glucuronosyltransferase 1A determines localization to the endoplasmic reticulum by both static retention and retrieval mechanisms Lydia Barre´, Jacques Magdalou, Patrick Netter, Sylvie F
Trang 1glucuronosyltransferase 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: ouzzine@medecine.uhp-nancy.fr
(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.
Trang 2retention⁄ retrieval motif CVLF has been described for a
splice variant SV1 of the voltage- and Ca2+-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.
Trang 3which 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 locmedi-ated in the CT
of the stop transfer sequence, we constructed two mutant proteins: CD4–TMD⁄ CTser in which the lysines of the KSKTH motif were mutated to serine residues, and CD4–TMD⁄ CTmyc 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 ⁄
CTserprotein 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⁄ CTserprotein leaked from the ER into the latter compartment in the secretory pathway A similar behaviour was observed in the case of CD4– TMD⁄ CTmyc (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.
Trang 4analysis of cells expressing CD4–TMD⁄ CTser 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⁄ CTser 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⁄ CTserfrom 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 repro-duced
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 (TMD21) or 26 (TMD26) transmembrane residues, respectively (Fig 1) CD4–TMD21and CD4–TMD26proteins 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.
Trang 5CD4–TMD26 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–TMD26 protein leaked from
the ER and moved forward in the secretory pathway
Similar results were obtained for CD4–TMD21protein
(data not shown)
Immunofluorescence analysis of cells expressing
CD4–TMD26, 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–TMD26 was also located in the ER
and the perinuclear region corresponding to Golgi
complex, as shown by immunofluorescence analysis
(Fig 5B) Indeed, the CD4–TMD26 staining pattern
overlapped with the ER and Golgi markers, calnexin
and GM130, respectively (Fig 5B) In the case of
CD4–TMD21, 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.
Trang 6indicates 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.
Trang 7Experimental procedures
Chemicals were from Merck (Darmstadt, Germany) or
Sigma (St Louis, MO, USA) Vent DNA polymerase,
restriction enzymes, Endo H, PGNase F and Phototope
-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,
a 5¢ 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–
TMD21 and pCD4–TMD26 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 1VIG
FLLAVVLTVAFITF17 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⁄ CTser
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 chipri-meric 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⁄ CTmyc
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
2 mm 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⁄ Pi and suspended in sucrose–Hepes buffer (0.25 m sucrose, 5 mm Hepes, pH 7.4) containing Complete MiniTM 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
4C The pellet was resuspended by Dounce homogeniza-tion in sucrose–Hepes buffer The protein concentrahomogeniza-tion 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 37C in 50 mm 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 ImmobilonP membrane Proteins were then immunostained using a polyclonal anti-CD4 sera and Pho-totope-HRP labelled anti-rabbit secondary sera for
Trang 8chemi-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 ⁄ Pi for
20 min Cells were permeabilized or not by treatment with
0.1% (w⁄ v) Triton X-100 ⁄ NaCl ⁄ Pisolution for 4 min After
extensive washing in 0.2% (w⁄ v) gelatin in NaCl ⁄ Pi, 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⁄ Piand
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
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