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Originally published In Press as doi:10.1074/jbc.M602248200 on July 29, 2006

J. Biol. Chem., Vol. 281, Issue 40, 30063-30071, October 6, 2006
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Ubiquitination of MHC Class I Heavy Chains Is Essential for Dislocation by Human Cytomegalovirus-encoded US2 but Not US11*

Gerco C. Hassink, Martine T. Barel, Sjaak B. Van Voorden, Marjolein Kikkert, and Emmanuel J. Wiertz1

From the Department of Medical Microbiology, Leiden University Medical Center, Box 9600, 2300 RC Leiden, The Netherlands

Received for publication, March 9, 2006 , and in revised form, July 17, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The human cytomegalovirus-encoded glycoproteins US2 and US11 target newly synthesized major histocompatibility complex class I heavy chains for degradation by mediating their dislocation from the endoplasmic reticulum back into the cytosol, where they are degraded by proteasomes. A functional ubiquitin system is required for US2- and US11-dependent dislocation of the class I heavy chains. It has been assumed that the class I heavy chain itself is ubiquitinated during the dislocation reaction. To test this hypothesis, all lysines within the class I heavy chain were substituted. The lysine-less class I molecules could no longer be dislocated by US2 despite the fact that the interaction between the two proteins was maintained. Interestingly, US11 was still capable of dislocating the lysine-less heavy chains into the cytosol. Ubiquitination does not necessarily require lysine residues but can also occur at the N terminus of a protein. To investigate the potential role of N-terminal ubiquitination in heavy chain dislocation, a lysine-less ubiquitin moiety was fused to the N terminus of the class I molecule. This lysine-less fusion protein was still dislocated in the presence of US11. Ubiquitination could not be detected in vitro, either for the lysine-less heavy chains or for the lysine-less ubiquitin-heavy chain fusion protein. Our data show that although dislocation of the lysineless class I heavy chains requires a functional ubiquitin system, the heavy chain itself does not serve as the ubiquitin acceptor. This finding sheds new light on the role of the ubiquitin system in the dislocation process.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A key step in the mammalian defense against viral infections is the presentation of virus-encoded peptides by MHC2 class I molecules to cytotoxic T-cells. The peptides presented by the class I molecules are frequently derived from newly synthesized proteins, degraded by proteasomes (1, 2). In case of a virus infection, a proportion of the peptides will be of viral origin. This system allows efficient detection and elimination of the infected cells by cytotoxic T-cells. HCMV encodes two glycoproteins, US2 and US11, which cause rapid degradation of newly synthesized MHC class I heavy chains by mediating their retrograde transport or "dislocation" from the ER into the cytosol, where they are degraded by proteasomes (3, 4). In this way, HCMV eludes the surveying cytotoxic T-cells.

The disposal of ER proteins via dislocation into the cytosol, followed by proteasomal degradation, is a common feature of all eukaryotic cells. The US2- and US11-mediated degradation of MHC class I molecules has been instrumental in elucidating several mechanistic aspects of this pathway (3-16). A wide variety of ER-luminal proteins are degraded through dislocation to the cytosol. They include mutated and/or misfolded proteins such as CFTR{Delta}F508 and CPY*, oxidized proteins, and noncomplexed, redundant subunits of multimeric complexes such as TCR{alpha} chains (reviewed in Refs. 17-19), MHC class I heavy chains (20), and MHC class II {alpha} and beta chains (21, 22). In addition, the expression of various proteins is regulated through dislocation and proteasomal degradation, e.g. 3-hydroxy-3-methylglutaryl-coenzyme A reductase and apo-lipoprotein B100 (reviewed in Ref. 17).

How exactly ER proteins are exported to the cytosol is still poorly understood. It has been suggested that misfolded proteins are transported to the cytosol via the same channel through which they entered the ER, the Sec61 translocation complex (3, 23-29). Other potential constituents of a "dislocon" are Derlin-1-3 and multimembrane-spanning E3 ligases such as HRD1, GP78, and TEB4 (16, 30-33). In the cytosol, glycoproteins are stripped of their N-linked glycans by an N-glycanase (34-37) and subsequently degraded by the proteasome in a ubiquitin-dependent manner.

A functional ubiquitin system is not only essential for the final proteasomal degradation, it has also been shown to be indispensable for the initial dislocation of ER proteins (7, 29, 38-46). Dislocation of CPY* and 3-hydroxy-3-methylglutarylcoenzyme A reductase, for example, depends on the ubiquitin ligase Hrd1p (29, 41-43). The mammalian ortholog of Hrd1p, HRD1, facilitates the dislocation of CD3{delta} and TCR{alpha} (45). Furthermore, the US11-dependent dislocation of class I heavy chains (7, 44), the dislocation of TCR{alpha} (38, 40), as well as the dislocation of the µs chain of IgM (46) and IR322 (39) all depend on a functional ubiquitin system.

Initially, lysine residues within the dislocation substrate were considered to be the main target for ubiquitination, but it is becoming increasingly clear that this is not necessarily true in all cases. Replacement of all the lysines within the TCR{alpha} chain or substitution of the lysines within the cytoplasmic tail of the MHC class I heavy chain by arginines does not affect the dislocation of these proteins (13, 38, 47). The lysine residues within the cytoplasmic tail of MHC class I are also dispensable for the ubiquitin-dependent dislocation by the {gamma}-herpesvirus 68-encoded Mk3 protein, a viral E3 ligase (48). These findings are surprising since the lysines within the cytoplasmic tail of these proteins would appear to be the most obvious targets for the cytosolic ubiquitin system. In other cases, however, cytoplasmic lysines are essential ubiquitination targets, as is illustrated by the human immunodeficiency virus Vpu-induced dislocation of CD4, which depends on lysine residues within the cytosolic domain of CD4 (49). The dislocation of the soluble ER-lumenal proteins CPY* and RI332 also depends on the ubiquitination system (39, 50, 51). In those cases, luminal residues are only accessible to the ubiquitin ligation machinery after (partial) dislocation, implying that the initial steps of the retrotranslocation reaction do not require ubiquitination of the substrate per se.

To acquire further insight into the role of the ubiquitin system in the retrograde transport of ER (glyco-)proteins, we utilized the extremely efficient dislocation of MHC class I heavy chains in the context of HCMV US2 and US11. In this model, the dislocation and the subsequent degradation of the heavy chains can be evaluated as two separate events, both temporally and spatially. We investigated whether the actual retrotranslocation requires the conjugation of ubiquitin to the target protein itself. To this end, a series of class I heavy chain mutants was constructed in which all lysine residues were substituted. To evaluate the possible contribution of the N terminus of the substrate, a lysine-less, non-cleavable ubiquitin moiety was fused to the N terminus of the class I heavy chains. Although the lysine-less class I molecules were no longer dislocated by US2, retrograde movement took place as normal in the presence of US11. Dislocation of the mutant heavy chains nevertheless required a functional ubiquitin system. Although these results confirm the essential contribution of the ubiquitin system to glycoprotein dislocation, they indicate at the same time that retrotranslocation of the substrate can take place in the absence of ubiquitination of the substrate itself.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—Constructs expressing lysine-less MHC class I molecules were prepared by PCR using primers containing lysine to arginine codon mutations (see below); HLA A2*0201 was used as a template (44). The following primers were used: 5'-GAGTGGGCCCTCACTCTCCGTGTCTCC-3' (antisense) and 5'-GGAGACACGGAGAGTGAGGGCCCACTC-3' (sense) (bp 258-284), 5'-CCTCTCTCAGGGCGATGTAATCCCTGCCGTCG-3' (antisense) and 5'-CGGCAGGGATTACATCGCCCTGAGAGAGG-3' (sense) (bp 426-457), 5'-CGCCTCCCACCTGTGCCTGGTGGTC-3' (antisense) and 5'-CAGACCACCAGGCACAGGTGGGAGG-3' (sense) (bp 493-519), 5'-TGCGTTCTGGGGGCGTCCGTGCGCTGCAGCGTCTCCCTCCCGTTC-3' (antisense) and 5'GAACGGGAGGGAGACGCTGCAGCGCACGGACGCCCCCAGAACGCA-3' (sense) (bp 591-635), 5'-ACCACAGCCGCCCACCTCTGGAAGG-3' (antisense) and 5'-GAACCTTCCAGAGGTGGGCGGCTG-3' (sense) (bp 788-815), 5'-GGGTGAGGGGCCTGGGCAAACC-3' (antisense) and 5'-AGGGTTTGCCCAGGCCCCTCAC-3' (sense) (bp 863-886), 5'-CTCCTCTTCTATCTGAGCTCCTCCTCCTCCA-3' (antisense) and 5'-GGAGGAGGAGCTCAGATAGAAGAGGAGGGAGCT-3' (sense) (bp 994-130), and flanking sequences 5'-AGACCTCGAGCAGCTGTCTCACACTCTACAAGC-3' (antisense) and 5'-TAATACGACTCACTATAGGGAGA-3' (sense).

The PCR products were fused in three additional rounds of PCR. The resulting lysine-less MHC class I constructs were cloned into the pCR2 vector using the TOPO technology (Invitrogen) and subsequently subcloned into the pLZRS-EGFP vector.3 Ubiquitin-HLA fusion constructs were generated using a fusion PCR. Ubiquitin with the glycine at position 76 mutated to valine, and its lysine-less version (52) (kindly provided by Dr. Nico Dantuma, Stockholm), were fused to wild-type or lysine-less HLA-A2. US2 (53) was subcloned into the pLZRS vector expressing an internal ribosome entry site element and truncated nerve growth factor receptor ({Delta}NGFR), using GATEWAY technology (Invitrogen).

Antibodies—MR24, a polyclonal antiserum directed against the luminal {alpha}3 domain of HLA-A2 (44), and the polyclonal anti-US11 and anti-HRD1 rabbit sera have been described previously (44, 45). Ubiquitin was detected using monoclonal antibody P4D1 (Santa Cruz Biotechnology). Anti-US2 rabbit serum (54) was kindly donated by Dr. T. Jones (Pearl River, NY). Anti-NGFR antibody was provided by Dr. M. Heemskerk (Leiden). Rabbit serum against Derlin-1 (14) was kindly donated by Dr. T. Rapoport (Boston) and Dr. Y. Ye (Washington, D.C.). The monoclonal antibody 20.4, directed against the nerve growth factor receptor (NGFR), was obtained from ATCC (cell line HB-8737).

Cells—The hamster cell lines E36 and E36ts20 (the latter referred to as TS20) (55) were maintained at 33 °C in minimum essential medium (Invitrogen) supplemented with 10% (v/v) fetal calf serum (Greiner, Alphen a/d Rijn, The Netherlands), penicillin (100 units/ml), and streptomycin (100 µg/ml; Invitrogen).

Retroviral Transduction and Fluorescence-activated Cell Sorting—Replication-deficient recombinant retroviruses were produced using the pLZRS-EGFP or -{Delta}NGFR vectors and the Phoenix-A producer cells as described.3 Stable cell lines were generated by transduction of wild-type or US11-expressing TS20 cells (44) with recombinant retrovirus encoding wild-type or mutant MHC class I molecules as well as EGFP. US2-expressing cells were generated by transduction of TS20 cells with retrovirus expressing US2 and {Delta}NGFR. A retrovirus encoding only EGFP served as a control. EGFP- and {Delta}NGFR-positive cells were sorted using a FACSVantage cell sorter (BD Biosciences). {Delta}NGFR-positive cells were stained with anti-NGFR antibody 20.4 (ATCC cell line HB-8737) and goat anti-mouseTexas Red conjugate (Jackson, WA).

Pulse-Chase Analysis, Immunoprecipitation, and SDS-PAGE—For metabolic labeling/pulse-chase experiments, cells were starved in RPMI 1640 medium (BioWhittaker) without methionine and cysteine at either 33 °C or 40 °C for ~1 h. For experiments performed at the non-permissive temperature, TS20 cells were preincubated at 40 °C for 2-4 h, prior to the pulsechase experiment. Starvation and pulse-chase incubations were all performed at the same temperature. Where indicated, proteasome inhibitor ZL3H (Peptide Institute, Osaka, Japan) was used, added during starvation at a final concentration of 20 µM. The cells were metabolically labeled with 250 µCi of 35S-labeled Redivue Promix (a mixture of L-[35S]methionine and L-[35S] cysteine) (Amersham Biosciences) per 107 cells in starvation medium. For chase samples, radioactive medium was replaced with RPMI 1640 medium supplemented with 1 mM methionine and 0.1 mM cystine (Sigma).

The cells were lysed in Nonidet P-40-containing lysis buffer as described (44). Immunoprecipitations were performed at 4 °C on cleared lysates for 2-4 h using specific antiserum and protein A-Sepharose beads (Amersham Biosciences). The beads were washed with wash buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% (v/v) Nonidet P-40) supplemented with 0.1% SDS. The digitonin lysis buffer contained 1% (w/v) digitonin, 50 mM Tris/HCl (pH 7.5), 5 mM MgCl2, 150 mM NaCl, 1 mM leupeptin, and 1 mM 4-(2-aminoethyl) benzenesul-fonyl fluoride (Roche Applied Science, Woerden, The Netherlands). The washed beads were boiled in Laemmli sample buffer (40 mM Tris/HCl, pH 8.0, 4 mM EDTA, 8%(w/v) SDS, 40%(v/v) glycerol, 10%(v/v) beta-mercaptoethanol, and 0.1% bromphenol blue) for 5-10 min. For reimmunoprecipitations, Laemmli samples containing 10 mM dithiothreitol were diluted with equal amounts of 10 mM iodoacetic acid and subsequently diluted with Nonidet P-40 lysis mix to a final volume of 1 ml. The samples were subjected to immunoprecipitation with the antibodies indicated. Samples were loaded onto SDS-polyacrylamide gels and exposed to a storage phosphorimaging screen, which was scanned in a Personal Molecular Imager FX and analyzed with Quantity One software (Bio-Rad, Veenendaal, The Netherlands).

Subcellular Fractionation—Fractionation of cells was performed essentially as described (4). In brief, about 107 cells were starved and labeled for 30 min with 35S-Redivue Promix as described above. Cells were washed and resuspended in 1 ml of homogenization buffer (0.25 M sucrose, 10 mM triethanolamine, 10 mM potassium acetate, 1 mM EDTA, pH 7.6, supplemented with protease inhibitors leupeptin (0.1 mM) and 4-(2-aminoethyl)benzenesulfonyl fluoride (10 mM)). Cells were placed on ice and homogenized using a Dounce homogenizer with a tight-fitting pestle (50 strokes). The homogenate was spun at 1,000 x g at 4 °C in an Eppendorf centrifuge for 10 min. The pellet was saved, and the supernatant was spun at 10,000 x g at 4 °C for 30 min. The pellet was again saved, and the supernatant spun at 100,000 x g at 4 °C for 1 h. The latter two centrifugations were performed in a TLA 120.2 fixed-angle rotor, operated in a Beckman Optima2 TLX ultracentrifuge. All pellets were resuspended in Nonidet P-40 lysis buffer. HLA-A2 and US11 were immunoprecipitated simultaneously from the solubilized pellets and the 100,000 x g supernatant and separated by SDS-PAGE.

In Vitro Transcription and Translation and In Vitro Ubiquitination Assays—HLA-A0201 in pCDNA3 or TOPO plasmids (Invitrogen) were linearized with XhoI and HindIII, respectively, and used for in vitro transcription with T7 polymerase (Promega). Transcripts were translated in the presence of L-[35S]methionine in rabbit reticulocyte lysate without microsomes. Reaction mixtures of 25 µl were incubated at 30 °C for 45 min. 16-µl aliquots of these samples were denatured by adding 34 µl of phosphate-buffered saline containing 1% SDS and 10 mM dithiothreitol and boiling at 95 °C for 5 min. After the addition of 1 ml of non-denaturing buffer (1% Triton X-100, 50 mM Tris/HCl pH 7.5, 300 mM NaCl, 5 mM EDTA, 0.02% sodium azide) and 10 mM iodoacetic acid, immunoprecipitations were performed as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
US2-mediated Dislocation of MHC Class I Heavy Chains Is Ubiquitin-dependent—Previous studies have shown that the dislocation of MHC class I heavy chains by US11 requires a functional ubiquitin system (44). It has become clear that US2- and US11-dependent dislocation of class I heavy chains differ in several ways. For example, US2 and US11 differ in their preference for HLA class I haplotypes and cytoplasmic tail composition of the heavy chains (53, 56, 57). The fact that US11-mediated dislocation of class I heavy chains requires Derlin-1, but US2-mediated dislocation does not, indicates that the mechanism of the dislocation reaction is also different (14, 15). Recently, it was found that signal peptide peptidase activity is critical for class I heavy chain dislocation by US2 but not by US11 (58).

To investigate whether, like US11, US2-mediated dislocation requires a functional ubiquitin system, MHC class I heavy chains were co-expressed with US2 in TS20 cells, which have a temperature-sensitive mutation within the ubiquitin activating enzyme E1 (55). The dislocation and degradation of human class I heavy chains can be recapitulated in the hamster TS20 cells (7, 44). In the absence of the US proteins, the human class I molecules can be detected at the surface of the TS20 cells.

Degradation of the class I heavy chains was compared at the permissive (33 °C) and non-permissive temperature (40 °C), in the presence of proteasome inhibitor. At the permissive temperature, dislocation of the class I heavy chains proceeded as normal (Fig. 1A, lanes 2-4). Removal of the single N-linked glycan from the class I heavy chains by cytosolic N-glycanase resulted in the emergence of a deglycosylated degradation intermediate (lanes 2-4). The latter migrates at the same level as class I heavy chains, obtained at the end of the pulse, treated with N-glycanase in vitro (lane 1). At the non-permissive temperature, the breakdown intermediate was not observed, indicating that dislocation of the class I heavy chains was inhibited (Fig. 1A, lanes 5-7).

Some loss of A2wt was observed in TS20 cells at 40 °C. Most likely, this is due to degradation via an alternative pathway. This is supported by the observation that degradation occurred in the presence of proteasome inhibitor. Moreover, no deglycosylated heavy chain degradation intermediate was observed in the course of the chase despite the presence of proteasome inhibitor (Fig. 1A, compare lanes 2-4 and 5-7). In conclusion, these results show that a functional ubiquitin system is essential for the dislocation of MHC class I heavy chains in the presence of US2.


Figure 1
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  		FIGURE 1.
Dislocation of MHC class I molecules by US2 is dependent on the ubiquitin system and requires lysine residues within the class I heavy chains. A, first, TS20 cells expressing wild-type HLA-A2 (A2WT) and US2 were incubated at either 33 °C or 40 °C for 2 h. Next, the cells were starved, pulselabeled with 35S Met/Cys for 10 min, and chased for 30 and 90 min. The latter steps were all performed in the presence of proteasome inhibitor (ZL3H) at the indicated temperatures. HLA-A2 molecules were immunoprecipitated with the MR24 anti-heavy chain antibody and separated on SDS-polyacrylamide gel (10%). The presence or absence of carbohydrate side chains is indicated as +CHO or -CHO, respectively. The t = 0 sample was treated with N-glycosidase F as described (endoF; displayed in the first lane). B, TS20 cells expressing either A2WT or A2K->R were transduced with control (c) or US2-expressing retroviruses. Cells were pulse-labeled for 10 min and chased for 30 and 90 min at 33 °C with or without the proteasome inhibitor ZL3H. MHC class I heavy chains were immunoprecipitated using the MR24 antiserum. Samples were displayed on SDS-PAGE (10%). C, graph depicting the quantified results of the pulse-chase analysis shown in panel A (results obtained in the absence of proteasome inhibitor). D, A2WT and A2K->R-expressing TS20-US2 cells were metabolically labeled for 60 min with [35S]Met/[35S]Cys in presence of the proteasome inhibitor ZL3H or at 40 °C. The cells were lysed in the presence of digitonin as described and subjected to immunoprecipitations (IP) with the HC-A2 anti-heavy chain antibody. After denaturation of the first immunoprecipitate, reimmunoprecipitations (ReIP) were performed with HC-A2 and an antiserum directed against US2.

 
Lysine-less MHC Class I Heavy Chains Are Not Dislocated by US2—MHC class I heavy chains carry a number of lysine residues that may serve as acceptor sites for ubiquitin in the course of the dislocation and degradation process. To investigate whether these lysines are required for the ubiquitin-dependent dislocation by US2, we constructed a recombinant HLA A*0201 molecule, A2K->R, in which all 13 lysines were substituted for arginines. TS20 cells were transduced with retroviruses expressing the A2K->R or A2WT heavy chains. In addition, the cells were transduced with a retrovirus expressing US2 or with a control virus. The stability of the wild-type and recombinant class I heavy chains was evaluated in pulse-chase experiments (Fig. 1B). The A2WT heavy chains were relatively stable in the absence of US2 (Fig. 1A, upper left panel, lanes 2-4). When US2 was expressed in these cells, the A2WT heavy chains were degraded rapidly (Fig. 1B, lower left panel, lanes 2-4). In the presence of the proteasome inhibitor ZL3H, a deglycosylated degradation intermediate appeared, migrating at the level of endoglycosidase F-treated heavy chains (compare lanes 1 and 5-7).

Like the A2WT heavy chains, the A2K->R molecules were stable in cells not expressing US2 (Fig. 1B, upper right panel). Contrary to the A2WT molecules, however, the A2K->R heavy chains were not degraded in the presence of US2 (Fig. 1B, lower panels, compare lanes 2-4 and 9-11). Note that some deglycosylated heavy chains emerged in control and US2 cells in the presence of proteasome inhibitor after 90 min of chase (lane 14). Apparently, some dislocation of A2K->R takes place in the TS20 cells regardless of the presence of US2. Most likely, this is related to the fact that the mutant heavy chains are recognized by the quality control system of the hamster cells and are targeted for degradation. This is a very slow process, which is not comparable with the rapid and efficient degradation of heavy chains observed in the presence of US2 (lower panel, lanes 2-4).

A quantification of the degradation of full-length heavy chains in the absence of proteasome inhibitor is shown in Fig. 1C. These results indicate that the lysine residues within MHC class I heavy chains are essential for the US2-mediated dislocation of these chains.

US2 Can Interact with Lysine-less MHC Class I Heavy Chains—The stabilization of the A2K->R molecules in the presence of US2 might be the result of a lack of interaction of the lysine-less heavy chains with US2. Although the lysines had been replaced with arginines, which have very similar characteristics as far as side chain composition, structure, and polarity are concerned, it cannot be excluded that these alterations affected the interaction with US2. Therefore, we investigated whether US2 was still capable of interacting with the lysine-less class I heavy chains in reimmunoprecipitation experiments (Fig. 1D). The experiment has been performed in the presence of proteasome inhibitor or at 40 °C to prevent degradation of the class I heavy chains. Both in the presence of proteasome inhibitor and at 40 °C, US2 could be reimmunoprecipitated from A2K->R heavy chain immunoprecipitates (lane 4). These data indicate that US2 is capable of interacting with HLA-A2 molecules lacking lysine residues. From these results, we conclude that the lysine-less MHC class I heavy chains were not insensitive to US2-mediated degradation as a result of their inability to interact with the viral protein.


Figure 2
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  		FIGURE 2.
US11-mediated dislocation of MHC class I molecules does not depend on lysine residues within the class I heavy chains. A, US11-positive TS20 cells expressing either A2WT or A2K->R were pulsed and chased as described in the legend for Fig. 1. MHC class I heavy chains were immunoprecipitated using the MR24 antiserum. Samples were analyzed on SDS-PAGE (10%). Note: The increase of MHC class I heavy chains in lane 14 is due to a loading error. B, quantification of the pulse-chase experiments performed in the absence of proteasome inhibitor (panel A, lanes 2-4 and 9-11; glycosylated heavy chains only).

 


Figure 3
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  		FIGURE 3.
US11-mediated dislocation of A2K->R is ubiquitin-dependent. TS20 cells expressing A2K->R or A2WT and US11 were preincubated at either 33 °C or 40 °C for 2 h. Subsequently, the cells were starved, pulse-labeled with [35S]Met/[35S]Cys for 10 min, and chased for 30 and 90 min at the temperatures indicated. The experiment was performed in absence of proteasome inhibitor. MHC class I heavy chains and US11 were immunoprecipitated and displayed on SDS-PAGE (10%).

 
MHC Class I Heavy Chains Lacking Lysine Residues Are Dislocated by US11 in a Ubiquitin-dependent Manner—Next, we investigated whether the lysine-less class I heavy chains could be dislocated in the presence of US11. For this purpose, US11 was co-expressed with either A2WT or A2K->R heavy chains in TS20 cells. Wild-type heavy chains were rapidly degraded in the US11-expressing cells (Fig. 2A, lanes 2-4). Interestingly, unlike US2, US11 was capable of inducing dislocation of the A2K->R molecules (Fig. 2A, lanes 9-11; Fig. 2B). Deglycosylated heavy chains emerged in the US11-expressing cells in the absence of ZL3H(lanes 8-11), indicating that proteasomal degradation of the dislocated A2K->R molecules was inhibited. However, the inhibition of degradation was not as efficient as in the presence of proteasome inhibitor (compare lanes 9-11 and 12-14). Note that in the presence of proteasome inhibitor, the dislocation became the ratelimiting step, resulting in delayed appearance of the deglycosylated degradation intermediate (compare conversion rates of glycosylated (upper band) with deglycosylated (lower band) heavy chains in lanes 9-11 and 12-14).

These results clearly show that, in contrast to US2, US11 can dislocate heavy chains that lack lysine residues. To investigate whether the dislocation of the lysine-less heavy chains requires a functional ubiquitin system nonetheless, we compared the degradation of the lysineless class I molecules in the TS20 cells at 33 and 40 °C (Fig. 3). At 33 °C, the A2K->R molecules were dislocated, and again, deglycosylated heavy chains emerged in the absence of proteasome inhibitor (lanes 1-3). At 40 °C, the dislocation of lysine-less class I molecules was inhibited completely. Likewise, the A2WT heavy chains were stabilized at the non-permissive temperature (lanes 8-10). US11 co-precipitated with the A2K->R and A2WT class I heavy chains at 40 °C (lanes 4-6 and 8-10). To confirm that the stabilization of class I heavy chains at 40 °C was indeed due to the lack of a functional ubiquitin system and not to specific effects of the elevated temperature, similar experiments were performed in the parental E36 cells, which have an intact ubiquitin system. In all cases tested, heavy chain dislocation and degradation proceeded as normal in the E36 cells at 40 °C (data not shown). In conclusion, these observations indicate that a functional ubiquitin system is essential for US11-dependent dislocation of lysine-less MHC class I heavy chains.

In the Absence of Proteasome Inhibitor, a Soluble Degradation Intermediate of A2K->R Can Be Observed in the Cytosol of US11-expressing Cells—The AAA ATPase p97-Ufd-Npl4 complex is required for membrane extraction of (partly) dislocated ER proteins that are still associated with the ER membrane. This final step might require ubiquitination of the substrate (8, 9, 39, 46). Deglycosylated lysine-less degradation intermediates of MHC class I molecules are detectable in the US11 cells (Fig. 2A and 3), but it is unknown whether these are still associated with the ER membrane. To investigate whether these deglycosylated heavy chains had been fully released into the cytosol, a cell fractionation experiment was performed (Fig. 4). Note that the experiment with the A2K->R-expressing cells was performed in the absence of proteasome inhibitor. Deglycosylated A2K->R and A2WT accumulated in the 105 x g supernatants representing the cytosolic fractions (Fig. 4, lanes 4 and 8), confirming that lysine-less class I heavy chains are indeed extracted from the ER membrane.


Figure 4
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  		FIGURE 4.
A soluble cytosolic A2K->R degradation intermediate appears in US11-postive cells. US11-positive TS20 cells expressing either A2WT or A2 K->R were labeled with [35S]Met/[35S]Cys for 30 min and subjected to subcellular fractionation using differential centrifugation as described. Pellet fractions were solubilized in Nonidet P-40 lysis mix. MHC class I heavy chains were immunoprecipitated from the pellet fractions and the 105 x g supernatant (Sup) and displayed on SDS-PAGE (10%). The experiments with the A2WT-expressing cells, but not the A2K->R-expressing cells, were performed in the presence of ZL3H. The 103 x g pellet fractions contain many unbroken cells, explaining the large amounts of class I heavy chains present in those fractions.

 
US11-mediated Dislocation of MHC Class I Does Not Require N-terminal Ubiquitination—Theoretically, the lysine-less class I heavy chain could still be ubiquitinated at its N terminus. Therefore, the role of N-terminal ubiquitination was investigated in more detail using various ubiquitin-heavy chain fusion proteins. Either wild-type ubiquitin or a lysine-less ubiquitin moiety was fused to the N terminus of the class I heavy chains. Previously, it has been shown that US11-mediated dislocation of class I molecules requires polyubiquitination (13). Attachment of lysine-less ubiquitin to the N terminus of the class I heavy chain will prevent the formation of multiubiquitin chains at this site. The C-terminal glycine 76 of ubiquitin was replaced by a valine, creating a version that is no longer sensitive to deubiquitinating enzymes (52, 59-61). Four fusion proteins were constructed, consisting of ubiquitin (Ub) or lysine-less ubiquitin (UbK->R) fused with its C terminus to the N terminus of either wild-type or lysine-less HLA-A2. The fusion proteins were stably expressed in TS20 cells using recombinant retroviruses.

In pulse-chase experiments performed with US11-negative control cells, the UbK->R-A2K->R fusion protein remained stable throughout the chase (Fig. 5A, upper left panel, lanes 2-4). In US11-expressing cells, UbK->R-A2K->R was dislocated and degraded (Fig. 5A, lower left panel, lanes 2-4). This indicates that polyubiquitination at the N terminus of the class I molecules is not essential for US11-mediated dislocation. Dislocation of UbK->R-A2K->R resulted in the immediate emergence of the deglycosylated breakdown intermediate despite the absence of proteasome inhibitor in the experiment (Fig. 5A, lanes 2-4). Apparently, like A2K->R, UbK->R-A2K->R can be dislocated into the cytosol but is degraded less efficiently, resulting in a temporary accumulation of deglycosylated heavy chains.

UbWT-A2K->R, UbK->R-A2WT, and UbWT-A2WT were all dislocated and degraded in the presence of US11 (Fig. 5A, lanes 9-11; Fig. 5B). For the UbWT-A2K->R fusion protein, very little deglycosylated class I was observed (Fig. 5A, lanes 9-11). This indicates that the N-terminal ubiquitin successfully created a ubiquitin fusion degradation substrate (52, 62). In the absence of US11, all fusion proteins were stable throughout the chase, showing that ubiquitin fusion by itself did not induce dislocation of the class I heavy chains (Fig. 5, A and B, upper panels).

The data presented so far are based on the assumption that A2K->R and UbK->R-A2K->R cannot serve as ubiquitination substrates. To test this premise, ubiquitin conjugation to A2K->R and UbK->R-A2K->R was evaluated in vitro. Translation of the MHC class I heavy chain constructs in the absence of microsomal membranes renders their hydrophobic transmembrane domains accessible to cytosolic chaperones and quality control mechanisms (18, 19), thereby promoting their degradation through the ubiquitin-proteasome pathway. Fig. 5C, left panel, shows a direct load of translated wild-type and lysine-less heavy chains. The wild-type construct produces an additional smear of high molecular weight material. Immunoprecipitation with an anti-MHC class I heavy chain antibody revealed that this smear contained class I molecules (middle panel). Subsequent reimmunoprecipitation with a poly-ubiquitin-specific antibody identified this material as ubiquitin-conjugated class I heavy chains (right panel). Ubiquitinated heavy chains were not detectable for A2K->R (Fig. 5C, lanes 2, 4, and 6).

Ubiquitination of UbK->R-A2K->R and UbWT-A2K->R was evaluated in the experiment presented in Fig. 5D. Although translation of UbK->R-A2K->R did not yield ubiquitinated material, UbWT-A2K->R resulted in a ladder of bands corresponding to heavy chains carrying one or more ubiquitin moieties. These experiments show that neither the lysine-less MHC class I heavy chains nor the UbK->R-A2K->R fusion protein serve as ubiquitination substrates in vitro.

Although these in vitro assays differ in many respects from the ubiquitination reactions taking place in intact cells, the in vitro ubiquitination experiments were nevertheless helpful in assessing the presence of ubiquitin acceptor sites within the class I heavy chains. In addition, the presence of ubiquitinated A2K->R or UbK->RA2K->R molecules has been investigated in intact cells. No such molecules were detectable (data not shown).

Altogether, these results indicate that dislocation of MHC class I heavy chains in the context of US11 does not require ubiquitination of the heavy chains themselves. However, ubiquitination promotes the degradation of dislocated heavy chains by proteasomes. Fusion of ubiquitin to the class I molecules does not accelerate their dislocation, supporting the conclusion that N-terminal ubiquitination does not play a role in US11-initiated extraction of heavy chains from the ER membrane. The finding that ubiquitinated A2K->R cannot be detected in vitro suggests that the N terminus is not a favorable ubiquitination site.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we used the HCMV US2- and US11-mediated dislocation of MHC class I heavy chains as a model to investigate the role of the ubiquitin system in the dislocation and degradation of ER proteins. We show that the lysines within the class I molecule are essential for dislocation in the context of US2. In contrast, in US11-dependent dislocation, ubiquitination of the lysines or the N terminus of the class I heavy chain does not play a role. US2 and US11, however, both require a functional E1 enzyme to effect dislocation of MHC class I molecules.


Figure 5
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  		FIGURE 5.
Dislocation of MHC class I-ubiquitin fusion proteins by US11. A, TS20 and TS20-US11 cells expressing UbK->R-A2K->R or UbWT-A2K->R fusion proteins were pulse-labeled for 10 min with [35S]Met/[35S]Cys and chased for 30 and 90 min at 33 °C with or without the proteasome inhibitor ZL3H. HLA-A2 molecules with (+CHO) or without (-CHO) carbohydrate side chain were immunoprecipitated with the MR24 anti-heavy chain serum and analyzed on an SDS-polyacrylamide gel (10%). The t = 0 samples were treated with N-glycanase F (endoF). B, experiment as in panel A, using cells expressing UbK->R-A2WT or UbWT-A2WT fusion proteins. C, A2WT and A2K->R constructs were translated in vitro in the presence of [35S]methionine in the absence of microsomes. One-third of the translation product was loaded on gel directly (left panel). Two-thirds of the translation product were subjected to immunoprecipitation with anti heavy chain antibody (MR24). One-half of the immunoprecipitate was loaded on gel (middle panel), and the other half was subjected to a subsequent reimmunoprecipitation with the monoclonal antibody P4D1, recognizing polyubiquitin chains (right panel). D, UbK->R-A2K->R and UbWT-A2K->R constructs were translated in vitro and loaded onto a 10% SDS-polyacrylamide gel.

 
Although substitution of lysine residues within the MHC class I molecules does not prohibit their dislocation by US11, it does affect the efficiency of degradation once the heavy chains have reached the cytosol. As a result, deglycosylated heavy chains become visible in the cytosol in the absence of proteasome inhibitor (Figs. 2A, 3, and 6). The breakdown intermediates appear early after synthesis. Inhibition of the catalytic activity of the proteasome results in a different pattern, with the deglycosylated heavy chains emerging at later time points during the chase (Fig. 2A). This difference in kinetics may be explained by the fact that the substitution of lysines does not block heavy chain dislocation but mainly affects the degradation of dislocated molecules by the proteasome. In contrast, inhibition of the proteolytic activity of the proteasome also seems to cause a retardation of heavy chain dislocation, resulting in the delayed appearance of the heavy chains in the cytosol and accumulation at later time points. Thus, whereas heavy chain degradation is the ratelimiting step in the case of lysineless substrates, dislocation appears to be the rate-limiting step when the catalytic activity of the proteasome is blocked. It is interesting to note that proteasome inhibitors completely inhibit the retrograde movement of other ER dislocation substrates, including tyrosinase (63), ribophorin 332 (39), proparathyroid hormone-related protein (64), TCR{alpha} (65), CD3{delta} (66, 67), and MHC class II {alpha} and beta chains (21, 22). For those substrates, dislocation and proteasomal degradation may be more closely linked than for US11-mediated degradation of class I heavy chains.

Contrary to US11, US2 requires lysines within the MHC class I heavy chain for its dislocation into the cytosol. The stabilization of the A2K->R molecules in the presence of US2 might be the result of a lack of interaction of the lysine-less heavy chains with US2. According to crystal structure data, two lysine residues occur within the US2 interaction region of the class I heavy chain. One of these, K176, is directly in contact with US2 (68). In the A2 mutants used in our study, however, the lysines were replaced with arginines, which have very similar characteristics as far as side chain composition, structure, and polarity are concerned. Our data indicate that US2 can indeed still interact with the A2K->R heavy chains. In addition, it has been shown that substitution of the lysine residues at positions 127 and 144 of HLA-A2 does not affect the ability of US2 to retain this MHC class I mutant (69). The question remains: how do lysines within the class I molecules contribute to US2-mediated dislocation? Previously, it has been shown that lysines within the cytoplasmic tail of class I heavy chains, and even the entire cytoplasmic tail, are dispensable for dislocation by US2 (47, 53, 56). Combined, these findings suggest that ER-located lysine residues of the heavy chains play a role in dislocation by US2. Theoretically, US2 could induce a partial dislocation or "prolapse" of part of the heavy chain into the cytosol, resulting in cytosolic deposition of luminal lysine residues. Although this possibility cannot be excluded, this scenario is unlikely. Alternatively, ubiquitin could serve as a ratcheting molecule during the extraction of the class I heavy chains. Previous studies have indicated that a ratcheting function of polyubiquitin alone is insufficient for dislocation of class I heavy chains by US11 (13). Whether this is also the case for US2-mediated retrotranslocation of heavy chains remains to be established.


Figure 6
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  		FIGURE 6.
Formation of the dislocation complex mediating retrograde transport of MHC class I heavy chains in the context of HCMV US11.

 
It has become clear that the dislocation of glycoproteins from the ER involves a wide array of proteins. The precise composition of the "dislocation complex" is unclear as yet and could, in fact, be flexible, depending on which substrate has to be relocated to the cytosol. In addition to Derlin-1, VCP (p97)-interacting protein (VIMP), and p97, E3 ligases may also form a part of the dislocation complex (16, 30, 31). In the case of MHC class I dislocation by US11, the reaction commences with the interaction of the viral glycoprotein with the newly synthesized class I heavy chain (Fig. 6). US11 links the class I heavy chain to the complex of Derlin-1 and the small membrane-spanning molecule VIMP (14, 15). The next step might be the recruitment of translocon subunits (3, 23-29) and ER-resident ubiquitin ligase complexes such as HERP-HRD1-SEL1L and GP78 (16, 30, 31). The transmembrane regions of the multimembrane-spanning E3 enzymes may form a conduit around the class I heavy chain, possibly together with Derlin-1 and other membrane proteins, as has been hypothesized for the twin-arginine translocons occurring in peroxisomes and plant thylakoid membranes (70, 71). The advantage of forming a dislocon after the degradation substrate has been identified is that it will be adapted to the nature of the substrate (11, 12). Such an ad hoc arrangement would also allow the inclusion of transmembrane regions of substrates such as CFTR{Delta}F508 and apolipoprotein B100 (reviewed in Ref. 17). Finally, it is possible that the p97-Ufd1-Npl4 complex and the proteasome are attracted to, or even stabilize, the dislocation complex (8, 9). Using the ATPase activity of p97 and/or the proteasome, the dislocation substrate may be extracted from the ER membrane. In the course of this process, the heavy chain is unfolded and deglycosylated by N-glycanase. The latter has been found to bind to Derlin-1 (37, 72). The fact that the dislocation reaction is dependent on a functional ubiquitin system, combined with the observation that US11-mediated dislocation of the MHC class I molecule does not involve ubiquitination of the class I heavy chain itself, suggests that the dislocation reaction requires ubiquitination of adaptor proteins that recruit other components into the dislocation complex. Thus, the ubiquitinated proteins may serve as a recognition signal for the p97-Ufd1-Npl4 complex, and possibly, for other components implicated in the retrograde movement of substrates into the cytosol.


   FOOTNOTES
 
* This study was supported by Grant RUL 1998-1791 from the Dutch Cancer Society (to G. C. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 31-71-526-3932; Fax: 31-71-524-8148; E-mail: wiertz{at}lumc.nl.

2 The abbreviations used are: MHC, major histocompatibility complex; NGFR, nerve growth factor receptor; {Delta}NGFR, truncated NGFR; ER, endoplasmic reticulum; HCMV, human cytomegalovirus; TCR, T-cell receptor; EGFP, enhanced green fluorescent protein; Ub, ubiquitin; E1, ubiquitin-activating enzyme; E3, ubiquitin-protein isopeptide ligase; WT, wild type; CHO, Chinese hamster ovary; CPY*, a mutant form of carboxyl peptidase Y. Back

3 G. Nolan, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Hidde Ploegh, Brendan Lilley, Tom Rapoport, and Yihong Ye for generously providing reagents and Kees Franke for constructing the GATEWAY vectors.



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X. Wang, R. A. Herr, W.-J. Chua, L. Lybarger, E. J.H.J. Wiertz, and T. H. Hansen
Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce ERAD of MHC-I by viral E3 ligase mK3
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 All ASBMB Journals   Molecular and Cellular Proteomics 
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Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
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