Mitochondrial respiration links TOR complex 2 signaling to calcium regulation and autophagy

ABSTRACT

The target of rapamycin (TOR) kinase is a conserved regulator of cell growth and functions within 2 different protein complexes, TORC1 and TORC2, where TORC2 positively controls macroautophagy/autophagy during amino acid starvation. Under these conditions, TORC2 signaling inhibits the activity of the calcium-regulated phosphatase calcineurin and promotes the general amino acid control (GAAC) response and autophagy. Here we demonstrate that TORC2 regulates calcineurin by controlling the respiratory activity of mitochondria. In particular, we find that mitochondrial oxidative stress affects the calcium channel regulatory protein Mid1, which we show is an essential upstream activator of calcineurin. Thus, these findings describe a novel regulation for autophagy that involves TORC2 signaling, mitochondrial respiration, and calcium homeostasis.

The initiation of autophagy is linked to nutrient availability by distinct signaling pathways and is responsive to both environmental stress as well as intracellular cues, including organelle homeostasis. The rapamycin-sensitive TORC1 kinase complex is a well-established negative regulator of autophagy that functions in response to nitrogen availability. More recently, we and others have identified the rapamycin-insensitive TORC2 kinase complex as an independent positive regulator of autophagy, specifically under amino acid limited growth conditions. In this capacity, TORC2 functions via its downstream target kinase, Ypk1, to repress the activity of the calcium and Cmd1/calmodulin-dependent phosphatase calcineurin, whose activity inhibits the general amino acid control (GAAC) response required for autophagy following amino acid starvation. Specifically, calcineurin inhibits activation of the conserved EIF2S1/eIF2α kinase, Gcn2, as well as subsequent translation of the Gcn4 transcription factor, which targets several autophagy-related genes. Thus, in cells deficient for Ypk1 activity (ypk1Δ cells), calcineurin becomes activated and both the GAAC response and autophagy are inhibited.

Mitochondrial respiratory function also regulates amino acid starvation-induced autophagy by a mechanism that is independent of TORC1; however, its relationship to TORC2 has remained unexplored. In our recent publication, we identified mitochondria as a crucial missing link between TORC2-Ypk1 activity and the regulation of calcineurin, using the model yeast S. cerevisiae. The entry point for this study was to test whether mitochondrial function collaborates with TORC2-Ypk1 signaling to promote autophagy following amino acid starvation. Accordingly, we examined the consequence of disrupting mitochondrial function in ypk1Δ cells by deleting mitochondrial DNA to create respiratory-deficient ypk1Δ rho⁰ cells. We used western blot analysis to examine GFP-Atg8, which associates with autophagosomes and becomes degraded in the vacuole to release free GFP, as a quantitative readout of autophagy flux. Here we observed that autophagy is restored to wild-type (WT)-like levels in ypk1Δ rho⁰ cells under amino acid-starvation conditions. We confirmed that rescue of autophagy is due specifically to inhibiting mitochondrial respiration by deleting individual genes encoding mitochondrial electron transport chain (ETC) components, which similarly rescues autophagy in ypk1Δ cells. Importantly, we observed that calcineurin activity, which is constitutively elevated in ypk1Δ cells, is also returned to WT-like levels in ETC-deficient ypk1Δ cells, consistent with a model wherein mitochondria act downstream of TORC2-Ypk1 to regulate calcineurin (Fig. 1). Also consistent with this model is the observation that the GAAC response is similarly rescued in respiratory-deficient ypk1Δ cells during amino acid starvation.

Figure 1.

A model for TORC2-Ypk1 regulation of mitochondria and autophagy during amino acid starvation. Under amino acid-starvation conditions (-AA), TORC2-Ypk1 signaling promotes autophagy by suppressing mitochondria-derived ROS. Thus, in the absence of TORC2-Ypk1 signaling, as occurs in ypk1Δ cells, ROS act via the calcium channel regulatory protein Mid1 to activate calcineurin. Once activated, calcineurin suppresses the GAAC response and prevents autophagy flux.

How is mitochondrial respiration linked to calcineurin activity? Clues to this question came when we investigated the role of candidate calcium channel-related proteins on the activity of calcineurin in ypk1Δ cells. We found that a calcium channel regulatory protein, Mid1, a component of the high-affinity calcium uptake system (HACS), is required for activation of calcineurin and that deletion of MID1 restores autophagy to WT levels in ypk1Δ cells. The HACS also includes Cch1, which is proposed to function as a calcium channel within the plasma membrane, where it is regulated by Mid1 to facilitate calcium entry into the cell. Interestingly, we detected no effect on autophagy when CCH1 was deleted from ypk1Δ cells, suggesting that regulation of calcineurin by Mid1 occurs independently from the canonical HACS pathway. In support of this conclusion, we determined using fluorescence microscopy that Mid1 localizes to both the plasma membrane as well as the endoplasmic reticulum, and our data indicate that interactions between mitochondria and the endoplasmic reticulum may be important for Mid1 activity and for regulation of calcineurin as well as autophagy.

We probed further the relationship between mitochondria and Mid1 using the anti-arrhythmic drug amiodarone, which in yeast increases intracellular calcium by a mechanism that specifically requires the activity of Mid1. It had been observed previously that heightened Mid1 activity sensitizes cells to amiodarone and we observed that ypk1Δ cells are hypersensitive to sublethal doses of the drug. This hypersensitivity is reversed in ypk1Δ mid1Δ cells, however, consistent with Mid1 activity becoming increased in the absence of TORC2-Ypk1 signaling. Importantly, we observed that hypersensitivity of ypk1Δ cells to amiodarone is also reduced in combination with mitochondrial ETC mutants. Moreover, we found that hypersensitivity to the drug is reduced when ypk1Δ cells are pretreated with N-acetyl-L-cysteine, a scavenger for reactive oxygen species (ROS). These findings are consistent with additional data indicating that mitochondria-produced ROS are necessary for activation of calcineurin in ypk1Δ cells. Because Mid1 does not itself influence mitochondrial activity, including the accumulation of ROS, we conclude from these results that mitochondria act upstream of Mid1 to activate calcineurin (Fig. 1).

In total, our findings establish new functional connections between TORC2-Ypk1 signaling, mitochondrial respiratory activity, and the regulation of calcineurin during amino acid starvation, which ultimately affect the GAAC response and autophagy. A novel feature of this regulation is involvement of the calcium channel regulator Mid1, which we argue plays a key role in linking mitochondrial-ROS to calcineurin activity. Understanding how Mid1 activity responds to ROS remains an important question. Intriguingly, Mid1 possesses several conserved cysteine residues that are important for its function and thus may be involved directly in an oxidative stress response, as these residues are often targets for oxidation-dependent modification. Thus, one interesting possibility is that Mid1 responds directly to ROS to facilitate calcium signaling. The source(s) of calcium involved in this pathway, as well as the identity of other proteins that may be involved in calcium transport or signaling, remain to be identified.

Finally, our findings highlight the importance of TORC2-Ypk1 signaling in regulating mitochondrial respiratory activity in yeast. In this regard, there are emerging parallels in higher eukaryotes, both during development as well as in disease, including cancer, where mitochondrial function is controlled by the kinase AKT, the mammalian ortholog of Ypk1, in conjunction with insulin signaling as well as phosphoinositide 3-kinase and MTORC2. Importantly, these events can result in changes in the production and/or accumulation of ROS. Thus, it may well turn out that in higher eukaryotes, mitochondrial ROS regulated by MTORC2 may also affect both calcium signaling as well as autophagy.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by National Institutes of Health (NIH) grant GM086387 and NIH T-32 Training Grant in Molecular and Cellular Biology.