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Functional dissection of HCMV US11 in mediating the degradation of MHC class I molecules
BBRC Biochemical and Biophysical Research Communications 330 (2005) 1262–1267 www.elsevier.com/locate/ybbrc
Functional dissection of HCMV US11 in mediating the degradation of MHC class I molecules Seong-Ok Lee a, Sujin Hwang a, Junghyun Park b, Boyoun Park a, Bong-Suk Jin a, Sungwook Lee a, Eunkyung Kim a, Sunglim Cho a, Youngkyun Kim a, Kwangmin Cho b, Jinwook Shin b, Kwangseog Ahn a,* a
Department of Biological Sciences, Seoul National University, Seoul 151-747, Republic of Korea b College of Life Sciences, Korea University, Seoul 136-701, Republic of Korea Received 10 March 2005 Available online 25 March 2005
Abstract The human cytomegalovirus (HCMV) gene product US11 dislocates MHC I heavy chains from the endoplasmic reticulum (ER) and targets them for proteasomal degradation in the cytosol. To identify the structural and functional domains of US11 that mediate MHC class I molecule degradation, we constructed truncated mutants and chimeric proteins, and analyzed these to determine their intracellular localization and their ability to degrade MHC class I molecules. We found that only the luminal domain of US11 was essential to confer ER localization to the protein but that the ability to degrade MHC class I molecules required both the transmembrane domain and the luminal domain of US11. By analyzing a series of point mutants of the transmembrane domain, we were also able to identify Gln192 and Gly196 as being crucial for the functioning of US11, suggesting that these residues may play a critical role in interacting with the components of the protein degradation machinery. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Protein degradation; Proteasome; MHC class I molecules; Human cytomegalovirus; Endoplasmic reticulum; Protein trafficking
Human cytomegalovirus (HCMV) is a b-herpesvirus that infects 70–80% of the population of the Western world [1]. It causes benign but persistent infections and contributes to the high morbidity and mortality of immuno-compromised patients, most notably organ transplant recipients and AIDS patients [2,3]. To evade cytotoxic T lymphocytes (CTLs), HCMV encodes several proteins that can independently down-modulate the surface expression of the major histocompatibility complex (MHC) class I molecules. One of these, US3, is an immediate-early HCMV protein that sequesters MHC class I molecules in the endoplasmic reticulum (ER) by preventing the transport of the assembled *
Corresponding author. Fax: +82 2 872 1993. E-mail address: [email protected] (K. Ahn).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.112
MHC class I molecules to the cell surface [4–6]. The HCMV US6 gene product prevents peptide loading of the MHC class I molecules by inhibiting transporter associated with antigen processing (TAP)-mediated peptide translocation into the ER [7–10], while the US2 and US11 gene products induce rapid export of the newly synthesized MHC class I heavy chains (HCs) out of the ER into the cytosol where the HCs are subsequently degraded by the proteasome [11,12]. The HCMV products US2 and US11 show similar general modes of action but differ in their abilities to attack allelic class I HCs and the conformation of the HC molecules [13–16]. The exact mechanism that US11 deploys to disrupt MHC class I HC cell-surface expression is unclear. It has been shown to resemble the ER-associated degradation (ERAD) by which mammalian cells dispose of
S.-O. Lee et al. / Biochemical and Biophysical Research Communications 330 (2005) 1262–1267
misfolded proteins or unassembled subunits of oligomeric proteins. Misfolded or unassembled proteins are retrotranslocated from the ER back to the cytosol via the Sec61 complex [17–19]. Some studies in yeast and mammalian cells have reported that the proteins may be transported through the transmembrane protein Derlin-1/Der1p [20–22]. Ubiquitin is then attached to the ERAD substrates in the cytosol [23,24]. The AAA ATPase p97/cdc48 and its associated cofactors, Ufd1 and Npl4, recognize poly-ubiquitinated proteins, extract them from ER membrane, and escort the ubiquitin-conjugated proteins to the proteasome [22,25–28]. In this study, we examined the structural requirements necessary for US11 to mediate the selective degradation of MHC class I molecules, with the aim of gaining an insight into the mechanisms of viral pathogenesis and proteasome-mediated protein degradation. We found that the transmembrane domain is not essential for the retention of US11 in the ER where it functions but that a specific amino acid residue at the transmembrane domain is critical for the functioning of US11.
Materials and methods Plasmid constructs. Plasmid-expressing truncated mutants and chimeric proteins were constructed as shown in Fig. 1. The respective DNA fragments were obtained by either restriction digestion or PCR amplification. Single amino acid mutations were introduced by the
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PCR-mediated oligonucleotide-directed protocol with a pcDNA3.1 construct containing the US11 as a template. The following oligonucleotides were used: MM186 Æ 187VV (CG CAG TAT ACG CTG GTG GTG GTG GCA GTG ATT), Q192V (G ATG ATG GTG GCA GTG ATT GTA GTG TTT TGG GGG CTG), F194V (CTG GTG GCA GTG ATT CAA GTG GTT TGG GGG CTG TAT GTG), W195V (G GCA GTG ATT CAA GTG TTT GTG GGG CTG TAT GTG AAA GG), G196V (G ATT CAA GTG TTT TGG GTG CTG TAT GTG AAA GGT TGG), and Y198V (GTG TTT TGG GGG CTG GTT GTG AAA GGT TGG CTG C). After mutagenesis, all constructs were confirmed by DNA sequencing. Cell lines and cell culture. HeLa and U373-MG astrocytoma cell lines were obtained from American Type Culture Collection (Manassas, VA) and cultured in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FBS (HyClone Laboratories, Logan, Utah), 2 mM L-glutamine, 50 U/ml penicillin, and 50 lg/ml streptomycin. To establish stable cell lines expressing US11 or US11 mutants, we cloned each cDNA into the pcDNA3.1 mammalian expression vector (Invitrogen, Carlsbad, CA) and transfected the resulting constructs into U373-MG cells by the calcium phosphate precipitation method. Stable clones were selected by adding 0.5 mg/ml G418 (Life Technologies). Antibodies. The K455 antibody recognizes the MHC class I HC and b2-microglobulin (b2m) in both assembled and non-assembled forms. Monoclonal antibody (MAb) W6/32 recognizes only the complex of HC and b2m. Polyclonal antiserum (anti-US11) for detecting US11 was raised against the synthetic peptides corresponding to the luminal N-terminal portion (residues 16 to 35) of the proteins [29]. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (IgG) was purchased from the Jackson ImmunoResearch Laboratories (West Grove, PA). Metabolic labeling and immunoprecipitation. Cells were preincubated in methionine-free DMEM with or without proteasome inhibitor. The proteasome inhibitor (LLnL; Sigma, St. Louis, MO) was dissolved in ethanol at a concentration of 50 mg/ml for use as a 1:1000
Fig. 1. (A) Schematic diagram of US11, US110, US100, and UCU. TMD, transmembrane domain; CTD, cytoplasmic domain. (B) HeLa cells expressing the US11 gene product and the mutants indicated were stained with the polyclonal US11-antisera and DAPI, and analyzed for intracellular localization by immunofluorescence microscopy. (C) U373-MG cells stably expressing US11, US110, and US100 were labeled with [35S]methionine for 30 min and chased for 120 min. Cell lysates were immunoprecipitated with US11-antisera, and the immunoprecipitates were digested for 16 h with (+) or without ( ) endo H. (D) U373-MG stable cells were incubated with proteasome inhibitor LLnL for 2 h, labeled with [35S]methionine for 30 min, lysed in 0.5% NP-40 buffer, and then immunoprecipitated by the antibodies indicated. The samples were separated by 10% SDS–polyacrylamide gel electrophoresis.
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dilution. Cells were starved for 40 min in medium lacking methionine, labeled with 0.1 mCi/ml [35S]methionine (TranS-label; NEN Life Science, Boston, MA) for 30 min, and chased in regular medium for the indicated time. Following two washes with cold phosphate-buffered saline (PBS), the cells were lysed using 1% or 0.5% Nonidet P (NP)-40 (Sigma–Aldrich) in PBS for 30 min at 4 °C. Immunoprecipitation was performed as described previously [5]. For endoglycosidase H treatment, the immunoprecipitates were digested with 3 mU endoglycosidase H (Roche, Indianapolis, IN) for 16 h at 37 °C in 50 mM NaOAc (pH 5.6), 0.3% sodium dodecyl sulfate (SDS), and 150 mM b-mercaptoethanol. Flow cytometry and immunofluorescence microscopy. Cells were washed twice with PBS containing 1% bovine serum albumin (BSA) and then incubated for 1 h at 4 °C with W6/32. Normal mouse IgG was used as a negative control. The cells were washed twice with cold PBS containing 1% BSA and then stained with FITC-conjugated goat anti-mouse IgG for 40 min. A total of 10,000-gated events were collected by the FACSCalibur cytometer (BD Biosciences, San Jose, CA) and analyzed with CellQuest software (BD Biosciences). For the immunofluorescence analysis, cells were seeded onto glass coverslips 24 h before fixation with 3.7% formaldehyde in PBS. The fixed cells were permeabilized with 0.1% Triton X-100 and incubated with the primary antibody. Bound antibody was visualized with FITC-conjugated secondary antibody.
Results and discussion The luminal domain of US11 is sufficient to confer ER localization to the protein and interact with the MHC class I heavy chain US11 is a type-I transmembrane protein of the ER that does not contain any identifiable ER localization signals such as a KDEL motif at the carboxyl-terminal
end. In order to identify which domain of US11 is responsible for the retention of US11 in the ER, we generated truncated mutant forms of US11 and a chimeric molecule in which the transmembrane of US11 was replaced with the corresponding region of human CD4 (Fig. 1A). We examined the subcellular localization of the truncated mutants by indirect immunofluorescence microscopy. Wild-type US11 expressed in HeLa cells exhibited a typical ER staining pattern (Fig. 1B). Similar ER distribution patterns were observed for cells expressing US110 and US100, indicating that the luminal domain of US11 has ER retention properties. To further ascertain whether only the luminal domain of US11 is sufficient for protein retention in the ER, intracellular transport of the truncated mutants was monitored by assaying the sensitivity of their glycans to endoglycosidase H treatment after pulse-chase labeling. U373-MG cells stably expressing the truncated mutant proteins were pulse-labeled with [35S]methionine and then chased for 120 min. Both US110 and US100, similar to the wild-type US11, remained sensitive to endo H digestion (Fig. 1C). These results confirmed that only the luminal domain of US11 is essential for protein retention in the ER and that neither the transmembrane nor the cytoplasmic domain of US11 is required for this function. We also examined possible complex formation between mutant proteins and MHC class I molecules by means of co-immunoprecipitation using appropriate antibodies in comparison to the wild-type protein. Using anti-HC antibody, we observed co-precipitation with
Fig. 2. (A) U373-MG cells stably expressing US11, US110, US100, and UCU were stained with mAb W6/32, followed by FITC-conjugated antimouse IgG. (B) U373-MG stable cells and their parental cell line (mock) were lysed in NP-40 buffer and the lysates were subjected to SDS–PAGE under reducing conditions. After blotting to nitrocellulose, MHC class I heavy chains were detected with the K455 antibody.
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MHC class I molecules for both truncated mutants (Fig. 1D), suggesting that the luminal domain of US11 is also involved in the direct interaction with MHC class I molecules. The transmembrane domain of US11 is indispensable for inducing the degradation of MHC class I molecules To define the minimal structural domain of US11 for mediating class I molecule degradation, we examined whether the US11 mutants could down-regulate the surface level of MHC class I molecules by flow cytometry. The expression of MHC class I molecules on the cell surface was significantly reduced in wild-type US11- and US110-expressing cells but not in US100- and UCU-expressing cells (Fig. 2A). In order to address the question that the down-regulation of MHC class I molecules at the cell surface resulted from their degradation inside the cells, we then observed the total expression of MHC class I molecules in cell lysates using a polyclonal K455. The level of MHC class I molecules in the US100expressing cells was very similar to that found in the control cells (Fig. 2B, compare lanes 1 and 4). In contrast, MHC class I molecules had almost completely disappeared from the US11- and US110-expressing cells (lanes 2 and 3), suggesting that degradation of the MHC class I molecules had been induced in the latter. These results indicated that the surface down-regulation of MHC class I molecules is the consequence of a degradation of MHC class I molecules within the cell and that the transmembrane domain of US11 is required for inducing this degradation. Identifying amino acid residues in the transmembrane domain of US11 for mediating MHC class I degradation
Fig. 3. (A) Amino acid sequence of the transmembrane domain of US11. Residues in boldface were replaced with valine. (B) HeLa cells transiently expressing the indicated mutants were analyzed for intracellular localization by immunofluorescence microscopy. (C) HeLa cells transiently expressing the indicated mutants were labeled with [35S]methionine for 30 min and chased for 120 min. Cell lysates were immunoprecipitated with polyclonal US11-antisera and the immunoprecipitates were digested for 16 h with (+) or without ( ) endo H.
To characterize more precisely the sequence responsible for US11-mediated MHC class I molecule degradation, we replaced potential candidate residues in the transmembrane region with valine (Fig. 3A). For each position, the appropriate codon was altered such that the mutant encoded a valine residue. Fluorescence microscopy analysis of the constructs transiently expressed in HeLa cells showed that all of the mutants but G196V displayed the ER staining pattern (Fig. 3B). Endo-H analysis also supported the localization of the mutants in the ER (Fig. 3C). Thus, the spot-like staining pattern of G196V might be indicative of its localization in the particular sub-compartment of the ER. These results suggested that the substitution of an amino acid in the transmembrane domain does not affect subcellular localization of the US11 protein. To investigate whether US11 transmembrane point mutants induce the down-regulation of cell-surface MHC class I molecules, we monitored the cell-surface expression of MHC class I molecules by flow cytometry
with mAb W6/32. Interestingly, a reduction in the surface expression of MHC class I molecules was observed with the expression of MM186VV, Q192V, F194V, W195V, and Y1998V. The down-regulation of MHC class I molecules at the cell surface was also observed following the expression of G196V despite the abnormal subcellular localization of the latter (Fig. 4A). To correlate the down-regulation of MHC class I molecules with the intracellular events, we quantitated the level of MHC class I molecules in the total cell lysates. We found that, unlike other mutants, Q192V could not induce the degradation of MHC class I molecules (Fig. 4B), thereby demonstrating that the glutamine residue at position 192 is critical for the functioning of US11. If we also take into account that MHC class I molecules are not degraded in Q192V-expressing cells but simply fail to reach the cell surface, these molecules would appear to be sequestered in intracellular compartments.
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[4]
[5]
[6]
[7]
[8] Fig. 4. (A) HeLa cells transiently expressing the indicated mutants were stained with MAb W6/32 followed by FACS analysis. (B) HeLa cells transiently expressing the indicated mutants were lysed in 1% NP40 buffer. The proteins resolved on nitrocellulose membrane were blotted with K455.
[9]
[10]
In conclusion, we have demonstrated that the luminal domain of US11 is essential to confer ER localization to the protein and that the transmembrane domain of US11 is indispensable for mediating MHC class I molecule degradation. We have also identified glutamine at amino acid position 192 in the transmembrane domain as being critical for mediating the degradation of MHC class I molecules. Although we have been unable to elucidate the molecular mechanism, the glutamine residue at position 192 may play an important role in interacting with the components of the protein degradation machinery.
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[13]
[14]
Acknowledgments This work was supported by Korean Research Foundation Grant, KRF-2003-015-C00479. Seong-Ok Lee, Eunkyung Kim, and Youngkyun Kim were supported by the BK21 Program of the ministry of Education and Human Resources Development.
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S.-O. Lee et al. / Biochemical and Biophysical Research Communications 330 (2005) 1262–1267 [21] R. Hitt, D.H. Wolf, Der1p, a protein required for degradation of malfolded soluble proteins of the endoplasmic reticulum: topology and Der1-like proteins, FEMS Yeast Res. 4 (2004) 721–729. [22] Y. Ye, H.H. Meyer, T.A. Rapoport, The AAA ATPase Cdc48/ p97 and its partners transport proteins from the ER into the cytosol, Nature 414 (2001) 652–656. [23] U. Schubert, L.C. Anton, I. Bacik, J.H. Cox, S. Bour, J.R. Bennink, M. Orlowski, K. Strebel, J.W. Yewdell, CD4 glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway, J. Virol. 72 (1998) 2280– 2288. [24] C.E. Shamu, C.M. Story, T.A. Rapoport, H.L. Ploegh, The pathway of US11-dependent degradation of MHC class I heavy chains involves a ubiquitin-conjugated intermediate, J. Cell Biol. 147 (1999) 45–58. [25] N.W. Bays, S.K. Wilhovsky, A. Goradia, K. Hodgkiss-Harlow, R.Y. Hampton, HRD4/NPL4 is required for the proteasomal
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Functional dissection of HCMV US11 in mediating the degradation of MHC class I molecules Seong-Ok Lee a, Sujin Hwang a, Junghyun Park b, Boyoun Park a, Bong-Suk Jin a, Sungwook Lee a, Eunkyung Kim a, Sunglim Cho a, Youngkyun Kim a, Kwangmin Cho b, Jinwook Shin b, Kwangseog Ahn a,* a
Department of Biological Sciences, Seoul National University, Seoul 151-747, Republic of Korea b College of Life Sciences, Korea University, Seoul 136-701, Republic of Korea Received 10 March 2005 Available online 25 March 2005
Abstract The human cytomegalovirus (HCMV) gene product US11 dislocates MHC I heavy chains from the endoplasmic reticulum (ER) and targets them for proteasomal degradation in the cytosol. To identify the structural and functional domains of US11 that mediate MHC class I molecule degradation, we constructed truncated mutants and chimeric proteins, and analyzed these to determine their intracellular localization and their ability to degrade MHC class I molecules. We found that only the luminal domain of US11 was essential to confer ER localization to the protein but that the ability to degrade MHC class I molecules required both the transmembrane domain and the luminal domain of US11. By analyzing a series of point mutants of the transmembrane domain, we were also able to identify Gln192 and Gly196 as being crucial for the functioning of US11, suggesting that these residues may play a critical role in interacting with the components of the protein degradation machinery. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Protein degradation; Proteasome; MHC class I molecules; Human cytomegalovirus; Endoplasmic reticulum; Protein trafficking
Human cytomegalovirus (HCMV) is a b-herpesvirus that infects 70–80% of the population of the Western world [1]. It causes benign but persistent infections and contributes to the high morbidity and mortality of immuno-compromised patients, most notably organ transplant recipients and AIDS patients [2,3]. To evade cytotoxic T lymphocytes (CTLs), HCMV encodes several proteins that can independently down-modulate the surface expression of the major histocompatibility complex (MHC) class I molecules. One of these, US3, is an immediate-early HCMV protein that sequesters MHC class I molecules in the endoplasmic reticulum (ER) by preventing the transport of the assembled *
Corresponding author. Fax: +82 2 872 1993. E-mail address: [email protected] (K. Ahn).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.112
MHC class I molecules to the cell surface [4–6]. The HCMV US6 gene product prevents peptide loading of the MHC class I molecules by inhibiting transporter associated with antigen processing (TAP)-mediated peptide translocation into the ER [7–10], while the US2 and US11 gene products induce rapid export of the newly synthesized MHC class I heavy chains (HCs) out of the ER into the cytosol where the HCs are subsequently degraded by the proteasome [11,12]. The HCMV products US2 and US11 show similar general modes of action but differ in their abilities to attack allelic class I HCs and the conformation of the HC molecules [13–16]. The exact mechanism that US11 deploys to disrupt MHC class I HC cell-surface expression is unclear. It has been shown to resemble the ER-associated degradation (ERAD) by which mammalian cells dispose of
S.-O. Lee et al. / Biochemical and Biophysical Research Communications 330 (2005) 1262–1267
misfolded proteins or unassembled subunits of oligomeric proteins. Misfolded or unassembled proteins are retrotranslocated from the ER back to the cytosol via the Sec61 complex [17–19]. Some studies in yeast and mammalian cells have reported that the proteins may be transported through the transmembrane protein Derlin-1/Der1p [20–22]. Ubiquitin is then attached to the ERAD substrates in the cytosol [23,24]. The AAA ATPase p97/cdc48 and its associated cofactors, Ufd1 and Npl4, recognize poly-ubiquitinated proteins, extract them from ER membrane, and escort the ubiquitin-conjugated proteins to the proteasome [22,25–28]. In this study, we examined the structural requirements necessary for US11 to mediate the selective degradation of MHC class I molecules, with the aim of gaining an insight into the mechanisms of viral pathogenesis and proteasome-mediated protein degradation. We found that the transmembrane domain is not essential for the retention of US11 in the ER where it functions but that a specific amino acid residue at the transmembrane domain is critical for the functioning of US11.
Materials and methods Plasmid constructs. Plasmid-expressing truncated mutants and chimeric proteins were constructed as shown in Fig. 1. The respective DNA fragments were obtained by either restriction digestion or PCR amplification. Single amino acid mutations were introduced by the
1263
PCR-mediated oligonucleotide-directed protocol with a pcDNA3.1 construct containing the US11 as a template. The following oligonucleotides were used: MM186 Æ 187VV (CG CAG TAT ACG CTG GTG GTG GTG GCA GTG ATT), Q192V (G ATG ATG GTG GCA GTG ATT GTA GTG TTT TGG GGG CTG), F194V (CTG GTG GCA GTG ATT CAA GTG GTT TGG GGG CTG TAT GTG), W195V (G GCA GTG ATT CAA GTG TTT GTG GGG CTG TAT GTG AAA GG), G196V (G ATT CAA GTG TTT TGG GTG CTG TAT GTG AAA GGT TGG), and Y198V (GTG TTT TGG GGG CTG GTT GTG AAA GGT TGG CTG C). After mutagenesis, all constructs were confirmed by DNA sequencing. Cell lines and cell culture. HeLa and U373-MG astrocytoma cell lines were obtained from American Type Culture Collection (Manassas, VA) and cultured in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FBS (HyClone Laboratories, Logan, Utah), 2 mM L-glutamine, 50 U/ml penicillin, and 50 lg/ml streptomycin. To establish stable cell lines expressing US11 or US11 mutants, we cloned each cDNA into the pcDNA3.1 mammalian expression vector (Invitrogen, Carlsbad, CA) and transfected the resulting constructs into U373-MG cells by the calcium phosphate precipitation method. Stable clones were selected by adding 0.5 mg/ml G418 (Life Technologies). Antibodies. The K455 antibody recognizes the MHC class I HC and b2-microglobulin (b2m) in both assembled and non-assembled forms. Monoclonal antibody (MAb) W6/32 recognizes only the complex of HC and b2m. Polyclonal antiserum (anti-US11) for detecting US11 was raised against the synthetic peptides corresponding to the luminal N-terminal portion (residues 16 to 35) of the proteins [29]. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (IgG) was purchased from the Jackson ImmunoResearch Laboratories (West Grove, PA). Metabolic labeling and immunoprecipitation. Cells were preincubated in methionine-free DMEM with or without proteasome inhibitor. The proteasome inhibitor (LLnL; Sigma, St. Louis, MO) was dissolved in ethanol at a concentration of 50 mg/ml for use as a 1:1000
Fig. 1. (A) Schematic diagram of US11, US110, US100, and UCU. TMD, transmembrane domain; CTD, cytoplasmic domain. (B) HeLa cells expressing the US11 gene product and the mutants indicated were stained with the polyclonal US11-antisera and DAPI, and analyzed for intracellular localization by immunofluorescence microscopy. (C) U373-MG cells stably expressing US11, US110, and US100 were labeled with [35S]methionine for 30 min and chased for 120 min. Cell lysates were immunoprecipitated with US11-antisera, and the immunoprecipitates were digested for 16 h with (+) or without ( ) endo H. (D) U373-MG stable cells were incubated with proteasome inhibitor LLnL for 2 h, labeled with [35S]methionine for 30 min, lysed in 0.5% NP-40 buffer, and then immunoprecipitated by the antibodies indicated. The samples were separated by 10% SDS–polyacrylamide gel electrophoresis.
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dilution. Cells were starved for 40 min in medium lacking methionine, labeled with 0.1 mCi/ml [35S]methionine (TranS-label; NEN Life Science, Boston, MA) for 30 min, and chased in regular medium for the indicated time. Following two washes with cold phosphate-buffered saline (PBS), the cells were lysed using 1% or 0.5% Nonidet P (NP)-40 (Sigma–Aldrich) in PBS for 30 min at 4 °C. Immunoprecipitation was performed as described previously [5]. For endoglycosidase H treatment, the immunoprecipitates were digested with 3 mU endoglycosidase H (Roche, Indianapolis, IN) for 16 h at 37 °C in 50 mM NaOAc (pH 5.6), 0.3% sodium dodecyl sulfate (SDS), and 150 mM b-mercaptoethanol. Flow cytometry and immunofluorescence microscopy. Cells were washed twice with PBS containing 1% bovine serum albumin (BSA) and then incubated for 1 h at 4 °C with W6/32. Normal mouse IgG was used as a negative control. The cells were washed twice with cold PBS containing 1% BSA and then stained with FITC-conjugated goat anti-mouse IgG for 40 min. A total of 10,000-gated events were collected by the FACSCalibur cytometer (BD Biosciences, San Jose, CA) and analyzed with CellQuest software (BD Biosciences). For the immunofluorescence analysis, cells were seeded onto glass coverslips 24 h before fixation with 3.7% formaldehyde in PBS. The fixed cells were permeabilized with 0.1% Triton X-100 and incubated with the primary antibody. Bound antibody was visualized with FITC-conjugated secondary antibody.
Results and discussion The luminal domain of US11 is sufficient to confer ER localization to the protein and interact with the MHC class I heavy chain US11 is a type-I transmembrane protein of the ER that does not contain any identifiable ER localization signals such as a KDEL motif at the carboxyl-terminal
end. In order to identify which domain of US11 is responsible for the retention of US11 in the ER, we generated truncated mutant forms of US11 and a chimeric molecule in which the transmembrane of US11 was replaced with the corresponding region of human CD4 (Fig. 1A). We examined the subcellular localization of the truncated mutants by indirect immunofluorescence microscopy. Wild-type US11 expressed in HeLa cells exhibited a typical ER staining pattern (Fig. 1B). Similar ER distribution patterns were observed for cells expressing US110 and US100, indicating that the luminal domain of US11 has ER retention properties. To further ascertain whether only the luminal domain of US11 is sufficient for protein retention in the ER, intracellular transport of the truncated mutants was monitored by assaying the sensitivity of their glycans to endoglycosidase H treatment after pulse-chase labeling. U373-MG cells stably expressing the truncated mutant proteins were pulse-labeled with [35S]methionine and then chased for 120 min. Both US110 and US100, similar to the wild-type US11, remained sensitive to endo H digestion (Fig. 1C). These results confirmed that only the luminal domain of US11 is essential for protein retention in the ER and that neither the transmembrane nor the cytoplasmic domain of US11 is required for this function. We also examined possible complex formation between mutant proteins and MHC class I molecules by means of co-immunoprecipitation using appropriate antibodies in comparison to the wild-type protein. Using anti-HC antibody, we observed co-precipitation with
Fig. 2. (A) U373-MG cells stably expressing US11, US110, US100, and UCU were stained with mAb W6/32, followed by FITC-conjugated antimouse IgG. (B) U373-MG stable cells and their parental cell line (mock) were lysed in NP-40 buffer and the lysates were subjected to SDS–PAGE under reducing conditions. After blotting to nitrocellulose, MHC class I heavy chains were detected with the K455 antibody.
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MHC class I molecules for both truncated mutants (Fig. 1D), suggesting that the luminal domain of US11 is also involved in the direct interaction with MHC class I molecules. The transmembrane domain of US11 is indispensable for inducing the degradation of MHC class I molecules To define the minimal structural domain of US11 for mediating class I molecule degradation, we examined whether the US11 mutants could down-regulate the surface level of MHC class I molecules by flow cytometry. The expression of MHC class I molecules on the cell surface was significantly reduced in wild-type US11- and US110-expressing cells but not in US100- and UCU-expressing cells (Fig. 2A). In order to address the question that the down-regulation of MHC class I molecules at the cell surface resulted from their degradation inside the cells, we then observed the total expression of MHC class I molecules in cell lysates using a polyclonal K455. The level of MHC class I molecules in the US100expressing cells was very similar to that found in the control cells (Fig. 2B, compare lanes 1 and 4). In contrast, MHC class I molecules had almost completely disappeared from the US11- and US110-expressing cells (lanes 2 and 3), suggesting that degradation of the MHC class I molecules had been induced in the latter. These results indicated that the surface down-regulation of MHC class I molecules is the consequence of a degradation of MHC class I molecules within the cell and that the transmembrane domain of US11 is required for inducing this degradation. Identifying amino acid residues in the transmembrane domain of US11 for mediating MHC class I degradation
Fig. 3. (A) Amino acid sequence of the transmembrane domain of US11. Residues in boldface were replaced with valine. (B) HeLa cells transiently expressing the indicated mutants were analyzed for intracellular localization by immunofluorescence microscopy. (C) HeLa cells transiently expressing the indicated mutants were labeled with [35S]methionine for 30 min and chased for 120 min. Cell lysates were immunoprecipitated with polyclonal US11-antisera and the immunoprecipitates were digested for 16 h with (+) or without ( ) endo H.
To characterize more precisely the sequence responsible for US11-mediated MHC class I molecule degradation, we replaced potential candidate residues in the transmembrane region with valine (Fig. 3A). For each position, the appropriate codon was altered such that the mutant encoded a valine residue. Fluorescence microscopy analysis of the constructs transiently expressed in HeLa cells showed that all of the mutants but G196V displayed the ER staining pattern (Fig. 3B). Endo-H analysis also supported the localization of the mutants in the ER (Fig. 3C). Thus, the spot-like staining pattern of G196V might be indicative of its localization in the particular sub-compartment of the ER. These results suggested that the substitution of an amino acid in the transmembrane domain does not affect subcellular localization of the US11 protein. To investigate whether US11 transmembrane point mutants induce the down-regulation of cell-surface MHC class I molecules, we monitored the cell-surface expression of MHC class I molecules by flow cytometry
with mAb W6/32. Interestingly, a reduction in the surface expression of MHC class I molecules was observed with the expression of MM186VV, Q192V, F194V, W195V, and Y1998V. The down-regulation of MHC class I molecules at the cell surface was also observed following the expression of G196V despite the abnormal subcellular localization of the latter (Fig. 4A). To correlate the down-regulation of MHC class I molecules with the intracellular events, we quantitated the level of MHC class I molecules in the total cell lysates. We found that, unlike other mutants, Q192V could not induce the degradation of MHC class I molecules (Fig. 4B), thereby demonstrating that the glutamine residue at position 192 is critical for the functioning of US11. If we also take into account that MHC class I molecules are not degraded in Q192V-expressing cells but simply fail to reach the cell surface, these molecules would appear to be sequestered in intracellular compartments.
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[4]
[5]
[6]
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[8] Fig. 4. (A) HeLa cells transiently expressing the indicated mutants were stained with MAb W6/32 followed by FACS analysis. (B) HeLa cells transiently expressing the indicated mutants were lysed in 1% NP40 buffer. The proteins resolved on nitrocellulose membrane were blotted with K455.
[9]
[10]
In conclusion, we have demonstrated that the luminal domain of US11 is essential to confer ER localization to the protein and that the transmembrane domain of US11 is indispensable for mediating MHC class I molecule degradation. We have also identified glutamine at amino acid position 192 in the transmembrane domain as being critical for mediating the degradation of MHC class I molecules. Although we have been unable to elucidate the molecular mechanism, the glutamine residue at position 192 may play an important role in interacting with the components of the protein degradation machinery.
[11]
[12]
[13]
[14]
Acknowledgments This work was supported by Korean Research Foundation Grant, KRF-2003-015-C00479. Seong-Ok Lee, Eunkyung Kim, and Youngkyun Kim were supported by the BK21 Program of the ministry of Education and Human Resources Development.
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