Calreticulin-dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules
2009; Springer Nature; Volume: 28; Issue: 23 Linguagem: Inglês
10.1038/emboj.2009.296
ISSN1460-2075
AutoresChristopher M. Howe, Malgorzata Garstka, Mohammed S. Al‐Balushi, Esther Ghanem, Antony N. Antoniou, Susanne Fritzsche, Gytis Jankevicius, Nasia Kontouli, Clemens Schneeweiß, Anthony P. Williams, Tim Elliott, Sebastian Springer,
Tópico(s)Adenosine and Purinergic Signaling
ResumoArticle8 October 2009free access Calreticulin-dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules Christopher Howe Christopher Howe Cancer Sciences Division, University of Southampton School of Medicine, Southampton, UK Search for more papers by this author Malgorzata Garstka Malgorzata Garstka Department of Biochemistry and Cell Biology, Jacobs University, Bremen, GermanyPresent address: Division of Tumor Biology, The Netherlands Cancer Institute, P.O. Box 90203, 1066BE Amsterdam, The Netherlands Search for more papers by this author Mohammed Al-Balushi Mohammed Al-Balushi Department of Biochemistry and Cell Biology, Jacobs University, Bremen, Germany Department of Microbiology and Immunology, Sultan Qaboos University, Muscat, Oman Search for more papers by this author Esther Ghanem Esther Ghanem Department of Biochemistry and Cell Biology, Jacobs University, Bremen, Germany Search for more papers by this author Antony N Antoniou Antony N Antoniou Cancer Sciences Division, University of Southampton School of Medicine, Southampton, UKPresent address: Department of Immunology & Molecular Pathology, Windeyer Institute of Medical Science, University College London, London W1P 6DB, UK Search for more papers by this author Susanne Fritzsche Susanne Fritzsche Department of Biochemistry and Cell Biology, Jacobs University, Bremen, Germany Search for more papers by this author Gytis Jankevicius Gytis Jankevicius Department of Biochemistry and Cell Biology, Jacobs University, Bremen, Germany Search for more papers by this author Nasia Kontouli Nasia Kontouli Cancer Sciences Division, University of Southampton School of Medicine, Southampton, UK Search for more papers by this author Clemens Schneeweiss Clemens Schneeweiss Department of Biochemistry and Cell Biology, Jacobs University, Bremen, Germany Search for more papers by this author Anthony Williams Anthony Williams Cancer Sciences Division, University of Southampton School of Medicine, Southampton, UK Search for more papers by this author Tim Elliott Corresponding Author Tim Elliott Cancer Sciences Division, University of Southampton School of Medicine, Southampton, UKJoint senior authors. Search for more papers by this author Sebastian Springer Corresponding Author Sebastian Springer Department of Biochemistry and Cell Biology, Jacobs University, Bremen, GermanyJoint senior authors. Search for more papers by this author Christopher Howe Christopher Howe Cancer Sciences Division, University of Southampton School of Medicine, Southampton, UK Search for more papers by this author Malgorzata Garstka Malgorzata Garstka Department of Biochemistry and Cell Biology, Jacobs University, Bremen, GermanyPresent address: Division of Tumor Biology, The Netherlands Cancer Institute, P.O. Box 90203, 1066BE Amsterdam, The Netherlands Search for more papers by this author Mohammed Al-Balushi Mohammed Al-Balushi Department of Biochemistry and Cell Biology, Jacobs University, Bremen, Germany Department of Microbiology and Immunology, Sultan Qaboos University, Muscat, Oman Search for more papers by this author Esther Ghanem Esther Ghanem Department of Biochemistry and Cell Biology, Jacobs University, Bremen, Germany Search for more papers by this author Antony N Antoniou Antony N Antoniou Cancer Sciences Division, University of Southampton School of Medicine, Southampton, UKPresent address: Department of Immunology & Molecular Pathology, Windeyer Institute of Medical Science, University College London, London W1P 6DB, UK Search for more papers by this author Susanne Fritzsche Susanne Fritzsche Department of Biochemistry and Cell Biology, Jacobs University, Bremen, Germany Search for more papers by this author Gytis Jankevicius Gytis Jankevicius Department of Biochemistry and Cell Biology, Jacobs University, Bremen, Germany Search for more papers by this author Nasia Kontouli Nasia Kontouli Cancer Sciences Division, University of Southampton School of Medicine, Southampton, UK Search for more papers by this author Clemens Schneeweiss Clemens Schneeweiss Department of Biochemistry and Cell Biology, Jacobs University, Bremen, Germany Search for more papers by this author Anthony Williams Anthony Williams Cancer Sciences Division, University of Southampton School of Medicine, Southampton, UK Search for more papers by this author Tim Elliott Corresponding Author Tim Elliott Cancer Sciences Division, University of Southampton School of Medicine, Southampton, UKJoint senior authors. Search for more papers by this author Sebastian Springer Corresponding Author Sebastian Springer Department of Biochemistry and Cell Biology, Jacobs University, Bremen, GermanyJoint senior authors. Search for more papers by this author Author Information Christopher Howe1,‡, Malgorzata Garstka2,‡, Mohammed Al-Balushi2,3, Esther Ghanem2, Antony N Antoniou1, Susanne Fritzsche2, Gytis Jankevicius2, Nasia Kontouli1, Clemens Schneeweiss2, Anthony Williams1, Tim Elliott 1 and Sebastian Springer 2 1Cancer Sciences Division, University of Southampton School of Medicine, Southampton, UK 2Department of Biochemistry and Cell Biology, Jacobs University, Bremen, Germany 3Department of Microbiology and Immunology, Sultan Qaboos University, Muscat, Oman ‡These authors contributed equally to this work *Corresponding authors: Biochemistry and Cell Biology, School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany. Tel.: +49 421 200 3243; Fax: +49 421 200 3249; E-mail: [email protected] Sciences Division, Somers Cancer Research Building, University of Southampton School of Medicine, Mailpoint 824, Southampton General Hospital, Southampton SO16 6YD, UK. Tel.: +44 2380 796193; Fax: +44 2380 795152; E-mail: [email protected] The EMBO Journal (2009)28:3730-3744https://doi.org/10.1038/emboj.2009.296 Present address: Division of Tumor Biology, The Netherlands Cancer Institute, P.O. Box 90203, 1066BE Amsterdam, The Netherlands PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin-deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub-optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER–Golgi intermediate compartment (ERGIC) and the cis-Golgi, together with sub-optimally loaded class I molecules. Calreticulin that lacks its C-terminal KDEL retrieval sequence assembles with the peptide-loading complex but neither retrieves sub-optimally loaded class I molecules from the cis-Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide. Introduction Major histocompatibility complex (MHC) class I molecules are used by the immune system to survey the intracellular proteome of all nucleated cells for the presence of viruses, parasites, and tumor-specific antigens. They bind peptide fragments of eight to ten amino acids in the lumen of the endoplasmic reticulum (ER) and transport them to the plasma membrane, where they are presented to cytotoxic T lymphocytes (CTLs). Specific high-affinity peptides that fulfil the canonical binding requirements for length and sequence increase the conformational stability of class I molecules, leading to long-lived peptide-class I complexes in vivo (Elliott et al, 1991), which are generally required for efficient CTL responses. The binding of high-affinity peptides to class I molecules within the ER is an iterative process that depends on an assembly of proteins called the peptide-loading complex (PLC) (Sadasivan et al, 1996). This comprises the transporter associated with antigen processing (TAP) heterodimer that translocates peptides from the cytosol to the ER lumen in an ATP-dependant manner; the soluble lectin chaperone calreticulin (CRT); the protein disulphide isomerases ERp57 and PDI (Gao et al, 2002; Garbi et al, 2006; Park et al, 2006); and the MHC class I-specific accessory protein, tapasin (Sadasivan et al, 1996). MHC class I molecules that have not yet bound optimal high-affinity peptide (and which are called here sub-optimally loaded or peptide-receptive) cycle between the ER and the cis side of the Golgi apparatus; they can proceed to the cell surface only when they become bound to a high-affinity peptide (Garstka et al, 2007). Although the molecular mechanism of the regulation of class I trafficking and quality control is unclear, we and others have proposed that the PLC, or some of its components, may accompany sub-optimally loaded class I molecules to the ER–Golgi intermediate compartment (ERGIC) or the cis-Golgi and help retrieve it to the ER (Wright et al, 2004). In this scenario, the retrieval of the PLC or of its individual members may be mediated by the putative ER retrieval sequences found in calreticulin (KDEL in the single-letter amino acid code; Opas et al, 1991), tapasin (KKXX; Ortmann et al, 1997), and possibly ERp57 (QEDL; Bennett et al, 1988; Mazzarella et al, 1994). Importantly, such signals are not apparent in MHC class I heavy chains themselves. Calreticulin binds proteins that carry the glycan-processing intermediate Glc1Man7−9GlcNAc2 (Ware et al, 1995; Spiro et al, 1996; Vassilakos et al, 1998). This glycan moiety is maintained on unfolded polypeptides in the ER by a glucosyltransferase that is specific for unfolded proteins, leading to the binding of the protein to calreticulin (and/or calnexin) until it has acquired its folded conformation (Sousa et al, 1992; Hammond et al, 1994; Peterson and Helenius, 1999; Ritter et al, 2005; Caramelo and Parodi, 2007). Direct protein–protein interactions also contribute to the chaperone activity of calreticulin and thus to MHC class I folding (Ireland et al, 2008). The role of calreticulin in MHC class I antigen presentation has been studied in calreticulin-deficient cells derived from knockout mouse embryos. Interestingly, in such cells, MHC class I molecules are transported to the cell surface loaded with low-affinity peptides. As the PLC can form in the absence of calreticulin, this suggests a function for calreticulin in the quality control of class I–peptide complexes. In addition, calreticulin-deficient cells cannot efficiently retain sub-optimally loaded class I molecules inside the cell; instead, class I molecules move to the surface with accelerated kinetics (Gao et al, 2002). This has led us to propose that calreticulin may determine the localization of sub-optimally loaded class I molecules. In this study, we investigate the role of calreticulin in the intracellular localization of sub-optimally loaded MHC class I molecules. We find that calreticulin does not prevent them from exiting the ER, but that it travels to the Golgi apparatus where it co-localizes with them and that its retrieval from there to the ER—by means of its own C-terminal KDEL retrieval sequence—is required for their retrieval to the ER and for efficient presentation of a model antigen. Remarkably, this function of calreticulin seems to be independent of its role in the PLC. Results Loss of the KDEL sequence leads to the secretion of calreticulin To investigate the role of calreticulin in the intracellular localization of MHC class I molecules, we used the murine embryonic fibroblast cell line K42, which carries a homozygous deletion of calreticulin (Nakamura et al, 2001), to express two variants of calreticulin: a deletion of the C-terminal KDEL retrieval sequence (termed CRT-HAΔKDEL), and a mutation of the KDEL sequence to KDEV (CRT-HAKDEV) (Andres et al, 1990; Haugejorden et al, 1991). Both proteins contained an internal hemagglutinin (HA) tag for detection at the very C terminus of CRT-HAΔKDEL and just before the KDEL sequence in CRT-HAKDEV. As controls, we used the constructs CRT-KDEL (wild-type calreticulin) and CRT-HAKDEL (wild-type calreticulin with an HA tag inserted before the KDEL sequence; Figure 1A). Transfected cells were cloned to near-homogeneous expression of the co-transduced truncated nerve growth factor receptor, resulting in a uniform expression of calreticulin constructs (data not shown), and were then assessed for expression levels of calreticulin constructs by western blotting (Figure 1B). None of the constructs was found to be produced at levels significantly above that of endogenous calreticulin in the syngeneic wild-type K41. Importantly, the steady-state levels of CRT-HAKDEV and CRT-HAΔKDEL proteins were only 33 and 19% of CRT-HAKDEL, respectively, suggesting that they may be secreted from the cells. Figure 1.The calreticulin retention mutant HAΔKDEL is deficient in antigen presentation. (A) Rat calreticulin (CRT) constructs used in this study. Hemagglutinin (HA) tags were inserted as indicated. (B) Expression of the calreticulin constructs in K42 cells. Immunoblot of cell lysates probed with anti-calreticulin (left panel) and anti-HA (right panel) antibodies. Glycerinaldehyde phosphate dehydrogenase (GAPDH) was used as a loading control. In this and the following panels, KDEL, HAKDEL, HAΔKDEL, etc. stand for K42 cells expressing the respective calreticulin construct. The bar chart shows the ratio of calreticulin to GAPDH signals, normalized to unity for HAKDEL independently in each panel, to provide a comparison between the values in the two panels. (C) The HAΔKDEL construct is secreted into the supernatant medium. Immunoblot from cell lysate (lys) and cell supernatant (sn) collected at the start of the experiment (day 0), after one (day 1), and after two days (day 2). Calreticulin and constructs were detected by western blotting with anti-calreticulin (for K41 and KDEL) or anti-HA antibodies. Numbers on the right show the ratio of supernatant to lysate signals on day 2. Equivalent amounts of lysate and supernatant were applied in all samples (see the Materials and methods section), but as the supernatants were analysed on different membranes, direct comparison of the band strengths between blots is not possible. (D) FACS analysis of the surface presentation of SIINFEKL peptide on H-2Kb (detected using MAb 25D1.16) at different GFP–ubiquitin–SIINFEKL expression levels (x-axis). Eight sectors used for quantification in (E) are separated by vertical lines. (E) Analysis of data from 1D. For each of the eight GFP intensity sectors of each sample in 1D, the mean intensity of MAb 25.D1.16 staining was calculated and plotted against the GFP mean fluorescence. Controls are K42 cells transfected with GFP only (no peptide) and K42 stained without first antibody. Download figure Download PowerPoint To estimate the loss of intracellular retention of the calreticulin variants, we measured their accumulation in the culture supernatant (Figure 1C). Although CRT-KDEL and CRT-HAKDEL were efficiently retained inside the cells, CRT-HAΔKDEL was secreted, as observed previously by Sönnichsen et al (1994). In contrast, CRT-HAKDEV was only secreted to a small extent, suggesting that the C-terminal KDEV sequence retains part of the KDEL function. This was surprising because in other studies, a KDEL-to-KDEV change abolished the retention of some proteins (Tang et al, 1992). We conclude that the HA tag does not interfere with the retention of calreticulin, and that KDEL-mediated retrieval is required for keeping calreticulin inside the cell. Loss of the KDEL sequence of calreticulin leads to impaired presentation of a model antigen We next asked how the loss of the KDEL sequence of calreticulin would affect the loading of peptides onto class I molecules, and how this would compare with the lack of the entire calreticulin protein. To measure the presentation of an endogenous model peptide, we expressed a chimeric construct of the green fluorescent protein (GFP) and the H-2Kb-binding peptide, SIINFEKL, bridged by ubiquitin. When the construct is produced in cells, the SIINFEKL peptide is released by the ubiquitin C-terminal hydrolase. The peptide is then available for transport into the ER, loading onto class I molecules, and subsequent presentation at the cell surface; its original amount in the cell can be deduced from the intensity of GFP fluorescence (Neijssen et al, 2005). We detected the complex of SIINFEKL and H-2Kb at the cell surface through flow cytometry with the SIINFEKL-specific monoclonal antibody 25D1.16 (Porgador et al, 1997), and we simultaneously recorded GFP fluorescence in these cells. This enabled us to relate the amount of surface presentation of the SIINFEKL peptide to its intracellular concentration, and thus calculate the efficiency of its presentation through class I molecule (Figure 1D and E). Although in the group of cells with the lowest GFP expression, the signals for all calreticulin-expressing cells showed the same background (which is expected as the antibody only recognizes the introduced SIINFEKL peptide), we found that when the peptide was expressed, K41 cells were far more efficient than K42 at processing and presenting SIINFEKL, consistent with published data (Gao et al, 2002). CRT-HAKDEL restored the presentation of SIINFEKL to 60–80% of the wild type at different intracellular peptide levels, with the incomplete restoration seen perhaps due to the species difference between rat calreticulin and mouse cells, or the genetic variation between K41 and K42 cells outside the calreticulin locus. Importantly, in striking contrast to the full-length construct, CRT-HAΔKDEL did not increase the loading above that seen for K42. Thus, the KDEL Golgi-to-ER retrieval sequence of calreticulin is essential for optimal peptide loading over a range of endogenous peptide concentrations. Interestingly, the debilitating effect of the loss of calreticulin (or its KDEL tail) on peptide loading can be overcome by an increased antigen dose, as a two-fold increase in GFP–ubiquitin–peptide expression leads to a restoration of the surface levels of the peptide–class I complex. This may be the reason why some (presumably the most abundant) antigens are calreticulin-independent for their presentation to T cells (Gao et al, 2002). Loss of the KDEL sequence of calreticulin does not impair the PLC One possible reason why the KDEL sequence of calreticulin is necessary for efficient antigen presentation is that its absence may impair the incorporation of calreticulin into the PLC and/or the function of the PLC in general, which would in turn affect peptide loading and class I localization. To assess the composition of the PLC in the presence of different calreticulin variants, we lysed the cells in digitonin (which preserves the weak interactions in the loading complex (Diedrich et al, 2001)) and immunoprecipitated the PLC with an anti-TAP1 antibody. The co-precipitating MHC class I heavy chain, tapasin, ERp57, and calreticulin were then detected by western blotting. Figure 2 shows that CRT-HAΔKDEL was detected in the PLC at normal levels, showing that the KDEL sequence is not required for the incorporation of calreticulin into the PLC. When we investigated the association of ERp57, a direct binding partner of calreticulin, with the PLC, we observed variable levels in K42 cells, but never in K41 cells or in K42 transfected with calreticulin constructs (Figure 2A). This variation in K42 cells may be due to a loss of stability of the PLC when calreticulin is absent, such as a more rapid equilibrium of ERp57 association to and dissociation from tapasin in K42 cells. Indeed, when we inhibited disulphide exchange using the alkylating agent methyl methanethiosulfonate (MMTS) before lysis to stabilize the PLC, the majority of ERp57 was consistently detected as a disulphide-bonded conjugate with tapasin as reported previously (Peaper et al, 2005), both in K41 cells and in K42 cells expressing the calreticulin constructs (Figure 2B). Despite the difference in the steady-state levels of CRT-HAKDEL and CRT-HAΔKDEL (Figure 1B), calreticulin was precipitated in equal amounts from the PLC of cells expressing either of the constructs (Figure 2A). We conclude that the assembly of a functional PLC occurs even in the absence of the C-terminal KDEL sequence of calreticulin, and that presence of calreticulin in the PLC alone does not warrant efficient peptide loading of class I molecules. Figure 2.The calreticulin mutant HAΔKDEL is incorporated normally into the peptide-loading complex. (A) Immunoprecipitation of peptide-loading complexes from lysates of the indicated cell lines (left panel: anti-TAP1 antiserum, detection of the component proteins by immunoblotting). A sample of each cell lysate (corresponding to 104 cells) was analysed in the same way (right panel). (B) ERp57 is present in the PLC of CRT-ΔKDEL containing cells as a disulphide-bonded conjugate with tapasin. Cells were pre-treated with the alkylating agent MMTS before lysis, and anti-TAP1 immunoprecipitates were separated by non-reducing (to detect the disulphide-bonded ERp57–tapasin conjugate) or reducing SDS–PAGE as indicated, followed by immunoblotting. Download figure Download PowerPoint Calreticulin deficiency does not alter the packaging of class I into COPII vesicles Calreticulin can retain associated proteins inside the cell (Molinari et al, 2004). We therefore hypothesized that the effect of calreticulin on class I trafficking and antigen presentation may, at least in part, be due to such a localizing effect of calreticulin on sub-optimally loaded class I, that is, calreticulin may redirect those class I molecules that are associated with it to the ER. Since sub-optimally loaded class I molecules cycle between ER and cis-Golgi (Garstka et al, 2007), we hypothesized that calreticulin may act either at the level of exit of class I molecules from the ER, impeding their packaging into COPII vesicles (which form at ER exit sites and transport proteins between the ER and the ERGIC (Lee and Miller, 2007)); or else, that it may act at the level of the Golgi, promoting the packaging of sub-optimally loaded class I molecules into retrograde COPI vesicles for retrieval to the ER. To distinguish between these possibilities, we asked whether packaging of endogenous H-2Db and H-2Kb class I molecules into COPII vesicles was affected by the lack of calreticulin. Microsomal membranes were prepared from radiolabelled K42 CRT-KDEL and K42 cells, incubated with cytosol as a source of COPII components and with ATP and GTP to stimulate COPII vesicle formation, and COPII vesicles were isolated by differential centrifugation as described previously by Garstka et al (2007). The vesicles were lysed overnight in the presence or absence of specific peptide (FAPGNYPAL, which binds to both Db and Kb molecules). In the absence of exogenously added peptide, peptide-receptive class I molecules dissociate into heavy chain and β2m (Townsend et al, 1989), whereas when peptide is added, they are converted to the peptide-bound form. We then immunoprecipitated class I molecules with the conformation-specific monoclonal antibodies: B22.249 (for peptide-bound H2-Db) and Y3 (for peptide-bound H2-Kb), treated the samples with endoglycosidase F1 (EndoF1) (which has the same substrate specificity as EndoH; (Trimble and Tarentino, 1991)), and detected class I molecules by SDS–PAGE and autoradiography (Figure 3A). Vesicle samples lysed in the presence of peptide gave the total amount of class I (peptide occupied plus peptide receptive), whereas vesicles lysed in the absence of peptide gave only those class I molecules that had been loaded with high-affinity peptide before vesicle budding (Garstka et al, 2007). To compare and average multiple experiments, we standardized all packaging efficiencies to those of the Na+/K+ ATPase, a protein of the plasma membrane that is exported well from the ER. The class I molecules from the vesicle fraction were indeed contained in COPII vesicles (and not in another kind of vesicle) because in the presence of the dominant inhibitor of COPII budding, Sar1 (T39N, Barlowe et al, 1994), much smaller amounts were immunoprecipitated. Figure 3.Calreticulin does not influence packaging of class I molecules into COPII vesicles. (A) COPII vesicles (lanes 1–7) were generated in an in vitro reaction from K42 CRT-KDEL (top three panels) or K42 cells (bottom three panels). Controls in this reaction were the omission of cytosol (lane 4) or ATP (lane 5), or addition of dominant-negative Sar1 (T39N; lanes 6 and 7). COPII vesicles or the corresponding donor microsome membranes (after the reaction; lane 8–11) were lysed with detergent (in the presence of 10 μM peptide as indicated in lanes 3, 7, and 9), and H-2Db, H-2Kb, and Na+/K+ ATPase were sequentially immunoprecipitated from the lysates. Immunoprecipitates were treated with EndoF1 (lanes 2–9), PNGase (lane 10), or no glycosidase as indicated. Lane 7 was moved to a different position of the gel to facilitate comparisons (and is flanked by white lines to indicate this fact). The numbers below the panels are quantifications of the EndoF1-sensitive (pre-cis-Golgi) bands. The position of the class I molecules with undigested glycans on the gels (visible especially in the last lane in the donor microsomes) is indicated with an asterisk. One out of three independent experiments is shown here. (B) COPII packaging efficiency of H-2Db and H-2Kb in K42 CRT-KDEL cells is the same for peptide-occupied class I (black bars) and total class I molecules (white bars). Evaluation of the three independent experiments from (A). Packaging efficiencies of the peptide-occupied (lane 2 over lane 8) and total (lane 3 over lane 9) class I molecules were normalized to the packaging efficiency of the Na+/K+ ATPase in same reactions (see the Materials and methods section for a detailed description). The error bars show the s.e.m. values. (C) COPII packaging of all forms of H-2Db and H-2Kb is higher in K42 CRT-KDEL (black bars) than in K42 cells (white bars). Evaluation of the three independent experiments mentioned in (A). Packaging efficiencies of total class I molecules (lane 3 over lane 9) were normalized to the packaging efficiency of the Na+/K+ ATPase in same reactions, and are shown here compared between K42 CRT-KDEL and K42 (see the Materials and methods section for a detailed description). The error bars show the s.e.m. values. Download figure Download PowerPoint For both H-2Db and Kb, the rate of incorporation into the vesicles of the peptide-occupied forms was not significantly different from that of total class I molecules in K42 CRT-KDEL cells (Figure 3B). Thus, at the level of the ER, there is no distinction in the rate of COPII packaging of peptide-occupied and peptide-receptive forms of class I molecules. This agrees with our previous observations on HLA-A*0201, HLA-B*5101, and H-2 Kb (Garstka et al, 2007). As expected, in K42 cells, there was a much higher proportion (about 85%) of peptide-receptive class I molecules than in K42 CRT-KDEL (about 50%; in Figure 3A, the difference between band strengths in lanes 9 and 8 corresponds to the amount of peptide-receptive class I molecules). Nevertheless, these peptide-receptive molecules were not exported more efficiently in K42 cells, that is, in the absence of calreticulin; in contrast, in K42 cells, export of total Db and total Kb from the ER was actually decreased (Figure 3C). Interestingly, the export of Kb molecules from the ER was significantly more efficient than that of Db, mirroring the faster trafficking of H-2Kb in vivo. These findings demonstrate that calreticulin does not delay the export of peptide-receptive class I molecules from the ER. Calreticulin exits the ER and is found accumulating in the cis-Golgi where it co-localizes with peptide-receptive class I molecules We next investigated the alternative hypothesis that, in a manner analogous to the chaperone protein, BiP/GRP78 (Pelham, 1988; Schweizer et al, 1991; Hammond and Helenius, 1994), calreticulin may travel to the Golgi apparatus together with unfolded proteins and retrieve them back to the ER. In agreement with this proposition, we detected small amounts of endogenous calreticulin in COPII vesicles of K41 cells (Figure 4A). In contrast, using immunofluorescence microscopy, we were unable to detect post-ER localization of either endogenous calreticulin in K41 or of CRT-HAKDEL in K42 cells, perhaps because its retrieval is very efficient, resulting in a low steady-state concentration in the Golgi (Supplementary Figure S5). A GFP fusion of calreticulin (Snapp et al, 2006) that is easier to detect than the endogenous calreticulin, was accumulated in a post-ER compartment in COS-1 cells, and in K41 cells, an accumulation was visible, although weakly (Figure 4B). The detection of these weak accumulations in post-ER compartments was difficult because of the strong ER background. We reasoned that overexpression of sub-optimally loaded class I molecules may move a visible amount of endogenous calreticulin to a post-ER compartment, in analogy to the situation described by Hammond and Helenius (1994), in which overexpression of the vesicular stomatitis virus glycoprotein (VSV-G) led to a movement of BiP to the Golgi apparatus. This proposition was testable since we had found earlier that murine class I molecules, when overexpressed in primate fibroblast cells, are inefficiently loaded with peptide and
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