Role of SERCA1 Truncated Isoform in the Proapoptotic Calcium Transfer from ER to Mitochondria during ER Stress

Abstract

SUMMARY Among the new players at the endoplasmic reticulum (ER)-mitochondria interface regulating interorganelle calcium signaling, those specifically involved during ER stress are not known at present. We report here that the truncated variant of the sarcoendoplasmic reticulum Ca2+-ATPase 1 (S1T) amplifies ER stress through the PERK-eIF2α-ATF4-CHOP pathway. S1T, which is localized in the ER-mitochondria microdomains, determines ER Ca2+ depletion due to increased Ca2+ leak, an increased number of ER-mitochondria contact sites, and inhibition of mitochondria movements. This leads to increased Ca2+ transfer to mitochondria in both resting and stimulated conditions and activation of the mitochondrial apoptotic pathway. Interestingly, S1T knockdown was shown to prevent ER stress, mitochondrial Ca2+ overload, and subsequent apoptosis. Thus, by bridging ER stress to apoptosis through increased ER-mitochondria Ca2+ transfer, S1T acts as an essential determinant of cellular fate.

Calcium signalling-dependent mitochondrial dysfunction and bioenergetics regulation in respiratory chain Complex II deficiency

“…Earlier data have shown that ER Ca 2 þ depletion can be linked to the induction of the unfolded protein response (UPR). 18 However, our data revealed no induction of UPR in SDHA R554W cells (Supplementary Figure S3). These results may indicate that the increased cytosolic and mitochondrial agonist-evoked Ca 2 þ signals are largely linked to increased basal Ca 2 þ levels.…”

“…The light signal was collected and calibrated into [Ca 2 þ ] values, as earlier described. 18 On the basis of the experimental traces, the maximum rates of ER Ca 2 þ uptake were measured from the first derivative of the ascending refilling phase obtained after CaCl 2 addition, and the maximum rates of ER Ca 2 þ leak were measured from the first derivative of the curve on addition of TBuBHQ using Origin 5 software. Fitting of the curve was performed using Microsoft Excel software.…”

“…This parameter is referred to as the mitochondrial movement index. 18 Binarization and normalization were applied to avoid artefacts eventually arising from changes of intensity and focal plane.…”

“…Plasma membrane and microsomal fractions were isolated as already described. 18,40 SDS-PAGE was then performed. The signal was revealed using ECL Plus western-blot detection reagent (Amersham Biosciences, Orsay, France).…”

“…For the experiments shown in Figures 5c and d, membrane fraction obtained as already described 18 was used to reveal both SERCA and PMCA. Protein synthesis was inhibited using 100 mM CHX alone or in the presence of 300 mM 3-NP or 5 mM Oligomycin for the indicated time points.…”