Rajan Sankaranarayanan

Organelle membranes employ diverse lipids to relay key signals for efficient coordination of cellular processes. Diacylglycerol (DAG) is a simple yet critical lipid secondary messenger, but the regulatory mechanisms and functional implications for its distribution remain poorly understood. We have recently shown that Protein Kinase C (Pkc1) activation is driven by selective DAGs (C36:0, C36:1), whose levels are governed by Disco-interacting protein 2 (Dip2) (Shambhavi et al., 2025). Here, through genetic, chemical, and lipidomic screens, we show that the absence of Dip2 leads to specific DAG accumulation on the mitochondrial membrane and impacts its morphology, function, and quality control in yeast. Remarkably, the elevated DAGs in Δdip2 promote translocation of Pkc1 to mitochondria via its DAG-binding C1 domain, but not the HR1 domain, suggesting functional partitioning between the regulatory domains. We also show that only specific DAGs, not the bulk DAGs, are required for Pkc1’s targeting and inactivating Phospholipase C (Plc1) restores Pkc1 localisation and the associated mitochondrial defects. In addition, we identify that respiratory growth triggers specific DAG (C36:1) accumulation in the mitochondria, thereby promoting Pkc1 recruitment. Furthermore, we establish that the Psi1-Plc1-Dip2 axis is required for survival under respiratory growth conditions. Taken together, our study uncovers a novel, Dip2-mediated, unconventional Pkc1 signalling axis for maintaining mitochondrial homeostasis under nutrient transition and highlights how distinct lipid fingerprints enable precise control over organellar homeostasis.

Mitochondria originated through endosymbiosis of an Alphaproteobacterium within an Asgard archaeal host, with ATP export to the cytosol being a key driver for the organelle integration. The mitochondrial ATP/ADP carrier (AAC), a member of the SLC25 family, performs this critical function, whose evolutionary origin was not known due to the absence of any known prokaryotic homologues and was therefore termed a eukaryotic innovation. Here, using protein tertiary structure search combined with comprehensive sequence analyses, we identify conserved bacterial inner membrane transporters, CysZ and YihY, as putative homologues of mitochondrial AAC. CysZ and YihY are structurally similar to AAC, albeit with a circular permutation of one of the six transmembrane helices. Strikingly, we could identify the conserved MCF motif—a characteristic feature of the SLC25 family—in the bacterial sulfate transporter CysZ, suggesting a common ancestry. Together, our results identify a bacterial origin for the mitochondrial ATP exporter, thus resolving a long-standing question in mitochondrial evolution and a key step required for the emergence of eukaryotic cell complexity.

Mitochondria originated through endosymbiosis of an Alphaproteobacterium within an Asgard archaeal host, with ATP export to the cytosol being a key driver for the organelle integration. The mitochondrial ATP/ADP carrier (AAC), a member of the SLC25 family, performs this critical function, whose evolutionary origin was not known due to the absence of any known prokaryotic homologues and was therefore termed a eukaryotic innovation. Here, using protein tertiary structure search combined with comprehensive sequence analyses, we identify conserved bacterial inner membrane transporters, CysZ and YihY, as putative homologues of mitochondrial AAC. CysZ and YihY are structurally similar to AAC, albeit with a circular permutation of one of the six transmembrane helices. Strikingly, we could identify the conserved MCF motif—a characteristic feature of the SLC25 family—in the bacterial sulfate transporter CysZ, suggesting a common ancestry. Together, our results identify a bacterial origin for the mitochondrial ATP exporter, thus resolving a long-standing question in mitochondrial evolution and a key step required for the emergence of eukaryotic cell complexity.

Diacylglycerols (DAGs) are used for metabolic purposes and are tightly regulated secondary lipid messengers in eukaryotes. DAG subspecies with different fatty-acyl chains are proposed to be involved in the activation of distinct PKC isoforms, resulting in diverse physiological outcomes. However, the molecular players and the regulatory origin for fine-tuning the PKC pathway are unknown. Here, we show that Dip2, a conserved DAG regulator across Fungi and Animalia, has emerged as a modulator of PKC signalling in yeast. Dip2 maintains the level of a specific DAG subpopulation, required for the activation of PKC-mediated cell wall integrity pathway. Interestingly, the canonical DAG-metabolism pathways, being promiscuous, are decoupled from PKC signalling. We demonstrate that these DAG subspecies are sourced from a phosphatidylinositol pool generated by the acyl-chain remodelling pathway. Furthermore, we provide insights into the intimate coevolutionary relationship between the regulator (Dip2) and the effector (PKC) of DAG-based signalling. Hence, our study underscores the establishment of Dip2-PKC axis about 1.2 billion years ago in Opisthokonta, which marks the rooting of the first specific DAG-based signalling module of eukaryotes.

Diacylglycerols (DAGs) are used for metabolic purposes and are tightly regulated secondary lipid messengers in eukaryotes. DAG subspecies with different fatty-acyl chains are proposed to be involved in the activation of distinct PKC isoforms, resulting in diverse physiological outcomes. However, the molecular players and the regulatory origin for fine-tuning the PKC pathway are unknown. Here, we show that Dip2, a conserved DAG regulator across Fungi and Animalia, has emerged as a modulator of PKC signalling in yeast. Dip2 maintains the level of a specific DAG subpopulation, required for the activation of PKC-mediated cell wall integrity pathway. Interestingly, the canonical DAG-metabolism pathways, being promiscuous, are decoupled from PKC signalling. We demonstrate that these DAG subspecies are sourced from a phosphatidylinositol pool generated by the acyl-chain remodelling pathway. Furthermore, we provide insights into the intimate coevolutionary relationship between the regulator (Dip2) and the effector (PKC) of DAG-based signalling. Hence, our study underscores the establishment of Dip2-PKC axis about 1.2 billion years ago in Opisthokonta, which marks the rooting of the first specific DAG-based signalling module of eukaryotes.

Proofreading during translation of the genetic code is a key process for not only translation quality control but also for its modulation under stress conditions to provide fitness advantage. A major class of proofreading modules represented by editing domains of alanyl-tRNA synthetase (AlaRS-Ed) and threonyl-tRNA synthetase (ThrRS-Ed) features a common fold and an invariant Zn²⁺ binding motif across life forms. Here, we reveal the structural basis and functional consequence along with the necessity for their operational dichotomy, i.e., the metal ion is ubiquitous in one and inhibitor for the other. The universally conserved Zn²⁺ in AlaRS-Ed protects its proofreading activity from reactive oxygen species (ROS) to maintain high fidelity Ala-codons translation, necessary for cell survival. On the other hand, mistranslation of Thr-codons is well tolerated by the cells, thereby allowing for a ROS-based modulation of ThrRS-Ed’s activity. A single residue rooted over ~3.5 billion years of evolution has been shown to be primarily responsible for the functional divergence. The study presents a remarkable example of how protein quality control is integrated with redox signalling through leveraging the tunability of metal binding sites from the time of last universal common ancestor (LUCA).

Diacylglycerols (DAGs) are used for metabolic purposes and are tightly regulated secondary lipid messengers in eukaryotes. DAG subspecies with different fatty-acyl chains are proposed to be involved in the activation of distinct PKC isoforms, resulting in diverse physiological outcomes. However, the molecular players and the regulatory origin for fine-tuning the PKC pathway are unknown. Here, we show that Dip2, a conserved DAG regulator across Fungi and Animalia, has emerged as a modulator of PKC signalling in yeast. Dip2 maintains the level of a specific DAG subpopulation, required for the activation of PKC-mediated cell wall integrity pathway. Interestingly, the canonical DAG-metabolism pathways, being promiscuous, are decoupled from PKC signalling. We demonstrate that these DAG subspecies are sourced from a phosphatidylinositol pool generated by the acyl-chain remodelling pathway. Furthermore, we provide insights into the intimate coevolutionary relationship between the regulator (Dip2) and the effector (PKC) of DAG-based signalling. Hence, our study underscores the establishment of Dip2-PKC axis about 1.2 billion years ago in Opisthokonta, which marks the rooting of the first specific DAG-based signalling module of eukaryotes.

Outliers in scientific observations are often ignored and mostly remain unreported. However, presenting them is always beneficial since they could reflect the actual anomalies that might open new avenues. Here, we describe two examples of the above that came out of the laboratories of two of the pioneers of nucleic acid research in the area of protein biosynthesis, Paul Berg and Donald Crothers. Their work on the identification of D-aminoacyl-tRNA deacylase (DTD) and ‘Discriminator hypothesis’, respectively, were hugely ahead of their time and were partly against the general paradigm at that time. In both of the above works, the smallest and the only achiral amino acid turned out to be an outlier as DTD can act weakly on glycine charged tRNAs with a unique discriminator base of ‘Uracil’. This peculiar nature of glycine remained an enigma for nearly half a century. With a load of available information on the subject by the turn of the century, our work on ‘chiral proofreading’ mechanisms during protein biosynthesis serendipitously led us to revisit these findings. Here, we describe how we uncovered an unexpected connection between them that has implications for evolution of different eukaryotic life forms.

Aldehydes, being an integral part of carbon metabolism, energy generation, and signalling pathways, are ingrained in plant physiology. Land plants have developed intricate metabolic pathways which involve production of reactive aldehydes and its detoxification to survive harsh terrestrial environments. Here, we show that physiologically produced aldehydes, i.e., formaldehyde and methylglyoxal in addition to acetaldehyde, generate adducts with aminoacyl-tRNAs, a substrate for protein synthesis. Plants are unique in possessing two distinct chiral proofreading systems, D-aminoacyl-tRNA deacylase1 (DTD1) and DTD2, of bacterial and archaeal origins, respectively. Extensive biochemical analysis revealed that only archaeal DTD2 can remove the stable D-aminoacyl adducts on tRNA thereby shielding archaea and plants from these system-generated aldehydes. Using Arabidopsis as a model system, we have shown that the loss of DTD2 gene renders plants susceptible to these toxic aldehydes as they generate stable alkyl modification on D-aminoacyl-tRNAs, which are recycled only by DTD2. Bioinformatic analysis identifies the expansion of aldehyde metabolising repertoire in land plant ancestors which strongly correlates with the recruitment of archaeal DTD2. Finally, we demonstrate that the overexpression of DTD2 offers better protection against aldehydes than in wild type Arabidopsis highlighting its role as a multi-aldehyde detoxifier that can be explored as a transgenic crop development strategy.

Aldehydes, being an integral part of carbon metabolism, energy generation and signalling pathways, are ingrained in plant physiology. Land plants have developed intricate metabolic pathways which involve production of reactive aldehydes and its detoxification to survive harsh terrestrial environments. Here, we show that physiologically produced aldehydes i.e., formaldehyde and methylglyoxal in addition to acetaldehyde, generate adducts with aminoacyl-tRNAs, a substrate for protein synthesis. Plants are unique in possessing two distinct chiral proofreading systems, D-aminoacyl-tRNA deacylase1 (DTD1) and DTD2, of bacterial and archaeal origins, respectively. Extensive biochemical analysis revealed that only archaeal DTD2 can remove the stable D-aminoacyl adducts on tRNA thereby shielding archaea and plants from these system-generated aldehydes. Using Arabidopsis as a model system, we have shown that the loss of DTD2 gene renders plants susceptible to these toxic aldehydes as they generate stable alkyl modification on D-aminoacyl-tRNAs, which are recycled only by DTD2. Bioinformatic analysis identifies the expansion of aldehyde metabolising repertoire in land plant ancestors which strongly correlates with the recruitment of archaeal DTD2. Finally, we demonstrate that the overexpression of DTD2 offers better protection against aldehydes than in wild-type Arabidopsis highlighting its role as a multi-aldehyde detoxifier that can be explored as a transgenic crop development strategy.