A citrate efflux transporter important for manganese distribution and phosphorus uptake in rice.

The plant citrate transporters, functional in mineral nutrient uptake and homeostasis, usually belong to the multidrug and toxic compound extrusion transporter family. We identified and functionally characterized a rice (Oryza sativa) citrate transporter, OsCT1, which differs from known plant citrate transporters and is structurally close to rice silicon transporters. Domain analysis depicted that OsCT1 carries a bacterial citrate-metal transporter domain, CitMHS. OsCT1 showed citrate efflux activity when expressed in Xenopus laevis oocytes and is localized to the cell plasma membrane. It is highly expressed in the shoot and reproductive tissues of rice, and its promoter activity was visible in cells surrounding the vasculature. The OsCT1 knockout (KO) lines showed a reduced citrate content in the shoots and the root exudates, whereas overexpression (OE) line showed higher citrate exudation from their roots. Further, the KO and OE lines showed variations in the manganese (Mn) distribution leading to changes in their agronomical traits. Under deficient conditions (Mn-sufficient conditions followed by 8 days of 0 µm MnCl2 · 4H2 O treatment), the supply of manganese towards the newer leaf was found to be obstructed in the KO line. There were no significant differences in phosphorus (P) distribution; however, P uptake was reduced in the KO and increased in OE lines at the vegetative stage. Further, experiments in Xenopus oocytes revealed that OsCT1 could efflux citrate with Mn. In this way, we provide insights into a mechanism of citrate-metal transport in plants and its role in mineral homeostasis, which remains conserved with their bacterial counterparts.

Identification of MATE Family and Characterization of GmMATE13 and GmMATE75 in Soybean's Response to Aluminum Stress.

The multidrug and toxic compound extrusion (MATE) proteins are coding by a secondary transporter gene family, and have been identified to participate in the modulation of organic acid exudation for aluminum (Al) resistance. The soybean variety Glycine max "Tamba" (TBS) exhibits high Al tolerance. The expression patterns of MATE genes in response to Al stress in TBS and their specific functions in the context of Al stress remain elusive. In this study, 124 MATE genes were identified from the soybean genome. The RNA-Seq results revealed significant upregulation of GmMATE13 and GmMATE75 in TBS upon exposure to high-dose Al3+ treatment and both genes demonstrated sequence homology to citrate transporters of other plants. Subcellular localization showed that both proteins were located in the cell membrane. Transgenic complementation experiments of Arabidopsis mutants, atmate, with GmMATE13 or GmMATE75 genes enhanced the Al tolerance of the plant due to citrate secretion. Taken together, this study identified GmMATE13 and GmMATE75 as citrate transporter genes in TBS, which could improve citrate secretion and enhance Al tolerance. Our findings provide genetic resources for the development of plant varieties that are resistant to Al toxicity.

Role of sodium dependent SLC13 transporter inhibitors in various metabolic disorders.

The sodium dependent SLC13 family transporters comprise of five genes SLC13A1, SLC13A2 (NaDC1), SLC13A3 (NaDC3), SLC13A4 and SLC13A5 (NaCT). Among them, NaDC1, NaDC3 and NaCT are sodium dependent transporters belonging to family of dicarboxylates (succinate, malate, α-ketoglutarate) and tricarboxylates (citrate). The mouse and the human NaCT structures have still not been crystallized, therefore structural information is taken from the related bacterial transporter of VcINDY. Citrate in the cytosol works as a precursor for the fatty acid synthesis, cholesterol, and low-density lipoproteins. The excess citrate from the matrix is translocated to the cytosol for fatty acid synthesis through these transporters and thus controls the energy balance by downregulating the glycolysis, tricarboxylic acid (TCA), and fatty acid breakdown. These transporters play an important role in regulating various metabolic diseases including cancer, diabetes, obesity, fatty liver diseases and CNS disorders. These di and tricarboxylate transporters are emerging as new targets for metabolic disorders such as obesity and diabetes. The mutation in the function of the NaCT causes several neurological diseases including neonatal epilepsy and impaired brain development whereas mutation of genes coding for citrate transport present in the liver may provide positive effect. Therefore, continued efforts from the earlier work on citrate transporters are required for the development of citrate inhibitors. This review discusses the structure, function, and regulation of the NaCT transporter. The review also highlights citrate role in diagnosing diseases such as cancer, diabetes, fatty liver, and diabetes. The therapeutic perspective of synthetic inhibitors against NaCT transporters is succinctly summarized.

Mitochondrial Citrate Transporters CtpA and YhmA Are Required for Extracellular Citric Acid Accumulation and Contribute to Cytosolic Acetyl Coenzyme A Generation in Aspergillus luchuensis mut. kawachii.

Aspergillus luchuensis mut. kawachii (A. kawachii) produces a large amount of citric acid during the process of fermenting shochu, a traditional Japanese distilled spirit. In this study, we characterized A. kawachii CtpA and YhmA, which are homologous to the yeast Saccharomyces cerevisiae mitochondrial citrate transporters Ctp1 and Yhm2, respectively. CtpA and YhmA were purified from A. kawachii and reconstituted into liposomes. The proteoliposomes exhibited only counterexchange transport activity; CtpA transported citrate using countersubstrates, especially cis-aconitate and malate, whereas YhmA transported citrate using a wider variety of countersubstrates, including citrate, 2-oxoglutarate, malate, cis-aconitate, and succinate. Disruption of ctpA and yhmA caused deficient hyphal growth and conidium formation with reduced mycelial weight-normalized citrate production. Because we could not obtain a ΔctpA ΔyhmA strain, we constructed an S-tagged ctpA (ctpA-S) conditional expression strain in the ΔyhmA background using the Tet-On promoter system. Knockdown of ctpA-S in ΔyhmA resulted in a severe growth defect on minimal medium with significantly reduced acetyl coenzyme A (acetyl-CoA) and lysine levels, indicating that double disruption of ctpA and yhmA leads to synthetic lethality; however, we subsequently found that the severe growth defect was relieved by addition of acetate or lysine, which could remedy the acetyl-CoA level. Our results indicate that CtpA and YhmA are mitochondrial citrate transporters involved in citric acid production and that transport of citrate from mitochondria to the cytosol plays an important role in acetyl-CoA biogenesis in A. kawachiiIMPORTANCE Citrate transport is believed to play a significant role in citrate production by filamentous fungi; however, details of the process remain unclear. This study characterized two citrate transporters from Aspergillus luchuensis mut. kawachii Biochemical and gene disruption analyses showed that CtpA and YhmA are mitochondrial citrate transporters required for normal hyphal growth, conidium formation, cytosolic acetyl-CoA synthesis, and citric acid production. The characteristics of fungal citrate transporters elucidated in this study will help expand our understanding of the citrate production mechanism and facilitate the development and optimization of industrial organic acid fermentation processes.

AhFRDL1-mediated citrate secretion contributes to adaptation to iron deficiency and aluminum stress in peanuts.

Although citrate transporters are involved in iron (Fe) translocation and aluminum (Al) tolerance in plants, to date none of them have been shown to confer both biological functions in plant species that utilize Fe-absorption Strategy I. In this study, we demonstrated that AhFRDL1, a citrate transporter gene from peanut (Arachis hypogaea) that is induced by both Fe-deficiency and Al-stress, participates in both root-to-shoot Fe translocation and Al tolerance. Expression of AhFRDL1 induced by Fe deficiency was located in the root stele, but under Al-stress expression was observed across the entire root-tip cross-section. Overexpression of AhFRDL1 restored efficient Fe translocation in Atfrd3 mutants and Al resistance in AtMATE-knockout mutants. Knocking down AhFRDL1 in the roots resulted in reduced xylem citrate and reduced concentrations of active Fe in young leaves. Furthermore, AhFRDL1-knockdown lines had reduced root citrate exudation and were more sensitive to Al toxicity. Compared to an Al-sensitive variety, enhanced AhFRDL1 expression in an Fe-efficient variety contributed to higher levels of Al tolerance and Fe translocation by promoting citrate secretion. These results indicate that AhFRDL1 plays a significant role in Fe translocation and Al tolerance in Fe-efficient peanut varieties under different soil-stress conditions. Given its dual biological functions, AhFRDL1 may serve as a useful genetic marker for breeding for high Fe efficiency and Al tolerance.

Decrease of citric acid produced by Aspergillus niger through disruption of the gene encoding a putative mitochondrial citrate-oxoglutarate shuttle protein.

The transporter that exports citric acid (CA) generated in mitochondria to the cytosol is an important component of the CA production machinery of Aspergillus niger. In this report, we cloned and identified the gene cocA, encoding a 33.7-kDa putative mitochondrial citrate-oxoglutarate shuttle protein of the CA hyper-producer A. niger WU-2223L. The amount of CA produced by a representative cocA disruptant (35 g/L) was significantly lower than that produced by strain WU-2223L (63 g/L) after culture for 12 days under CA production conditions, and the phenotype of the cocA disruptant differed in part from that of strain WU-2223L. A cocA disruptant complemented with cocA exhibited the same phenotypes as those of strain WU-2223L. This report is the first to show that cocA and its protein product clearly contribute to substantial CA production by A. niger, and provides a significant insight into microbial organic acid production by fermentation. Abbreviations: CA: citric acid; CD medium: Czapek-Dox medium; CS: citrate synthase; CTP: citrate transport protein; HR: homologous recombination; MCF: mitochondrial carrier family; RT-PCR: reverse-transcription PCR; TCA: tricarboxylic acid.

Two MATE Transporters with Different Subcellular Localization are Involved in Al Tolerance in Buckwheat.

Buckwheat (Fagopyrum esculentum) shows high tolerance to aluminum (Al) toxicity, but the molecular mechanisms responsible for this high Al tolerance are still poorly understood. Here, we investigated the involvement of two MATE (multi-drug and toxic compound extrusion) genes in Al tolerance. Both FeMATE1 and FeMATE2 showed efflux transport activity for citrate, but not for oxalate when expressed in Xenopus oocytes. A transient assay with buckwheat leaf protoplasts using green fluorescent protein (GFP) fusion showed that FeMATE1 was mainly localized to the plasma membrane, whereas FeMATE2 was localized to the trans-Golgi and Golgi. The expression of FeMATE1 was induced by Al only in the roots, but that of FeMATE2 was up-regulated in both the roots and leaves. Furthermore, the expression of both genes only responded to Al toxicity, but not to other stresses including low pH, cadmium (Cd) and lanthanum (La). Heterologous expression of FeMATE1 or FeMATE2 in the Arabidopsis mutant atmate partially rescued its Al tolerance. Expression of FeMATE1 also partially recovered the Al-induced secretion of citrate in the transgenic lines, whereas expression of FeMATE2 did not complement the citrate secretion. Further physiological analysis showed that buckwheat roots also secreted citrate in addition to oxalate in response to Al in a dose-responsive manner. Taken together, our results indicate that FeMATE1 is involved in the Al-activated citrate secretion in the roots, while FeMATE2 is probably responsible for transporting citrate into the Golgi system for the internal detoxification of Al in the roots and leaves of buckwheat.

Quantitative metabolic flux analysis reveals an unconventional pathway of fatty acid synthesis in cancer cells deficient for the mitochondrial citrate transport protein.

The mitochondrial citrate transport protein (CTP), encoded by SLC25A1, accommodates bidirectional trafficking of citrate between the mitochondria and cytosol, supporting lipid biosynthesis and redox homeostasis. Genetic CTP deficiency causes a fatal neurodevelopmental syndrome associated with the accumulation of L- and D-2-hydroxyglutaric acid, and elevated CTP expression is associated with poor prognosis in several types of cancer, emphasizing the importance of this transporter in multiple human pathologies. Here we describe the metabolic consequences of CTP deficiency in cancer cells. As expected from the phenotype of CTP-deficient humans, somatic CTP loss in cancer cells induces broad dysregulation of mitochondrial metabolism, resulting in accumulation of lactate and of the L- and D- enantiomers of 2-hydroxyglutarate (2HG) and depletion of TCA cycle intermediates. It also eliminates mitochondrial import of citrate from the cytosol. To quantify the impact of CTP deficiency on metabolic flux, cells were cultured with a set of 13C-glucose and 13C-glutamine tracers with resulting data integrated by metabolic flux analysis (MFA). CTP-deficient cells displayed a major restructuring of central carbon metabolism, including suppression of pyruvate dehydrogenase (PDH) and induction of glucose-dependent anaplerosis through pyruvate carboxylase (PC). We also observed an unusual lipogenic pathway in which carbon from glucose supplies mitochondrial production of alpha-ketoglutarate (AKG), which is then trafficked to the cytosol and used to supply reductive carboxylation by isocitrate dehydrogenase 1 (IDH1). The resulting citrate is cleaved to produce lipogenic acetyl-CoA, thereby completing a novel pathway of glucose-dependent reductive carboxylation. In CTP deficient cells, IDH1 inhibition suppresses lipogenesis from either glucose or glutamine, implicating IDH1 as a required component of fatty acid synthesis in states of CTP deficiency.

Plasma Membrane Na⁺-Coupled Citrate Transporter (SLC13A5) and Neonatal Epileptic Encephalopathy.

SLC13A5 is a Na⁺-coupled transporter for citrate that is expressed in the plasma membrane of specific cell types in the liver, testis, and brain. It is an electrogenic transporter with a Na⁺:citrate3- stoichiometry of 4:1. In humans, the Michaelis constant for SLC13A5 to transport citrate is ~600 µM, which is physiologically relevant given that the normal concentration of citrate in plasma is in the range of 150-200 µM. Li⁺ stimulates the transport function of human SLC13A5 at concentrations that are in the therapeutic range in patients on lithium therapy. Human SLC13A5 differs from rodent Slc13a5 in two important aspects: the affinity of the human transporter for citrate is ~30-fold less than that of the rodent transporter, thus making human SLC13A5 a low-affinity/high-capacity transporter and the rodent Slc13a5 a high-affinity/low-capacity transporter. In the liver, SLC13A5 is expressed exclusively in the sinusoidal membrane of the hepatocytes, where it plays a role in the uptake of circulating citrate from the sinusoidal blood for metabolic use. In the testis, the transporter is expressed only in spermatozoa, which is also only in the mid piece where mitochondria are located; the likely function of the transporter in spermatozoa is to mediate the uptake of citrate present at high levels in the seminal fluid for subsequent metabolism in the sperm mitochondria to generate biological energy, thereby supporting sperm motility. In the brain, the transporter is expressed mostly in neurons. As astrocytes secrete citrate into extracellular medium, the potential function of SLC13A5 in neurons is to mediate the uptake of circulating citrate and astrocyte-released citrate for subsequent metabolism. Slc13a5-knockout mice have been generated; these mice do not have any overt phenotype but are resistant to experimentally induced metabolic syndrome. Recently however, loss-of-function mutations in human SLC13A5 have been found to cause severe epilepsy and encephalopathy early in life. Interestingly, there is no evidence of epilepsy or encephalopathy in Slc13a5-knockout mice, underlining the significant differences in clinical consequences of the loss of function of this transporter between humans and mice. The markedly different biochemical features of human SLC13A5 and mouse Slc13a5 likely contribute to these differences between humans and mice with regard to the metabolic consequences of the transporter deficiency. The exact molecular mechanisms by which the functional deficiency of the citrate transporter causes epilepsy and impairs neuronal development and function remain to be elucidated, but available literature implicate both dysfunction of GABA (γ-aminobutyrate) signaling and hyperfunction of NMDA (N-methyl-d-aspartate) receptor signaling. Plausible synaptic mechanisms linking loss-of-function mutations in SLC13A5 to epilepsy are discussed.

Mitochondrial Citrate Transporter-dependent Metabolic Signature in the 22q11.2 Deletion Syndrome.

The congenital disorder 22q11.2 deletion syndrome (22qDS), characterized by a hemizygous deletion of 1.5-3 Mb on chromosome 22 at locus 11.2, is the most common microdeletion disorder (estimated prevalence of 1 in 4000) and the second risk factor for schizophrenia. Nine of ∼30 genes involved in 22qDS have the potential of disrupting mitochondrial metabolism (COMT, UFD1L, DGCR8, MRPL40, PRODH, SLC25A1, TXNRD2, T10, and ZDHHC8). Deficits in bioenergetics during early postnatal brain development could set the basis for a disrupted neuronal metabolism or synaptic signaling, partly explaining the higher incidence in developmental and behavioral deficits in these individuals. Here, we investigated whether mitochondrial outcomes and metabolites from 22qDS children segregated with the altered dosage of one or several of these mitochondrial genes contributing to 22qDS etiology and/or morbidity. Plasma metabolomics, lymphocytic mitochondrial outcomes, and epigenetics (histone H3 Lys-4 trimethylation and 5-methylcytosine) were evaluated in samples from 11 22qDS children and 13 age- and sex-matched neurotypically developing controls. Metabolite differences between 22qDS children and controls reflected a shift from oxidative phosphorylation to glycolysis (higher lactate/pyruvate ratios) accompanied by an increase in reductive carboxylation of α-ketoglutarate (increased concentrations of 2-hydroxyglutaric acid, cholesterol, and fatty acids). Altered metabolism in 22qDS reflected a critical role for the haploinsufficiency of the mitochondrial citrate transporter SLC25A1, further enhanced by HIF-1α, MYC, and metabolite controls. This comprehensive profiling served to clarify the biochemistry of this disease underlying its broad, complex phenotype.

Functional Analysis of a MATE Gene OsFRDL2 Revealed its Involvement in Al-Induced Secretion of Citrate, but a Lower Contribution to Al Tolerance in Rice.

The multidrug and toxic compound extrusion (MATE) transporters represent a large transporter family in plants, but the role of most genes in this family has not been examined. We functionally characterized a MATE family member, OsFRDL2, in rice (Oryza sativa). OsFRDL2 showed an efflux transport activity for citrate when it was expressed in both Xenopus oocytes and cultured tobacco cells. OsFRDL2 was mainly expressed in the roots and its expression was not induced by iron (Fe) deficiency, but it was rapidly up-regulated by aluminum (Al). Furthermore, the expression of OsFRDL2 was regulated by ART1, a C2H2-type zinc-finger transcription factor for Al tolerance. OsFRDL2 protein was localized at unidentified vesicles in the cytosol, but not co-localized with either mitochondria or peroxisomes when expressed in both onion epidermal cells and cultured tobacco cells. Knockout of OsFRDL2 decreased Al-induced secretion of citrate from the roots, but did not affect the internal citrate concentration. The Al-induced inhibition of root elongation was similar between the OsFRDL2 knockout line and its wild-type rice. Knockout of OsFRDL2 did not affect the translocation of Fe from the roots to the shoots. A double mutant between osfrdl2 and osfrdl4 or osfrdl1 did not further decrease the Al-induced citrate secretion and Fe translocation compared with the single mutant. Collectively, our results indicate that although OsFRDL2 is involved in the Al-induced secretion of citrate, its contribution to high Al tolerance is relatively small in rice.

The growing research toolbox for SLC13A5 citrate transporter disorder: a rare disease with animal models, cell lines, an ongoing Natural History Study and an engaged patient advocacy organization.

TESS Research Foundation (TESS) is a patient-led nonprofit organization seeking to understand the basic biology and clinical impact of pathogenic variants in the SLC13A5 gene. TESS aims to improve the fundamental understanding of citrate's role in the brain, and ultimately identify treatments and cures for the associated disease. TESS identifies, organizes, and develops collaboration between researchers, patients, clinicians, and the pharmaceutical industry to improve the lives of those suffering from SLC13A5 citrate transport disorder. TESS and its partners have developed multiple molecular tools, cellular and animal models, and taken the first steps toward drug discovery and development for this disease. However, much remains to be done to improve our understanding of the disorder associated with SLC13A5 variants and identify effective treatments for this devastating disease. Here, we describe the available SLC13A5 resources from the community of experts, to foundational tools, to in vivo and in vitro tools, and discuss unanswered research questions needed to move closer to a cure.

Overview of research in SLC13A5 citrate transporter disorder SLC13A5 citrate transporter disorder is an ultra-rare, neurodevelopmental disorder that severely impacts cognition and motor control. It is characterized by frequent, intractable seizures that develop hours or days after birth, low tone, global developmental delay, a unique, varied, and difficult to categorize movement disorder, limited expressive verbal capabilities, tooth abnormalities, and increased citrate in both the CNS and serum. Seizures are frequently medically intractable, patients are often on multiple antiseizure medications and have frequent emergency room visits and hospitalizations for status epilepticus. SLC13A5 citrate transporter disorder is caused by mutations in the SLC13A5 gene which encodes a sodium-dependent citrate transporter, NaCT. NaCT is responsible for transporting citrate, a key molecule in cellular metabolism, from the extracellular space into cells, especially in the central nervous system and the liver. NaCT has been extensively studied in multiple animal models and affects lifespan and loss of some transporter activity actually improves metabolic syndrome in all animal species tested so far while causing mild neurological dysfunction in rodents. Although not definitively proven, it is presumed that loss of neuronal cell citrate transporter activity in the brain is the cause of seizures. Since the discovery of the disorder in 2014, there has been a rapid expansion in characterization of the disease. This has been aided by development of multiple models and molecular tools for studying wild type and mutant SLC13A5 making it a tractable candidate for therapeutic development. TESS Research Foundation is dedicated to driving SLC13A5 research and supporting children and families living with the disorder. Here, we describe the available SLC13A5 resources from the community of experts, to foundational tools, to in vivo and in vitro tools, and discuss unanswered research questions needed to move closer to a cure.

Targeting Longevity Gene SLC13A5: A Novel Approach to Prevent Age-Related Bone Fragility and Osteoporosis.

Reduced expression of the plasma membrane citrate transporter SLC13A5, also known as INDY, has been linked to increased longevity and mitigated age-related cardiovascular and metabolic diseases. Citrate, a vital component of the tricarboxylic acid cycle, constitutes 1-5% of bone weight, binding to mineral apatite surfaces. Our previous research highlighted osteoblasts' specialized metabolic pathway facilitated by SLC13A5 regulating citrate uptake, production, and deposition within bones. Disrupting this pathway impairs bone mineralization in young mice. New Mendelian randomization analysis using UK Biobank data indicated that SNPs linked to reduced SLC13A5 function lowered osteoporosis risk. Comparative studies of young (10 weeks) and middle-aged (52 weeks) osteocalcin-cre-driven osteoblast-specific Slc13a5 knockout mice (Slc13a5cKO) showed a sexual dimorphism: while middle-aged females exhibited improved elasticity, middle-aged males demonstrated enhanced bone strength due to reduced SLC13A5 function. These findings suggest reduced SLC13A5 function could attenuate age-related bone fragility, advocating for SLC13A5 inhibition as a potential osteoporosis treatment.

Citrate transporter inhibitors: possible new anticancer agents.

The culmination of 80+ years of cancer research implicates the aberrant metabolism in tumor cells as a root cause of pathogenesis. Citrate is an essential molecule in intermediary metabolism, and its amplified availability to critical pathways in cancer cells via citrate transporters confers a high rate of cancer cell growth and proliferation. Inhibiting the plasma membrane and mitochondrial citrate transporters - whether individually, in combination, or partnered with complementary metabolic targets - in order to combat cancer may prove to be a consequential chemotherapeutic strategy. This review aims to summarize the use of different classes of citrate transporter inhibitors for anticancer activity, either individually or as part of a cocktail.

Identification and genetic characterization of mitochondrial citrate transporters in Aspergillus niger.

Aspergillus niger is a major cell factory for citric acid production, and the process of citrate export from mitochondria to cytoplasm is predicted to be one of rate-limiting steps in citric acid accumulation. Currently, the mitochondrial citrate transporters (Ctps) in A. niger are not fully characterized. Here, six putative Ctp encoding genes (ctpA to ctpF) were identified based on their homology with a mitochondrial citrate transporter ScCtp1 from Saccharomyces cerevisiae. Disruption of individual ctpA to ctpF caused varying degrees of decline in citric acid accumulation at different fermentation stages, whereas a mutant strain S1696 with disruption of all six ctps showed complete loss of citiric acid production. S1696 also exhibited delayed growth, reduced conidia formation, and decreased pigmentogenesis. Exogenous addition of citrate partially restored the conidia formation and pigmentogenesis in S1696 mutant. Reintroduction of individual ctps (ctpA to ctpF) into S1696 at the amyA locus showed that ctpA, ctpB, and ctpD restored the citric acid titers to 88.5, 93.8, and 94.6% of the parent strain, respectively. Additionally, the formation of conidia and pigment production was partially restored after reintroduction of ctpA, ctpB, or ctpD. Overexpression of respective ctpA, ctpB, and ctpD in the parent strain resulted in increases in citric acid accumulation by 32.8, 19.3, and 24.2%, respectively. These results demonstrate that CtpA, CtpB, and CtpD play important roles in citric acid transport across the mitochondrial membrane and function in a redundant manner. Enhancement of citric acid transport process can serve as a target for boosting citric acid accumulation in A. niger.

Functional Verification of the Citrate Transporter Gene in a Wine Lactic Acid Bacterium, Lactiplantibacillus plantarum.

Organic acid metabolism by lactic acid bacteria plays a significant role in improving wine quality. During this process, the uptake of extracellular organic acids by the transporters is the first rate-limiting step. However, up to now, there is very little published research on the functional verification of organic acid transporter genes in wine lactic acid bacteria. In this study, a predicted citrate transporter gene JKL54_04345 (citP) by protein homology analysis was knocked out using a CRISPR/Cas9-based gene-editing system, and then complemented using the modified pMG36e vectors in a major wine lactic acid bacterium, Lactiplantibacillus plantarum XJ25, to verify its function in citrate metabolism for the first time. The results showed that the gene knockout mutant XJ25-ΔcitP lost the ability to utilize citric acid, while the gene complement mutant XJ25-ΔcitP-pMG36ek11-citP fully recovered the ability of citric acid utilization. Meanwhile, citP knockout and complement barely affected the utilization of l-malic acid. These indicated that citP in L. plantarum functioned as a citrate transporter and was the only gene responsible for citrate transporter. In addition, two modified plasmid vectors used for gene supplement in L. plantarum showed distinct transcription efficiency. The transcription efficiency of citP in XJ25-ΔcitP-pMG36ek11-citP mutant was 4.01 times higher than that in XJ25-ΔcitP-pMG36ek-citP mutant, and the utilization rate of citric acid in the former was 3.95 times higher than that in the latter, indicating that pMG36ek11 can be used as a high-level expression vector in lactic acid bacteria.

Characterization of GmMATE13 in its contribution of citrate efflux and aluminum resistance in soybeans.

Citrate exudation mediated by a citrate transporter of the MATE protein family is critical for resisting aluminum (Al) toxicity in soybeans. However, the expression patterns of citrate transporter genes differ under Al stress. Thus, exploring the responsive pattern of GmMATEs in response to Al stress is of great importance to understand the Al resistance mechanism in soybeans. In the present study, the phylogenetic analysis, transcriptionally expressed pattern, and function of GmMATE13 were investigated. The results show that soybean GmMATE13 is highly homologous to known citrate transporter proteins from other plants. Under Al exposure, the transcript abundance of GmMATE13 was increased during a 24 h Al treatment period. The expression of GmMATE13 is specifically induced by Al exposure, but not by the status of Fe, Cu, Cd, or La. Moreover, it was also highly increased when soybean seedlings were grown on acidic soil with a high Al content. Subcellular localization showed that GmMATE13 was localized on the plasma membrane when it was transiently expressed in Arabidopsis protoplasts. Investigation of tissue localization of GmMATE13 expression by investigating GUS activity staining under control of the GmMATE13 promoter showed that it was mainly expressed in the central cylinder in the root tips of the soybean under Al-free conditions, yet extended to cortical and epidermis cells under Al stress. Finally, overexpressing GmMATE13 in soybean hairy roots enhanced Al resistance by increasing citrate efflux. Collectively, we conclude that GmMATE13 is a promising candidate to improve the resistance of soybean to Al toxicity in acidic soil.

A Novel and Cross-Species Active Mammalian INDY (NaCT) Inhibitor Ameliorates Hepatic Steatosis in Mice with Diet-Induced Obesity.

Mammalian INDY (mINDY, NaCT, gene symbol SLC13A5) is a potential target for the treatment of metabolically associated fatty liver disease (MAFLD). This study evaluated the effects of a selective, cross-species active, non-competitive, non-substrate-like inhibitor of NaCT. First, the small molecule inhibitor ETG-5773 was evaluated for citrate and succinate uptake and fatty acid synthesis in cell lines expressing both human NaCT and mouse Nact. Once its suitability was established, the inhibitor was evaluated in a diet-induced obesity (DIO) mouse model. DIO mice treated with 15 mg/kg compound ETG-5773 twice daily for 28 days had reduced body weight, fasting blood glucose, and insulin, and improved glucose tolerance. Liver triglycerides were significantly reduced, and body composition was improved by reducing fat mass, supported by a significant reduction in the expression of genes for lipogenesis such as SREBF1 and SCD1. Most of these effects were also evident after a seven-day treatment with the same dose. Further mechanistic investigation in the seven-day study showed increased plasma ß-hydroxybutyrate and activated hepatic adenosine monophosphate-activated protein kinase (AMPK), reflecting findings from Indy (-/-) knockout mice. These results suggest that the inhibitor ETG-5773 blocked citrate uptake mediated by mouse and human NaCT to reduce liver steatosis and body fat and improve glucose regulation, proving the concept of NaCT inhibition as a future liver treatment for MAFLD.

Molecular Mechanism of Citrate Efflux by the Mitochondrial Citrate Transporter CT in Filamentous Fungus Mucor circinelloides WJ11.

The mitochondrial citrate transporter (MCT) plays an important role in citrate efflux from the mitochondria in eukaryotes, and hence provides a direct correlation between carbohydrate metabolism and lipid synthesis. Our previous studies on transporters confirmed the presence of two MCTs (TCT and CT) in oleaginous Mucor circinelloides WJ11 associated with high lipid accumulation. However, the molecular mechanism of citrate efflux from the mitochondria by MCT in M. circinelloides is still unclear. To study the citrate transport mechanism of CT, the citrate transporter gene was expressed in Escherichia coli, and its product was purified. The citrate transport activity of the protein was studied in CT reconstituted liposomes. Our results showed high efficiency of CT for [14C] citrate/citrate exchange with K m 0.01 mM at 25°C. Besides citrate, other molecules such as oxaloacetate, malate, fumarate, succinate aconitate, oxoadipate, isocitrate, and glutamate also promote citrate transport. In addition, the ct overexpression and knockout plasmids were constructed and transferred into M. circinelloides WJ11, and the mitochondria were isolated, and the transport activity was studied. Our findings showed that in the presence of 10 mM malate, the mitochondria of ct-overexpressing transformant showed 51% increase in the efflux rate of [14C] citrate, whereas the mitochondria of the ct-knockout transformant showed 18% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. This study provided the first mechanistic evidence of citrate efflux from the mitochondria by citrate transporter in oleaginous filamentous fungus M. circinelloides, which is associated with high lipid accumulation.

Tricarboxylate Citrate Transporter of an Oleaginous Fungus Mucor circinelloides WJ11: From Function to Structure and Role in Lipid Production.

The citrate transporter protein (CTP) plays an important role in citrate efflux from the mitochondrial matrix to cytosol that has great importance in oleaginous fungi. The cytoplasmic citrate produced after citrate efflux serves as the primary carbon source for the triacylglycerol and cholesterol biosynthetic pathways. Because of the CTP's importance, our laboratory has extensively studied its structure/function relationships in Mucor circinelloides to comprehend its molecular mechanism. In the present study, the tricarboxylate citrate transporter (Tct) of M. circinelloides WJ11 has been cloned, overexpressed, purified, kinetically, and structurally characterized. The Tct protein of WJ11 was expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system for kinetic studies. Our results showed that Tct has a high affinity for citrate with Km 0.018 mM. Furthermore, the tct overexpression and knockout plasmids were created and transformed into M. circinelloides WJ11. The mitochondria of the tct-overexpressing transformant of M. circinelloides WJ11 showed a 49% increase in citrate efflux, whereas the mitochondria of the tct-knockout transformant showed a 39% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. To elucidate the structure-function relationship of this biologically important transporter a 3D model of the mitochondrial Tct protein was constructed using homology modeling. The overall structure of the protein is V-shaped and its 3D structure is dimeric. The transport stability of the structure was also assessed by molecular dynamics simulation studies. The activity domain was identified to form hydrogen bond and stacking interaction with citrate and malate upon docking. Tricarboxylate citrate transporter has shown high binding energy of -4.87 kcal/mol to citric acid, while -3.80 kcal/mol to malic acid. This is the first report of unraveling the structural characteristics of WJ11 mitochondrial Tct protein and understanding the approach of the transporting toward its substrate. In conclusion, the present findings support our efforts to combine functional and structural data to better understand the Tct of M. circinelloides at the molecular level and its role in lipid accumulation.