Amit R. Reddi

Probing in vivo Mn2+ speciation and oxidative stress resistance in yeast cells with electron-nuclear double resonance spectroscopy

Manganese is an essential transition metal that, among other functions, can act independently of proteins to either defend against or promote oxidative stress and disease. The majority of cellular manganese exists as low molecular-weight Mn2+ complexes, and the balance between opposing “essential” and “toxic” roles is thought to be governed by the nature of the ligands coordinating Mn2+. Until now, it has been impossible to determine manganese speciation within intact, viable cells, but we here report that this speciation can be probed through measurements of 1H and 31P electron-nuclear double resonance (ENDOR) signal intensities for intracellular Mn2+. Application of this approach to yeast (Saccharomyces cerevisiae) cells, and two pairs of yeast mutants genetically engineered to enhance or suppress the accumulation of manganese or phosphates, supports an in vivo role for the orthophosphate complex of Mn2+ in resistance to oxidative stress, thereby corroborating in vitro studies that demonstrated superoxide dismutase activity for this species.

The Overlapping Roles of Manganese and Cu/Zn SOD in Oxidative Stress Protection

In various organisms, high intracellular manganese provides protection against oxidative damage through unknown pathways. Herein we use a genetic approach in Saccharomyces cerevisiae to analyze factors that promote manganese as an antioxidant in cells lacking Cu/Zn superoxide dismutase (sod1 Delta). Unlike certain bacterial systems, oxygen resistance in yeast correlates with high intracellular manganese without a lowering of iron. This manganese for antioxidant protection is provided by the Nramp transporters Smf1p and Smf2p, with Smf1p playing a major role. In fact, loss of manganese transport by Smf1p together with loss of the Pmr1p manganese pump is lethal to sod1 Delta cells despite normal manganese SOD2 activity. Manganese-phosphate complexes are excellent superoxide dismutase mimics in vitro, yet through genetic disruption of phosphate transport and storage, we observed no requirement for phosphate in manganese suppression of oxidative damage. If anything, elevated phosphate correlated with profound oxidative stress in sod1 Delta mutants. The efficacy of manganese as an antioxidant was drastically reduced in cells that hyperaccumulate phosphate without effects on Mn SOD activity. Non-SOD manganese can provide a critical backup for Cu/Zn SOD1, but only under appropriate physiologic conditions.

Manganese Homeostasis in Saccharomyces cerevisiae

Manganese, whose name is derived from a Greek word for magic, is an essential trace element that is required by organisms across every kingdom of life.1 The magic of manganese lies not only in its ability to transform itself into an effector for a diverse range of redox and nonredox functions but also in its ability to appear and disappear from a variety of locations within a cell. Regarding the former, decades of chemical, biochemical, and biophysical characterization of manganese-containing complexes have revealed much about how primary and secondary coordination spheres temper the reactivity and function of the metal center.2,3 However, regarding the latter, the cellular transport and trafficking of manganese, much of the mystery persists.4–6 A diverse array of metalloproteins require manganese for function, including oxidoreductases, DNA and RNA poly-merases, peptidases, kinases, decarboxylases, and sugar transferases, which are present in a variety of cellular locales, such as the nucleus, mitochondria, cytosol, Golgi, and vacuole.3 Yet it is not completely clear how cells manage to import appropriate amounts of environmental manganese, transport the metal to the correct intracellular compartments, and distribute it to relevant biomacromolecules.4–6 Matters may be further complicated by environmental stresses that could lead to under- or overexposure of cells to manganese; too little manganese may inactivate manganese-requiring biological processes, whereas too much manganese is toxic.3–6 The latter is underscored by manganism, a Parkin-son’s disease-like condition in which overexposure to manganese leads to severe neurological damage.7–12 In such instances where cells are manganese stressed, either through deficiency or surplus, living systems are obligated to respond through the concerted regulation of manganese cell surface and intracellular transporters, as well as any putative manganese chaperones, so as to maintain healthy intracellular concentrations of the metal and correctly appropriate manganese to its cognate protein ligands.4–6 Much of our current, albeit limited understanding of manganese homeostatic mechanisms has been elucidated through molecular genetic studies of the budding yeast, Saccharomyces cerevisiae. As such, this review will largely focus on global manganese homeostatic pathways operative in the eukaryotic cell of S. cerevisiae, with references being made to analogous pathways in metazoans where applicable. Specifically, this review will highlight the mechanisms by which cell surface and intracellular manganese transporters import and distribute manganese, as well as how these homeostatic mechanisms respond to manganese deficiency or surplus. The manner in which cells mediate the uptake and distribution of manganese is dependent on the exposure of environmental manganese to the cell. As depicted in Figure 1, manganese exposure lies on a continuum between two environmental extremes, manganese deficiency and surplus, with manganese sufficiency occupying a place in the continuum between the two extremes.6 The range of intra-cellular manganese levels that constitute manganese sufficiency is quite large, nearly 2 orders of magnitude. In various studies done, yeast were seen to accumulate between 2–100 nmol of manganese/(10 × 109 cells), or 0.04–2.0 mM manganese (assuming a single yeast cell has a volume of 50 femtoliters), without any impact on cell growth.13–18 However, at levels below or above this, the stresses of manganese deficiency and manganese toxicity ensue, setting off a series of responses aimed at normalizing manganese levels. In general, cells respond to such manganese stress by upregulating or downregulating cell surface and intra-cellular transport systems. As described in detail below, regulation of manganese transport in yeast does not involve any known transcriptional pathway,6,13,19 such as those described for transporters of copper, zinc, and iron.20 Instead, all the known manganese regulatory pathways in yeast occur at the post-translational level through changes in transporter protein localization and turnover. In this review, we shall provide an overview of the various manganese transport systems in S. cerevisiae, how they function under diverse cellular conditions, and how certain transporters are regulated in response to manganese stress. Figure 1 Approximation of the intracellular accumulation of manganese in yeast as a function of varying concentrations of manganese supplemented to a minimal medium. Superimposed on the diagram is a growth isotherm of yeast cells as a function of manganese supplementation. ... 2. Nramp Manganese Transporters, Smf1p and Smf2p 2.1. Overview of the Nramp Family of Metal Ion Transporters The Nramp family constitutes a large class of metal-ion transporters that are widely conserved from bacteria to humans.21 Nramp transporters, which are localized either at the cell surface or in intracellular vesicles, drive the translocation of a wide range of divalent metal substrates, including manganese, iron, cobalt, copper, zinc, and cadmium, across membranes toward the cytosol by coupling the flow of protons and metals.4,6,21–32 The first of these to be mechanistically studied was mammalian DMT1 (divalent metal transporter). When expressed in oocytes, Dmt1 was found to cotransport Fe2+ and H+ with a stoichiometry of 1:1 at pH 7.0 and physiological membrane potentials of −90 to −30 mV, demonstrating that Nramp transporters are indeed proton-metal cotransporters.32 DMT1 was also seen to exhibit “driving force slippage”, where at increased membrane potentials or proton concentrations metal and proton uptake becomes decoupled, resulting in significant deviations (slippage) from metal/proton stoichiometries of 1:1.32 Driving force slippage is thought to “put the brakes” on excessive metal ion uptake and prevent toxic metal ion overload under conditions when protons are in excess.24,25 Although it is unusual for cations (protons) to drive the translocation of other cations (divalent transition metals), Nelson and colleagues observed an influence of anions on the currents generated by DMT1.24,27 Their results indicated that metal transport is also dependent on Cl− or other small anions and that the divalent metals may in fact be cotransported with Cl− due to its abundance.27 All Nramp transporters possess a highly conserved core of 10 transmembrane domains, with yeast and mammalian Nramps possessing 11 and 12 transmembrane domains, respectively, as well as a highly conserved metal transport motif in a cytoplasmic loop between transmembrane domain 8 and 9.25 The current lack of high-resolution structural information has made it difficult to delineate the pathway and residues critical for metal and proton translocation. However, mutagenesis studies of bacterial, yeast, and mammalian Nramps, which have been extensively reviewed elsewhere,25 are beginning to uncover the key residues that enable metal binding/translocation, proton coupling, and substrate slippage. Mn2+ is a relatively hard Lewis acid, and based solely on Pearson’s Hard–Soft Acid Base Theory, one would expect the Nramp transporters to contain hard bases such as proteinaceous carboxylates to coordinate metal.3 Indeed, the Nramp transporters possess a number of conserved glutamate and aspartate-based carboxylate ligands that, when mutated, abrogate transport activity of the protein. For example, three anionic and highly conserved residues, D93, E154, and D192 in transmembrane domains 1, 3 and 4, respectively, are essential for metal transport activity in human DMT1, and the same is true for the analogous residues in bacterial and yeast Nramp transporters.25,33

The effect of phosphate accumulation on metal ion homeostasis in Saccharomyces cerevisiae

Much of what is currently understood about the cell biology of metals involves their interactions with proteins. By comparison, little is known about interactions of metals with intracellular inorganic compounds such as phosphate. Here we examined the role of phosphate in metal metabolism in vivo by genetically perturbing the phosphate content of Saccharomycescerevisiae cells. Yeast pho80 mutants cannot sense phosphate and have lost control of phosphate uptake, storage, and metabolism. We report here that pho80 mutants specifically elevate cytosolic and nonvacuolar levels of phosphate and this in turn causes a wide range of metal homeostasis defects. Intracellular levels of the hard-metal cations sodium and calcium increase dramatically, and cells become susceptible to toxicity from the transition metals manganese, cobalt, zinc, and copper. Disruptions in phosphate control also elicit an iron starvation response, as pho80 mutants were seen to upregulate iron transport genes. The iron-responsive transcription factor Aft1p appears activated in cells with high phosphate content in spite of normal intracellular iron levels. The high phosphate content of pho80 mutants can be lowered by mutating Pho4p, the transcription factor for phosphate uptake and storage genes. Such lowering of phosphate content by pho4 mutations reversed the high calcium and sodium content of pho80 mutants and prevented the iron starvation response. However, pho4 mutations only partially reversed toxicity from heavy metals, representing a novel outcome of phosphate dysregulation. Overall, these studies underscore the importance of maintaining a charge balance in the cell; a disruption in phosphate metabolism can dramatically impact on metal homeostasis.

Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.

Significance All heme-dependent functions require the mobilization of labile heme (LH), of which there is little understanding of its nature and dynamics. To probe LH pools, we developed genetically encoded fluorescent heme sensors and deployed them in the unicellular eukaryote Saccharomyces cerevisiae. We find that LH is relatively abundant in the cytosol, but exceedingly low in the mitochondria and nucleus. Further, we find that LH can be mobilized by signaling molecules like nitric oxide. We also find that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase constitutes a major heme buffer and is responsible for regulating the activity of a heme-dependent transcription factor. Altogether, our work will have profound implications for understanding the mechanisms of heme utilization. Heme is an essential cofactor and signaling molecule. Heme acquisition by proteins and heme signaling are ultimately reliant on the ability to mobilize labile heme (LH). However, the properties of LH pools, including concentration, oxidation state, distribution, speciation, and dynamics, are poorly understood. Herein, we elucidate the nature and dynamics of LH using genetically encoded ratiometric fluorescent heme sensors in the unicellular eukaryote Saccharomyces cerevisiae. We find that the subcellular distribution of LH is heterogeneous; the cytosol maintains LH at ∼20–40 nM, whereas the mitochondria and nucleus maintain it at concentrations below 2.5 nM. Further, we find that the signaling molecule nitric oxide can initiate the rapid mobilization of heme in the cytosol and nucleus from certain thiol-containing factors. We also find that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase constitutes a major cellular heme buffer, and is responsible for maintaining the activity of the heme-dependent nuclear transcription factor heme activator protein (Hap1p). Altogether, we demonstrate that the heme sensors can be used to reveal fundamental aspects of heme trafficking and dynamics and can be used across multiple organisms, including Escherichia coli, yeast, and human cell lines.

Analysis of Hypoxia and Hypoxia-Like States through Metabolite Profiling

Background In diverse organisms, adaptation to low oxygen (hypoxia) is mediated through complex gene expression changes that can, in part, be mimicked by exposure to metals such as cobalt. Although much is known about the transcriptional response to hypoxia and cobalt, little is known about the all-important cell metabolism effects that trigger these responses. Methods and Findings Herein we use a low molecular weight metabolome profiling approach to identify classes of metabolites in yeast cells that are altered as a consequence of hypoxia or cobalt exposures. Key findings on metabolites were followed-up by measuring expression of relevant proteins and enzyme activities. We find that both hypoxia and cobalt result in a loss of essential sterols and unsaturated fatty acids, but the basis for these changes are disparate. While hypoxia can affect a variety of enzymatic steps requiring oxygen and heme, cobalt specifically interferes with diiron-oxo enzymatic steps for sterol synthesis and fatty acid desaturation. In addition to diiron-oxo enzymes, cobalt but not hypoxia results in loss of labile 4Fe-4S dehydratases in the mitochondria, but has no effect on homologous 4Fe-4S dehydratases in the cytosol. Most striking, hypoxia but not cobalt affected cellular pools of amino acids. Amino acids such as aromatics were elevated whereas leucine and methionine, essential to the strain used here, dramatically decreased due to hypoxia induced down-regulation of amino acid permeases. Conclusions These studies underscore the notion that cobalt targets a specific class of iron proteins and provide the first evidence for hypoxia effects on amino acid regulation. This research illustrates the power of metabolite profiling for uncovering new adaptations to environmental stress.

Regulation of intracellular heme trafficking revealed by subcellular reporters

Significance The intracellular and extracellular trafficking of heme, a hydrophobic and potentially cytotoxic cofactor in proteins such as hemoglobin, remains an underexplored area. While cellular heme can be derived exogenously or from de novo synthesis, it is unclear if there is differential trafficking of heme from these two sources. To critically examine this possibility, we developed peroxidase-based enzymatic reporters for heme and deployed them in subcellular compartments in mammalian cells and in several tissues in the Caenorhabditis elegans animal model. Our results show that extracellular and endogenous heme is trafficked to virtually all intracellular compartments via distinct cellular routes and that interorgan heme transport is essential for systemic regulation of heme homeostasis in C. elegans. Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.

Regulation of Manganese Antioxidants by Nutrient Sensing Pathways in Saccharomyces cerevisiae

In aerobic organisms, protection from oxidative damage involves the combined action of enzymatic and nonproteinaceous cellular factors that collectively remove harmful reactive oxygen species. One class of nonproteinaceous antioxidants includes small molecule complexes of manganese (Mn) that can scavenge superoxide anion radicals and provide a backup for superoxide dismutase enzymes. Such Mn antioxidants have been identified in diverse organisms; however, nothing regarding their physiology in the context of cellular adaptation to stress was known. Using a molecular genetic approach in Bakers’ yeast, Saccharomyces cerevisiae, we report that the Mn antioxidants can fall under control of the same pathways used for nutrient sensing and stress responses. Specifically, a serine/threonine PAS-kinase, Rim15p, that is known to integrate phosphate, nitrogen, and carbon sensing, can also control Mn antioxidant activity in yeast. Rim15p is negatively regulated by the phosphate-sensing kinase complex Pho80p/Pho85p and by the nitrogen-sensing Akt/S6 kinase homolog, Sch9p. We observed that loss of either of these upstream kinase sensors dramatically inhibited the potency of Mn as an antioxidant. Downstream of Rim15p are transcription factors Gis1p and the redundant Msn2/Msn4p pair that typically respond to nutrient and stress signals. Both transcription factors were found to modulate the potency of the Mn antioxidant but in opposing fashions: loss of Gis1p was seen to enhance Mn antioxidant activity whereas loss of Msn2/4p greatly suppressed it. Our observed roles for nutrient and stress response kinases and transcription factors in regulating the Mn antioxidant underscore its physiological importance in aerobic fitness.

Heme Mobilization in Animals: A Metallolipid’s Journey

Heme is universally recognized as an essential and ubiquitous prosthetic group that enables proteins to carry out a diverse array of functions. All heme-dependent processes, from protein hemylation to heme signaling, require the dynamic and rapid mobilization of heme to hemoproteins present in virtually every subcellular compartment. The cytotoxicity and hydrophobicity of heme necessitates that heme mobilization is carefully controlled at the cellular and systemic level. However, the molecules and mechanisms that mediate heme homeostasis are poorly understood. In this Account, we provide a heuristic paradigm with which to conceptualize heme trafficking and highlight the most recent developments in the mechanisms underlying heme trafficking. As an iron-containing tetrapyrrole, heme exhibits properties of both transition metals and lipids. Accordingly, we propose its transport and trafficking will reflect principles gleaned from the trafficking of both metals and lipids. Using this conceptual framework, we follow the flow of heme from the final step of heme synthesis in the mitochondria to hemoproteins present in various subcellular organelles. Further, given that many cells and animals that cannot make heme can assimilate it intact from nutritional sources, we propose that intercellular heme trafficking pathways must exist. This necessitates that heme be able to be imported and exported from cells, escorted between cells and organs, and regulated at the organismal level via a coordinated systemic process. In this Account, we highlight recently discovered heme transport and trafficking factors and provide the biochemical foundation for the cell and systems biology of heme. Altogether, we seek to reconceptualize heme from an exchange inert cofactor buried in hemoprotein active sites to an exchange labile and mobile metallonutrient.

Heme Gazing: Illuminating Eukaryotic Heme Trafficking, Dynamics, and Signaling with Fluorescent Heme Sensors

Heme (iron protoporphyrin IX) is an essential protein prosthetic group and signaling molecule required for most life on Earth. All heme-dependent processes require the dynamic and rapid mobilization of heme from sites of synthesis or uptake to hemoproteins present in virtually every subcellular compartment. The cytotoxicity and hydrophobicity of heme necessitate that heme mobilization be carefully controlled to mitigate the deleterious effects of this essential toxin. Indeed, a number of disorders, including certain cancers, cardiovascular diseases, and aging and age-related neurodegenerative diseases, are tied to defects in heme homeostasis. However, the molecules and mechanisms that mediate heme transport and trafficking, and the dynamics of these processes, are poorly understood. This is in large part due to the lack of physical tools for probing cellular heme. Herein, we discuss the recent development of fluorescent probes that can monitor and image kinetically labile heme with respect to its mobilization and role in signaling. In particular, we will highlight how heme gazing with these tools can uncover new heme trafficking factors upon being integrated with genetic screens and illuminate the concentration, subcellular distribution, and dynamics of labile heme in various physiological contexts. Altogether, the monitoring of labile heme, along with recent biochemical and cell biological studies demonstrating the reversible regulation of certain cellular processes by heme, is challenging us to reconceptualize heme from being a static cofactor buried in protein active sites to a dynamic and mobile signaling molecule.