Requirements for the biogenesis of [2Fe-2S] proteins in the human and yeast cytosol.

The biogenesis of iron-sulfur (Fe/S) proteins entails the synthesis and trafficking of Fe/S clusters, followed by their insertion into target apoproteins. In eukaryotes, the multiple steps of biogenesis are accomplished by complex protein machineries in both mitochondria and cytosol. The underlying biochemical pathways have been elucidated over the past decades, yet the mechanisms of cytosolic [2Fe-2S] protein assembly have remained ill-defined. Similarly, the precise site of glutathione (GSH) requirement in cytosolic and nuclear Fe/S protein biogenesis is unclear, as is the molecular role of the GSH-dependent cytosolic monothiol glutaredoxins (cGrxs). Here, we investigated these questions in human and yeast cells by various in vivo approaches. [2Fe-2S] cluster assembly of cytosolic target apoproteins required the mitochondrial ISC machinery, the mitochondrial transporter Atm1/ABCB7 and GSH, yet occurred independently of both the CIA system and cGrxs. This mechanism was strikingly different from the ISC-, Atm1/ABCB7-, GSH-, and CIA-dependent assembly of cytosolic-nuclear [4Fe-4S] proteins. One notable exception to this cytosolic [2Fe-2S] protein maturation pathway defined here was yeast Apd1 which used the CIA system via binding to the CIA targeting complex through its C-terminal tryptophan. cGrxs, although attributed as [2Fe-2S] cluster chaperones or trafficking proteins, were not essential in vivo for delivering [2Fe-2S] clusters to either CIA components or target apoproteins. Finally, the most critical GSH requirement was assigned to Atm1-dependent export, i.e. a step before GSH-dependent cGrxs function. Our findings extend the general model of eukaryotic Fe/S protein biogenesis by adding the molecular requirements for cytosolic [2Fe-2S] protein maturation.

Comparative study of susceptibility to methylmercury cytotoxicity in cell types composing rat peripheral nerves: a higher susceptibility of dorsal root ganglion neurons.

Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.

Heliorhodopsin-mediated light-modulation of ABC transporter.

Heliorhodopsins (HeRs) have been hypothesized to have widespread functions. Recently, the functions for few HeRs have been revealed; however, the hypothetical functions remain largely unknown. Herein, we investigate light-modulation of heterodimeric multidrug resistance ATP-binding cassette transporters (OmrDE) mediated by Omithinimicrobium cerasi HeR. In this study, we classifiy genes flanking the HeR-encoding genes and identify highly conservative residues for protein-protein interactions. Our results reveal that the interaction between OcHeR and OmrDE shows positive cooperatively sequential binding through thermodynamic parameters. Moreover, light-induced OcHeR upregulates OmrDE drug transportation. Hence, the binding may be crucial to drug resistance in O. cerasi as it survives in a drug-containing habitat. Overall, we unveil a function of HeR as regulatory rhodopsin for multidrug resistance. Our findings suggest potential applications in optogenetic technology.

Loss of the yeast transporter Agp2 upregulates the pleiotropic drug-resistant pump Pdr5 and confers resistance to the protein synthesis inhibitor cycloheximide.

The transmembrane protein Agp2, initially shown as a transporter of L-carnitine, mediates the high-affinity transport of polyamines and the anticancer drug bleomycin-A5. Cells lacking Agp2 are hyper-resistant to polyamine and bleomycin-A5. In these earlier studies, we showed that the protein synthesis inhibitor cycloheximide blocked the uptake of bleomycin-A5 into the cells suggesting that the drug uptake system may require de novo synthesis. However, our recent findings demonstrated that cycloheximide, instead, induced rapid degradation of Agp2, and in the absence of Agp2 cells are resistant to cycloheximide. These observations raised the possibility that the degradation of Agp2 may allow the cell to alter its drug resistance network to combat the toxic effects of cycloheximide. In this study, we show that membrane extracts from agp2Δ mutants accentuated several proteins that were differentially expressed in comparison to the parent. Mass spectrometry analysis of the membrane extracts uncovered the pleiotropic drug efflux pump, Pdr5, involved in the efflux of cycloheximide, as a key protein upregulated in the agp2Δ mutant. Moreover, a global gene expression analysis revealed that 322 genes were differentially affected in the agp2Δ mutant versus the parent, including the prominent PDR5 gene and genes required for mitochondrial function. We further show that Agp2 is associated with the upstream region of the PDR5 gene, leading to the hypothesis that cycloheximide resistance displayed by the agp2Δ mutant is due to the derepression of the PDR5 gene.

Principles of peptide selection by the transporter associated with antigen processing.

Our ability to fight pathogens relies on major histocompatibility complex class I (MHC-I) molecules presenting diverse antigens on the surface of diseased cells. The transporter associated with antigen processing (TAP) transports nearly the entire repertoire of antigenic peptides into the endoplasmic reticulum for MHC-I loading. How TAP transports peptides specific for MHC-I is unclear. In this study, we used cryo-EM to determine a series of structures of human TAP, both in the absence and presence of peptides with various sequences and lengths. The structures revealed that peptides of eight or nine residues in length bind in a similarly extended conformation, despite having little sequence overlap. We also identified two peptide-anchoring pockets on either side of the transmembrane cavity, each engaging one end of a peptide with primarily main chain atoms. Occupation of both pockets results in a global conformational change in TAP, bringing the two halves of the transporter closer together to prime it for isomerization and ATP hydrolysis. Shorter peptides are able to bind to each pocket separately but are not long enough to bridge the cavity to bind to both simultaneously. Mutations that disrupt hydrogen bonds with the N and C termini of peptides almost abolish MHC-I surface expression. Our findings reveal that TAP functions as a molecular caliper that selects peptides according to length rather than sequence, providing antigen diversity for MHC-I presentation.

Emerging Role of ABC Transporters in Glia Cells in Health and Diseases of the Central Nervous System.

ATP-binding cassette (ABC) transporters play a crucial role for the efflux of a wide range of substrates across different cellular membranes. In the central nervous system (CNS), ABC transporters have recently gathered significant attention due to their pivotal involvement in brain physiology and neurodegenerative disorders, such as Alzheimer's disease (AD). Glial cells are fundamental for normal CNS function and engage with several ABC transporters in different ways. Here, we specifically highlight ABC transporters involved in the maintenance of brain homeostasis and their implications in its metabolic regulation. We also show new aspects related to ABC transporter function found in less recognized diseases, such as Huntington's disease (HD) and experimental autoimmune encephalomyelitis (EAE), as a model for multiple sclerosis (MS). Understanding both their impact on the physiological regulation of the CNS and their roles in brain diseases holds promise for uncovering new therapeutic options. Further investigations and preclinical studies are warranted to elucidate the complex interplay between glial ABC transporters and physiological brain functions, potentially leading to effective therapeutic interventions also for rare CNS disorders.

Near Infrared Autofluorescence Lifetime Imaging of Human Retinal Pigment Epithelium Using Adaptive Optics Scanning Light Ophthalmoscopy.

Purpose: To demonstrate the first near-infrared adaptive optics fluorescence lifetime imaging ophthalmoscopy (NIR-AOFLIO) measurements in vivo of the human retinal pigment epithelial (RPE) cellular mosaic and to visualize lifetime changes at different retinal eccentricities. Methods: NIR reflectance and autofluorescence were captured using a custom adaptive optics scanning light ophthalmoscope in 10 healthy subjects (23-64 years old) at seven eccentricities and in two eyes with retinal abnormalities. Repeatability was assessed across two visits up to 8 weeks apart. Endogenous retinal fluorophores and hydrophobic whole retinal extracts of Abca4-/- pigmented and albino mice were imaged to probe the fluorescence origin of NIR-AOFLIO. Results: The RPE mosaic was resolved at all locations in five of seven younger subjects (<35 years old). The mean lifetime across near-peripheral regions (8° and 12°) was longer compared to near-foveal regions (0° and 2°). Repeatability across two visits showed moderate to excellent correlation (intraclass correlation: 0.88 [τm], 0.75 [τ1], 0.65 [τ2], 0.98 [a1]). The mean lifetime across drusen-containing eyes was longer than in age-matched healthy eyes. Fluorescence was observed in only the extracts from pigmented Abca4-/- mouse. Conclusions: NIR-AOFLIO was repeatable and allowed visualization of the RPE cellular mosaic. An observed signal in only the pigmented mouse extract infers the fluorescence signal originates predominantly from melanin. Variations observed across the retina with intermediate age-related macular degeneration suggest NIR-AOFLIO may act as a functional measure of a biomarker for in vivo monitoring of early alterations in retinal health.

Identification and functional characterization of ABC transporters for selenium accumulation and tolerance in soybean.

ATP-binding cassette (ABC) transporters were crucial for various physiological processes like nutrition, development, and environmental interactions. Selenium (Se) is an essential micronutrient for humans, and its role in plants depends on applied dosage. ABC transporters are considered to participate in Se translocation in plants, but detailed studies in soybean are still lacking. We identified 196 ABC genes in soybean transcriptome under Se exposure using next-generation sequencing and single-molecule real-time sequencing technology. These proteins fell into eight subfamilies: 8 GmABCA, 51 GmABCB, 39 GmABCC, 5 GmABCD, 1 GmABCE, 10 GmABCF, 74 GmABCG, and 8 GmABCI, with amino acid length 121-3022 aa, molecular weight 13.50-341.04 kDa, and isoelectric point 4.06-9.82. We predicted a total of 15 motifs, some of which were specific to certain subfamilies (especially GmABCB, GmABCC, and GmABCG). We also found predicted alternative splicing in GmABCs: 60 events in selenium nanoparticles (SeNPs)-treated, 37 in sodium selenite (Na2SeO3)-treated samples. The GmABC genes showed differential expression in leaves and roots under different application of Se species and Se levels, most of which are belonged to GmABCB, GmABCC, and GmABCG subfamilies with functions in auxin transport, barrier formation, and detoxification. Protein-protein interaction and weighted gene co-expression network analysis suggested functional gene networks with hub ABC genes, contributing to our understanding of their biological functions. Our results illuminate the contributions of GmABC genes to Se accumulation and tolerance in soybean and provide insight for a better understanding of their roles in soybean as well as in other plants.

Genome wide identification and expression profiling of ATP binding cassette (ABC) transporters gene family in lentil (Lens culinaris Medikus) under aluminium stress condition.

Adenosine triphosphate-binding cassette transporters (ABC transporters) are involved in regulating plant growth, development and tolerance to environmental stresses. In this study, a total of 138 ABC transporter genes were identified in the lentil genome that were classified into eight subfamilies. Four lentil ABC transporters from subfamily B and I were clustered together with the previously characterized ABC transporter proteins related to aluminium (Al) detoxification. Lentil ABC transporter genes were distributed across the chromosomes. Tandem duplication was the main driving force for expansion of the ABC gene family. Collinearity of lentil with soybean indicated that ABC gene family is closely linked to Glycine max. ABC genes in the same subfamily showed similar gene structure and conserved motifs. The ABC promoter regions harboured a large number of plant hormones and multiple stress responsive cis-regulatory elements. The qRT-PCR showed that ABC genes had varied expression in roots of lentil at different time points under Al stress. This is the first report on genome wide identification and expression analyses of genes encoding ABC transporter genes in lentil which has provided in-depth insight for future research on evolution and elucidation of molecular mechanisms for aluminium tolerance.

The substrate-binding domains of the osmoregulatory ABC importer OpuA transiently interact.

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.

Structural insights in to the atypical type-I ABC Glucose-6-phosphate importer VCA0625-27 of Vibrio cholerae.

Sugar phosphates are potential sources of carbon and phosphate for bacteria. Despite that the process of internalization of Glucose-6-Phosphate (G6P) through plasma membrane remained elusive in several bacteria. VCA0625-27, made of periplasmic ligand binding protein (PLBP) VCA0625, an atypical monomeric permease VCA0626, and a cytosolic ATPase VCA0627, recently emerged as hexose-6-phosphate uptake system of Vibrio cholerae. Here we report high resolution crystal structure of VCA0625 in G6P bound state that largely resembles AfuA of Actinobacillus pleuropneumoniae. MD simulations on VCA0625 in apo and G6P bound states unraveled an 'open to close' and swinging bi-lobal motions, which are diminished upon G6P binding. Mutagenesis followed by biochemical assays on VCA0625 underscored that R34 works as gateway to bind G6P. Although VCA0627 binds ATP, it is ATPase deficient in the absence of VCA0625 and VCA0626, which is a signature phenomenon of type-I ABC importer. Further, modeling, docking and systematic sequence analysis allowed us to envisage the existence of similar atypical type-I G6P importer with fused monomeric permease in 27 other gram-negative bacteria.

Substrate uptake patterns shape niche separation in marine prokaryotic microbiome.

Marine heterotrophic prokaryotes primarily take up ambient substrates using transporters. The patterns of transporters targeting particular substrates shape the ecological role of heterotrophic prokaryotes in marine organic matter cycles. Here, we report a size-fractionated pattern in the expression of prokaryotic transporters throughout the oceanic water column due to taxonomic variations, revealed by a multi-"omics" approach targeting ATP-binding cassette (ABC) transporters and TonB-dependent transporters (TBDTs). Substrate specificity analyses showed that marine SAR11, Rhodobacterales, and Oceanospirillales use ABC transporters to take up organic nitrogenous compounds in the free-living fraction, while Alteromonadales, Bacteroidetes, and Sphingomonadales use TBDTs for carbon-rich organic matter and metal chelates on particles. The expression of transporter proteins also supports distinct lifestyles of deep-sea prokaryotes. Our results suggest that transporter divergency in organic matter assimilation reflects a pronounced niche separation in the prokaryote-mediated organic matter cycles.

Compound heterozygous ABCA12 variants identified in a Chinese patient with congenital ichthyosiform erythroderma: Advancing genotype-phenotype correlations and literature review.

BACKGROUND: Ichthyosis is a common keratotic skin disease with high clinical, etiological and genetic heterogeneity. There are four types of non-syndromic hereditary ichthyoses, among which autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of recessive Mendelian disorders. ARCI present with different phenotypes and ABCA12 pathogenic variants have been shown to cause complex ARCI phenotypes, including harlequin ichthyosis (HI), lamellar ichthyosis (LI) and congenital ichthyosiform erythroderma (CIE). METHODS: A sporadic male patient, clinically diagnosed with CIE, was enrolled in this study. Exome sequencing was combined with Sanger sequencing to confirm the diagnosis and identify the pathogenic variants. In silico predictions were made using multiple software programs, and the identified variants were interpreted using the ACMG guidelines. A review of all literature reported ABCA12 variants was performed to explore genotype-phenotype correlations. RESULTS: Compound heterozygous ABCA12 variants [c.5381+1G>A and c.5485G>C (p.Asp1829His)] (NM_173076) were identified. The two variants were not detected in the public database. c.5381+1G>A is predicted to affect ABCA12 mRNA splicing and Asp1829 is highly conserved among various species. In silico analysis suggested that these two variants were responsible for the phenotype of the patient. Genotype-phenotype correlation analysis showed that biallelic truncation variants and/or exon/amino acid deletions in ABCA12 are the most common causes of HI. Biallelic missense variants are most common in LI and CIE. CONCLUSIONS: The compound heterozygous ABCA12 variants caused the CIE phenotype observed in the patient. The spectrum of ABCA12 pathogenic variants were broaden. Genotype-phenotype correlation analysis provided detailed evidence which can be used in future prenatal diagnosis and can inform the need for genetic counselling for patients with ABCA12-related ARCIs.

An ATP13A1-assisted topogenesis pathway for folding multi-spanning membrane proteins.

Many multi-spanning membrane proteins contain poorly hydrophobic transmembrane domains (pTMDs) protected from phospholipid in mature structure. Nascent pTMDs are difficult for translocon to recognize and insert. How pTMDs are discerned and packed into mature, muti-spanning configuration remains unclear. Here, we report that pTMD elicits a post-translational topogenesis pathway for its recognition and integration. Using six-spanning protein adenosine triphosphate-binding cassette transporter G2 (ABCG2) and cultured human cells as models, we show that ABCG2's pTMD2 can pass through translocon into the endoplasmic reticulum (ER) lumen, yielding an intermediate with inserted yet mis-oriented downstream TMDs. After translation, the intermediate recruits P5A-ATPase ATP13A1, which facilitates TMD re-orientation, allowing further folding and the integration of the remaining lumen-exposed pTMD2. Depleting ATP13A1 or disrupting pTMD-characteristic residues arrests intermediates with mis-oriented and exposed TMDs. Our results explain how a "difficult" pTMD is co-translationally skipped for insertion and post-translationally buried into the final correct structure at the late folding stage to avoid excessive lipid exposure.

Comparative 3D genome analysis between neural retina and retinal pigment epithelium reveals differential cis-regulatory interactions at retinal disease loci.

BACKGROUND: Vision depends on the interplay between photoreceptor cells of the neural retina and the underlying retinal pigment epithelium (RPE). Most genes involved in inherited retinal diseases display specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis-regulatory elements (CREs) in 3D nuclear space. RESULTS: To understand the role of differential chromatin architecture in establishing tissue-specific expression at inherited retinal disease loci, we mapped genome-wide chromatin interactions using in situ Hi-C and H3K4me3 HiChIP on neural retina and RPE/choroid from human adult donor eyes. We observed chromatin looping between active promoters and 32,425 and 8060 candidate CREs in the neural retina and RPE/choroid, respectively. A comparative 3D genome analysis between these two retinal tissues revealed that 56% of 290 known inherited retinal disease genes were marked by differential chromatin interactions. One of these was ABCA4, which is implicated in the most common autosomal recessive inherited retinal disease. We zoomed in on retina- and RPE-specific cis-regulatory interactions at the ABCA4 locus using high-resolution UMI-4C. Integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish revealed tissue-specific CREs interacting with ABCA4. CONCLUSIONS: Through comparative 3D genome mapping, based on genome-wide, promoter-centric, and locus-specific assays of human neural retina and RPE, we have shown that gene regulation at key inherited retinal disease loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes at retinal disease loci, but also delineate the search space for non-coding genomic variation underlying unsolved inherited retinal diseases.

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides.

Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.

Phytomediated stress modulates antioxidant status, induces overexpression of CYP6M2, Hsp70, α-esterase, and suppresses the ABC transporter in Anopheles gambiae (sensu stricto) exposed to Ocimum tenuiflorum extracts.

The incorporation of phytoactive compounds in the management of malarial vectors holds promise for the development of innovative and efficient alternatives. Nevertheless, the molecular and physiological responses that these bioactive substances induce remain underexplored. This present study investigated the toxicity of different concentrations of aqueous and methanol extracts of Ocimum tenuiflorum against larvae of Anopheles gambiae (sensu stricto) and unraveled the possible underlying molecular pathways responsible for the observed physiological effects. FTIR and GCMS analyses of phytoactive compounds in aqueous and methanol crude extracts of O. tenuiflorum showed the presence of OH stretching vibration, C = C stretching modes of aromatics and methylene rocking vibration; ring deformation mode with high levels of trans-ß-ocimene, 3,7-dimethyl-1,3,6-octatriene in aqueous extract and 4-methoxy-benzaldehyde, 1,3,5-trimethyl-cyclohexane and o-cymene in methanol extract. The percentage mortality upon exposure to methanol and aqueous extracts of O. tenuiflorum were 21.1% and 26.1% at 24 h, 27.8% and 36.1% at 48 h and 36.1% and 45% at 72 h respectively. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), down-regulation of ABC transporter, overexpression of CYP6M2, Hsp70, and α-esterase, coupled with significantly increased levels of SOD, CAT, and GSH, were observed in An. gambiae (s.s.) exposed to aqueous and methanol extracts of O. tenuiflorum as compared to the control. Findings from this study have significant implications for our understanding of how An. gambiae (s.s.) larvae detoxify phytoactive compounds.

An ATP-binding cassette transporter, OsABCB24, is involved in female gametophyte development and early seed growth in rice.

Female gametogenesis has been rarely studied due to gametophyte lethality and the unavailability of related genetic resources. In this study, we identified a rice ATP-binding cassette transporter, OsABCB24, whose null function displayed a significantly reduced seed setting rate by as much as 94%-100% compared with that of the wild type (WT). The reciprocal cross of WT and mutant plants demonstrated that the female reproductive organs in mutants were functionally impaired. Confocal microscopy observations revealed that, although megasporogenesis remained unaffected in CRISPR/Cas9 osabcb24 mutants, the formation of female gametophytes was interrupted. Additionally, the structure of the syncytial nucleus was impaired during the initial stages of endosperm formation. Histochemical analysis showed that OsABCB24 was preferentially expressed at the conjunction of receptacle and ovary, spanning from the functional megaspore stage to the two-nucleate embryo sac stage. Further, OsABCB24 was identified as an endoplasmic reticulum membrane-localized protein. Notably, the overexpression of OsABCB24 triggered a 1.5- to 2-fold increase in grain production compared to the WT. Our findings showed that OsABCB24 plays a key role in both female gametophyte development and the early development of seeds.

Structure and activity of the septal peptidoglycan hydrolysis machinery crucial for bacterial cell division.

The peptidoglycan (PG) layer is a critical component of the bacterial cell wall and serves as an important target for antibiotics in both gram-negative and gram-positive bacteria. The hydrolysis of septal PG (sPG) is a crucial step of bacterial cell division, facilitated by FtsEX through an amidase activation system. In this study, we present the cryo-EM structures of Escherichia coli FtsEX and FtsEX-EnvC in the ATP-bound state at resolutions of 3.05 Å and 3.11 Å, respectively. Our PG degradation assays in E. coli reveal that the ATP-bound conformation of FtsEX activates sPG hydrolysis of EnvC-AmiB, whereas EnvC-AmiB alone exhibits autoinhibition. Structural analyses indicate that ATP binding induces conformational changes in FtsEX-EnvC, leading to significant differences from the apo state. Furthermore, PG degradation assays of AmiB mutants confirm that the regulation of AmiB by FtsEX-EnvC is achieved through the interaction between EnvC-AmiB. These findings not only provide structural insight into the mechanism of sPG hydrolysis and bacterial cell division, but also have implications for the development of novel therapeutics targeting drug-resistant bacteria.

Integrated omics of Saccharomyces cerevisiae CENPK2-1C reveals pleiotropic drug resistance and lipidomic adaptations to cannabidiol.

Yeast metabolism can be engineered to produce xenobiotic compounds, such as cannabinoids, the principal isoprenoids of the plant Cannabis sativa, through heterologous metabolic pathways. However, yeast cell factories continue to have low cannabinoid production. This study employed an integrated omics approach to investigate the physiological effects of cannabidiol on S. cerevisiae CENPK2-1C yeast cultures. We treated the experimental group with 0.5 mM CBD and monitored CENPK2-1C cultures. We observed a latent-stationary phase post-diauxic shift in the experimental group and harvested samples in the inflection point of this growth phase for transcriptomic and metabolomic analysis. We compared the transcriptomes of the CBD-treated yeast and the positive control, identifying eight significantly overexpressed genes with a log fold change of at least 1.5 and a significant adjusted p-value. Three notable genes were PDR5 (an ABC-steroid and cation transporter), CIS1, and YGR035C. These genes are all regulated by pleiotropic drug resistance linked promoters. Knockout and rescue of PDR5 showed that it is a causal factor in the post-diauxic shift phenotype. Metabolomic analysis revealed 48 significant spectra associated with CBD-fed cell pellets, 20 of which were identifiable as non-CBD compounds, including fatty acids, glycerophospholipids, and phosphate-salvage indicators. Our results suggest that mitochondrial regulation and lipidomic remodeling play a role in yeast's response to CBD, which are employed in tandem with pleiotropic drug resistance (PDR). We conclude that bioengineers should account for off-target product C-flux, energy use from ABC-transport, and post-stationary phase cell growth when developing cannabinoid-biosynthetic yeast strains.