Biology subjects

Laxman, S.

Publications and source records attributed to Laxman, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Rad53/Chk2 couples mitochondrial outputs and ROS with G2/M cell cycle progression

Cell-cycle progression requires coordination with cellular metabolism, yet how checkpoint kinases contribute to this coordination during normal growth remains poorly understood. Here, we uncover an unexpected role for the checkpoint kinase Rad53/Chk2 in coupling mitochondrial and redox homeostasis to G2/M progression. In prototrophic S. cerevisiae, Rad53 loss (rad53{Delta}) disrupts cell-cycle progression and causes cells to accumulate in G2/M, alongside extensive metabolic remodelling with increased tricarboxylic acid (TCA) cycle flux. Mitochondrial proteomics reveals altered relative abundances of individual electron transport chain proteins, and mitochondrial membrane potential increases without increased oxygen consumption or ATP generation. This altered mitochondrial state is associated with elevated reactive oxygen species (ROS) and oxidative stress. Genetic analyses establish that mitochondrial phenotypes require Rad53 kinase activity and are distinct from the loss of SML1. Antioxidant treatment rescues cell-division, budding defects and the accumulation of abnormally sized cells, establishing ROS as a primary cause of cell-cycle defects. Thus, Rad53 couples mitochondrial and redox homeostasis to G2/M progression during normal growth.

cell biology↗

bioRxiv2026

A conserved Pho4-Pho84 axis ubiquitously maintains intracellular phosphate homeostasis in yeast

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.

systems biology↗

bioRxiv2026

The yeast mitochondrial Porin represses Snf1/AMP Kinase signaling to attenuate viral replication

Although fungi are broadly infected with mycoviruses, the antiviral mechanisms fungal cells use to oppose viral replication are not well understood. Here we discover a new mitochondrially controlled signaling mechanism in the budding yeast Saccharomyces cerevisiae that limits replication of L-A, an RNA mycovirus that endemically infects this organism. We show that Por1, the mitochondrial voltage dependent anion channel, prevents hyper-replication of L-A in stationary phase cells that have exhausted media nutrients. By investigating known stationary phase regulators, we find that deletion of the AMP-activated Kinase homolog SNF1 reverses hyper-replication of L-A observed in por1{Delta} cells. This epistatic relationship suggests that Por1 negatively regulates Snf1 in stationary phase cells and derepressed Snf1 promotes L-A hyper-replication. We confirm this model, first demonstrating that POR1 prevents the accumulation of activated Snf1 throughout stationary phase. By investigating Snf1 signaling targets we show that this POR1-SNF1 regulatory mechanism acts in stationary phase cells to limit amino acid availability that sustain L-A replication. POR1-SNF1 signaling represents a novel physiological control mechanism to limit viral replication in a eukaryotic cell.

genetics↗

bioRxiv2025

Evolutionary analysis of Trehalose breakdown pathways

Trehalose is a widely prevalent, abundant 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 have not been 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 ~4000 prokaryotic and eukaryotic genomes. Our study suggests the presence of trehalase in the Last Eukaryotic Common Ancestor (LECA), 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 trehalose phosphorylases (TP) in addition to trehalase. In contrast, trehalose breakdown in prokaryotes is highly sporadic but can occur via multiple, independently evolved pathways, including trehalase, the trehalose-specific PTS and trehalose phosphorylase. 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.

genomics↗

bioRxiv2025

Cell clusters are programmed towards a reductive metabolic 1 state by adherence junctions

Solitary cells form stable clusters via cell-cell adhesion using adherens junctions. The role of these junctions in early cell-state changes as cells form clusters is unclear. Here, we uncover that the formation of cadherin junctions as cells cluster drives a ubiquitous metabolic reprogramming. This reprogramming enhances the pentose phosphate pathway (PPP) and NADPH production to augment a reductive state. Consequently, cell clusters stabilized by cadherin junctions have reduced intracellular reactive oxygen species (ROS), are resistant to exogenous ROS-inducing agents, and have reduced apoptotic markers. Mechanistically, this metabolic reprogramming is driven by the cadherin-dependent activation of NRF2. Blocking the cadherin junction-dependent metabolic program reverses clustered cells to resemble the solitary cell state, increasing cell death and enhancing sensitivity to exogenous ROS. These insights suggest a biochemical basis for adherens junctions mediating a reductive metabolic program as solitary cells form clusters, with implications for understanding multicellular organization and collective cell behavior.

cell biology↗

bioRxiv2025

3D confinement physically regulates cell cycle progression in budding yeast

Our understanding of cell division control traditionally comes via using liquid broths or 2D flat-plate cultures -- which cannot recapitulate the complex visco-elasto-plastic properties of natural habitats such as tissues, mucus, and soil. Consequently, how such regimes of physical confinement influence proliferative growth remains unknown. Here, using engineered, mechanically tunable and transparent growth matrices, we directly visualize yeast budding and division across 3D viscoelastic regimes. We discover that elevated physical confinement drastically prolongs budding intervals without any physiological defects or activating global regulatory programs. Remarkably, extended cell division times are not associated with transcriptional or proteomic signatures of mechanosensation or cell cycle regulation. Rather, 3D confinement physically constrains the volumetric growth of incipient buds -- manifesting as delayed cell cycle progression. Hence, our findings establish that physical constrainment regulates eukaryotic cell division.

biophysics↗

bioRxiv2025

Quantitative, temporal dynamics of the cellular amino acid economy reveals public vs private goods and enables syntrophic yeast community design.

The biosynthetic capacity of the cell governs the production and exchange of amino acids. Amino acids have diverse metabolic origins and intracellular requirements. Therefore, to understand the cellular amino acid economy we require a quantitative understanding of intracellular and extracellular their amounts, and how distinct amino acids change temporally during growth. Using the unicellular eukaryote, yeast, we establish a quantitative blueprint of the intracellular and extracellular amino acid economy, including the fluxes of production, secretion and consumption across different growth phases. Certain amino acids dominate the intracellular pool, and proportions of distinct amino acids continuously change during growth. The extracellular pool is notably distinct from the intracellular pool in terms of composition and dynamics. Only some public amino acids are subsequently consumed, while others are secreted in surplus of cellular demands. Five amino acids are intracellular and privatized, and continuously utilized to support diverse metabolism. Through this, we establish pairs of highly effective synthetic, exchange-based communities of public good auxotrophs. Our results suggest organizing frameworks addressing the dynamic intracellular and inter-cellular amino acid economy and its trade, and informs the rational engineering of syntrophic cell communities.

systems biology↗

bioRxiv2025

Gcn4 mediates SAM allocations and methylation balance between phospholipids and histones

Methionine availability alters the methylation status of various cellular molecules, and thereby controls metabolic state, via its metabolite S-adenosyl methionine (SAM). In yeast cells, supplementing excess methionine drives cell growth and proliferation, as well as phospholipid biogenesis. High methionine stabilizes the transcription factor Gcn4 (Atf4), which drives an anabolic transcription program to sustain high growth. Here, we investigated whether Gcn4 regulates the allocation of SAM towards distinct methylation outputs, during this methionine-dependent anabolic program. We find that methionine induced Gcn4 increases the expression of phospholipid methyltransferases during methionine supplementation. In{Delta} gcn4 cells, the expression of these methyltransferases significantly decreases, and results in reduced SAM allocations towards phospholipid methylation, alongside increased intracellular SAM, disrupting the balance in the SAM/SAH ratio in these cells. Excess SAM pools are re-allocated to histones, resulting in their hyper-methylation. Collectively, we uncover that Gcn4 modulates SAM allocations towards phospholipid methyltransferases, and the overall methyl allocation between phospholipids and histones. Our study reveals a novel role for Gcn4 in SAM dependent phospholipid biosynthesis in cells growing in methionine sufficiency.

cell biology↗

bioRxiv2025

Plasma membrane folate transport in fungi and plants is mediated by members of the oligopeptide transporter (OPT) family

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 transporter, OPT1, was synthetically sick 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 glutathione, we used alanine-scanning mutants of the residues in the transmembrane domains of the channel to identify the residues required specifically for the uptake of folates and distinct from those required for glutathione. We further examined the oligopeptide transporter family of other organisms for the presence of folate transporters. In C. albicans, CaOPT1, the orthologue of S. cerevisiae OPT1 efficiently transported folate but not glutathione, while the previously characterized glutathione transporter, CaOPT7 could not transport folate. Aspergillus fumigatus has eight homologues of the oligopeptide transporter family, of which OptB and OptH could transport folates. In the plant Arabidopsis thaliana, the Opt1 homologs AtOpt2, AtOpt4, and AtOpt6 could 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.

genetics↗

bioRxiv2025

Seo1p, a high affinity, plasma membrane transporter of the gamma-Glu-met dipeptide in yeasts and fungi

{gamma}-Glu dipeptides are ubiquitous in nature, and yet their metabolism and transport are poorly understood. Here we investigate this using the dipeptide {gamma}-Glu-met in Saccharomyces cerevisiae. {gamma}-Glu-met was efficiently utilized by S. cerevisiae and its degradation was dependent on both the glutathione degrading cytosolic Dug2p/Dug3p complex, and the vacuolar Ecm38p. Using a transcriptomics approach, followed by a genetic screen, we identified Seo1p, an orphan transporter of yeast, as the transporter of {gamma}-Glu-met. Uptake studies confirmed Seo1p as a high affinity (Km =48uM), highly specific transporter of {gamma}-Glu-met since other analogs like n-Glu-met, {gamma}-Glu-leu, {gamma}-Glu-cys, {gamma}-Glu-met-gly, methionine and methionine sulfoxide were not transported by Seo1p. Candida spp. also encoded a functional Seo1p. A second transporter, Opt2p, identified in the screen, was also investigated. However, Opt2p was not primarily involved in {gamma}-Glu-met uptake. Its deletion affected vacuolar morphology, that 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.

genetics↗

bioRxiv2024

Diurnal variation in skeletal muscle mitochondrial function dictates time of day-dependent differences in exercise capacity

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 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 evidence, we illustrate that loss of Sirtuin4 (SIRT4), a well-known mitochondrial regulator, abrogates diurnal variation in mitochondrial functions and consequently abolishes time-of-day dependent exercise performance. Therefore, our findings unequivocally demonstrate the pivotal role of baseline skeletal-muscle mitochondrial functions in dictating diurnal exercise capacity.

physiology↗

bioRxiv2024

Decapping activators Edc3 and Scd6 act redundantly with Dhh1 in post-transcriptional repression of starvation-induced pathways

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 act redundantly, in parallel with Dhh1 and in cooperation with Pat1, to adjust gene expression to nutrient availability by controlling mRNA decapping and decay.

genetics↗

bioRxiv2024

Active control of mitochondrial network morphology by metabolism driven redox state

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 genetically or chemically, 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 controls mitochondrial form.

cell biology↗

bioRxiv2023

Metabolic adaptation pilots the differentiation of human hematopoietic cells

A continuous supply of energy and metabolic intermediates is an essential prerequisite for survival and 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. A mechanistic basis of this hypothesis is provided by the established link between core energy metabolism and epigenetic covalent modifications of chromatin. This hypothesis 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.

cell biology↗

bioRxiv2023

A demand-based framework explains prioritization strategies upon transient limitations of different amino acids.

Cells require disparate amounts of distinct amino acids, which themselves have discrete biosynthetic costs. However, it remains unclear if and how cells respond differently to their scarcity. To explore this, we re-organized amino acids into distinct groups based on their metabolic origins. Subsequently, using yeast we assessed responses to transient disruptions in amino acid supply, and uncover diverse restoration responses for distinct amino acids. Cells hierarchically prioritize restoring glutamate-, sulfur-, pentose-phosphate- and pyruvate-derived amino acids. Particularly, the strongest response is to the glutamate-derived amino acid arginine. We find that the extent and priority of the restoration response is determined by the composite demand for an amino acid, coupled with low individual biosynthetic costs of that amino acid. We propose that cells employ a conserved strategy guided by the law of demand, to prioritize amino acids restoration upon transient limitation.

systems biology↗

bioRxiv2023

The PP2A-like phosphatase Ppg1 mediates assembly of the Far complex to balance gluconeogenic outputs and adapt to glucose depletion

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 requirement of PP2A-like phosphatase Ppg1 for appropriate carbon allocations towards gluconeogenic outputs - trehalose, glycogen, UDP-glucose, UDP-GlcNAc - specifically after glucose depletion. This homeostatic 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 as localized signaling hubs. We show that the Far complex assembly is Ppg1 catalytic activity-dependent. The assembled Far complex is required to maintain gluconeogenic outputs after glucose depletion. Glucose in turn regulates Far complex abundance. This Ppg1-mediated Far complex assembly, and dependent control of gluconeogenic outputs enhances 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 enable cells metabolically adapt to nutrient fluctuations.

systems biology↗

bioRxiv2022

Aedes aegypti eggs use rewired polyamine and lipid metabolism to survive extreme desiccation

Upon extreme water loss, some organisms pause their life cycles and escape death, in a process called anhydrobiosis. While widespread in microbes, this is uncommon in animals. Mosquitoes of the Aedes genus are vectors for several viral diseases in humans. These mosquitoes lay eggs that survive extreme desiccation and this property greatly enhances geographical expansion of these insects. The molecular principles of egg survival and hatching post-desiccation in these insects remain obscure. In this report, we find that eggs of Aedes aegypti, in contrast to those of Anopheles stephensi, are true anhydrobiotes. Aedes embryos acquire desiccation tolerance at a late developmental stage. We uncover unique proteome-level changes in Aedes embryos during desiccation. These changes reflect a metabolic state with reduced central carbon metabolism, and precise rewiring towards polyamine production, altered lipid levels and enhanced lipid utilization for energy. Using inhibitor-based approaches targeting these processes in blood-fed mosquitoes that lay eggs, we infer a two-step process of anhydrobiosis in Aedes eggs, where polyamine accumulation as well as lipid breakdown confer desiccation tolerance, and rapid lipid breakdown fuels energetic requirements enabling the revival of mosquito larvae post rehydration.

biochemistry↗

bioRxiv2022

Phosphate budgeting to mitochondria controls glucose-mediated mitochondrial repression

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{Delta} ubp3 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{Delta} ubp3 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.

biochemistry↗