Wt-CFTR and G551D-CFTR were expressed in Hela cells which do not endogenously express CFTR and their function was measured by 
. The expression of the CFTR protein was assessed by western blotting. As shown in the mature fully glycosylated CFTR and core-glycosylated CFTR were expressed in both cell types. Therefore, we found that in our cell model the mutant protein can fold and traffic normally to the plasma membrane. This was in accordance with previous results showing that G551D-CFTR exhibits no trafficking defects 
. We investigated the cAMP-dependent halide flux through Wt-CFTR and G551D-CFTR () in the stably transfected cells. We found that it was deeply decreased in G551D-CFTR cells, as previously described 
. Therefore, we showed that viral tranfection of the cells led to the expression of Wt-CFTR and G551D-CFTR and that their expression and function were in accordance with previous results, indicating that the cell model could be used for further experiments.
A reference 2-DE (pH 4–7) of 6 gels performed with proteins complex linked to G551D-CFTR obtained by immunoprecipitation is presented in . The image data analysis detected 165 protein spots in the complex. The spot showing the higher expression was further analyzed (A, on ). It was excised from the gel, trypsined and analyzed by MS. The results indicate that the spot corresponded to calumenin (). The MS/MS results leading to calumenin identification are summarized in . The MS spectrum and the coverage of the calumenin sequence are presented in .
Calumenin belongs to the G551D-CFTR’s complex.
Identification of spots A by Mass Spectrometry.
MS/MS results for Calumenin identification.
Calumenin is a multiple EF-hand Ca2+
-binding protein located in the ER. It belongs to the CREC family of Ca2+
-binding proteins. It is also found in the secretory pathway and can be secreted to the extracellular space. It interacts with different ligands and it inhibits several proteins in the ER membrane such as the ryanodine receptor (RyR2) and the Ca2+
-transporting ATPase (SERCA2). Some other functions concern participation in the secretory process, ER lumenal chaperone activity and signal transduction. It is also involved in a large variety of disease processes (for review 
) and it was shown to be involved in the Ca2+
homeostasis of the ER, due to a direct interaction with SERCA2 and RyR2. Indeed, an inhibition of SERCA2-mediated Ca2+
uptake into SR in cardiomyocyte due to an interaction with calumenin was observed 
. To assess the expression of calumenin in G551D-CFTR expressing cells, western blots (n
5) were performed to compare its basal level with that of wt-CFTR expressing cells. As shown in , no difference was observed between Wt- or G551D-CFTR expressing cells suggesting that it is not involved in a difference of the Ca2+
homeostasis between Wt- and G551D-CFTR cells. Whereas, calumenin was detected in the CFTR’s ER-Associated Folding Proteome and seems to be modulated in Fdel508-CFTR expressing cells, its direct interaction with CFTR has never clearly been established 
. Therefore, we assessed its interaction with CFTR by coimmunoprecipitation. As shown in , we found that calumenin was bound to CFTR and that it was more abundant in the G551D-CFTR complex than in the Wt-CFTR complex. The reverse coimmunoprecipitation indicated that the amount of bound G551D-CFTR onto calumenin was higher than the amount of bound Wt-CFTR (not shown). Because we found that calumenin binds to the mature G551D-CFTR form (170 kDa) and because human calumenin is found in the ER as well as in the Golgi complex 
, our results suggest that it is likely involved in the CFTR maturation.
More calumenin is bound onto G551D-CFTR than onto Wt-CFTR.
Grp78/Bip is a major sensor for the Unfolded Protein Response (UPR) triggering in the ER. It is modulated as well as calumenin in F508del-CFTR expressing cells and we previously showed that it is involved in the observed UPR in CF cells 
. Therefore, we assessed Grp78 expression in G551D-CFTR expressing cells, using Fdel508-CFTR cells as a positive control. As shown in , we observed an increased Grp78 expression in G551D-CFTR cells when compared to Wt-CFTR expressing cells. The quantitation indicated that its expression was not significantly different to that observed in Fdel508-CFTR cells, suggesting the UPR triggering (). Because it was previously shown that G551D-CFTR is not retained in the ER 
this result suggests that UPR, or at least an ER stress, may be triggered in G551D-CFTR expressing cells due to another mechanism involving calumenin.
UPR is likely triggered in G551D-CFTR expressing cells.
Because the calumenin - CFTR interaction in the ER of cells had never been shown before and because some extra bands were observed when calumenin was detected in the cells using western blottings, we had to further provide evidence showing this interaction. For this purpose, Surface Plasmon Resonance was used 
. The sensorgrams obtained when pure calumenin was linked on a sensorchip and pure CFTR was injected (0.15 to 1.5 nM), showing an interaction followed by a dissociation phase which was not zero when the injection was stopped indicated that the interaction occured (). The obtained value for the dissociation constant was 3.8×10−12
M showing a strong interaction. The opposite experiment in which purified CFTR was linked on the sensorchip was also performed (not shown). The SPR experiments confirming the direct interaction permitted to rule out the possibility of an unspecific binding during co-immunoprecipitation experiments. The calumenin - CFTR interaction was also evaluated with the online Search Tool for the Retrieval of Interacting Genes/Protein (STRING 9.0, http://string-db.org
) to predict both direct and indirect interactions 
. As shown in CFTR was found to be a calumenin’s network. Among the predicted calumenin’s partners which are listed in , some are depicted and characterized by biochemical experiments such as RyR and ATP2A2 
. In order to show that in our cell model calumenin is located within the ER, as described in other cell types, immunofluorescence was performed. The nuclei of the cells as well as the ER and calumenin were labelled in wt- and G551D-CFTR expressing cells (). The results indicated that in our cell model calumenin is indeed localized in the ER and that therefore the described interaction between Calumenin and wt- or G551D-CFTR takes place within the ER.
Evidence of the direct calumenin-CFTR interaction.
Predicted Functional Partners for Calumenin.
The calumenin – CFTR interaction takes place in the ER.
Our results suggest that the G551D-CFTR’s maturation/trafficking is different from the one of Wt-CFTR because it likely involves more calumenin. Furthermore, we suggest that UPR is likely triggered in G551D-CFTR expressing cells. Nevertheless, the UPR pathway may be different than the one observed in Fdel508-CFTR cells because the G551D-CFTR protein is not retained in the ER. In conclusion, we show here a direct calumenin - wt-CFTR and calumenin - G551D-CFTR interaction. Because we found the interaction within the ER and because the amount of bound calumenin is likely higher with the mutated CFTR, we propose that the G551D-CFTR protein may be directed to a specific trafficking pathway. The exact role of calumenin upon UPR in G551D-CFTR expressing cells is now under investigation in our laboratory as well as its possible role as a chaperone for the most frequent mutation found in CF (F508del) which is retained in the ER. Furthermore, according to the Bip/Grp78 expression, we propose that UPR may be triggered in G551D-CFTR expressing cells, independently of any retention of the mutated CFTR in the ER.