a stop codon and XhoI site. To generate pCD4–

TMD
21
and pCD4–TMD
26
expression vectors with the
hydrophobic segment of the TMD extended from 17 to 21
and 26 residues, respectively, amino acids LALA and
LALALALAL were inserted into TMD sequence
1
VIG
FLLAVVLTVAFITF
17
between Val9 and Leu10 residues
by two rounds SOE-PCR [16] using pCD4–TMD as tem-
plate. Mutants were systematically checked by sequencing.
Schematic representation of the constructs is shown in
Fig. 1.
Site-directed mutagenesis
Mutation of lysine residues of the cytoplasmic tail motif
KSKTH to serine residues was performed by PCR using a
sense primer 5¢-GGATCCGTGATTGGTTTCCTCTTG-3¢
containing a BamHI site and UGT1A6 nucleotides 1467–
1482 and an antisense primer 5¢-CTCGAGTCAATGG
GTACTGGAACTGTGGGCTTTCTT-3¢ introducing the
mutations and encoding for the last six residues of human
UGT1A6 (1593–1576) followed by a stop codon and XhoI
site. The PCR product was cloned into BamHI and XhoI
sites of pTM1 expression vector in frame with CD4 ecto-
domain to generate pCD4–TMD ⁄ CT
ser

vector.
Extension of the length of the UGT1A cytoplasmic tail
from 25 to 36 residues by the addition of myc-tag sequence
(EQKLISEEDLN) was achieved by PCR using a sense pri-
mer as above and a chimeric oligonucleotide encoding the
last six residues of UGT1A6 and a myc-tag sequence fol-
lowed by a stop codon and XhoI site as the antisense pri-
mer. The PCR product was ligated into BamHI and XhoI
sites of pTM1 expression vector to generate CD4–
TMD ⁄ CT
myc
plasmid (Fig. 1).
Expression in HeLa cells and endoglycosidase
digestions
HeLa cells were grown in Dulbecco’s modified Eagle’s med-
ium supplemented with 10% (v ⁄ v) fetal calf serum and
2mm glutamine. DNA transfection of different plasmid
constructs and isolation of recombinant colonies were per-
formed as described [22]. Colonies expressing similar levels
of recombinant proteins were selected by immunoblot ana-
lysis. Cells were then cultured and harvested at confluency,
washed with NaCl ⁄ P
i
and suspended in sucrose–Hepes
buffer (0.25 m sucrose, 5 mm Hepes, pH 7.4) containing
Complete Mini
TM
protease inhibitors (Roche Molecular
Biochemicals, Indianapolis, IN, USA). Cells were lysed by
three 5-s sonication (Vibra Cell, Bioblock Scientific, Illirch,

France) and centrifuged at 12 000 g for 20 min. Membranes
were pelleted from the supernatant for 1 h at 100 000 g at
4 °C. The pellet was resuspended by Dounce homogeniza-
tion in sucrose–Hepes buffer. The protein concentration of
the homogenate was evaluated by the method of Bradford
[23]. Membrane proteins (50 lg) were boiled for 10 min in
denaturing buffer (0.5% (w ⁄ v) sodium dodecyl sulfate, 1%
(v ⁄ v) 2-mercaptoethanol), then digested with Endo H or
PNGase F for 2 h at 37 °Cin50mm sodium citrate buffer
(pH 5.5) or 50 mm sodium phosphate buffer (pH 7.5) con-
taining 1% NP-40, respectively. Endo H cleaves aspara-
gine-linked high mannose structures generating a peptide
with one attached N-acetylglucosamine residue. PNGase F
is an amidase that cleaves between the innermost N-acetyl-
glucosamine and the asparagine residue removing all types
of N-glycan chains from glycopeptides and glycoproteins.
Endoglycosidase-digested and nontreated samples were elec-
trophoresed on 10% (w ⁄ v) SDS ⁄ polyacrylamide gels and
transferred to ImmobilonPÒ membrane. Proteins were then
immunostained using a polyclonal anti-CD4 sera and Pho-
totopeÒ-HRP labelled anti-rabbit secondary sera for chemi-
L. Barre
´
et al. Retention of human UGT1A in endoplasmic reticulum
FEBS Journal 272 (2005) 1063–1071 ª 2005 FEBS 1069
luminescence detection (Cell Signaling Technology, Beverly,
MA, USA).
Immunofluorescence microscopy
Immunofluorescence was performed as described by Louvard
et al. [24]. Briefly, cells were grown on glass coverslips and

fixed with 3% (w ⁄ v) paraformaldehyde in NaCl ⁄ P
i
for
20 min. Cells were permeabilized or not by treatment with
0.1% (w ⁄ v) Triton X-100 ⁄ NaCl ⁄ P
i
solution for 4 min. After
extensive washing in 0.2% (w ⁄ v) gelatin in NaCl ⁄ P
i
, cells
were stained with fluoresceine isothiocyanate (FITC)-conju-
gated anti-CD4 sera. Immunostaining of marker proteins of
the Golgi apparatus and ER compartment were then carried
out using monoclonal antibodies raised against GM-130 and
calnexin, respectively, and rhodamine-conjugated secondary
antibodies. Finally, cells were washed in NaCl ⁄ P
i
and moun-
ted on microscope slides. Confocal laser scanning microscopy
was performed using a Leica TCS SP2 equipped with an
acousto-optical beamsplitter. Excitation was achieved in
sequential scan mode between frame by the 488 nm line from
an Ar laser (for FITC) and the 543 nm line from an HeNe
laser [for tetramethylrhodamine isothiocyanate (TRITC)].
Fluorescence emissions were recorded within an Airy disk
confocal pinhole setting (2.3 A
˚
). Three-dimensional images
were compiled into a single-view projection using LCS3D
image processing software (Leica Microsystems, Mannheim,

Germany).
Acknowledgements
This work was supported by grants from Fonds National
pour la Science, Ligue Re
´
gionale Contre le Cancer,
Re
´
gion Lorraine, Communaute
´
Urbaine du Grand
Nancy and Institut Fe
´
de
´
ratif de Recherche 111 (Bio-
inge
´
nierie). Dr N. Venkatesan is gratefully acknowledged
for critical reading of the manuscript and Dr D. Dumas
for performing confocal laser scanning microscopy.
References
1 Munro S & Pelham HR (1987) A C-terminal signal pre-
vents secretion of luminal ER proteins. Cell 48, 899–907.
2 Nilsson T, Jackson M & Peterson PA (1989) Short cyto-
plasmic sequences serve as retention signals for trans-
membrane proteins in the endoplasmic reticulum. Cell
58, 707–718.
3 Lewis MJ & Pelham HR (1990) A human homologue of
the yeast HDEL receptor. Nature 348, 162–163.

4 Cosson P & Letourneur F (1994) Coatomer interaction
with di-lysine endoplasmic reticulum retention motifs.
Science 263, 1629–1631.
5 Teasdale RD & Jackson MR (1996) Signal-mediated
sorting of membrane proteins between the endoplasmic
reticulum and the Golgi apparatus. Annu Rev Cell Dev
Biol 12, 27–54.
6 Zarei MM, Eghbali M, Alioua A, Song M, Knaus HG,
Stefani E & Toro L (2004) An endoplasmic reticulum
trafficking signal prevents surface expression of a vol-
tage- and Ca
2+
-activated K
+
channel splice variant.
Proc Natl Acad Sci USA 101, 10072–10077.
7 Nishikawa S & Nakano A (1993) Identification of a
gene required for membrane protein retention in the
early secretory pathway. Proc Natl Acad Sci USA 90,
8179–8183.
8 Pedrazzini E, Villa A & Borgese N (1996) A mutant
cytochrome b5 with a lengthened membrane anchor
escapes from the endoplasmic reticulum and reaches the
plasma membrane. Proc Natl Acad Sci USA 93, 4207–
4212.
9 Yang M, Ellenberg J, Bonifacino JS & Weissman AM
(1997) The transmembrane domain of a carboxyl-
terminal anchored protein determines localization to the
endoplasmic reticulum. J Biol Chem 272, 1970–1975.
10 Sato M, Sato K & Nakano A (1996) Endoplasmic reti-

culum localization of Sec12p is achieved by two
mechanisms: Rer1p-dependent retrieval that requires the
transmembrane domain and Rer1p-independent
retention that involves the cytoplasmic domain. J Cell
Biol 134, 279–293.
11 Rayner JC & Pelham HR (1997) Transmembrane
domain-dependent sorting of proteins to the ER and
plasma membrane in yeast. EMBO J 16, 1832–1841.
12 Munro S (1995) A comparison of the transmembrane
domains of Golgi and plasma membrane proteins. Bio-
chem Soc Trans 23, 527–530.
13 Harding D, Fournel-Gigleux S, Jackson MR &
Burchell B (1988) Cloning and substrate specificity of
a human phenol UDP-glucuronosyltransferase
expressed in COS-7 cells. Proc Natl Acad Sci USA 85,
8381–8385.
14 Ebner T & Burchell B (1993) Substrate specificities of
two stably expressed human liver UDP-glucuronosyl-
transferases of the UGT1 gene family. Drug Metab
Dispos 21, 50–55.
15 Ritter JK, Chen F, Sheen YY, Tran HM, Kimura S,
Yeatman MT & Owens IS (1992) A novel complex
locus UGT1 encodes human bilirubin, phenol, and other
UDP-glucuronosyltransferase isozymes with identical
carboxyl termini. J Biol Chem 267, 3257–3261.
16 Radominska-Pandya A, Pokrovskaya ID, Xu J, Little
JM, Jude AR, Kurten RC & Czernik PJ (2002) Nuclear
UDP-glucuronosyltransferases: identification of
UGT2B7 and UGT1A6 in human liver nuclear mem-
branes. Arch Biochem Biophys 399, 37–48.

17 Ouzzine M, Magdalou J, Burchell B & Fournel-Gigleux
S (1999) An internal signal sequence mediates the
targeting, translocation and retention of human
Retention of human UGT1A in endoplasmic reticulum L. Barre
´
et al.
1070 FEBS Journal 272 (2005) 1063–1071 ª 2005 FEBS
UDP-glucuronosyltransferase (UGT1A6) into the endo-
plasmic reticulum. J Biol Chem 274, 31401–31409.
18 Jackson MR, Nilsson T & Peterson PA (1993) Retrieval
of membrane proteins to the endoplasmic reticulum.
J Cell Biol 121, 317–333.
19 Borgese N, Colombo S & Pedrazzini E (2003) The tale
of tail-anchored proteins: coming from the cytosol and
looking for a membrane. J Cell Biol 161, 1013–1019.
20 Honsho M, Mitoma JY & Ito A (1998) Retention of
cytochrome b5 in the endoplasmic reticulum is trans-
membrane and luminal domain-dependent. J Biol Chem
273, 20860–20866.
21 Parker AK, Gergely FV & Taylor CW (2004) Targeting
of inositol 1,4,5-trisphosphate receptors to the endoplas-
mic reticulum by multiple signals within their transmem-
brane domains. J Biol Chem 279, 23797–23805.
22 Ouzzine M, Magdalou J, Burchell B & Fournel-Gigleux
S (1999) Expression of functionally active human hepa-
tic UDP-glucuronosyltransferase (UGT1A6) lacking the
N-terminal signal sequence in the endoplasmic reticu-
lum. FEBS Lett 454, 187–191.
23 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein util-

izing the principle of protein–dye binding. Anal Biochem
72, 248–254.
24 Louvard D, Reggio H & Warren G (1982) Antibodies
to the Golgi complex and the rough endoplasmic
reticulum. J Cell Biol 92, 92–107.
L. Barre
´
et al. Retention of human UGT1A in endoplasmic reticulum
FEBS Journal 272 (2005) 1063–1071 ª 2005 FEBS 1071