Adopting the rapamycin trapping assay to track the trafficking of murine MHC class I alleles, H-2K(b)

Abstract

Background: 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.

Results: 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.

Conclusions: 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-2K(b) 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.

Fig. 1

Endogenous H-2K^b accumulate in a juxtanuclear region and can reach the cell surface. MEF cells were fixed, permeabilized, and double stained for H-2K^b with anti-P8 serum and for one of the early secretory organelles, either ER (anti-PDI), ERGIC (anti-p58), or cis-Golgi (GM130). Prior to fixation, cells were either kept at 37 °C or incubated for 2 h at 15 °C or 20 °C, panels b and c, respectively. Most of the cells showed, in addition to the ER pattern, a juxtanuclear accumulation (a, arrows) that partially overlapped with the ERGIC and cis-Golgi stains. Some of the cells also showed weak cell surface stain (asterisk). Nuclei were stained with Draq5 as depicted in blue. All samples are shown in a bright-field mode to delineate the overall structure of the stained cells. Region of interest (ROI) from selected cells is quantified and a scatter plot with pearson’s coefficient is given.Scale bars, 10 μm

Fig. 2

Recombinant GFP-K^b also form a post-ER accumulation. MEF cells transiently transfected with GFP-K^b were fixed, permeabilized, and stained with antibodies against PDI (a), p58 (b), or GM130 (c). Prior to fixation, cells were either kept at 37 °C or incubated for 1.5 h at 15 °C (b) or 20 °C (c). Scatter plots with pearson’s coefficient depict the fluorescence intensities of the secondary Ab (Cy3) on the x-axis and the GFP fluorescence on the y-axis. Scale bars, 10 μm

Fig. 3

H-2K^b–GFP molecules exit the ER in COPII vesicles. COPII vesicles were generated by an in vitro reaction from MEF cells. Controls for this reaction were the omission of cytosol (lane 3), ATP (lane 4), or addition of dominant-negative Sar1 (T39N; lane 5). COPII vesicles or the corresponding donor microsomal membranes (after the reaction; lane 6–8) were lysed with detergent (in the presence of 10 μM peptide as indicated in lanes 1 and 7). H-2K^b–GFP (upper panel) and Na⁺/K⁺ATPase (lower panel) were sequentially immunoprecipitated from the lysates with anti-GFP serum and α6F antibodies, respectively as depicted in panel a. Immunoprecipitates were treated with EndoF1 (lanes 1–7). The band intensities were quantified and plotted in panel (b). One out of three experiments is shown here

Fig. 4

Examining the proper size of recombinant Venus-FRB-K^b molecules in 293 T cells. Venus-FRB-K^b showed an estimate size of 80KDa and associated with both Venus-FRB-Tapasin (lane 2) and wild type tapasin (lane 3)

Fig. 5

Cycling of Venus-FRB-Kb from post-ER compartment back to the ER. Venus-FRB-Kb is trapped in the ER by Cerulean-FKBP-Ii upon addition of rapaymcin. The fluorescence intensity of Venus-FRB-Kb accumulation from the first panel disappears upon the addition of rapamycin (1 mM) for 2 h (second panel). Trapping of Venus-FRB-Kb molecules does not occur in the controls where rapamycin is absent (first and third panels) or in the presence of Cerulean-Iip35 that lacks the FKBP domain (fourth panel). Scale bars, 10 μm

Fig. 6

Integrity of the Golgi apparatus in the presence of rapamycin. The Golgi structure is maintained even after six hours incubation of Jurkat cells with cycloheximide and rapamycin. Jurkat cells triple transfected with Venus-FRB-Kb Cerulean-FKBP-Ii, and GalT-mCherry show intact Golgi even after six hours treatment with cycloheximide and rapamycin. Scale bars, 10 μm

Fig. 7

Live cell imaging for Venus-FRB-Kb cycling. (a) Six different snapshots of live Jurkat cells coexpressing Venus-FRB-Kb and Cerulean-FKBP-Ii (double-transfected red and green cell = RG) or expressing only one of the constructs (only Ii in red = R cell) were taken from a 45 min movie (images were captured at a 2-min interval; snapshots are revealed at different allocated time points). Each image consists of four compiled z-stacks, where each ∆z = 1 μm. Before recording the movie, cells were transferred to a 37 °C heat stage in CO₂-independent buffer and incubated first with cycloheximide (10 μg/ml) for 10 min and then with rapamycin (1 μM). Pearson’s coefficient was calculated from each image by defining a circular region of interest (ROI) around the Golgi area where the green fluorescence intensity of Venus-FRB-Kb accumulates (an arrow pointing to the circle in the first panel). For the cell only expressing Cerulean-FKBP-Ii in red (R cell), the ROI was drawn around the entire cell. (b) The graph shows the time-dependent change in Pearson’s coefficient based on the variation of the green fluorescence intensity with respect to the red fluorescence in the chosen ROI. There is an increase in Pearson’s coefficient for the RG but not for the control (R) cell. (c) There is a 25 % increase in the percentage of cells that show Venus-FRB-Kb localization in the ER upon addition of rapamycin (*p < 0.02). The error bars correspond to the SEM values from 2 sets of experiments (n > 250)

Fig. 8

Preliminary data on the trafficking of H-2Kb from post-ER compartment directly to the cell surface. MEF cells were double transfected with Venus-FRB-K^b and Cerulean-FKBP-GPI. The invariant chain Ii is retained in the ER, whereas GPI can reach the cell surface. Cells treated with cycloheximide and rapamycin without addition of peptides showed Venus-FRB-K^b in the ER with a faint delineation of the cell surface. From the intensity RGB profile, the pseudo-color curves do not overlay. Cells treated with 10 μM of SIINFEKL peptides showed a clear cell surface stain that was more intense in the presence of rapamycin. The RGB profile depicts an overlay of the fluorophore curves at the same distance. Scale bars, 10 μm