Inorganic phosphate (Pi) is central to fundamental cellular processes and the metabolic economy, and is constantly acquired in order to maintain intracellular Pi levels. While much is known about cellular adaptation during Pi starvation, how intracellular Pi is maintained in Pi-replete conditions remains unclear. Here, using Saccharomyces cerevisiae, we uncover an essential role for the Pho4 transcription factor in maintaining intracellular Pi under Pi-replete conditions, via the high affinity Pho84 transporter. Basal Pho4-dependent output is required for intracellular Pi maintenance, and the loss of Pho4 results in decreased intracellular Pi. We uncover that the Pho4 dependent, high affinity Pi transporter Pho84 is the primary transporter required for this intracellular Pi maintenance in phosphate replete conditions, and is not compensated by other transporters. The loss of Pho4 or Pho84 decreases intracellular Pi, with reduced ATP and glycolysis, and decreased growth. Through comparative genomic and phylogenetic analyses we establish that Pho84 is universally conserved across fungi, and Pho84 alone is orthologous to the plant high-affinity phosphate transporter PHT1. Thus, Pho84 is a primary determinant of intracellular Pi homeostasis during phosphate replete growth, and Pi acquisition in replete conditions is built around high-affinity phosphate transport. These findings reiterate the importance of Pi acquisition via high-affinity transport for metabolic homeostasis, with implications for microbial fermentation-based applications.

The mitochondrial inner membrane (IMM) is depicted as a scaffold housing the enzymes of the electron transport chain, allowing electron transfer and generation of the electrochemical gradient that drives respiration and adenosine triphosphate (ATP) synthesis. However, evidence from imaging, structural biology, and novel chemical probes reveals that the IMM is not a mere passive backdrop: its physical properties change with the metabolic state, and together these changes influence mitochondrial function. Here, we summarize how IMM microviscosity, lipid packing, and mesoscale organization might shape electron transfer kinetics, reactive oxygen species (ROS) production, and mitochondrial form/function. We discuss new chemical probes that reveal IMM organization, alongside novel chemical/genetic approaches to alter IMM organization and mitochondrial function. Finally, we outline current challenges in measuring or modulating the state of the IMM, suggesting approaches required to address how the IMM state is coupled with electron entry routes, ROS dynamics, and mitochondrial morphology.

The actin cytoskeletal network is closely associated with mitochondria and performs crucial functions in mitochondrial movement, inheritance, and fission-fusion. Although its role in mitochondrial division is established, the specific contributions of actin-binding proteins (ABPs) remain unclear. Here, we report the role of tropomyosin, an ABP, in modulating mitochondrial morphology and dynamics. We demonstrate that loss of TPM1 and TPM2 in Saccharomyces cerevisiae differentially alters mitochondrial morphology. Tpm1 deletion results in fragmented mitochondria, whereas Tpm2 deletion produces an elongated tubular morphology. Through live-cell imaging, we show the localization of both paralogs to mitochondria, providing direct evidence of their association with the organelle. Microscopy-based analysis of fission-fusion frequencies revealed no change in the Tpm1 deletion, whereas Tpm2 deletion showed a decrease in these events, with a concomitant increase in the fusion factor Mgm1. Further, we characterized the overall health of mitochondria in the Tpm deletion mutants. Fragmented mitochondria in the Tpm1 deletion were hyperpolarized and exhibited increased mass and activity with elevated OCR, ATP levels, and basal ROS. In contrast, the tubular morphology of the Tpm2 deletion did not impair mitochondrial health. Overall, our findings suggest that Tpm modulates mitochondrial morphology and dynamics through its association with the actin cytoskeletal network.

A cell functions as a metabolic economy that parses information on nutrient availability and internal metabolic flux and then converts that to state outcomes that maintain or shift homeostasis. Here, we discuss how the nutrient-signaling machinery in cells follows an hourglass (or bow tie) design architecture, using modular signal integrators. In eukaryotic cells, this architecture is exemplified by two evolutionarily conserved, core complexes: mechanistic target of rapamycin complex 1 (mTORC1)/TORC1 and AMP-activated protein kinase (AMPK). This bow-tie design of core signal integrators enables cells to condense, fan-in, and integrate noisy metabolic information. These integrators then meaningfully transduce this condensed information into durable cell state transitions by fanning-out resource allocations toward distinct outputs that are all within the possibilities of the existing metabolic economy. Through this design, cells can incorporate diverse metabolite- or flux-sensing modules, secondary integrators, and localization or higher-order assemblies to alter response kinetics over time and space. We discuss how these inherent design constraints result in robust yet versatile cellular decision-making that can drive cell-to-cell heterogeneity. Finally, we highlight how genetic mutations in this machinery disrupt information processing through the bow tie, shifting homeostasis toward disease states.

Trehalose is a widely prevalent disaccharide that acts as a cellular stress protectant and functions as an energy source that enters central carbon metabolism when broken down. The evolution and distribution of trehalose breakdown pathways across kingdoms of life is not well studied, and therefore the ability of different organisms to consume trehalose as a carbon source is unknown. In this study, we build a comprehensive evolutionary analysis of the four known trehalose breakdown pathways-trehalase (acid, neutral, glycosyl hydrolase 15), trehalose phosphorylases (TP, treP), and trehalose specific phosphotransferases (PTS), by studying their distributions across ∼3,800 prokaryotic and eukaryotic genomes. Our study suggests the presence of trehalase in the last eukaryotic common ancestor, and reveals near-universal presence of trehalase in eukaryotes, except in all birds where trehalase was lost in the first bird ancestor. Fungi alone retain additional TPs in addition to trehalase. In contrast, while trehalose breakdown in prokaryotes is highly sporadic, it can occur via multiple, independently evolved pathways, including trehalase, the trehalose-specific PTS and TP. Finally, we observe that a subset of fast-growing Gammaproteobacteria retain the trehalose specific PTS, the loss of which reduces growth in Escherichia coli. Overall, our findings uncover the evolutionary landscape of trehalose breakdown, and use of this versatile disaccharide as an energy reserve in different kingdoms of life.

Exercise impinges on almost all physiological processes at an organismal level and is a potent intervention to treat various diseases. Exercise performance is well established to display diurnal rhythm, peaking during the late active phase. However, the underlying molecular/metabolic factors and mitochondrial energetics that possibly dictate time-of-day exercise capacity remain unknown. Here, we have unraveled the importance of diurnal variation in mitochondrial functions as a determinant of skeletal muscle exercise performance. Our results show that exercise-induced muscle metabolome and mitochondrial energetics are distinct at ZT3 and ZT15. Importantly, we have elucidated key diurnal differences in mitochondrial functions that are well correlated with disparate time-of-day-dependent exercise capacity. Providing causal mechanistic evidence, we illustrate that loss of Sirtuin4 (SIRT4), a well-known mitochondrial regulator, abrogates mitochondrial diurnal variation and consequently abolishes time-of-day-dependent muscle output. Therefore, our findings unequivocally demonstrate the pivotal role of baseline skeletal muscle mitochondrial functions in dictating diurnal exercise capacity.

Molecular control over cell division is traditionally studied using liquid broths or 2D flat-plate cultures — neither of which recapitulate the complex visco-elasto-plastic properties of 3D natural habitats such as tissues, mucus, and soil. Consequently, how such regimes of physical confinement influence proliferative growth remains unknown. Here, by engineering mechanically tunable and transparent growth matrices, we directly visualize yeast budding across 3D viscoelastic regimes. We discover that elevated physical confinement drastically prolongs budding intervals without causing physiological defects. Remarkably, reduced proliferative rates are not associated with transcriptional signatures of mechanosensation or cell cycle dysregulation. Rather, 3D confinement physically constrains the volumetric growth of incipient buds — manifesting as delayed cell cycle progression. Hence, our findings establish a fundamentally unique form of physical regulation over eukaryotic cell division. ### Competing Interest Statement The authors have declared no competing interest. National Centre for Biological Sciences, https://ror.org/03gf8rp76

Folates are essential for all organisms. They are acquired either through de novo biosynthesis or from the diet. Yeast, fungi, and plants make their own folates, and it has not been clear if plasma membrane folate transporters exist in these organisms. Using a synthetic lethal screen in Saccharomyces cerevisiae, we observed that deletions in a gene encoding the previously identified glutathione (GSH) transporter, OPT1, exhibited severe growth defects with a disruption in folate biosynthesis. Uptake experiments confirmed that Opt1p/Hgt1p can transport folinic acid and the naturally abundant methyl tetrahydrofolate. As S. cerevisiae Opt1p was able to transport both folate and GSH, we used alanine-scanning mutants in the transmembrane domains of the channel pore to identify the residues required specifically for the uptake of folates and distinct from those required for GSH. We further examined the oligopeptide transporter (OPT) family of other organisms for the presence of folate transporters. In C. albicans, CaOPT1, the ortholog of S. cerevisiae OPT1, efficiently transported folate but not GSH, while the previously characterized GSH transporter, CaOPT7, could not transport folate. Aspergillus fumigatus has eight homologs of the OPT family, of which OptB and OptH transport folates. In the plant Arabidopsis thaliana, the Opt1 homologs AtOpt2, AtOpt4, and AtOpt6 transport folates. This discovery of folate transporters across fungi and plants fills a critical gap in our understanding of folate metabolism and can benefit the exploitation of these pathways in pathogenic fungi and in plants.

Balancing SAM allocations for methylation reactions, and maintaining the SAM/SAH ratio is crucial for cellular homeostasis. How cells balance the allocation of methyl pools between different sinks, remains under studied. In this study using S. cerevisiae , we identify a role of the amino acid response regulator Gcn4 (Atf4) in balancing the methyl allocations between phospholipids and histones when methionine is abundant. Here, when SAM/SAH ratio increases during methionine supplementation, Gcn4-dependent outputs critically regulate the appropriate allocation of methyl pools to phospholipids and histones. Gcn4 regulates phospholipid methylation by controlling Ino2 levels, which is a primary transcriptional regulator of phospholipid biogenesis. In the absence of Gcn4, Ino2 levels decrease, leading to the downregulation of the PE methyltransferases Cho2 and Opi3. This downregulation of these methyltransferases reduces SAM consumption for phospholipid biosynthesis, and in turn elevates the SAM/SAH ratio in the cell. The elevated SAM pools are subsequently re-allocated towards histone hyper-methylation. Our study reveals the novel role of Gcn4 as a regulator of phospholipid biosynthesis during methionine sufficiency, highlighting its role in appropriate methyl allocations in cells. This Gcn4-dependent check on methylation is therefore necessary to enable cell proliferation when SAM pools are abundant. ### Competing Interest Statement The authors have declared no competing interest. Department of Science and Technology, INSPIRE Faculty Fellowship (RS), DST/INSPIRE/04/2020/002317 DST-ANRF-Core Research Grant, Government of India, CRG/2023/007183 DBT S. Ramachandran National Bioscience Award for Career Development, Department of Biotechnology, Govt of India Institute of Bioinformatics and Applied Biotechnology, Bangalore Department of IT-BT, Government of Karnataka, India DBT-Wellcome Trust India Alliance Senior Fellowship, IA/S/21/2/505922

Effective clearance of Mycobacterium tuberculosis (Mtb) requires targeting drug-tolerant populations within host macrophages. Here, we show that macrophage metabolic states govern redox heterogeneity and drug response in intracellular Mtb. Using a redox-sensitive fluorescent reporter (Mrx1-roGFP2), flow cytometry, and transcriptomics, we found that macrophages with high oxidative phosphorylation (OXPHOS) and low glycolysis harbor reductive, drug-tolerant Mtb, whereas glycolytically active macrophages generate mitochondrial ROS via reverse electron transport, imposing oxidative stress on Mtb and enhancing drug efficacy. Computational and genetic analyses identified NRF2 as a key regulator linking host metabolism to bacterial redox state and drug tolerance. Pharmacological reprogramming of macrophages with the FDA-approved drug meclizine (MEC) shifted metabolism towards glycolysis, suppressed redox heterogeneity, and reduced Mtb drug tolerance in macrophages and mice. MEC exhibited no adverse interactions with frontline anti-TB drugs. These findings demonstrate the therapeutic potential of host metabolic reprogramming to overcome Mtb drug tolerance.

Mitochondria are dynamic organelles that constantly change morphology. What controls mitochondrial morphology however remains unresolved. Using actively respiring yeast cells growing in distinct carbon sources, we find that mitochondrial morphology and activity are unrelated. Cells can exhibit fragmented or networked mitochondrial morphology in different nutrient environments independent of mitochondrial activity. Instead, mitochondrial morphology is controlled by the intracellular redox state, which itself depends on the nature of electron entry into the electron transport chain (ETC)-through complex I/II or directly to coenzyme Q/cytochrome c. In metabolic conditions where direct electron entry is high, reactive oxygen species (ROS) increase, resulting in an oxidized cytosolic environment and rapid mitochondrial fragmentation. Decreasing direct electron entry into the ETC by genetic or chemical means, or reducing the cytosolic environment rapidly restores networked morphologies. Using controlled disruptions of electron flow to alter ROS and redox state, we demonstrate minute-scale, reversible control between networked and fragmented forms in an activity-independent manner. Mechanistically, the fission machinery through Dnm1 responds in minute-scale to redox state changes, preceding the change in mitochondrial form. Thus, the metabolic state of the cell and its consequent cellular redox state actively control mitochondrial form.

Degradation of many yeast mRNAs involves decapping by the Dcp1:Dcp2 complex. Previous studies on decapping activators Edc3 and Scd6 suggested their limited roles in mRNA decay. RNA-seq analysis of mutants lacking one or both proteins revealed that Scd6 and Edc3 have largely redundant activities in targeting numerous mRNAs for degradation that are masked in the single mutants. These transcripts are frequently targeted by decapping activators Dhh1 and Pat1, and the collective evidence suggests that Scd6/Edc3 act interchangeably to recruit Dhh1 to Dcp2. Ribosome profiling shows that redundancy between Scd6 and Edc3 and their functional interactions with Dhh1 and Pat1 extend to translational repression of particular transcripts, including a cohort of poorly translated mRNAs displaying interdependent regulation by all four factors. Scd6/Edc3 also participate with Dhh1/Pat1 in post-transcriptional repression of proteins required for respiration and catabolism of alternative carbon sources, which are normally expressed only in limiting glucose. Simultaneously eliminating Scd6/Edc3 increases mitochondrial membrane potential and elevates metabolites of the tricarboxylic acid and glyoxylate cycles typically observed only during growth in low glucose. Thus, Scd6/Edc3 acts redundantly, in parallel with Dhh1 and in cooperation with Pat1, to adjust gene expression to nutrient availability by controlling mRNA decapping and decay.

Crabtree-positive yeasts rapidly consume glucose via glycolysis, making it difficult to experimentally estimate their actual glycolytic rate or flux. We present a stable isotope labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based protocol to quantitatively estimate glycolytic and related carbon metabolic fluxes using Saccharomyces cerevisiae. This approach defines time windows to capture glucose metabolic intermediate production before label saturation, enabling a comparison of glycolytic flux changes across different cells. This protocol provides a reliable, quantitative approach to study dynamic metabolic fluxes in these cells. For complete details on the use and execution of this protocol, please refer to Vengayil et al., 2024.1.

Mitochondria are dynamic organelles with essential roles in energetics and metabolism. Several metabolites are common to both the cytosolic and mitochondrial fractions of the cell. The compartmentalization of metabolites within the mitochondria allows specialized uses for mitochondrial metabolism. Inorganic phosphate (Pi) is one such critical metabolite required for ATP synthesis, via glycolysis and mitochondrial oxidative phosphorylation. Estimating total cellular Pi levels cannot distinguish the distribution of Pi pools across different cellular compartments, such as the cytosol and mitochondria, and therefore separate the contributions made toward glycolysis or other cytosolic metabolic processes vs. mitochondrial outputs. Quantifying Pi pools in mitochondria can therefore be very useful toward understanding mitochondrial metabolism and phosphate homeostasis. Here, we describe a protocol for the fairly rapid, efficient isolation of mitochondria from Saccharomyces cerevisiae by immunoprecipitation for quantitative estimation of mitochondrial and cytosolic Pi pools. This method utilizes magnetic beads to capture FLAG-tagged mitochondria (Tom20-FLAG) from homogenized cell lysates. This method provides a valuable tool to investigate changes in mitochondrial phosphate dynamics. Additionally, this protocol can be coupled with LC-MS approaches to quantitatively estimate mitochondrial metabolites and proteins and can be similarly used to assess other metabolite pools that are partitioned between the cytosol and mitochondria. Key features • This protocol describes how to isolate mitochondria from Saccharomyces cerevisiae for quantitative estimation of inorganic phosphate or other metabolites. • Mitochondria are efficiently isolated by immunoprecipitation using magnetic beads, bypassing the need for time-consuming density-based centrifugation. • This method can be integrated into LC-MS-based workflows to quantify mitochondrial metabolites and proteins.

γ-Glu dipeptides are ubiquitous in nature, and yet their metabolism and transport are poorly understood. Here we investigate this using the dipeptide γ-Glu-met in Saccharomyces cerevisiae. γ-Glu-met was efficiently utilized by S. cerevisiae, and using a transcriptomics approach, followed by a genetic screen, we identified Seo1p, an orphan transporter of yeast, as the transporter of γ-Glu-met. Uptake studies confirmed Seo1p as a high-affinity (Km = 48 μM), highly specific transporter of γ-Glu-met, as other analogs like n-Glu-met, γ-Glu-leu, γ-Glu-cys, γ-Glu-met-gly, methionine, and methionine sulfoxide were not transported by Seo1p. The expression of SEO1 was also repressed by these sulfur sources in the medium, but it was derepressed in the presence of γ-Glu-met. Seo1p homologs were present in yeast and fungi, and both Candida auris and Candida albicans were found to encode a functional Seo1p. The intracellular degradation of γ-Glu-met was investigated and found to be dependent on both the glutathione degrading cytosolic Dug2p/Dug3p complex, and the vacuolar γ-glutamyl transpeptidase, Ecm38p. Opt2p, a member of the oligopeptide transporter family, was also identified in the screen, and deletions in OPT2 led to an inability to grow on γ-Glu-met. However, Opt2p was not primarily involved in γ-Glu-met uptake. Its deletion affected vacuolar biogenesis, which interfered with the degradation of the peptide through Ecm38p. These studies demonstrate how organisms have evolved dedicated pathways for the uptake of these unusual peptides.

A teaching course based on seemingly improbable curiosities could attract students' interest in the magic of biochemical pathways and their evolution.

Many cells in high glucose repress mitochondrial respiration, as observed in the Crabtree and Warburg effects. Our understanding of biochemical constraints for mitochondrial activation is limited. Using a Saccharomyces cerevisiae screen, we identified the conserved deubiquitinase Ubp3 (Usp10), as necessary for mitochondrial repression. Ubp3 mutants have increased mitochondrial activity despite abundant glucose, along with decreased glycolytic enzymes, and a rewired glucose metabolic network with increased trehalose production. Utilizing Δubp3 cells, along with orthogonal approaches, we establish that the high glycolytic flux in glucose continuously consumes free Pi. This restricts mitochondrial access to inorganic phosphate (Pi), and prevents mitochondrial activation. Contrastingly, rewired glucose metabolism with enhanced trehalose production and reduced GAPDH (as in Δubp3 cells) restores Pi. This collectively results in increased mitochondrial Pi and derepression, while restricting mitochondrial Pi transport prevents activation. We therefore suggest that glycolytic-flux dependent intracellular Pi budgeting is a key constraint for mitochondrial repression.

A continuous supply of energy is an essential prerequisite for survival and represents the highest priority for the cell. We hypothesize that cell differentiation is a process of optimization of energy flow in a changing environment through phenotypic adaptation. The mechanistic basis of this hypothesis is provided by the established link between core energy metabolism and epigenetic covalent modifications of chromatin. This theory predicts that early metabolic perturbations impact subsequent differentiation. To test this, we induced transient metabolic perturbations in undifferentiated human hematopoietic cells using pharmacological inhibitors targeting key metabolic reactions. We recorded changes in chromatin structure and gene expression, as well as phenotypic alterations by single-cell ATAC and RNA sequencing, time-lapse microscopy and flow cytometry. Our observations suggest that these metabolic perturbations are shortly followed by alterations in chromatin structure, leading to changes in gene expression. We also show that these transient fluctuations alter the differentiation potential of the cells.

Cells contain disparate amounts of distinct amino acids, each of which has different metabolic and chemical origins, but the supply cost vs demand requirements of each is unclear. Here, using yeast we quantify the restoration-responses after disrupting amino acid supply, and uncover a hierarchically prioritized restoration strategy for distinct amino acids. We comprehensively calculate individual amino acid biosynthetic supply costs, quantify total demand for an amino acid, and estimate cumulative supply/demand requirements for each amino acid. Through this, we discover that the restoration priority is driven by the gross demand for an amino acid, which is itself coupled to low supply costs for that amino acid. Demand from metabolic requirements dominate the demand-pulls for an amino acid, as exemplified by the largest restoration response upon disrupting arginine supply. Collectively, this demand-driven framework that drives the amino acid economy can identify novel amino acid responses, and help design metabolic engineering applications. How cells prioritize restoring amino acids when supply is limited is unknown. Here, using budding yeast, the authors build an economic framework to explain prioritization strategies and find that the amino acid economy is driven by demand for low-cost amino acids.

To sustain growth in changing nutrient conditions, cells reorganize outputs of metabolic networks and appropriately reallocate resources. Signaling by reversible protein phosphorylation can control such metabolic adaptations. In contrast to kinases, the functions of phosphatases that enable metabolic adaptation as glucose depletes are poorly studied. Using a Saccharomyces cerevisiae deletion screen, we identified the PP2A-like phosphatase Ppg1 as required for appropriate carbon allocations towards gluconeogenic outputs—trehalose, glycogen, UDP-glucose, UDP-GlcNAc—after glucose depletion. This Ppg1 function is mediated via regulation of the assembly of the Far complex—a multi-subunit complex that tethers to the ER and mitochondrial outer membranes forming localized signaling hubs. The Far complex assembly is Ppg1 catalytic activity-dependent. Ppg1 regulates the phosphorylation status of multiple ser/thr residues on Far11 to enable the proper assembly of the Far complex. The assembled Far complex is required to maintain gluconeogenic outputs after glucose depletion. Glucose in turn regulates Far complex amounts. This Ppg1-mediated Far complex assembly, and Ppg1-Far complex dependent control of gluconeogenic outputs enables adaptive growth under glucose depletion. Our study illustrates how protein dephosphorylation is required for the assembly of a multi-protein scaffold present in localized cytosolic pools, to thereby alter gluconeogenic flux and enable cells to metabolically adapt to nutrient fluctuations.