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.

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 Km 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.

The Cumulative Amount of Exuded Citrate Controls Its Efficiency to Mobilize Soil Phosphorus

Root exudation of citrate is discussed as mechanism to mobilize P from the soils' solid phase. Microbial processes can mitigate the mobilization efficiency of citrate. Due to higher microbial activity in topsoils compared to subsoils, we hypothesized a lower mobilization efficiency of exuded citrate in topsoils than in the subsoils. As a model system we used microdialysis (MD) probes and we followed diffusive fluxes of citrate from the perfusate into the soil and of phosphate from the soil into the dialysate in three soil horizons (Oa, Ah, Bw) of a Fagus sylvatica L. stand Cambisol. Three different MD perfusates with a KCl background concentration have been used: control, 1, and 3 mmol L−1 citric acid. Fluxes have been measured after 24, 48, and 144 h. The high-citrate perfusate increased the cumulative 144 h P-influx by a factor of 8, 13, and 113 in the Oa, Ah, and Bw horizon, respectively. With the high-citrate treatment, P mobilization efficiency decreased over time, whereas for the low citrate, P mobilization efficiency had a maximum at day 2. Minimum P mobilization efficiency of citrate was 1:25,000 mol phosphate per mol citrate in the Oa horizon between days 2 and 6, and maximum was 1:286 in the Bw-horizon during day 2. An increasing citrate efflux over time indicated an increasing sink term for citrate in the soil due to microbial decay or immobilization processes. Cumulative phosphate influx could be fitted to cumulative citrate efflux and soil horizon in a logarithmic model explaining 87% of the variability. For the first time, we could follow the localized P-uptake with citrate exudation over several days. Cumulative citrate efflux as the main control of P-mobilization has been barely discussed yet, however, it could explain some gaps in the role of carboxylates in the rhizosphere. Batch experiments are not capable to elucidate microscale dynamic competition for phosphate and carboxylates. MD is a promising tool for spatially explicit investigation of phosphate–citrate exchange, since such detailed insights in are not possible with batch experiments. In combination with the analysis of microbial properties, this technique has a huge potential to identify mobilization processes in soils as induced by citrate.

Rhizosphere priming of two near-isogenic wheat lines varying in citrate efflux under different levels of phosphorus supply

Backgrounds and Aims The rhizosphere priming effect (RPE) has been explained from the perspective of microbial responses to root exudates and nutrient availability. This study introduced a chemical process that could also contribute to RPE: root exudates (organic acid ligands) could liberate mineral-protected carbon (C) in soil for microbial degradation. Methods Wheat (Triticum aestivum L.) near-isogenic lines varying in citrate efflux were grown for 6 weeks in a C4 soil supplied with either low (10 μg g–1) or high P (40 μg g–1). Total below-ground CO2 was trapped and partitioned for determination of soil organic C decomposition and RPE using a stable isotopic tracing technique. Mineral dissolution was examined by incubating soil with citric ligand at a series of concentrations. Key Results High P increased RPE (81 %), shoot (32 %) and root biomass (57 %), root-derived CO2-C (20 %), microbial biomass C (28 %) and N (100%), soil respiration (20 %) and concentrations of water-extractable P (30 %), Fe (43 %) and Al (190 %), but decreased inorganic N in the rhizosphere. Compared with Egret-Burke, wheat line Egret-Burke TaMATE1B with citrate efflux had lower inorganic N, microbial biomass C (16 %) and N (30 %) in the rhizosphere but greater RPE (18 %), shoot biomass (12 %) and root-derived CO2-C (low P 36 %, high P 13 %). Egret-Burke TaMATE1B also had higher concentrations of water-extractable P, Fe and Al in the rhizosphere, indicating the release of mineral-protected C. In addition, citrate ligand facilitated Fe and Al release from soil, with their concentrations rising with increasing ligand concentration and incubation time. Conclusions While high P supply increased microbial growth and RPE possibly due to higher total root exudation, citrate efflux from the root might have facilitated the liberation of mineral-bound C, leading to the higher RPE under Egret-Burke TaMATE1B. Mineral dissolution may be an important process that regulates RPE and should be considered in future RPE research.

The Drosophila mitochondrial citrate carrier regulates L-2-hydroxyglutarate accumulation by coupling the tricarboxylic acid cycle with glycolysis

The oncometabolites D- and L-2-hydroxyglutarate (2HG) broadly interfere with cellular metabolism, physiology, and gene expression. A key regulator of 2HG metabolism is the mitochondrial citrate carrier (CIC), which, when mutated, promotes excess D-/L-2HG accumulation. The mechanism by which CIC influences 2HG levels, however, remains unknown. Here we studied the Drosophila gene scheggia (sea), which encodes the fly CIC homolog, to explore the mechanisms linking mitochondrial citrate efflux to L-2HG metabolism. Our findings demonstrate that decreased Drosophila CIC activity results in elevated glucose catabolism and increased lactate production, thereby creating a metabolic environment that inhibits L-2HG degradation.