There were errors published in J. Cell Sci. 124, 2143Sci. 124, -2152 In the section given below, PtdIns(3,4,5)P 3 was on four occasions incorrectly printed instead of the correct Ins(1,4,5)P 3 .We apologise for this mistake. Increased mitochondrial Ca2+ drives the adaptive metabolic boost observed during early phases of ER stress Increases in mitochondrial respiration and ATP production are often consequences of increases in mitochondrial Ca 2+ (Green and Wang, 2010). In order to determine whether early phases of ER stress induced by tunicamycin increased mitochondrial Ca 2+ , we treated cells expressing cytosolic or mitochondrial aequorins with histamine [which evokes Ins(1,4,5)P 3 -dependent Ca2+ release] and compared their mitochondrial Ca 2+ uptake. We observed that histamine led to a mitochondrial Ca 2+ uptake that was significantly higher in tunicamycinpretreated cells (P<0.05; 4 hours) than in untreated cells (Fig. 6A). Cytosolic Ca 2+ increased similarly in tunicamycin-treated and untreated cells (Fig. 6B). These results indicate that the differences in mitochondrial Ca 2+ levels are not due to altered Ca 2+ release mediated by the Ins(1,4,5)P 3 receptor but to an enhanced mitochondrial Ca 2+ uptake, presumably due to the increased apposition of ER and mitochondrial Ca 2+ channels. By using a different dye, Fura-2, we monitored the peak cytosolic Ca 2+ levels after thapsigargin addition, reflecting the kinetics of Ca 2+ release after sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) inhibition. After 4 hours of tunicamycin treatment, the thapsigargin-induced Ca 2+ peak was increased, and it was further elevated by inhibition of mitochondrial Ca 2+ uptake using Ru360 (Fig. 6C). These results suggest that, besides the Ins(1,4,5)P 3 -receptor-mediated direct Ca 2+ transfer from the ER to neighboring mitochondria, an additional phenomenon associated with the early phases of ER stress involves Ca 2+ leak from the ER, also resulting in mitochondrial Ca 2+ uptake. Indeed, no mitochondrial Ca 2+ uptake following the thapsigargin-induced Ca 2+ leak was observed in Mfn2-knockout cells (Fig. 6D), which is reflected by the lack of effect of Ru360. This result further indicates that juxtaposition of mitochondria with the ER is necessary for the mitochondrial Ca 2+ uptake evoked by Ca 2+ leak during early phases of ER stress.Finally, to test whether mitochondrial Ca 2+ levels control the metabolic mitochondrial boost, we measured oxygen consumption rates resulting from OXPHOS in the presence of the Ins(1,4,5)P 3 receptor inhibitor xestospongin B or the mitochondrial Ca 2+ uptake inhibitor RuRed. We observed that both xestospongin B and RuRed decreased the rate of oxygen consumption after tunicamycin treatment (Fig. 7A,B), which confirms that increased mitochondrial Ca 2+ uptake, resulting from ER-mitochondrial coupling, is necessary for the metabolic response observed during early phases of ER stress. Therefore, in order to evaluate whether the early metabolic boost forms part of an adaptive response triggere...

The mitochondria-associated membrane (MAM) is a domain of the endoplasmic reticulum (ER) that mediates the exchange of ions, lipids and metabolites between the ER and mitochondria. ER chaperones and oxidoreductases are critical components of the MAM. However, the localization motifs and mechanisms for most MAM proteins have remained elusive. Using two highly related ER oxidoreductases as a model system, we now show that palmitoylation enriches ER-localized proteins on the MAM. We demonstrate that palmitoylation of cysteine residue(s) adjacent to the membrane-spanning domain promotes MAM enrichment of the transmembrane thioredoxin family protein TMX. In addition to TMX, our results also show that calnexin shuttles between the rough ER and the MAM depending on its palmitoylation status. Mutation of the TMX and calnexin palmitoylation sites and chemical interference with palmitoylation disrupt their MAM enrichment. Since ER-localized heme oxygenase-1, but not cytosolic GRP75 require palmitoylation to reside on the MAM, our findings identify palmitoylation as key for MAM enrichment of ER membrane proteins.

The mitochondria-associated membrane (MAM) has emerged as an endoplasmic reticulum (ER) signaling hub that accommodates ER chaperones, including the lectin calnexin. At the MAM, these chaperones control ER homeostasis but also play a role in the onset of ER stress-mediated apoptosis, likely through the modulation of ER calcium signaling. These opposing roles of MAM-localized chaperones suggest the existence of mechanisms that regulate the composition and the properties of ER membrane domains. Our results now show that the GTPase Rab32 localizes to the ER and mitochondria, and we identify this protein as a regulator of MAM properties. Consistent with such a role, Rab32 modulates ER calcium handling and disrupts the specific enrichment of calnexin on the MAM, while not affecting the ER distribution of protein-disulfide isomerase and mitofusin-2. Furthermore, Rab32 determines the targeting of PKA to mitochondrial and ER membranes and through its overexpression or inactivation increases the phosphorylation of Bad and of Drp1. Through a combination of its functions as a PKA-anchoring protein and a regulator of MAM properties, the activity and expression level of Rab32 determine the speed of apoptosis onset.

Protein secretion from the endoplasmic reticulum (ER) requires the enzymatic activity of chaperones and oxidoreductases that fold polypeptides and form disulfide bonds within newly synthesized proteins. The best-characterized ER redox relay depends on the transfer of oxidizing equivalents from molecular oxygen through ER oxidoreductin 1 (Ero1) and protein disulfide isomerase to nascent polypeptides. The formation of disulfide bonds is, however, not the sole function of ER oxidoreductases, which are also important regulators of ER calcium homeostasis. Given the role of human Ero1α in the regulation of the calcium release by inositol 1,4,5-trisphosphate receptors during the onset of apoptosis, we hypothesized that Ero1α may have a redox-sensitive localization to specific domains of the ER. Our results show that within the ER, Ero1α is almost exclusively found on the mitochondria-associated membrane (MAM). The localization of Ero1α on the MAM is dependent on oxidizing conditions within the ER. Chemical reduction of the ER environment, but not ER stress in general leads to release of Ero1α from the MAM. In addition, the correct localization of Ero1α to the MAM also requires normoxic conditions, but not ongoing oxidative phosphorylation.