The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of growth. Mammalian TOR complex 2 (mTORC2) regulates AGC kinase family members and is implicated in various disorders, including cancer and diabetes. Here we report that mTORC2 is localized to the endoplasmic reticulum (ER) subcompartment termed mitochondria-associated ER membrane (MAM). mTORC2 localization to MAM was growth factor-stimulated, and mTORC2 at MAM interacted with the IP 3 receptor (IP3R)-Grp75-voltage-dependent anion-selective channel 1 ER-mitochondrial tethering complex. mTORC2 deficiency disrupted MAM, causing mitochondrial defects including increases in mitochondrial membrane potential, ATP production, and calcium uptake. mTORC2 controlled MAM integrity and mitochondrial function via Akt mediated phosphorylation of the MAM associated proteins IP3R, Hexokinase 2, and phosphofurin acidic cluster sorting protein 2. Thus, mTORC2 is at the core of a MAM signaling hub that controls growth and metabolism.

could be demonstrated directly under physiological conditions by using a combined approach of single channel recordings and high resolution confocal microscopy (21).These studies suggest that, despite the short lasting [Ca 2ϩ ] mito increase, mitochondria can generate sustained microdomains of low Ca 2ϩ in the subplasmalemmal region. However, it is unclear whether mitochondria act as a Ca 2ϩ sink or as a Ca 2ϩ relay mechanism, or whether the increase in mitochondrial Ca 2ϩ per se is an essential step in the activation of the CCE as suggested recently (22). Furthermore, inter-organelle Ca 2ϩ cross-talk between the ER and the mitochondria * This work was supported by the Austrian Science Funds P14586-PHA and SFB 714 (to W. F. G.) and the Swiss National Funds 31-56902.99 (to N. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.¶ To whom correspondence should be addressed: Dept. concentration at the inner side of the patch membrane; CCE, capacitative Ca 2ϩ entry; CGP 37157, 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one; FCCP, carbonyl cyanide-4-trifluoromethoxyphenylhyrazone; IP 3 , inositol 1,4,5-triphosphate; mtDsRed, mitochondrial-targeted DsRed; P o , open state probability of single BK Ca channels; RP-mt, mitochondrialtargeted ratiometric-pericam; V applied , applied holding potential; V pm , effective potential of the patch; V wc , whole cell membrane potential; YC4-ER, ER-targeted yellow chameleon 4; GFP, green fluorescent protein; SERCA, sarco/endoplasmic reticulum calcium ATPase.

Mitochondria are ancient endosymbiotic guests that joined the cells in the evolution of complex life. While the unique ability of mitochondria to produce adenosine triphosphate (ATP) and their contribution to cellular nutrition metabolism received condign attention, our understanding of the organelle's contribution to Ca 2+ homeostasis was restricted to serve as passive Ca 2+ sinks that accumulate Ca 2+ along the organelle's negative membrane potential. This paradigm has changed radically. Nowadays, mitochondria are known to respond to environmental Ca 2+ and to contribute actively to the regulation of spatial and temporal patterns of intracellular Ca 2+ signaling. Accordingly, mitochondria contribute to many signal transduction pathways and are actively involved in the maintenance of capacitative Ca 2+ entry, the accomplishment of Ca 2+ refilling of the endoplasmic reticulum and Ca 2+ -dependent protein folding. Mitochondrial Ca 2+ homeostasis is complex and regulated by numerous, so far, genetically unidentified Ca 2+ channels, pumps and exchangers that concertedly accomplish the organelle's Ca 2+ demand. Notably, mitochondrial Ca 2+ homeostasis and functions are crucially influenced by the organelle's structural organization and motility that, in turn, is controlled by matrix/cytosolic Ca 2+ . This review intends to provide a condensed overview on the molecular mechanisms of mitochondrial Ca 2+ homeostasis (uptake, buffering and storage, extrusion), its modulation by other ions, kinases and small molecules, and its contribution to cellular processes as fundamental basis for the organelle's contribution to signaling pathways. Hence, emphasis is given to the structure-to-function and mobility-to-function relationship of the mitochondria and, thereby, bridging our most recent knowledge on mitochondria with the best-established mitochondrial function: metabolism and ATP production.

Endoplasmic reticulum (ER) Ca 2؉ refilling is an active process to ensure an appropriate ER Ca 2؉ content under basal conditions and to maintain or restore ER Ca 2؉ concentration during/after cell stimulation. The mechanisms to achieve successful ER Ca 2؉ refilling are multiple and built on a concerted action of processes that provide a suitable reservoir for Ca 2؉ sequestration into the ER. Despite mitochondria having been found to play an essential role in the maintenance of capacitative Ca 2؉ entry by buffering subplasmalemmal Ca 2؉ , their contribution to ER Ca 2؉ refilling was not subjected to detailed analysis so far. Thus, this study was designed to elucidate the involvement of mitochondria in Ca 2؉ store refilling during and after cell stimulation. ER Ca 2؉ refilling was found to be accomplished even during continuous inositol 1,4,5-trisphosphate (IP 3 )-triggered ER Ca 2؉ release by an agonist. Basically, ER Ca 2؉ refilling depended on the presence of extracellular Ca 2؉ as the source and sarcoplasmic/endoplasmic reticulum Ca 2؉ ATPase (SERCA) activity. Interestingly, in the presence of an IP 3 -generating agonist, ER Ca 2؉ refilling was prevented by the inhibition of trans-mitochondrial Ca 2؉ flux by CGP 37157 (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one) that precludes the mitochondrial Na ؉ /Ca 2؉ exchanger as well as by mitochondrial depolarization using a mixture of oligomycin and antimycin A. In contrast, after the removal of the agonist, ER refilling was found to be

Mitochondria modulate Ca2؉ signals by taking up, buffering, and releasing Ca 2؉ at key locations near Ca 2؉ release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca 2؉ homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca 2؉ released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1-fragmented mitochondria took up more slowly the Ca 2؉ entering across plasma membrane channels, because the Ca 2؉ ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca 2؉ entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/ rotenone reduced CCE by ϳ35%. These observations indicate that close contact to Ca 2؉ influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca 2؉ propagation within interconnected mitochondria.