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: 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. 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 . The stop transfer sequence of the human UDP- glucuronosyltransferase 1A determines localization to the endoplasmic reticulum by both static retention and retrieval. 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