- Research article
- Open Access
Adopting the rapamycin trapping assay to track the trafficking of murine MHC class I alleles, H-2Kb
© Ghanem and Al-Balushi. 2015
Received: 1 September 2015
Accepted: 14 December 2015
Published: 29 December 2015
In mammalian cells, the quality control (QC) of properly folded proteins is monitored in the early secretory pathway, particularly in the endoplasmic reticulum (ER). Several proteins, including our protein of interest, major histocompatibility complex class I (MHC class I), can bypass the first line of ER-QC and reside in post-ER compartments in an unfolded form. Such forms entail both monomeric and dimeric structures that are devoid of peptides and thus cannot fulfill the immunological function of antigen presentation at the cell surface. MHC class I structures become mature and properly folded once loaded with the appropriate peptides in the framework of the peptide loading complex (PLC). Despite the flood of information on the diverse trafficking behavior of different MHC class I alleles, there is still controversy on the actual trajectory followed by improperly folded murine MHC class I alleles, namely H-2Kb. In this study, we employ an in vitro rapamycin trapping assay, live cell imaging, and a biochemical COPII budding approach to further investigate the trafficking of H-2Kb beyond the level of the ER.
We confirm the egress of H-2Kb in an unfolded form to a post-ER compartment from where they can cycle back to the ER. Deciphering the exact identity of the post-ER compartment by laser scanning microscopy did not only point to the existence of the ERGIC and cis-Golgi compartments as residency areas for unfolded proteins, but also to the involvement of an addional compartment, that lies in close proximity and possesses high resemblance to the aforementioned compartments. Interestingly, we were capable of showing using the same rapamycin trapping assay that H-2Kb can undergo a potential maturation event during their cycling; this is attained upon addition of peptides and trapping of accumulated post-ER molecules at the cell surface.
Our findings deepen the understanding of H-2Kb trafficking outside the ER and pave the way to decipher the role and the trafficking of certain PLC chaperones, such as tapasin, throughout H-2Kb post-ER QC. Finally, we demonstrate the plausible usage of the rapamycin assay to assess the trafficking of defected proteins especially in diseases and under therapeutic studies.
Like other transmembrane glycoproteins, major histocompatibility complex (MHC)1 class I molecules are subject to cellular quality-control (QC) during folding and maturation. A substantial body of work has investigated the intracellular trafficking of MHC class I from its biosynthesis in the endoplasmic reticulum (ER) to its deposition at the cell surface for its proper function. To initiate the journey, the free heavy chain (FHC) of MHC class I interacts with the ER chaperone protein calnexin and with the protein disulfide isomerase ERp57 until it has folded and associated with the light chain, beta 2 microglobulin (β2m). In case they pass the initial folding hurdle, they can bind to the light chain, β2m, forming dimers that are recognized by the lectin chaperone, calreticulin; together with three other proteins, tapasin, ERp57, and TAP, they form the class I peptide loading complex (PLC) . Tapasin plays a crucial role in the maturation of MHC class I molecules by editing high affinity peptides onto MHC class I grooves. Tapasin also bridges MHC class I to TAP, the transporter associated with antigen processing, thereby enhancing peptide loading onto MHC class I.
It has been shown that dimers can leave the ER to the cis-Golgi from where they cycle back to the ER . Thus, another step of QC at the cis side of the Golgi apparatus is needed to hinder their egress to the cell surface and ultimately direct their route back to the ER. This retrograde pathway is highly mediated by calreticulin . During the ER-Golgi cycle, the PLC is believed to assist the heavy chain- β2m dimers to bind specific peptides into their binding groove . Susbequently, the peptide-HC-β2m complexes dissociate from the PLC and move as completely folded molecules through the secretory pathway to the cell surface to elicit an immune reponse by cytotoxic T cells (CTL) in case of infection.
Controlling the post-ER trafficking of MHC class I and their ER-Golgi cycle is very vital to the health of the cell. Any uncontrolled trafficking might lead to the escape of empty dimers or FHC to the cell surface contributing to certain diseases, such as spondyloarthropathy [5, 6]. Furthermore, empty dimers or monomers of MHC class I at the cell surface can bind peptides [7, 8] and become recognized by CTL. Thus, they can trigger the death of a healthy cell by binding to exogenous non-self peptides from the extracellular space.
Despite the flood of scientific studies that exist in the literature on the post-ER QC of partially folded MHC class I, there is no single mechanism underlying the behavior of different MHC class I allotypes during the ER-Golgi cycle. For instance, it is well reported that partially loaded human allotypes, HLA-B5 and HLA-A2, can reach the Golgi where they undergo peptide-loading and exit to the cell surface , whereas dimers of HLA-B27 accumulate in a subcompartment of the ER where they are destined for degradation by ubiquitination . Additionally, murine H-2Kd get sorted at the cis-Golgi cisternae , and can reach the cell surface even in a dimeric form. However, dimers or FHC of H2-Kb cannot make it to the cell surface [12, 13] though dimers- but not FHC- can reach post-ER compartment from where their trajectory either back to the ER, or to the cell surface, or to degradation is not fully discerned.
In this study, we employ the in vitro rapamycin-trapping assay to further investigate the destiny of H-2Kb trafficking from a post-ER compartment. The assay depends on the generation of recombinant proteins tagged to FK506 binding protein (FKBP) or FKBP-rapamycin binding protein (FRB) that can interact upon addition of rapamycin. Our results show that recombinant H-2Kb mimic the localization pattern of endogenous molecules, namely they are capable of exiting the ER to the ER- Golgi intermediate complex (ERGIC) and/or cis-Golgi compartments. Furthermore, recombinant H-2Kb fused with FKBP or FRB are able to cycle back to the ER as revealed by their trapping with an ER localized marker. Trapping of H-2Kb was also exhibited at the cell surface, demonstrating possible sorting of partially folded molecules from the Golgi to the cell surface. Our results demonstrate the plausible usage of the rapamycin assay to assess the trafficking of proteins especially in diseases and under therapeutic studies. Finally, it paves the way to further explore the peptide-rescue function of unloaded MHC class I in post-ER compartments through their interaction with certain members of the PLC complex.
Mammalian cells and transfections
Mouse embryonic fibroblasts (MEF), untransfected or stably expressing H-2Kb-GFP, were obtained from Michael Edidin (Johns Hopkins University, Maryland, US). Cells were cultured in DMEM and supplemented with 10 % heat-inactivated FCS and PSG (100 IU/ml penicillin, 100 mg/ml streptomycin, and 2 mM glutamine). Plain cells were transiently transfected using the FuGENE reagent (Roche Molecular Biochemicals, Indianapolis, IN). For single transfection, 50 μl OptiMEM were mixed with 2 μl FuGENE and 400 ng/μl DNA. For multiple transfection, 50 μl OptiMEM were mixed with 3 μl FuGENE and 200 ng/μl of each DNA. The OptiMEM/FuGENE/DNA mixture was kept at room temperature for 30 min and then added to cells that were already in 120 μl of fresh media. For the transfection to occur, the cells were incubated for 4 to 5 h with the FuGENE mixture. Afterwards, 400 μl of new media were added, and cells were incubated at 37 °C with 5 % CO2 overnight. The cell line, 293 T (ATCC® CRL-3216), is a highly transfectable derivative of human embryonic kidney 293 cells, and contains the SV40 T-antigen.
Electroporation of Jurkat cells
One million cells were centrifuged at 200 x g for 10 min at room temperature (RT). 2 μg of DNA were mixed in 82 μl Nucleofector solution V (Amaxa) and 18 μl of supplement solution 1 at RT. In addition, the Nucleofector and supplement solutions were only mixed in the quantities required for each experiment to make sure that freshly mixed solutions were always used. After centrifugation as above, the supernatant was discarded, and the pellet was resuspended in 100 μl of the DNA/solution mixture. The mixture was then transferred to a sterile Amaxa cuvette, and cells were transfected using program X-001. The cells were then left at RT for 10 min. Meanwhile, RPMI medium (1.5 ml) was equilibrated in the incubator to 37 °C. 500 μl of the equilibrated medium was then added into the cuvette (very smoothly the cells were drawn into and out of the pipette tip three times, such that all sedimented cells were resuspended). The transfected cells were then plated and transferred immediately to the incubator at 37 °C with 5 % CO2. After 24 h, 1 ml of the supernatant was discarded, again care was taken not to disturb the sedimented cells. The remaining 500 μl cells were resuspended and half of the amount was used for the first-day experiment. To the remaining cells, 1250 μl of fresh media was added, and they were left to grow for another 24 h for the second-day experiment. For the first-day experiment, 250 μl of (about 600,000 cells) were splitted into three different wells 83 μl/each of a 6-well Ibidi chamber (Ibidi, Munich, Germany). The wells of the chamber were coated with 0.1 % polylysine. L-polylysine (Sigma) for 40 min at RT, the wells were then washed with water, and dried in the incubator for 10 min. Higher density of cells was bathed in a lower volume of buffer to keep them in the field of vision. Then to each of the wells, we added 117 μl of CO2-independent buffer. Three different experiments were performed in parallel.
The following monoclonal antibodies (mAbs) were used: rabbit anti-GM130 (against the cytosolic tail of GM130 and marking mostly the cis-Golgi cisternae, purchased from BD Biosciences); rabbit anti-p58 (against p58 proteins in the ERGIC, a gift from Dr. Jaakko Saraste, Bergen, Norway); rabbit anti-PDI (against PDI proteins in the ER, a gift from Dr. Irina Majoul, Lübeck University, Germany); anti-P8 serum (against H-2Kb, a generous gift from Sjaak Neefjes, Netherlands Cancer Institute, Amsterdam), rabbit anti-EEA1 (as an early endosomal marker, BD Laboratories), α6F monoclonal antibody against Na-K-ATPase α1-subunit (α6F) , and the rabbit anti-murine tapasin antibody, 2668, against residues 11 to 34 .
Chemicals and peptides
The peptide SIINFEKL (SL8) (from ovalbumin, 257–264; purified by HPLC) was purchased from Biosyntan (Berlin, Germany), dissolved with a stock concentration of 2.5 mM, and used at a final concentration of 10 μM. Peptides were added to the cells, without electroporation, for one to two hours.
Cycloheximide (purchased from Applichem; lot 9 V001009) is used as an inhibitor of protein biosynthesis in eukaryotes by blocking translational elongation. was mixed in water to a stock concentration of 10 mg/ml. The percentage of viability of cells as tested by trypan blue was about 80 % in the presence of 50 μg/ml of cycloheximide. Thus, we used this concentration throughout all the conducted experiments. Rapamycin (Sigma; lot 039 K4015) was mixed in DMSO at a stock concentration of 1 mM and used at 1 μM. Rapamycin is a small chemical with several roles among which it is believed to mediate the interaction of FKBP and FRB domains through its interface.
To avoid any effects of rapamycin on the trapping of freshly synthesized molecules, cells were pretreated with cycloheximide for ten minutes prior to rapamycin addition.
Isolation of DNA
Plasmid preparations from small overnight cultures (1–5 ml) were either obtained using the QIAprep spin kit according to supplier’s protocol (Qiagen, Hilden, Germany), especially if the DNA was used for sequencing, or prepared using the rapid boiling miniprep method .
Immunofluorescence and temperature block
To arrest trafficking of class I molecules in the ERGIC, cells expressing green fluorescent protein (GFP)- H-2Kb fusion proteins were incubated for 2 h at 15 °C and then fixed for 15 min at 15 °C with 4 % paraformaldehyde in phosphate-buffered saline (PBS) . To accumulate class I molecules in the Golgi apparatus, cells were incubated for 2 h at 20 °C  and then fixed for 15 min at 20 °C with 4 % paraformaldehyde in PBS. After fixation, cells were permeabilized and stained with the indicated antibodies for one hour at room temperature. To detect H-2Kb cycling, cells were cotransfected with Ii and H-2Kb prior to their treatment with cycloheximide and rapamycin. Ii corresponds to the invariant chain associated with MHC class II moleucles and is usually used as an ER marker. Cells were then fixed and observed using a Zeiss LSM 510 confocal microscope. Cells that were cotransfected with FKBP or FRB constructs were grown in the chambers of plastic microscope slides (Ibidi, Martinsried, Germany) and fixed with paraformaldehyde without permeabilization in the wells. The cells were then observed directly in the chambers without any mounting using confocal microscope, LSM 510.
For quantification, the plugin JACoP version 2.0 was used in imageJ . The pearson’s coefficient of the intensities of the two channels was plotted by the red channel on the x-coordinate (organelle markers) and the green channel on the y-coordinate (H2-Kb). Pearson’s coefficient provides an approximate of the rate of linear association between the two used fluorochromes. Its values range between −1 and 1 with zero no correlation and negative values for negative correlation. Region of interest (ROI) from selected cells was cropped, then channels were split, and quantified using JACoP plugin.
COPII vesicle formation assay
The COPII generation assay was carried out as described by [3, 20, 21]. Briefly, mouse fibroblasts were radiolabelled with [35S]-methionine for 30 min and then harvested, resuspended in a low osmolarity buffer, and broken with 40 passes through a 22G syringe needle. After sedimenting nuclei and unbroken cells, the medium-speed pellet (15,000xg, 5 min) was washed twice and used as donor membranes for microsomes. Budding reactions consisted of microsomal membranes, pig brain cytosol, ATP regenerating system, and 0.2 mM GTP. Vesicles were isolated from the supernatant resulting from a 15,000 x g spin and sedimented by a 100 000 x g centrifugation, lysed in 1 % digitonin in 50 mM Tris-Cl (pH 7.5) and 150 mM NaCl, and radiolabelled proteins were then immunoprecipitated using rabbit serum specific for the GFP tag on H-2Kb (Fig. 4, lower panel), or as a control for budding with Na+/K+-ATPase antibodies (Fig. 4, upper panel). A peptide specific for H-2Kb (sequence SIINFEKL in the single letter amino acid code) was added to vesicles and to microsomes during lysis at 1 μM final concentration. Samples were treated with EndoF1 before SDS–PAGE, except for one sample that was used as a size reference band for class I heavy chain. Bands were quantified using ImageJ (1.44p) analysis tool an their relative densities were plotted using the highest peak of the budding reaction, donor membranes with peptides, as a standard.
293 t cells were transfected with Venus-FRB- Kb or cotransfected with Venus-FRB-tapasin and Venus-FRB-Kb. At 6 h post transfection, cells were labelled for 14 h with 35S-Met/Cys. Cells were lysed using 1 % digitonin. IP was perfomed with anti-H-2Kb (P8).
Results and discussion
Endogenous and recombinant H-2Kb molecules accumulate in a post-ER compartment
Taken together, our results so far suggest that the trafficking of a large pool of both endogenous and recombinant H-2Kb is jammed outside the ER. The calculated pearsons’ coefficients did not show complete correlation between the colocalized H-2Kb and organelle channels, but was slightly higher for the ERGIC than the cis-Golgi. This underlying fact points up the possibility of the involvement of another quality control compartment that might resemble as well the ERGIC and cis-Golgi compartments. This behavioral phenotype compounded with the cell surface expression of molecules might be restricted to MEF cells and is prone to variation based on the origin and overall characteristics of the tested cell line.
H-2Kb exit the ER in COPII vesicles
In vitro FKBP/FRB rapamycin-trapping assay is adopted to further confirm the trafficking of H-2Kb
EGh_FRB- H-2Kb_fwd (HindIII)
H-2Kb in ss-EGFP- FRB
EGh_FRB- H-2Kb_rev (BamHI)
H-2Kb in ss-EGFP- FRB
Murine tapasin in ss-EGFP-FRB
Murine tapasin in ss-EGFP-FRB
The disappearance of accumulated Venus-FRB-Kb over time is not due to the disruption of the Golgi apparatus
Live- cell imaging to examine the cycling of recombinant Venus-FRB-Kb
Egress of H-2Kb to the cell surface upon addition of peptides
Finally we assessed the localization of Venus-FRB-Kb in MEF cells. Similar to endogenous H-2Kb and of GFP-H-2Kb, they were mostly localized to the ER and showed an additional accumulation close to the nucleus with a weak delineation of the cell surface (data not shown). Cells were pretreated with cycloheximide for 10 min- to avoid cross-linking of the nascent proteins in the ER- prior to their one-hour incubation with or without rapamycin.. As expected, after treatment with rapamycin, 60 % of the cells lacked a juxtanuclear accumulation and mostly showed an ER-like pattern for Venus-FRB-Kb with few vesicular structures that could be late endosomes (data not shown). Trapping failed to occur in cells co-transfected with Cerulean-FKBP-Ii and Venus-Kb lacking the FRB domain (data not shown).
Several reports have examined proteins that undergo post-ER quality control; Examples include MHC class I partially-folded dimers that reach post-ER compartments from where they cycle back to the ER possibly by Bap31  or calreticulin ; mutants of influenza hemagglutinin that leave the ER to reach the plasma membrane for degradation by endocytosis ; the N153D mutant of tissue non-specific alkaline phosphatase (TNSALP) , which leaves the ER and is retained in the cis-Golgi until degradation; and the ts405 mutant form of the vesicular stomatitis virus G protein (VSVG), which cycles between the Golgi and ER until it is finally degraded by ERAD .
In this work, we used an exquisite rapamycin-trapping assay to detect and confirm the trafficking and sorting of MHC class I molecules, H-2Kb, from post-ER compartments.
We provide biochemical evidence that in MEF cells, H-2Kb exit the ER in COPII vesicles. This assay is well established and monitored earlier in Springer’s laboratory in Germany. The result provides independent biochemical evidence that indeed GFP-H2Kb can leave the ER, and that the sorting-QC that determines the intracellular transport of dimers is located in a post-ER compartment. Interestingly, GFP-Kb molecules were found in COPII vesicle fractions regardless of the presence of peptides.
In addition, we present microscopic evidence that the post-ER compartment where H-2Kb accumulate resembles closely the ERGIC and/or the cis-Golgi compartments. The central aim of this report was to assess the destiny of the trafficking of H-2Kb accumulated molecules. We were able to show that the majority of H-2Kb alleles can cycle back to the ER as revealed by the in vitro and in vivo trapping with an ER marker, Ii. Since not all the molecules showed similar cycling pattern, then we believe that there is a degradation pathway that releases the stress of accumulating immature proteins outside the ER. One possibility arising from the exit of unfolded proteins from the ER is their degradation in an ERGIC-expanded region  or in a QC compartment (QCC) . However, given that the PLC assists the heavy chain- β2m dimers to bind peptides onto the class I peptide binding groove  and based on what we have shown that TAP is active in post-ER compartment, then there is a substantial possibility of loading partially-folded molecules with peptides during their ER-Golgi cycle, as a rescuing event to further open the gate for protein channeling to the cell surface.
The latter assumption is supported by our data on the trapping of H-2Kb at the cell surface by GPI. We showed that cells incubated with peptides exhibited a cell surface stain that was upregulated in the presence of rapamycin. Our report paves the way for further work to decipher the trafficking of H-2Kb to the cell surface and to unravel the chaperones or proteins that mediate H-2Kb trafficking and stability at the cell surface.
Finally, this assay can be extended to examine the cycling of other ER chaperones, such as tapasin, that has also been shown to partially exit the ER especially in lymphocytes.
1 Abbreviations used: β 2 m, beta-2 microglobulin; BFA, brefeldin A; CTL, cytotoxic T lymphocytes; ER, endoplasmic reticulum; ERAD, ER-associated degradation; ER-QCC, endoplasmic reticulum quality control compartment; FHC, free heavy chains; FKBP, FK506 binding protein; FRB, FKPB-rapamycin binding protein; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; HC, (MHC class I molecule) heavy chain; MHC, Major histocompatibility complex; PLC, (MHC class I) peptide loading complex; QC, quality control.
The authors would like to thank Prof. Sebastian Springer for reagent donations and for helpful discussions and technical support. Also they would like to thank Prabudha Sangupta at NIH for providing the FKBP and FRB constructs and Nele Beutler at Duesseldorf for conducting the immunoprecipitation assays. This work was supported by the Deutscher Akademisher Austausch Dienst (DAAD).
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