Manganese is a physiologically relevant TORC1 activator in yeast and mammals

Abstract

The essential biometal manganese (Mn) serves as a cofactor for several enzymes that are crucial for the prevention of human diseases. Whether intracellular Mn levels may be sensed and modulate intracellular signaling events has so far remained largely unexplored. The highly conserved target of rapamycin complex 1 (TORC1, mTORC1 in mammals) protein kinase requires divalent metal cofactors such as magnesium (Mg2+) to phosphorylate effectors as part of a homeostatic process that coordinates cell growth and metabolism with nutrient and/or growth factor availability. Here, our genetic approaches reveal that TORC1 activity is stimulated in vivo by elevated cytoplasmic Mn levels, which can be induced by loss of the Golgi-resident Mn2+ transporter Pmr1 and which depend on the natural resistance-associated macrophage protein (NRAMP) metal ion transporters Smf1 and Smf2. Accordingly, genetic interventions that increase cytoplasmic Mn2+ levels antagonize the effects of rapamycin in triggering autophagy, mitophagy, and Rtg1-Rtg3-dependent mitochondrion-to-nucleus retrograde signaling. Surprisingly, our in vitro protein kinase assays uncovered that Mn2+ activates TORC1 substantially better than Mg2+, which is primarily due to its ability to lower the Km for ATP, thereby allowing more efficient ATP coordination in the catalytic cleft of TORC1. These findings, therefore, provide both a mechanism to explain our genetic observations in yeast and a rationale for how fluctuations in trace amounts of Mn can become physiologically relevant. Supporting this notion, TORC1 is also wired to feedback control mechanisms that impinge on Smf1 and Smf2. Finally, we also show that Mn2+-mediated control of TORC1 is evolutionarily conserved in mammals, which may prove relevant for our understanding of the role of Mn in human diseases.

Golgi in and out: multifaceted role and journey of manganese

“…In doing so, NRAMP2 activity overlaps with NRAMP1's to secure adequate Mn levels in the cell. Such cooperativity between PM uptake and Golgi release of Mn is conserved in yeast (Nicastro et al ., 2022) and could possibly result from the high demand for Golgi‐localized, Mn‐dependent enzymes, electing this compartment as a strong sink for Mn. An open question concerns the possible interplay between this pool of Golgi Mn and the vacuolar reservoir of Mn.…”

“…Supporting this view, Pottier et al (2022) identified a TGN-localized NRAMP isoform in poplar, PotriNRAMP3.1, the overexpression of which affects Mn translocation to the leaves. Their study suggests that The pivotal role of a Golgi-derived cytosolic pool of Mn was also demonstrated in yeast and mammals, where hyperactivation of the central metabolic regulator TORC1 has been shown to depend on binding to Mn 2+ in the cytosol (Nicastro et al, 2022). In yeast, TORC1 hyperactivation requires Mn transport by Smf2, which it regulates in a feedback loop.…”

“…The pivotal role of a Golgi‐derived cytosolic pool of Mn was also demonstrated in yeast and mammals, where hyperactivation of the central metabolic regulator TORC1 has been shown to depend on binding to Mn 2+ in the cytosol (Nicastro et al ., 2022). In yeast, TORC1 hyperactivation requires Mn transport by Smf2, which it regulates in a feedback loop.…”

Manganese Stress Tolerance Depends on Yap1 and Stress-Activated MAP Kinases

“…Calcineurin B-independent Yap1 down-regulation upon H 2 O 2 [ 39 ] or MnCl 2 addition suggests that manganese could drive the modulation of other phosphatase or kinase activities that promote Yap1 decay. Along this line, we recently found that manganese stimulates the enzymatic activity of the TORC1 complex in vitro and in vivo [ 43 ]. In any case, although calcineurin does not mediate MnCl 2 -triggered Yap1 decay this phosphatase is needed to activate the stress response transcription factor Crz1.…”

“…Yeast cells were grown overnight in the appropriate medium, diluted to A 600 = 0.3 and grown for 3 h at 26 °C prior to addition of MnCl 2 , H 2 O 2 or Cycloheximide (CHX). Protein extracts were isolated as previously described [ 43 ], separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE), using 8% ( w / v ) polyacrylamide (37.5:1), and transferred to nitrocellulose membranes (Hybond, GE Healthcare, Amersham, UK) according to standard protocols. The Slt2 TEY phosphorylation site was marked a rabbit anti-phospho-p44/42 antibody (Thr-202/Tyr-204; Cell Signaling, Danvers, MA, USA, catalog# 9101), Hog1 phosphorylation with a p38 MAPK Thr180/182 antibody (Cell Signaling, catalog# 9101), Slt2 with an anti-Slt2 mouse antibody (Santa Cruz, Dallas, TX, USA, catalog# sc-374440), GFP with a anti-GFP mouse antibody (JL-8, Clontech, Shiga, Japan, catalog# 632381), G6DPH by an anti-G6DPH rabbit antibody (Sigma, catalog# A9521) and Pgk1 with an anti-Pgk1 mouse antibody (Invitrogen, Waltham, MA, USA, catalog# 459250) prior to inmuno-detection with a secondary antibody.…”