Previously, we reported an engineered Saccharomyces cerevisiae CEN.PK113-1A derivative able to produce succinic acid (SA) from glycerol with net CO2 fixation. Apart from an engineered glycerol utilization pathway that generates NADH, the strain was equipped with the NADH-dependent reductive branch of the TCA cycle (rTCA) and a heterologous SA exporter. However, the results indicated that a significant amount of carbon still entered the CO2-releasing oxidative TCA cycle. The current study aimed to tune down the flux through the oxidative TCA cycle by targeting the mitochondrial uptake of pyruvate and cytosolic intermediates of the rTCA pathway, as well as the succinate dehydrogenase complex. Thus, we tested the effects of deletions of MPC1, MPC3, OAC1, DIC1, SFC1, and SDH1 on SA production. The highest improvement was achieved by the combined deletion of MPC3 and SDH1. The respective strain produced up to 45.5 g/L of SA, reached a maximum SA yield of 0.66 gSA/gglycerol, and accumulated the lowest amounts of byproducts when cultivated in shake-flasks. Based on the obtained data, we consider a further reduction of mitochondrial import of pyruvate and rTCA intermediates highly attractive. Moreover, the approaches presented in the current study might also be valuable for improving SA production when sugars (instead of glycerol) are the source of carbon.

Pendrin (SLC26A4) is an anion exchanger from the SLC26 transporter family which is mutated in human patients affected by Pendred syndrome, an autosomal recessive disease characterized by sensoneurinal deafness and hypothyroidism. Pendrin is also expressed in the kidney where it mediates the exchange of internal HCO3- for external Cl- at the apical surface of renal type B and non-A non-B-intercalated cells. Studies using pendrin knockout mice have first revealed that pendrin is essential for renal base excretion. However, subsequent studies have demonstrated that pendrin also controls chloride absorption by the distal nephron and that this mechanism is critical for renal NaCl balance. Furthermore, pendrin has been shown to control vascular volume and ultimately blood pressure. This review summarizes the current knowledge about how pendrin is linking renal acid-base regulation to blood pressure control.

Solute carrier (SLC) transporters are membrane-bound proteins that facilitate nutrient transport, and the movement across cellular membranes of various substrates ranging from ions to amino acids, metabolites and drugs. Recently, SLCs have gained increased attention due to their functional linkage to innate immunological processes such as the clearance of dead cells and anti-microbial defense. Further, the druggable nature of these transporters provides unique opportunities for improving outcomes in different immunological diseases. Although the SLCs represent the largest group of transporters and are often identified as significant hits in omics data sets, their role in immunology has been insufficiently explored. This is partly due to the absence of tools that allow identification of SLC expression in particular immune cell types and enable their comparison before embarking on functional studies. In this study, we used publicly available RNA-Seq data sets to analyze the transcriptome in adaptive and innate immune cells, focusing on differentially and highly expressed SLCs. This revealed several new insights: first, we identify differentially expressed SLC transcripts in phagocytes (macrophages, dendritic cells, and neutrophils) compared to adaptive immune cells; second, we identify new potential immune cell markers based on SLC expression; and third, we provide user-friendly online tools for researchers to explore SLC genes of interest (and the rest of the genes as well), in three-way comparative dot plots among immune cells. We expect this work to facilitate SLC research and comparative transcriptomic studies across different immune cells.

P4-ATPases in complex with Cdc50 subunits are lipid flippases that couple ATP hydrolysis with lipid transport to the cytoplasmic leaflet of membranes to create lipid asymmetry. Such vectorial transport has been shown to contribute to vesicle formation in the late secretory pathway. Some flippases are regulated by autoinhibitory regions that can be destabilized by protein kinase-mediated phosphorylation and possibly by binding of cytosolic proteins. In addition, the binding of lipids to flippases may also induce conformational changes required for the activity of these transporters. Here, we address the role of phosphatidylinositol-4-phosphate (PI4P) and the terminal autoinhibitory tails on the lipid flipping activity of the yeast lipid flippase Drs2-Cdc50. By functionally reconstituting the full-length and truncated forms of Drs2 in a 1:1 complex with the Cdc50 subunit, we provide compelling evidence that lipid flippase activity is exclusively detected for the truncated Drs2 variant and is dependent on the presence of the phosphoinositide PI4P. These findings highlight the critical role of phosphoinositides as lipid co-factors in the regulation of lipid transport by the Drs2-Cdc50 flippase.

The cellular compartment plays an essential role in organizing the complex and diverse biochemical reactions within the cell. By mimicking the function of such cellular compartments, the challenge of constructing artificial compartments has been taken up to develop new biochemical tools for efficient material production and diagnostics. The important features required for the artificial compartment are that it isolates the interior from the external environment and is further functionalized to control the transport of target chemicals to regulate the interior concentration of both substrate and reaction products. In this study, an artificial compartment with size-selective molecular transport function was constructed by using a DNA origami-guided liposome prepared by modifying the method reported by Perrault et al. This completely isolates the liposome interior, including the DNA origami skeleton, from the external environment and allows the assembly of a defined number of molecules of interest inside and/or outside the compartment. By incorporating a bacterial membrane protein, OmpF, into the liposome, the resulting artificial compartment was shown to transport only the molecule of interest with a molecular weight below 600 Da from the external environment into the interior of the compartment.

The Bile Acid Sodium Symporter (BASS) family transports a wide array of molecules across membranes, including bile acids in humans, and small metabolites in plants. These transporters, many of which are sodium-coupled, have been shown to use an elevator mechanism of transport, but exactly how substrate binding is coupled to sodium ion binding and transport is not clear. Here we solve the crystal structure at 2.3 Å of a transporter from Neisseria Meningitidis (ASBTNM) in complex with pantoate, a potential substrate of ASBTNM. The BASS family is characterised by two helices that cross-over in the centre of the protein in an arrangement that is intricately held together by two sodium ions. We observe that the pantoate binds, specifically, between the N-termini of two of the opposing helices in this cross-over region. During molecular dynamics simulations the pantoate remains in this position when sodium ions are present but is more mobile in their absence. Comparison of structures in the presence and absence of pantoate demonstrates that pantoate elicits a conformational change in one of the cross-over helices. This modifies the interface between the two domains that move relative to one another to elicit the elevator mechanism. These results have implications, not only for ASBTNM but for the BASS family as a whole and indeed other transporters that work through the elevator mechanism.

Selenium (Se) can counteract cadmium (Cd) toxicity in wheat, but the molecular mechanism of different Se forms reducing Cd uptake and accumulation in wheat seedlings remain unclear. Here, a hydroponic experiment was conducted to investigate the effects of three Se forms (selenite (Se(IV)), selenate (Se(VI)) and seleno-L-methionine (SeMet)) on Cd2+ influx, Cd subcellular distribution, and Cd accumulation in wheat seedlings, and the underlying molecular mechanisms were investigated through transcriptome analysis. Consequently, Se(IV) and Se(VI) addition significantly reduced root Cd concentration by 74.3% and 80.8%, respectively, and all Se treatments significantly decreased shoot Cd concentration by approximately 34.2%-74.9%, with Se(IV) addition having the most pronounced reducing effect. Transcriptome analysis showed the reduction of Cd accumulation after Se(IV) addition was mainly due to the downregulation of Cd uptake genes. The inhibition of Cd accumulation after Se(VI) addition was not only associated with the downregulation of Cd uptake genes, but also related to the sequestration of Cd in vacuole. For SeMet addition, the reduction of Cd accumulation was mainly related to the sequestration of Cd in vacuole as GSH-Cd. The above findings provide novel insights to understand the effects of different forms of Se on Cd uptake and accumulation and tolerance in wheat.

Copper is a trace element vital to many cellular functions. Yet its abnormal levels are toxic to cells, provoking a variety of severe diseases. The high affinity copper transporter 1 (CTR1), being the main in-cell copper [Cu(I)] entry route, tightly regulates its cellular uptake via a still elusive mechanism. Here, all-atoms simulations unlock the molecular terms of Cu(I) transport in eukaryotes disclosing that the two methionine (Met) triads, forming the selectivity filter, play an unprecedented dual role both enabling selective Cu(I) transport and regulating its uptake rate thanks to an intimate coupling between the conformational plasticity of their bulky side chains and the number of bound Cu(I) ions. Namely, the Met residues act as a gate reducing the Cu(I) import rate when two ions simultaneously bind to CTR1. This may represent an elegant autoregulatory mechanism through which CTR1 protects the cells from excessively high, and hence toxic, in-cell Cu(I) levels. Overall, our outcomes resolve fundamental questions in CTR1 biology and open new windows of opportunity to tackle diseases associated with an imbalanced copper uptake.

Lipid-conjugated pH sensors based on fluorophores coupled to lipids are a powerful tool for monitoring pH gradients in biological microcompartments and reconstituted membrane systems. This protocol describes the synthesis of pH sensors based on amine-reactive pHrodo esters and the amino phospholipid phosphatidylethanolamine. The major features of this sensor include efficient partitioning into membranes and strong fluorescence under acidic conditions. The protocol described here can be used as a template to couple other amine-reactive fluorophores to phosphatidylethanolamines. Graphical overview Synthesis of lipid-conjugated pH sensors based on amine-reactive fluorophore esters and the aminophospholipid phosphoethanolamine (PE).

Humans frequently consume plant-based foods in their daily life. Contamination of agricultural soils by heavy metals (HMs) is a major food and nutritional security issue. The crop plants grown in HM-contaminated agricultural soil may accumulate more HMs in their edible part, further transferring into the food chain. Consumption of HM-rich crops can cause severe health issues in humans. On the other hand, the low content of the essential HM in the edible part of the crop also causes health problems. Therefore, researchers must try to reduce the non-essential HM in the edible part of the crop plants and improve the essential HMs. Phytoremediation and biofortification are the two strategies for resolving this problem. The genetic component helps to improve the efficiency of phytoremediation and biofortification processes in plants. They help eliminate HMs from soil and improve essential HM content in crop plants. The membrane transporter genes (genetic components) are critical in these two strategies. Therefore, engineering membrane transporter genes may help reduce the non-essential HM content in the edible part of crop plants. Targeted gene editing by genome editing tools like CRISPR could help plants achieve efficient phytoremediation and biofortification. This article covers gene editing\'s scope, application, and implication to improve the phytoremediation and biofortification processes in non-crop and crop plants.