Manganese is a physiologically relevant TORC1 activator in yeast and mammals

Nicastro, Gaillard etal. eLife 2022;11:e80497. DOI: https://doi.org/10.7554/eLife.80497 1 of 20 Manganese is a physiologically relevant TORC1 activator in yeast andmammals Raffaele Nicastro1†, Hélène Gaillard2,3†, Laura Zarzuela2, MariePierre PéliGulli1, Elisabet FernándezGarcía2,3, Mercedes Tomé2, Néstor GarcíaRodríguez2,3, Raúl V Durán2, Claudio De Virgilio1*, Ralf Erik Wellinger2,3* 1University of Fribourg, Department of Biology, Fribourg, Switzerland; 2Centro Andaluz de Biología Molecular y Medicina Regenerativa - CABIMER, Consejo Superior de Investigaciones Científicas, Universidad de Sevilla, Seville, Spain; 3Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Seville, Spain 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 Golgiresident Mn2+ transporter Pmr1 and which depend on the natural resistanceassociated 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 Rtg1Rtg3dependent mitochondriontonucleus 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. Editor's evaluation Your manuscript uses budding yeast to uncover a new input for the central metabolic regulator TOR complex 1 (TORC1) – manganese (Mn) levels and also demonstHelene rates that this dependence on Mn is conserved in humans. The combination of both in vivo and in vitro approaches as well as the demonstration of conservation of this phenomenon make the manuscript both broad and deep. TORC1 is already clearly a central coordinator of multiple inputs to guide cellular decisions of catabolism vs anabolism. Information on an additional way to modulate its activity is highly influential on both basic cell biology as well as therapeutic research. RESEARCH ARTICLE *For correspondence: [email protected] (CDV); [email protected] (REW) †These authors contributed equally to this work Competing interest: The authors declare that no competing interests exist. Funding: See page 16 Preprinted: 09 December 2021 Received: 22 May 2022 Accepted: 18 July 2022 Published: 29 July 2022 Reviewing Editor: Maya Schuldiner, Weizmann Institute of Science, Israel Copyright Nicastro, Gaillard etal. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Research article Biochemistry and Chemical Biology | Genetics and Genomics Nicastro, Gaillard etal. eLife 2022;11:e80497. DOI: https://doi.org/10.7554/eLife.80497 2 of 20 Introduction Manganese (Mn) is a vital trace element that is required for the normal activity of the brain and nervous system by acting, among other mechanisms, as an essential, divalent metal cofactor for enzymes such as the mitochondrial enzyme superoxide dismutase 2 (Weisiger and Fridovich, 1973), the apical activator of the DNA damage response serine/threonine kinase ATM (Chan etal., 2000) or the Mn2+- activated glutamine synthetase (Wedler and Denman, 1984). However, Mn2+ becomes toxic when enriched in the human body (Couper, 1837). While mitochondria have been proposed as a preferential organelle where Mn2+ accumulates and unfolds its toxicity by increasing oxidative stress and thus mitochondrial dysfunction (Aguirre and Culotta, 2012), the molecular mechanisms of Mn2+ toxicity in humans are also related to protein misfolding, endoplasmic reticulum (ER) stress, and apoptosis (Harischandra etal., 2019). Mn2+ homeostasis is coordinated by a complex interplay between various metal transporters for Mn2+ uptake and intracellular Mn2+ distribution and represents an essential task of eukaryotic cells, which is also of vital importance specifically for neuronal cell health (Horning etal., 2015). Much of our knowledge on Mn2+ transport across the plasma membrane into the ER, the Golgi, endosomes, and vacuoles comes from studies in Saccharomyces cerevisiae (outlined in Figure1A). Typically, Mn2+ is shuttled across membranes by transporters that belong to the natural resistanceassociated macrophage protein (NRAMP) family, which are highly conserved metal transporters responsible for iron (Fe) and Mn2+ uptake (Supek etal., 1996). Not surprisingly, therefore, NRAMP orthologs have been found to crosscomplement functions in yeast, mice, and humans (Sacher etal., 2000). One of the beststudied NRAMPs is the yeast plasma membrane protein Smf1. Interestingly, extracellular Fe or Mn2+ supplementation triggers Bsd2 adaptor proteindependent, Rsp5mediated ubiquitination of Smf1, which initiates its sorting through the endocytic multivesicular body pathway and subsequent lysosomal degradation (Eguez et al., 2004; Liu and Culotta, 1999). The Smf1 paralogs Smf2 and Smf3 are less well studied, but Smf2 is predominantly localized at endosomes and its levels decrease under conditions of Mn or Fe overload (Liu etal., 1997; MorenoCermeño etal., 2010). Within cells, the Ptype ATPase Pmr1 (also known as Bsd1) represents a key transporter that shuttles Ca2+ and Mn2+ ions into the Golgi lumen. Its loss leads to increased levels of Mn2+ in the cytoplasm due to defective detoxification (Lapinskas etal., 1995). Noteworthy, several phenotypes associated with loss of Pmr1 have been shown to arise as a consequence of Mn2+ accumulation in the cytoplasm, including telomere shortening, genome instability, and bypass of the superoxide dismutase Sod1 requirement (Bolton etal., 2002; GarcíaRodríguez etal., 2012; Lapinskas etal., 1995; Lue etal., 2005). TORC1/mTORC1 is a central, highly conserved controller of cell growth and aging in eukaryotes. It coordinates the cellular response to multiple inputs, including nutritional availability, bioenergetic status, oxygen levels, and, in multicellular organisms, the presence of growth factors (Albert and Hall, 2015; Laplante and Sabatini, 2012). In response to these diverse cues, TORC1 regulates cell growth and proliferation, metabolism, protein synthesis, autophagy, and DNA damage responses (Liu and Sabatini, 2020; Sancak etal., 2008). In S. cerevisiae, which played a pivotal role in the discovery and dissection of the TOR signaling network (Heitman etal., 1991), TORC1 is mainly localized on the surfaces of vacuoles and endosomes (Betz and Hall, 2013; Hatakeyama etal., 2019) where it integrates, among other cues, amino acid signals through the Rag GTPases and Pib2 (Nicastro etal., 2017; Tanigawa etal., 2021). In mammals, amino acids also activate the Rag GTPases, which then recruit mTORC1 to the lysosomal surface where it can be allosterically activated by the small GTPase Rheb (Ras homolog expressed in brain) that mediates the presence of growth factors and sufficient energy levels (Binda etal., 2009; Sancak etal., 2008; Sancak etal., 2010; Wedaman etal., 2003). Interestingly, mTORC1 regulates cellular Fe homeostasis (Bayeva etal., 2012), but how it may be able to sense Fe levels remains largely unknown. In addition, although TORC1/mTORC1 requires divalent metal ions to coordinate ATP at its catalytic cleft (Brunn etal., 1996; Withers etal., 1997), it is currently not known whether these or any other trace elements may play a physiological or regulatory role in controlling its activity. In yeast, genetic evidence links high levels of cytoplasmic Mn2+ to TORC1 function, as loss of Pmr1 confers rapamycin resistance (Devasahayam etal., 2007). However, the underlying molecular mechanism(s) by which Mn2+ may mediate rapamycin resistance remains to be explored. Here, we show that Mn2+ uptake by NRAMP transporters modulates rapamycin resistance and that, in turn, TORC1 Research article Biochemistry and Chemical Biology | Genetics and Genomics Nicastro, Gaillard etal. eLife 2022;11:e80497. DOI: https://doi.org/10.7554/eLife.80497 3 of 20 A B F C D WT bsd2∆ smf2∆ bsd2∆ smf2∆ RAP [10 ng/ml] MnCl2 -1 mM WT pmr1∆ bsd2∆ MnCl2 -1 mM -1 mM RAP [10 ng/ml] E RAP [10 ng/ml]control WT pmr1∆ gdt1∆ smf1∆ smf2∆ atx2∆ spf1∆ mtm1∆ ccc1∆ pho84∆ pmr1∆ RAP [10 ng/ml]control pEMPTY pSMF2 pSLC11A1 pSLC11A2 pmr1∆ smf2∆ Vacuole Mitochondria Nucleus ER A B bsd PM PVE V acuole V V Mit oc h o ndria N u c l e u s ER Rsp5 Bsd2 Smf1 Smf2 Atx2 Ccc1 Pho84 Pmr1 Mtm1? Spf1 Mn2+ Mn M 2+ Mn2+ Mn M 2+ Mn M 2+ + WT pmr1∆ pmr1∆ smf2∆ smf2∆ CaCl2 -+ +-+-+- < M 1650 3000 4000 2000 1000 5000 6000 7000 8000 12000 [bp] Southern blot of genomic DNA Figure 1. Natural resistanceassociated macrophage protein (NRAMP) transporters link Mn2+- import to rapamycin resistance. (A)Schematical outline of yeast Mn2+ transporters and their intracellular localization. PM, plasma membrane; PVE, prevacuolar endosomes; ER, endoplasmic reticulum. Note that Bsd2 is a specific adaptor protein for Rsp5mediated Smf1 and Smf2 ubiquitination in response to Mn2+ overload. The Golgi Mn2+ transporter Gdt1 is omitted for clarity. (B–D)Growth on MnCl2 and/or rapamycincontaining medium (RAP). Tenfold dilutions of exponentially growing cells are shown. Strains and compound concentrations are indicated. Note that the medium used in (D)was supplemented with 10mM CaCl2. Data obtained in a medium without CaCl2 are shown in Figure1—figure supplement 3. (E)Southern blot analysis of telomere length. Genomic DNA was derived from cells grown in a medium supplemented or not with CaCl2 and cleaved by XhoI before agarose gel electrophoresis. The 1.3kb average length of telomeres from WT cells (dashed white line, black arrow) and size marker (M)are shown. (F)Growth of pmr1∆ smf2∆ double mutants transformed with plasmids expressing yeast Smf2, Mus musculus SLC11A1, or SLC11A2 on rapamycincontaining medium. Complementary data showing that pmr1∆ rapamycin resistance is not linked to Gap1 or Tor1 localization is provided in Figure1—figure supplement 1. Rapamycin sensitivity of bsd2∆ cells overexpressing the Vcx1M1 transporter are shown in Figure1—figure supplement 2. Figure 1 continued on next page Research article Biochemistry and Chemical Biology | Genetics and Genomics Nicastro, Gaillard etal. eLife 2022;11:e80497. DOI: https://doi.org/10.7554/eLife.80497 4 of 20 inhibition by rapamycin regulates NRAMP transporter availability. Moreover, intracellular Mn2+ excess antagonizes rapamycininduced autophagy, mitophagy, and Rtg13 transcription factor complexdependent retrograde response activation. Surprisingly, our in vitro analyses reveal that TORC1 protein kinase activity is strongly activated in the presence of MnCl2. In our attempts to understand the mechanisms underlying these observations, we discovered that Mn2+, when compared to Mg2+, significantly boosts the affinity of TORC1 for ATP. Combined, our findings also indicate that TORC1 activity is regulated by and regulates intracellular Mn2+ levels, defining Mn2+ homeostasis as a key factor in cell growth control. Importantly, our studies in human cells indicate that Mn2+- driven TORC1 activation is likely conserved throughout evolution, opening new perspectives for our understanding of Mn2+ toxicities and their role in neurodegenerative disorders and aging. Results NRAMP transporters regulate cytoplasmic Mn2+ levels and rapamycin resistance Yeast cells lacking the Golgilocalized Ptype ATPase Pmr1, which transports Ca2+ and Mn2+ ions from the cytoplasm to the Golgi lumen, are resistant to the TORC1 inhibitor rapamycin (Devasahayam etal., 2006; Devasahayam etal., 2007), a phenotype that is generally associated with increased TORC1 activity. In pmr1∆ mutants, defective Mn2+ shuttling at the Golgi leads to protein sorting defects and accumulation of the general amino acid permease Gap1 at the plasma membrane (Kaufman etal., 1994). In theory, this may translate into unrestrained uptake and intracellular accumulation of amino acids, and thus hyperactivation of TORC1. However, arguing against such a model, we found cells lacking both Pmr1 and Gap1 to remain resistant to low doses of rapamycin (Figure1—figure supplement 1A). We then asked whether loss of Pmr1 may affect the expression levels or cellular localization of TORC1. Our results indicated that GFPTor1 protein levels and localization to vacuolar and endosomal membranes remained unaltered in exponentially growing and rapamycintreated WT and pmr1∆ cells (Figure1—figure supplement 1B and C). Given the roughly fivefold increased intracellular Mn2+ levels in cells lacking Pmr1 (Lapinskas etal., 1995), we then considered the possibility that Mn2+ may have a more direct role in TORC1 activation. We, therefore, assessed the rapamycin sensitivity of cells lacking the adaptor protein Bsd2, which mediates Rsp5dependent degradation in response to high Mn2+ levels of both the plasma membraneand endosomal membraneresident NRAMP Mn2+ transporters Smf1 and Smf2, respectively (Figure1A; Liu etal., 1997). Interestingly, bsd2∆ cells were as sensitive to rapamycin as WT cells, but, unlike WT cells, could be rendered rapamycin resistant by the addition of 1mM MnCl2 in the growth medium (Figure1B and Figure1—figure supplement 2). This effect was strongly reduced in the absence of Smf2 (Figure1C), suggesting that Smf2dependent endosomal Mn2+ export and, consequently, cytoplasmic accumulation of Mn2+ may be required for rapamycin resistance under these conditions. In line with such a model, we found that overexpression of the vacuolar membraneresident Vcx1M1 transporter, which imports Mn2+ into the vacuolar lumen, suppressed the Mn2+- induced rapamycin resistance of bsd2∆ mutants and increased their sensitivity to rapamycin in the absence of extracellular MnCl2 supply (Figure1—figure supplement 2). The online version of this article includes the following source data and figure supplement(s) for figure 1: Source data 1. Uncropped autoradiography image shown in Figure1E. Source data 2. Raw autoradiography image shown in Figure1E. Figure supplement 1. pmr1∆ rapamycin resistance is not linked to Gap1 or Tor1 localization. Figure supplement 1—source data 1. Uncropped blot shown in Figure1—figure supplement 1C. Figure supplement 1—source data 2. Raw blot shown in Figure1—figure supplement 1C. Figure supplement 2. Growth of bsd2∆ mutants expressing Vcx1M1 from plasmid pVCX1M1 on MnCl2 and/or rapamycin (RAP) containing medium. Figure supplement 3. Natural resistanceassociated macrophage protein (NRAMP) transporters mediate rapamycin resistance of pmr1∆ mutants. Figure 1 continued Research article Biochemistry and Chemical Biology | Genetics and Genomics Nicastro, Gaillard etal. eLife 2022;11:e80497. DOI: https://doi.org/10.7554/eLife.80497 5 of 20 As schematically outlined in Figure1A, several metal transporters have been associated with Mn2+ transport in yeast, including those localized at the plasma membrane (Smf1 and Pho84), the endosomes (Smf2 and Atx2), the Golgi (Pmr1 and Gdt1), the vacuole (Ccc1 and Vcx1), the ER (Spf1), and possibly the mitochondria (Mtm1). To identify which of these transporters contributes to the rapamycin resistance of pmr1∆ cells, we next monitored the growth of double mutants lacking Pmr1 and any of the corresponding metal transporters in the presence of rapamycin (Figure1D). Interestingly, only loss of Smf1 or Smf2 reestablished rapamycin sensitivity in pmr1∆ cells, while loss of Gdt1 or Ccc1 even slightly enhanced the rapamycin resistance phenotype of pmr1∆ cells. Of note, growth was monitored on CaCl2supplemented media that improved the growth of pmr1∆ cells lacking Smf1 or Smf2 without affecting their sensitivity to rapamycin, which also rules out the possibility that Ca2+ is associated with the observed rapamycin resistance phenotype (Figure1D and Figure1—figure supplement 3). Taken together, our data indicate that increased intracellular Mn2+ levels in pmr1∆ cells lead to rapamycin resistance and that this phenotype can be suppressed either by reducing Mn2+ import through loss of Smf1 or, possibly, by increasing Mn2+ sequestration in endosomes through loss of Smf2 as previously suggested (Luk and Culotta, 2001). To confirm our prediction that loss of Smf2 reduces cytoplasmic Mn2+ levels, we measured MnCl2dependent telomere length shortening as an indirect proxy for cytoplasmic and nuclear Mn levels (GarcíaRodríguez etal., 2015). Accordingly, telomere length was significantly decreased in pmr1∆ cells (when compared to WT cells), increased in smf2∆ cells, and similar between pmr1∆ smf2∆ and WT cells (Figure1E). These data, therefore, corroborate our assumption that loss of Smf2 suppresses the high cytoplasmic and nuclear Mn2+ levels of pmr1∆ cells. The function of metal transporters is highly conserved across evolution as exemplified by the fact that the expression of the human Pmr1 ortholog, the secretory pathway Ca2+/Mn2+ ATPase ATP2C1/ SPCA1 (ATPase secretory pathway Ca2+ transporting 1), can substitute for Pmr1 function in yeast (Muncanovic etal., 2019). A similar degree of functional conservation from lower to higher eukaryotes exists for NRAMP transporters (Sacher etal., 2000). Because Smf2 is of specific interest in the context of the present study, we asked whether the orthologous mouse proteins, that is, the divalent metal transporter SLC11A1 (NRAMP1) and SLC11A2 (DMT1) isoforms (solute carrier family 11 member 1 and 2, respectively), can restore rapamycin resistance in pmr1∆ smf2∆ double mutants. Expression of SLC11A1 did not rescue the lack of Smf2, leading to poor growth even in the absence of rapamycin. However, the SLC11A2 isoform complemented Smf2 function in these assays (Figure1F), indicating that Pmr1 and Smf2 are evolutionarily conserved transporters that are required for Mn2+ homeostasis. Elevated levels of intracellular Mn2+ antagonize rapamycin-induced autophagy, mitophagy, and Rtg1-3 retrograde signaling Growth inhibition by rapamycin mimics starvation conditions and leads to the degradation and recycling of a wide spectrum of biological macromolecules via autophagy. In this context, the pmr1∆ mutant has previously been found to be defective in nutrient depletioninduced mitophagy (Kanki etal., 2010). We thus wondered if rapamycininduced autophagic processing may also be defective in pmr1∆ mutants. We took advantage of a GFPAtg8 fusion construct (Cheong and Klionsky, 2008) to monitor autophagy through GFPAtg8 synthesis and processing in cells that had been subjected to rapamycin treatment for up to 6hr. Rapamycininduced GFPAtg8 expression was strongly reduced in pmr1∆ cells and this phenotype was mitigated in the pmr1∆ smf2∆ double mutant (Figure2A and B), suggesting that the initiation of autophagy is compromised by elevated cytoplasmic Mn2+ levels. Next, we assessed mitophagy by following the expression and degradation of the GFPtagged mitochondrial membrane protein Om45 (Kanki etal., 2009). Rapamycin treatment led to a timedependent upregulation of Om45GFP protein levels in all tested strains (Figure2C and D). However, the accumulation of the cleaved GFP protein was strongly reduced in the pmr1∆ single mutant, while this effect was again suppressed in the pmr1∆ smf2∆ double mutant. Combined, our results therefore suggest that the intracellular Mn2+ flux modulates both rapamycininduced autophagy and mitophagy. To identify additional responses to elevated cytosolic Mn2+ levels, we took advantage of our previously published transcriptome analysis of pmr1∆ cells (GarcíaRodríguez etal., 2015). Careful analysis of these datasets revealed that the expression levels of genes activated by the heterodimeric Rtg1Rtg3 transcription factor are reduced in pmr1∆ cells (see Supplementary file 1). Since TORC1 Research article Biochemistry and Chemical Biology | Genetics and Genomics Nicastro, Gaillard etal. eLife 2022;11:e80497. DOI: https://doi.org/10.7554/eLife.80497 6 of 20 AWT pmr1∆ smf2∆ pmr1∆ smf2∆ RAP [h]02 460246 02460246 *GFP G-6-PDH GFP-Atg8 C RAP [h] *GFP G-6-PDH Om45-GFP F WT pmr1∆ smf2∆ pmr1∆ smf2∆ * ** E WT pmr1∆ smf2∆ pmr1∆ smf2∆ control RAP Rtg3-GFP 02460246 02460246 B *** ** *GFP [%] 0 20 40 60 80 100 WT pmr1∆ smf2∆ pmr1∆ smf2∆ D WT pmr1∆ smf2∆ pmr1∆ smf2∆ 01530015 30 01530015 30 G-6-PDH Rtg3-GFP RAP [min] WT pmr1∆ smf2∆ pmr1∆ smf2∆ WT pmr1∆ smf2∆ pmr1∆ smf2∆ GFP-Atg8 + *GFP [AU] 0 20 40 60 80 100 120 * * * *GFP [%] 0 5 10 15 20 25 30 ** Om45-GFP + *GFP [AU] 0 20 40 60 80 100 120 140* * WT pmr1∆ smf2∆ pmr1∆ smf2∆ 35 25 70 55 [kDa] 70 55 35 25 70 55 [kDa] 100 70 55 [kDa] WT pmr1∆smf2∆ pmr1∆ smf2∆ Rtg3-GFP [AU] 0 20 40 60 80 100 120 140 30150RAP [min] 301503015030150 *** * ** G Figure 2. Intracellular manganese (Mn) excess antagonizes rapamycininduced autophagy, mitophagy, and Rtg13 retrograde signaling. (A)Exponentially growing WT and indicated mutant strains expressing plasmidencoded GFPAtg8 were treated for up to 6hr with 200ng/ml rapamycin (RAP). GFPAtg8 and cleaved GFP (*GFP) protein levels were analyzed by immunoblotting. Glucose6phosphate dehydrogenase (G6PDH) levels were used as a loading control. (B)Quantification of GFPAtg8 and *GFP levels after a 6hr rapamycin treatment. Total GFP signal (Atg8GFP + *GFP) normalized to WT levels (left) and percentage of *GFP relative to the total GFP signal (right) are plotted. Data represent means ± SEM of independent experiments (n=4). Statistical analysis: twotailed ttest (paired for normalized data, unpaired for GFP* percentage). *p<0.05; **p<0.01; ***p<0.001. (C)Exponentially growing WT and indicated mutant strains expressing Om45GFP from the endogenous locus were treated and processed as in (A).(D)Quantification of Om45GFP and *GFP levels after a 6hr rapamycin treatment. Details as in (B)with n=3. (E)Representative fluorescence microscope images of WT, pmr1∆, smf2∆, and pmr1∆ smf2∆ mutants expressing an episomic Rtg3GFP reporter construct. Exponentially growing cells were treated or not (control) for 30min with 200ng/ml rapamycin (RAP). Scale bar represents 5µm. (F)Rtg3GFP expressing cells were treated for up to 30min with 200ng/ml rapamycin. Protein levels were analyzed by immunoblotting. G6PDH levels were used as a loading control. (G)Quantification of Rtg3GFP signals. Values normalized to WT levels after 15min of rapamycin treatment (highest signal) are plotted. Data represent means ± SEM of independent experiments (n=4). Statistical analysis: paired twotailed ttest. Figure 2 continued on next page Research article Biochemistry and Chemical Biology | Genetics and Genomics Nicastro, Gaillard etal. eLife 2022;11:e80497. DOI: https://doi.org/10.7554/eLife.80497 7 of 20 inhibits cytoplasmictonuclear translocation of Rtg1Rtg3 (RuizRoig etal., 2012), we monitored the localization and protein levels of Rtg3GFP in exponentially growing and rapamycintreated WT and pmr1∆ cells. Rapamycin treatment not only induced nuclear enrichment of Rtg3GFP as reported (Figure2E; RuizRoig etal., 2012) but also significantly increased the levels of Rtg3GFP (Figure2F and G). Loss of Pmr1, in contrast, significantly reduced the Rtg3GFP levels in exponentially growing and rapamycintreated cells, which also translated into barely visible levels of Rtg3GFP in the nucleus (Figure2E–G). Importantly, and in line with our finding that loss of Smf2 suppresses the high cytoplasmic and nuclear Mn2+ levels of pmr1∆ cells (see above), these latter defects in pmr1∆ cells were partially suppressed by loss of Smf2. Thus, our findings posit a model in which elevated cytoplasmic Mn2+ levels antagonize autophagy, mitophagy, and Rtg13dependent retrograde signaling presumably through activation of TORC1. MnCl2 stimulates TORC1 kinase activity in vitro The yeast Tor1 kinase is a member of the phosphatidylinositol 3kinase (PI3K)- related kinase (PIKK) family that can phosphorylate the human eukaryotic translation initiation factor 4E binding protein (eIF4BP/PHASI) in vitro (Alarcon etal., 1999). Curiously, it does so much more efficiently when the respective in vitro kinase assays contain Mn2+ rather than Mg2+ as the sole divalent cation, a property that it appears to share with PI3kinases (Carpenter etal., 1993; Dhand etal., 1994; Foukas etal., 2004). A similar preference for Mn2+ over Mg2+ has also been observed in mTOR kinase autophosphorylation assays (Brunn etal., 1996; Withers etal., 1997). Based on these and our observations, we decided to assess whether Mn2+ may act as a metal cofactor for TORC1 activity in vitro using TORC1 purified from yeast and a truncated form of Lst4 (Lst4Loop; Nicastro etal., 2021) as a substrate. In control experiments without divalent ions, TORC1 activity remained undetectable (Figure3A and B). The addition of MnCl2, however, not only stimulated TORC1 in vitro in a concentrationdependent manner, but also activated TORC1 dramatically more efficiently than MgCl2 (with 25fold lower levels of MnCl2 [38µM] than MgCl2 [980µM] promoting halfmaximal activation of TORC1; Figure3A and B). We next considered the possibility that Mn2+ is superior to Mg2+ in favoring the coordination of ATP in the catalytic cleft or TORC1. Supporting this idea, we found that Mn2+ significantly reduced the Km for ATP (5.3fold) of TORC1 (Figure3C and D). Moreover, even in the presence of saturating Mg2+ levels (i.e. 4mM), the addition of 160µM Mn2+ was able to enhance the VMAX almost twofold and decrease the Km for ATP of TORC1 from 50.7 to 34.4µM, which indicates that Mn2+ can efficiently compete with Mg2+ and thereby activate TORC1. Notably, the conditions used in our in vitro kinase assays are quite comparable to the in vivo situation: accordingly, intracellular Mg2+ levels in yeast are approximately around 2mM (van Eunen etal., 2010), while the Mn2+ levels range from 26µM in WT cells to 170µM in pmr1∆ cells (McNaughton etal., 2010). Our in vitro assays, therefore, provide a simple rationale for why pmr1∆ cells are resistant to rapamycin: elevated Mn2+ levels in pmr1∆ favorably boost the kinetic parameters of TORC1. TORC1 regulates NRAMP transporter protein levels To maintain appropriate intracellular Mn2+ concentrations, cells adjust Smf1 and Smf2 protein levels through Bsd2dependent and -independent, posttranslational modifications that ultimately trigger their vacuolar degradation through the endocytic multivesicular body pathway (Liu and Culotta, 1994; Liu etal., 1997). Because NRAMP transporters are important for Mndependent TORC1 activation, and because TORC1 is often embedded in regulatory feedback loops to ensure cellular homeostasis (Eltschinger and Loewith, 2016), we next asked whether rapamycinmediated TORC1 inactivation may affect the levels and/or localization of Smf1 and Smf2 using a strain that expresses Nterminally tagged Smf1 (GFPSmf1) under its own promoter from a nonendogenous locus or a strain that expresses Cterminally tagged Smf2 (Smf2GFP) from its endogenous locus (GarcíaRodríguez The online version of this article includes the following source data for figure 2: Source data 1. Quantification of blots for graphs shown in Figure2B–D–G. Source data 2. Uncropped blots shown in Figure2A–C–F and quantified in Figure2B–D. Source data 3. Raw blots shown in Figure2A–C–F and quantified in Figure2B–D. Figure 2 continued Research article Biochemistry and Chemical Biology | Genetics and Genomics Nicastro, Gaillard etal. eLife 2022;11:e80497. DOI: https://doi.org/10.7554/eLife.80497 8 of 20 etal., 2015; Renz etal., 2020). Rapamycin treatment triggered a strong increase in GFPSmf1 levels (Figure4A and B), which is likely due to transcriptional activation of Smf1 under these conditions as published earlier (Reinke etal., 2006). In line with this interpretation, we found the respective increase to be abolished when rapamycintreated cells were cotreated with the protein synthesis inhibitor cycloheximide (CHX). In addition, because GFPSmf1 appeared to be degraded at a similar rate in CHXtreated cells that were treated, or not, with rapamycin, our data further indicate that TORC1 does not control Smf1 levels through posttranslational control of Smf1 turnover. Interestingly, A B MgCl2 [µM] - 20 40 80 160 640320------ ---- - --204080 160 640320 MnCl2 [µM] Tor1 Kog1 Tco89 Lst4Loop 0 0.0 0.4 0.8 1.2 80 160 240 320 400 480 560 640 Divalent Ion [µM] MgCl2 MnCl2 1.2 3.6 11 33 100 Tor1 Kog1 Tco89 Lst4Loop MnCl2 [0.16 mM]MgCl2 [4 mM] MgCl2 [4 mM] MnCl2 [0.16 mM] ATP [µM] 1.2 3.6 11 33 1001.2 3.6 11 33 100 C Tor1 Kog1 Tco89 Lst4Loop Tor1 Kog1 Tco89 Lst4Loop D 020406080 100 ATP [µM] 0.0 0.4 0.8 1.2 Relative TORC1 activity [AU] MgCl2 MnCl2 MgCl2 MnCl2 KmATP=9.5 ±1.8 µM KmATP=34.4 ±3.1 µM KmATP=50.7 ±5.7 µM Relative TORC1 activity [AU] AutoradiographySYPRO Ruby staining AutoradiographySYPRO Ruby staining Figure 3. MnCl2 stimulates TORC1 kinase activity in vitro and in vivo. (A)In vitro TORC1 kinase assays using [γ-32P]-ATP, recombinant Lst4Loop as substrate, and increasing concentrations (twofold dilutions) of MgCl2 or MnCl2. Substrate phosphorylation was detected by autoradiography (lower blot) and SYPRO Ruby staining is shown as loading control (upper blot). (B)Quantification of the assay shown in (A).Curve fitting and parameter calculations were performed with GraphPad Prism. Data shown are means (± SEM, n=3). (C)In vitro kinase assays (as in A) using the indicated concentrations of MgCl2 and/or MnCl2 and increasing concentrations of ATP. Substrate phosphorylation was detected by autoradiography (lower blot) and SYPRO Ruby staining is shown as loading control (upper blot). (D)Quantification of the assay shown in (C).Data shown are means (± SEM, n=3). Curve fitting and parameter calculations were performed with GraphPad Prism. KmATP are shown for each curve. VMAX [MnCl2]=1.13 ± 0.06, VMAX [MgCl2 MnCl2]=0.89 ± 0.03, VMAX [MgCl2]=0.47 ± 0.02. The online version of this article includes the following source data for figure 3: Source data 1. Quantification of autoradiographies for graphs shown in Figure3B–D. Source data 2. Uncropped gels and autoradiographies shown in Figure3A–C and quantified in Figure3B–D. Source data 3. Raw gels and autoradiographies shown in Figure3A–C and quantified in Figure3B–D. Research article Biochemistry and Chemical Biology | Genetics and Genomics Nicastro, Gaillard etal. eLife 2022;11:e80497. DOI: https://doi.org/10.7554/eLife.80497 9 of 20 GFPSmf1 remained predominantly at the plasma membrane in rapamycintreated cells, even though these cells also accumulated more cleaved GFP in the vacuoles (Figure4C). We infer from these data that TORC1 inhibition activates Smf1 expression, likely as part of a feedback control loop through which TORC1 couples its activity to Mn2+ uptake. Rapamycintreated cells that were either cotreated or not with CHX exhibited Smf2GFP levels that steadily decreased at a similar rate, with cleaved GFP accumulating in parallel (Figure4D and E). Since this rate appeared to be higher than the one observed in cells treated with CHX alone, our data suggest that TORC1 antagonizes the turnover of Smf2. This was further corroborated by our fluorescence microscopy analyses revealing that the GFP signal in Smf2GFPexpressing cells shifted from late Golgi/endosomal foci (see GarcíaRodríguez etal., 2015) to a predominant signal within the vacuolar lumen when cells were treated with rapamycin (Figure4F). Whether this event is an indirect consequence of higher Mn2+ uptake under these conditions (see above), or potentially part of a local feedback control mechanism of endosomal TORC1 (Hatakeyama etal., 2019), remains to be addressed in future studies. A C D F GFP-Smf1 Adh1 *GFP Time [h]00.5 120120.500.51 2 RAP CHX RAP+CHX RAPcontrol GFP-Smf1 Vph1-mCherry Merge Time [h]00.5 120120.500.51 2 RAP CHX RAP+CHX Smf2-GFP Adh1 *GFP RAPcontrol Smf2-GFP Vph1-mCherry Merge B E 00.5 12 00.5 1200.5 12 Time [h] GFP-Smf1/Total [%] 0 20 40 60 80 00.5 12 00.5 1200.5 12 Time [h] 0 40 60 80 100 RAP CHX RAP+CHX RAP CHX RAP+CHX Smf2-GFP/Total [%] 20 **** *** **** ** *** **** *** ** 35 25 70 55 [kDa] 100 35 25 70 55 [kDa] 100 55 55 Figure 4. TORC1 regulates natural resistanceassociated macrophage protein (NRAMP) transporter levels. (A)GFPSmf1 expressing cells were cultivated for 5–6hr in a synthetic medium devoid of manganese sulfate before being treated with 200ng/ml rapamycin (RAP), 25µg/ml cycloheximide (CHX), or both compounds (RAP+ CHX) for the indicated times. GFPSmf1 and cleaved GFP (*GFP) protein levels were analyzed by immunoblotting using an antiGFP antibody. Alcohol dehydrogenase (Adh1) protein levels, probed with antiAdh1 antibodies, served as a loading control. (B)Quantification of GFPSmf1. Percentages of GFPSmf1 relative to the total GFP signal (GFPSmf1+ GFP) are plotted. Data represent means ± SEM of independent experiments (n=3). Statistical analysis: unpaired twotailed ttest. *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001. (C) Microscopic analysis of GFPSmf1 localization. Cells coexpressing GFPSmf1 and the vacuolar marker Vph1mCherry were grown exponentially in a manganesefree medium for 5–6hr, then treated with 200ng/ml rapamycin for 2hr. Scale bar represents 5µm. (D–E)Cells expressing Smf2GFP from its endogenous locus were grown, treated, and processed as in (A).(F)Microscopic analysis of Smf2GFP localization. Cells coexpressing Smf2GFP and the vacuolar marker Vph1mCherry were cultivated, treated, and examined as in (C). The online version of this article includes the following source data for figure 4: Source data 1. Quantification of blots for graphs shown in Figure4B–E. Source data 2. Uncropped blots shown in Figure4A–D and quantified in Figure4B–E. Source data 3. Raw blots shown in Figure4A–D and quantified in Figure4B–E. Research article Biochemistry and Chemical Biology | Genetics and Genomics Nicastro, Gaillard etal. eLife 2022;11:e80497. DOI: https://doi.org/10.7554/eLife.80497 16 of 20 Additional information Funding Funder Grant reference number Author Universidad de Sevilla 2020/00001326 Hélène Gaillard Ralf Erik Wellinger Junta de Andalucía P20-RT-01220 Ralf Erik Wellinger European Molecular Biology Organization STF-8685 Ralf Erik Wellinger Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung 310030_166474/184671 Claudio De Virgilio Ministerio de Ciencia, Innovación y Universidades PGC2018-096244-B-I00 Raúl V Durán Ministerio de Ciencia, Innovación y Universidades FPU19/04914 Laura Zarzuela The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions Raffaele Nicastro, Conceptualization, Formal analysis, Investigation, Methodology, Writing – review and editing; Hélène Gaillard, Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing – review and editing; Laura Zarzuela, Elisabet FernándezGarcía, Investigation; MariePierre PéliGulli, Néstor GarcíaRodríguez, Conceptualization, Investigation, Methodology, Writing – review and editing; Mercedes Tomé, Conceptualization, Investigation, Methodology; Raúl V Durán, Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Methodology, Writing – review and editing; Claudio De Virgilio, Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Methodology, Writing – original draft, Writing – review and editing; Ralf Erik Wellinger, Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing Author ORCIDs Raffaele Nicastro http://orcid.org/0000-0002-5420-2228 Hélène Gaillard http://orcid.org/0000-0002-5740-0641 MariePierre PéliGulli http://orcid.org/0000-0002-6908-7082 Néstor GarcíaRodríguez http://orcid.org/0000-0002-4049-1604 Claudio De Virgilio http://orcid.org/0000-0001-8826-4323 Ralf Erik Wellinger http://orcid.org/0000-0002-4421-6618 Decision letter and Author response Decision letter https://doi.org/10.7554/eLife.80497.sa1 Author response https://doi.org/10.7554/eLife.80497.sa2 Additional files Supplementary files • Supplementary file 1. RTG13 target genes downregulated in pmr1∆ cells. • Supplementary file 2. Plasmids used in this study. • Supplementary file 3. Yeast strains used in this study. • MDAR checklist Research article Biochemistry and Chemical Biology | Genetics and Genomics Nicastro, Gaillard etal. eLife 2022;11:e80497. DOI: https://doi.org/10.7554/eLife.80497 17 of 20 Data availability All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figure 1, Figure 1figure supplement 1, Figure 2, Figure 3, Figure 4 and Figure 5. The data set entitled 'Expression data of pmr1∆ mutants', originally published in GarcíaRodríguez et al (2012) and reused in this study is accessible at Gene Expression Omnibus under the accession code GSE29420. 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