Biology subjects

Shukla, A.

Publications and source records attributed to Shukla, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

SIRT1-IDR-Driven Temporal Gating of Sequential Transcription-Factor Recruitment Coordinates Fasting Adaptation

Precise temporal control of gene expression is critical for metabolic adaptation, yet how transcriptional regulators coordinate dynamic responses to nutrient stress remains poorly understood. Here, we identify the intrinsically disordered region (IDR) of SIRT1, encoded by exon-2 (E2), as a key determinant of transcriptional timing during fasting. Using global and conditional SIRT1{Delta}E2 mouse model, which mimics an endogenous transcript variant, we demonstrate that loss of the N-terminal IDR disrupts the hierarchical engagement of transcription factors (TFs), leading to premature and exaggerated activation of gluconeogenic genes. Mechanistically, the IDR acts as a "conformational timer," competitively gating SIRT1 interactions with CREB, FOXO1, and PPAR to ensure sequential TF recruitment and resolution. Absence of this regulatory module resulted in "runaway" CREB activity, aberrant hepatic glucose output, and impaired fasting-refeeding transitions, linking IDR-driven temporal control to systemic metabolic homeostasis. Our findings reveal a paradigm wherein IDRs in upstream metabolic sensors orchestrate transcriptional handovers, offering a molecular basis for the dynamic rewiring of gene networks during physiological transitions. This work expands the functional repertoire of disordered regions beyond TFs, positioning them as critical nodes in nutrient-responsive transcriptional circuits and potential targets for metabolic disorders.

physiology↗

bioRxiv2025

FastConformation: A Standalone ML-Based Toolkit for Modeling and Analyzing Protein Conformational Ensembles at Scale

Deep learning approaches like AlphaFold 2 (AF2) have revolutionized structural biology by accurately predicting the ground state structures of proteins. Recently, clustering and subsampling techniques that manipulate multiple sequence alignment (MSA) inputs into AlphaFold to generate conformational ensembles of proteins have also been proposed. Although many of these techniques have been made open source, they often require integrating multiple packages and can be challenging for researchers who have a limited programming background to employ. This is especially true when researchers are interested in subsampling to produce predictions of protein conformational ensembles, which require multiple computational steps. This manuscript introduces FastConformation, a Python-based application that integrates MSA generation, structure prediction via AF2, and interactive analysis of protein conformations and their distributions, all in one place. FastConformation is accessible through a user-friendly GUI suitable for non-programmers, allowing users to iteratively refine subsampling parameters based on their analyses to achieve diverse conformational ensembles. Starting from an amino acid sequence, users can make protein conformation predictions and analyze results in just a few hours on their local machines, which is significantly faster than traditional molecular dynamics (MD) simulations. Uniquely, by leveraging the subsampling of MSAs, our tool enables the generation of alternative protein conformations. We demonstrate the utility of FastConformation on proteins including the Abl1 kinase, LAT1 transporter, and CCR5 receptor, showcasing its ability to predict and analyze the protein conformational ensembles and effects of mutations on a variety of proteins. This tool enables a wide range of high-throughput applications in protein biochemistry, drug discovery, and protein engineering.

biophysics↗

bioRxiv2025

Spatial Prior-Guided Boundary and Region-Aware 2D Lesion Segmentation in Neonatal Hypoxic Ischemic Encephalopathy

Segmenting acute and hyper-acute brain lesions in neona-tal hypoxic-ischemic encephalopathy (HIE) from diffusion-weighted MRI (DWI) is critical for prognosis and treatment planning but remains challenging due to severe class imbalance and lesion variability. We propose a computationally efficient 2D segmentation framework leveraging ADC and ZADC maps as a three-channel input to UNet++ with an Inception-v4 encoder and scSE attention for enhanced spatial-channel recalibration. To address class critical imbalance and lack of volumetric context in 2D methods, we introduce a novel boundary-and-region-aware weighted loss integrating Tversky, Log-Hausdorff, and Focal losses. Our method surpasses state-of-the-art 2D approaches and achieves competitive performance against computationally intensive 3D architectures, securing a Dice Similarity Coefficient (DSC) of 0.6060, Mean Average Surface Distance (MASD) of 2.6484, and Normalized Surface Distance (NSD) of 0.7477. These results establish a new benchmark for neonatal HIE lesion segmentation, demonstrating superior detection of both acute and hyper-acute lesions while mitigating the challenge of loss collapse. The code is available at Neonatal-HIE-SPARSeg.

bioinformatics↗

bioRxiv2025

Endogenous structure of antimalarial target PfATP4 reveals new class of apicomplexan P-type ATPase modulators

The Plasmodium falciparum sodium efflux pump PfATP4 is a leading antimalarial target, but suffers from a lack of high-resolution structural information needed to identify functionally important features in conserved regions and guide rational design of next generation inhibitors. Here, we determine a 3.7[A] cryoEM structure of PfATP4 purified from CRISPR-engineered P. falciparum parasites, revealing a previously unknown, apicomplexan-specific binding partner, PfABP, which forms a conserved, likely modulatory interaction with PfATP4. The discovery of PfABP presents a new avenue for designing novel PfATP4 inhibitors.

microbiology↗

bioRxiv2025

Essential role of NLRC5 in cancer immune surveillance and cancer immunoediting

A key mechanism of immune escape from CD8+ T cell-mediated tumor control occurs via downregulation of NLRC5, the IFN{gamma}-induced transcriptional activator of MHC class-I. As NLRC5 deficiency does not abrogate CD8+ T cell development, we investigated whether NLRC5-dependent antitumor immune mechanisms are required for immune surveillance. Development of 3-methylcholanthrene (MCA)-induced endogenous fibrosarcoma was studied in Nlrc5-/- mice with Nlrc5+/+ and Rag1-/- mice serving as controls. Nlrc5-/- and Rag1-/- mice showed increased propensity to develop MCA-induced tumors with elevated growth rate compared to Nlrc5+/+ mice, and displayed significantly reduced survival. Tumors from Nlrc5+/+ and Nlrc5-/-mice, but not from Rag1-/- mice, contained necrotic areas and displayed T cell infiltration. Tumor cell lines established from MCA- induced tumors were evaluated for their sensitivity to immune-mediated growth control following implantation into immunocompetent C57BL/6 and immunodeficient Rag1-/- hosts. Tumors formed by Nlrc5+/+ tumor cell lines progressed unhindered in C57BL/6 hosts that reflected their immunoedited status, whereas cell lines from Nlrc5-/- and Rag1-/-tumors were efficiently controlled, indicating their non-immunoedited status. Proteomic analysis by mass spectrometry followed by pathway analysis revealed enrichment of granzyme-mediated cytolytic pathway in Nlrc5+/+ tumors that were absent in Nlrc5-/- tumors, which showed enrichment of humoral and innate immune pathways. Overall, our findings show that NLRC5 is required for robust tumor immune surveillance and tumor immunoediting and that compensatory humoral and innate immune mechanisms activated by the loss of NLRC5 are insufficient for cancer immune surveillance and cancer immunoediting.

immunology↗

bioRxiv2024

The wheat VIH2-3B, a functional PPIP5K controls the localization of fasciclin-like arabinogalactan protein

Inositol pyrophosphates (PP-InsPs) are important signalling molecules that participate in multiple physiological processes across a wide range of eukaryotes. Metabolic pathway kinases (VIP1/VIHs) leading to the production of PP-InsPs are now well characterized in yeast and plants. Previously, the wheat (Triticum aestivum L.) inositol pyrophosphate kinase (TaVIH2) was shown to encode a catalytic active kinase domain. Heterologous expression of TaVIH2 in Arabidopsis thaliana was shown to enhance drought tolerance by modulating the cell composition. In this study, we attempted to identify the interacting protein targets of wheat VIH2-3B using a yeast two-hybrid (Y2H) cDNA library screen, which led to the identification of 52 putative interactors that are primarily involved in cell wall-related functions. Notably, fasciclin-like arabinogalactan protein (FLA7), a glycosylphosphatidyl inositol (GPI)-anchored protein, emerged as the most frequently interacting partner. Further analysis using pulldown assays validated the interaction between TaVIH2-3B and TaFLA7 in vivo. Using the reporter fusion studies, we observed the localization of TaFLA7 to be a plasma membrane and this localization of the TaFLA7 was perturbed in the yeast vip1{Delta} strain. The expression of TaVIH2-3B bearing PPIP5K enzymatic activity in yeast mutants rescued the level of IP8 and restore the localisation of the TaFLA7 to the membrane. Expression analysis of TaFLA7 revealed a differential expression response to drought in wheat shoot tissues. TaFLA7 was also found to be highly expressed during grain development, particularly in the endosperm and seed coat during grain maturation. Taken together, these findings highlight the potential role of TaVIH2 in cell wall remodelling and stress response pathways, offering new insights into the functional roles of VIH proteins in plants.

plant biology↗

bioRxiv2024

Widespread destabilization of C. elegans microRNAs by the E3 ubiquitin ligase EBAX-1

MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to form complexes that direct mRNA repression. miRNAs are also the subject of regulation. For example, some miRNAs are destabilized through a pathway in which pairing to specialized transcripts recruits the ZSWIM8 E3 ubiquitin ligase, which polyubiquitinates AGO, leading to its degradation and exposure of the miRNA to cellular nucleases. Here, we found that 22 miRNAs in C. elegans are sensitive to loss of EBAX-1, the ZSWIM8 ortholog in nematodes, implying that these 22 miRNAs might be subject to this pathway of target-directed miRNA degradation (TDMD). The impact of EBAX-1 depended on the developmental stage, with the greatest effect on the miRNA pool (14.5%) observed in L1 larvae and the greatest number of different miRNAs affected (17) observed in germline-depleted adults. The affected miRNAs included the miR-35-42 family, as well as other miRNAs among the least stable in the worm, suggesting that TDMD is a major miRNA-destabilization pathway in the worm. The excess miR-35-42 molecules that accumulated in ebax-1 mutants caused increased repression of their predicted target mRNAs and underwent 3' trimming over time. In general, however, miRNAs sensitive to EBAX-1 loss had no consistent pattern of either trimming or tailing. Replacement of the 3' region of miR-43 substantially reduced EBAX-1 sensitivity, a result that differed from that observed previously for miR-35. Together, these findings broaden the implied biological scope of TDMD-like regulation of miRNA stability in animals, and indicate that a role for miRNA 3' sequences is variable in the worm.

molecular biology↗

bioRxiv2024

Inositol pyrophosphate catabolism by three families of phosphatases controls plant growth and development

Inositol pyrophosphates (PP-InsPs) are nutrient messengers whose cellular concentration must be tightly regulated. Diphosphoinositol pentakisphosphate kinases (PPIP5Ks) generate the active signaling molecule 1,5-InsP8. PPIP5Ks contain additional phosphatase domains involved in PP-InsP catabolism. Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSPs) and NUDIX phosphatases (NUDTs) also hydrolyze PP-InsPs. Here we dissect the relative contributions of the three different phosphatase families to plant PP-InsP catabolism and nutrient signaling. We report the biochemical characterization of inositol pyrophosphate phosphatases from Arabidopsis and Marchantia polymorpha. Overexpression of different PFA-DSP and NUDT enzymes affects PP-InsP levels and leads to stunted growth phenotypes in Arabidopsis. nudt17/18/21 knock-out mutants have altered PP-InsP pools and gene expression patterns, but no apparent growth defects. In contrast, Marchantia polymorpha Mppfa-dsp1ge, Mpnudt1ge and Mpvip1ge mutants display severe growth and developmental phenotypes associated with changes in cellular PP-InsP levels. Analysis of Mppfa-dsp1geand Mpvip1ge supports a role for PP-InsPs in Marchantia phosphate signaling, and additional functions in nitrate homeostasis and cell wall biogenesis. Simultaneous removal of two phosphatase activities enhances the observed growth phenotypes. Taken together, PPIP5K, PFA-DSP and NUDT inositol pyrophosphate phosphatases play important roles in growth and development by collectively shaping plant PP-InsP pools. Author summaryOrganisms must maintain adequate levels of nutrients in their cells and tissues. One such nutrient is phosphorus, an essential building block of cell membranes, nucleic acids and energy metabolites. Plants take up phosphorus in the form of inorganic phosphate and require sufficient cellular phosphate levels to support their growth and development. It has been shown that plants and other eukaryotic organisms "measure" cellular phosphate levels using inositol pyrophosphate signaling molecules. The concentration of inositol pyrophosphates serves as a proxy for the cellular concentration of inorganic phosphate, and therefore inositol pyrophosphate synthesis and degradation must be tightly regulated. Here, we report that three different families of enzymes contribute to the degradation of inositol pyrophosphates in plants. The different phosphatases together shape cellular inositol pyrophosphate pools and thereby affect inorganic phosphate levels. Loss-of-function mutants of the different enzymes display additional defects in nitrate levels and cell wall architecture, suggesting that inositol pyrophosphates regulate cellular processes beyond inorganic phosphate homeostasis.

plant biology↗

bioRxiv2024

Mutational Analysis of an Antimalarial Drug Target PfATP4

Among new antimalarials discovered over the past decade are multiple chemical scaffolds that target Plasmodium falciparum P-type ATPase (PfATP4). This essential protein is a Na+ pump responsible for the maintenance of Na+ homeostasis. PfATP4 belongs to the type 2D subfamily of P-type ATPases, for which no structures have been determined. To gain better insight into the structure/function relationship of this validated drug target, we generated a homology model of PfATP4 based on SERCA, a P2A-type ATPase, and refined the model using molecular dynamics in its explicit membrane environment. This model predicted several residues in PfATP4 critical for its function, as well as those that impart resistance to various PfATP4 inhibitors. To validate our model, we developed a genetic system involving merodiploid states of PfATP4 in which the endogenous gene was conditionally expressed, and the second allele was mutated to assess its effect on the parasite. Our model predicted residues involved in Na+ coordination as well as the phosphorylation cycle of PfATP4. Phenotypic characterization of these mutants involved assessment of parasite growth, localization of mutated PfATP4, response to treatment with known PfATP4 inhibitors, and evaluation of the downstream consequences of Na+ influx. Our results were consistent with modeled predictions of the essentiality of the critical residues. Additionally, our approach confirmed the phenotypic consequences of resistance-associated mutations as well as a potential structural basis for the fitness cost associated with some mutations. Taken together, our approach provides a means to explore the structure/function relationship of essential genes in haploid organisms. Significance StatementPlasmodium falciparum ATP4 (PfATP4) is a Na+ efflux pump and represents an important target for antimalarial drugs with nanomolar potency. However, the structure of PfATP4 is unknown, prompting the development of new methodologies to investigate the structure/function relationship. Here, we introduce a dynamic homology modeling approach to (a) identify key residues essential for PfATP4 function and (b) provide a structural basis to understand resistance-associated mutations. To validate these predictions, we developed a genetic system to manipulate the PfATP4 gene to assess the phenotypic consequences of such changes. Our results support the utility of combining homology modeling and genetics to gain functional insights into an antimalarial drug target.

microbiology↗

bioRxiv2023

Activity of Antibiotics against Burkholderia cepacia complex in Artificial Sputum Medium

Burkholderia cepacia complex (Bcc) is a collection of intrinsically drug-resistant gram-negative bacteria that cause life-threatening pulmonary disease in people with cystic fibrosis (CF). Standard antimicrobial susceptibility testing methods have poor predictive value for clinical outcomes in people with Bcc infections, likely due in part to the significant differences between in vitro testing conditions and the environment in which Bcc grow in the lungs of people with CF. We tested the activity of six commonly used antibiotics against two clinical Bcc strains grown to high density in an artificial sputum medium in order to assess their activity in conditions mimicking those found in vivo. There were major discrepancies between standard susceptibility results and activity in our model, with some antibiotics, including ceftazidime, showing minimal activity despite low MICs, while others, notably tobramycin, were more active in high-density growth conditions than in standard assays. This work underscores the urgent need to develop more clinically relevant susceptibility testing approaches for Bcc.

microbiology↗

bioRxiv2023

Discovery of Competent Chromatin Regions in Human Embryonic Stem Cells

The mechanisms underlying the ability of embryonic stem cells (ESCs) to rapidly activate lineage-specific genes during differentiation remain largely unknown. Through multiple CRISPR-activation screens, we discovered human ESCs have pre-established transcriptionally competent chromatin regions (CCRs) that support lineage-specific gene expression at levels comparable to differentiated cells. CCRs reside in the same topological domains as their target genes. They lack typical enhancer-associated histone modifications but show enriched occupancy of pluripotent transcription factors, DNA demethylation factors, and histone deacetylases. TET1 and QSER1 protect CCRs from excessive DNA methylation, while HDAC1 family members prevent premature activation. This "push and pull" feature resembles bivalent domains at developmental gene promoters but involves distinct molecular mechanisms. Our study provides new insights into pluripotency regulation and cellular plasticity in development and disease. One sentence summaryWe report a class of distal regulatory regions distinct from enhancers that confer human embryonic stem cells with the competence to rapidly activate the expression of lineage-specific genes.

developmental biology↗

bioRxiv2023

Terminator-dependent facilitated recycling of RNA polymerase III couples transcriptional activation and chromatin remodeling in vivo

Termination is a crucial step in generating the functional transcriptome of a cell. The short genes transcribed by RNA polymerase (pol) III are mostly found in the highly transcribed genomic loci. The mechanism responsible for their high transcription rate in vivo is not yet established. Transcription terminator-dependent facilitated recycling of pol III on naked DNA templates is reported to increase transcriptional output in vitro. We found that apart from defining the 3-end of transcript, the transcription terminator is essential for achieving high-level chromatin transcription by pol III in vitro and in vivo. Using terminator-deficient SNR6 gene templates or a recycling deficient pol III mutant, we show that the TFIIIC-dependent transcriptional activation of chromatin is a process with three closely linked components, viz. anti-repression to naked DNA levels with TFIIIC binding, TFIIIC-dependent chromatin remodeling for better accessibility of the sequence elements and terminator-directed full transcriptional activation. Measurement of pol III occupancy on different gene regions demonstrated a direct link between the high transcription rate and the terminator dependent recycling of pol III in vivo. This novel regulatory mechanism may be generally applicable to the highly transcribed genes in any cell and even for cancer management wherein pol III transcription is found highly elevated.

molecular biology↗

bioRxiv2022

Cauliflower mosaic virus disease spectrum uncovers novel susceptibility factor NCED9 in Arabidopsis thaliana

Viruses are intimately linked with their hosts and especially dependent on gene-for-gene interactions to establish successful infections. The genotype of their hosts thus has a strong influence on the outcome virus disease. On the host side, defence mechanisms like tolerance and resistance can occur within the same species leading to differing virus accumulation in relation to symptomology and plant fitness. The identification of novel resistance genes and susceptibility factors against viruses is an important part in understanding viral pathogenesis and securing food production. The model plant Arabidopsis thaliana displays a wide symptom spectrum in response to RNA virus infections and unbiased genome-wide association studies have proven a powerful tool to identify novel disease-genes. In this study we infected natural accessions of Arabidopsis thaliana with the pararetrovirus Cauliflower mosaic virus to study the phenotypic variations between accessions and their correlation with virus accumulation. Through genome-wide association mapping of viral accumulation differences, we identified several susceptibility factors for CaMV, the strongest of which was the abscisic acid synthesis gene NCED9. Further experiments confirmed the importance of abscisic acid homeostasis and its disruption for CaMV disease.

plant biology↗

bioRxiv2022

Transcriptome and biochemical analysis pinpoint multi-layered molecular processes associated with iron deficiency tolerance in hexaploid wheat

Iron (Fe) is an essential nutrient for plants that is indispensable for many physiological activities. Although few genotypes were identified with contrasting tolerance to Fe deficiency, the molecular insight into the distinct biochemical and transcriptional responses determining the trait is poorly known. This study aimed to identify the molecular and biochemical basis for the contrasting Fe deficiency tolerance in wheat genotype showing tolerance to Fe deficiency (cv. Kanchan-KAN) compared to susceptible (cv. PBW343-PBW) cultivar. Under Fe deficiency, the KAN show delayed chlorosis, high SPAD values and low malondialdehyde activity compared to PBW. The shoot transcriptomics studies show that a large set of genes for photosynthetic pathways were highly induced in PBW, suggesting its sensitivity to Fe deficiency. Although, under Fe deficiency, both the cultivars show distinct molecular re-arrangements, including high expression of genes involved in Fe uptake (including membrane transporters) and mobilization, the gene expression level was higher in KAN. Furthermore, the KAN cultivar also shows high ubiquitination activity in the shoot tissue suggesting a high turnover of proteins in the tolerant cultivar. These observations were also co-related with the high root phytosiderophores biosynthesis and its release that contributes to the enhanced Fe translocation index in KAN. Overall, our work provides the key link to understanding the mechanistic insight for the Fe deficiency tolerance in hexaploid wheat. This will enable wheat breeders to select genotypes for better Fe use efficiency for agriculture.

plant biology↗

bioRxiv2022

Learning Free Energy Pathways through Reinforcement Learning of Adaptive Steered Molecular Dynamics

In this paper, we develop a formulation to utilize reinforcement learning and sampling-based robotics planning to derive low free energy transition pathways between two known states. Our formulation uses Jarzynskis equality and the stiffspring approximation to obtain point estimates of energy, and construct an informed path search with atomistic resolution. At the core of this framework, is our first ever attempt we use a policy driven adaptive steered molecular dynamics (SMD) to control our molecular dynamics simulations. We show that both the reinforcement learning and robotics planning realization of the RL-guided framework can solve for pathways on toy analytical surfaces and alanine dipeptide.

biophysics↗

bioRxiv2022

Mechanisms of mRNA processing defects in inherited THOC6 intellectual disability syndrome

THOC6 is the genetic basis of autosomal recessive THOC6 Intellectual Disability Syndrome (TIDS). THOC6 facilitates the formation of the Transcription Export complex (TREX) tetramer, composed of four THO monomers. The TREX tetramer supports mammalian mRNA processing that is distinct from yeast TREX dimer functions. Human and mouse TIDS model systems allow novel THOC6-dependent TREX tetramer functions to be investigated. Biallelic loss-of-function (LOF) THOC6 variants do not influence the expression and localization of TREX members in human cells, but our data suggests reduced binding affinity of ALYREF. Impairment of TREX nuclear export functions were not detected in cells with biallelic THOC6 LOF. Instead, mRNA mis-splicing was observed in human and mouse neural tissue, revealing novel insights into THOC6-mediated TREX coordination of mRNA processing. We demonstrate that THOC6 is required for regulation of key signaling pathways in human corticogenesis that dictate the transition from proliferative to neurogenic divisions that may inform TIDS neuropathology.

molecular biology↗

bioRxiv2022

Enhancement of mitochondrial function fosters B cell immune memory

Differentiation of T and B cells to effector and memory cell fates are associated with extensive metabolic changes which are accompanied by altered mitochondrial dynamics. However, whether alterations in mitochondrial structure and function plays an active role in regulating effector versus memory cell fate decisions during immune responses remains unclear. Our studies here characterize changes in mitochondrial dynamics in activated B cells and show that increased mitochondrial mass and activity is a distinct feature of memory B cell lineage commitment in vivo. Using a directed screen of mitochondrial modulators, we identify mitochondrial fission inhibitor, Mdivi-1 as an agent that could enhance mitochondrial mass and function leading to augmented memory B cell differentiation. The enhanced memory B cell responses mediated by Mdivi-1, translated to more robust recall responses upon secondary antigen exposures. Moreover, Mdivi-1 when used in combination with subunit (SARS-CoV2) and inactivated (H1N1 influenza) vaccines led to remarkably improved vaccine efficacies and protection from lethal viral (H1N1) challenge. Single-cell transcriptomics revealed enhanced commitment to memory lineage differentiation in B cells following Mdivi-1 treatment. We propose that mitochondrial modulators such as Mdivi-1 are a novel class of "immune enhancers" that specifically reinforces immunological memory and could be broadly applied to improve the fidelity of immune responses and vaccine efficacies.

immunology↗

bioRxiv2022

Mitochondrially targeted proximity biotinylation and proteomic analysis in Plasmodium falciparum

Despite ongoing efforts to control malaria infection, progress in lowering the number of deaths and infections appears to have stalled. The continued high incidence of malaria infection and mortality is in part due to emergence of parasites resistant to frontline antimalarials. This highlights the need for continued identification of novel protein drug targets. Mitochondrial functions in Plasmodium falciparum, the deadliest species of human malaria parasite, are targets of validated antimalarials including atovaquone and proguanil (Malarone). Thus, there has been great interest in identifying other essential mitochondrial proteins as candidates for novel drug targets. Garnering an increased understanding of the proteomic landscape inside the P. falciparum mitochondrion will also allow us to learn about the basic biology housed within this unique organelle. We employed a proximity biotinylation technique and mass spectrometry to identify novel P. falciparum proteins putatively targeted to the mitochondrion. We fused the leader sequence of a mitochondrially targeted chaperone, Hsp60, to the promiscuous biotin ligase TurboID. Through these experiments, we generated a list of 122 "putative mitochondrial" proteins. To verify whether these proteins were indeed mitochondrial, we chose five candidate proteins of interest for localization studies using ectopic expression and tagging of each full-length protein. This allowed us to localize four candidate proteins of unknown function to the mitochondrion, three of which have previously been assessed to be essential. We suggest that phenotypic characterization of these and other proteins from this list of 122 could be fruitful in understanding the basic mitochondrial biology of these parasites and aid antimalarial drug discovery efforts.

microbiology↗