Explore the vast range of titles available.

Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site.

Background Regular aspirin use reduces colon adenoma and carcinoma incidence. UDP-glucuronosyltransferases (UGT) are involved in aspirin metabolism and clearance, and variant alleles in UGT1A6 have been shown to alter salicylic acid metabolism and risk of colon neoplasia. Methods In a randomized, cross-over, placebo-controlled trial of 44 healthy men and women, homozygous for UGT1A6*1 or UGT1A6*2, we explored differences between global epithelial and stromal expression, using Affymetrix U133 + 2.0 microarrays and tested effects of 60-day aspirin supplementation (325 mg/d) on epithelial and stromal gene expression and colon prostaglandin E2 (PGE2) levels. Results No statistically significant differences in gene expression were observed in response to aspirin or UGT1A6 genotype, but tissue PGE2 levels were lower with aspirin compared to placebo (p <0.001). Transcripts differentially expressed between epithelium and stroma (N = 4916, P <0.01, false discovery rate <0.001), included a high proportion of genes involved in cell signaling, cellular movement, and cancer. Genes preferentially expressed in epithelium were involved in drug and xenobiotic metabolism, fatty acid and lipid metabolism, apoptosis signaling, and ion transport. Genes preferentially expressed in stroma included those involved in inflammation, cellular adhesion, and extracellular matrix production. Wnt-Tcf4 pathway genes were expressed in both epithelium and stroma but differed by subcellular location. Conclusions These results suggest that, in healthy individuals, subtle effects of aspirin on gene expression in normal colon tissue are likely overwhelmed by inter-individual variability in microarray analyses. Differential expression of critical genes between colonic epithelium and stroma suggest that these tissue types need to be considered separately.

Cystic fibrosis (CF), the most common autosomal recessive disease in Caucasians, is due to mutations in the CFTR gene. F508del, the most frequent mutation in patients, impairs CFTR protein folding and biosynthesis. The F508del-CFTR protein is retained in the endoplasmic reticulum (ER) and its traffic to the plasma membrane is altered. Nevertheless, if it reaches the cell surface, it exhibits a Cl(-) channel function despite a short half-life. Pharmacological treatments may target the F508del-CFTR defect directly by binding to the mutant protein or indirectly by altering cellular proteostasis, and promote its plasma membrane targeting and stability. We previously showed that annexine A5 (AnxA5) directly binds to F508del-CFTR and, when overexpressed, promotes its membrane stability, leading to the restoration of some Cl(-) channel function in cells. Because Gonadotropin-Releasing Hormone (GnRH) increases AnxA5 expression in some cells, we tested it in CF cells. We showed that human epithelial cells express GnRH-receptors (GnRH-R) and that GnRH induces an AnxA5 overexpression and an increased Cl(-) channel function in F508del-CFTR cells, due to an increased stability of the protein in the membranes. Beside the numerous physiological implications of the GnRH-R expression in epithelial cells, we propose that a topical use of GnRH is a potential treatment in CF.

The origin and cellular complexity of eukaryotes represent a major enigma in biology. Current data support scenarios in which an archaeal host cell and an alphaproteobacterial (mitochondrial) endosymbiont merged together, resulting in the first eukaryotic cell. The host cell is related to Lokiarchaeota, an archaeal phylum with many eukaryotic features. The emergence of the structural complexity that characterizes eukaryotic cells remains unclear. Here we describe the ‘Asgard’ superphylum, a group of uncultivated archaea that, as well as Lokiarchaeota, includes Thor-, Odin- and Heimdallarchaeota. Asgard archaea affiliate with eukaryotes in phylogenomic analyses, and their genomes are enriched for proteins formerly considered specific to eukaryotes. Notably, thorarchaeal genomes encode several homologues of eukaryotic membrane-trafficking machinery components, including Sec23/24 and TRAPP domains. Furthermore, we identify thorarchaeal proteins with similar features to eukaryotic coat proteins involved in vesicle biogenesis. Our results expand the known repertoire of ‘eukaryote-specific’ proteins in Archaea, indicating that the archaeal host cell already contained many key components that govern eukaryotic cellular complexity. This work describes the Asgard superphylum, an assemblage of diverse archaea that comprises Odinarchaeota, Heimdallarchaeota, Lokiarchaeota and Thorarchaeota, offering insights into the earliest days of eukaryotic cells and their complex features. Archaea with eukaryotic tendencies Although the origin of eukaryotic cells from prokaryotic ancestors remains an enigma, it has become clear that the root of eukaryotes lies among a group of prokaryotes known as archaea. The recent identification of newly described archaea belonging to the Asgard superphylum, including Lokiarchaeota and Thorarchaeota, revealed a group of prokaryotes containing many proteins and genetic sequences that are otherwise found only in eukaryotes. Thijs Ettema and colleagues extend the search for eukaryotic roots by describing further additions to the Asgard superphylum: the Odinarchaeota and Heimdallarchaeota. The new Asgard genomes encode homologues of several components of eukaryotic membrane-trafficking machineries, suggesting that the archaeal ancestor of eukaryotes was well equipped to evolve the complex cellular features that are characteristic of eukaryotic cells.

Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter–drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR–Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC–drug associations for a number of clinically relevant compounds. A set of CRISPR–Cas9-based genetic screens in a haploid human cell line identifies more than 200 gene–drug associations involving solute carriers (SLCs), transporters important for the uptake and activity of cytotoxic drugs.

Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a “wrinkled” seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential.

Requirement of mineral elements in different plant tissues is not often consistent with their transpiration rate; therefore, plants have developed systems for preferential distribution of mineral elements to the developing tissues with low transpiration. Here we took silicon (Si) as an example and revealed an efficient system for preferential distribution of Si in the node of rice (Oryza sativa). Rice is able to accumulate more than 10% Si of the dry weight in the husk, which is required for protecting the grains from water loss and pathogen infection. However, it has been unknown for a long time how this hyperaccumulation is achieved. We found that three transporters (Lsi2, Lsi3, and Lsi6) located at the node are involved in the intervascular transfer, which is required for the preferential distribution of Si. Lsi2 was polarly localized to the bundle sheath cell layer around the enlarged vascular bundles, which is next to the xylem transfer cell layer where Lsi6 is localized. Lsi3 was located in the parenchyma tissues between enlarged vascular bundles and diffuse vascular bundles. Similar to Lsi6, knockout of Lsi2 and Lsi3 also resulted in decreased distribution of Si to the panicles but increased Si to the flag leaf. Furthermore, we constructed a mathematical model for Si distribution and revealed that in addition to cooperation of three transporters, an apoplastic barrier localized at the bundle sheath cells and development of the enlarged vascular bundles in node are also required for the hyperaccumulation of Si in rice husk.

Endocytosis is a critical step in the process by which many therapeutic nanomedicines reach their intracellular targets. Our understanding of cellular uptake mechanisms has developed substantially in the past five years. However, these advances in cell biology have not fully translated to the nanoscience and therapeutics literature. Misconceptions surrounding the role of different endocytic pathways and how to study these pathways are hindering progress in developing improved nanoparticle therapies. Here, we summarize the latest insights into cellular uptake mechanisms and pathways. We highlight limitations of current systems to study endocytosis, particularly problems with non-specific inhibitors. We also summarize alternative genetic approaches to robustly probe these pathways and discuss the need to understand how cells endocytose particles in vivo. We hope that this critical assessment of the current methods used in studying nanoparticle uptake will guide future studies at the interface of cell biology and nanomedicine. Successful nanomedicine approaches rely on the efficient cellular uptake of nanoparticles, yet endocytic mechanisms remain challenging to probe. In this Review the authors describe the different cellular endocytic pathways and provide a critical discussion of the available tools and systems for their study.

Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target to reduce excessive use of chemical fertilizers, with substantial benefits to farmers and the environment. In Arabidopsis (Arabidopsis thaliana), allocation of more NO₃⁻ to shoots was associated with higher NUE; however, the commonality of this process across plant species have not been sufficiently studied. Two Brassica napus genotypes were identified with high and low NUE. We found that activities of V-ATPase and V-PPase, the two tonoplast proton-pumps, were significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE genotype (814); and consequently, less vacuolar NO₃⁻ was retained in roots of Xiangyou15. Moreover, NO₃⁻ concentration in xylem sap, [¹⁵N] shoot:root (S:R) and [NO₃⁻] S:R ratios were significantly higher in Xiangyou15. BnNRT1.5 expression was higher in roots of Xiangyou15 compared with 814, while BnNRT1.8 expression was lower. In both B. napus treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity, vacuolar sequestration capacity (VSC) of NO⁻⁻ in roots substantially decreased. Expression of NRT1.5 was up-regulated, but NRT1.8 was down-regulated, driving greater NO₃⁻ long-distance transport fromroots to shoots. NUE in Arabidopsis mutants impaired in proton pumps was also significantly higher than in the wild type col-0. Taken together, these data suggest that decrease in VSC of NO₃⁻ in roots will enhance transport to shoot and essentially contribute to higher NUE by promoting NO₃⁻ allocation to aerial parts, likely through coordinated regulation of NRT1.5 and NRT1.8.

Rapid pollen tube growth requires uptake of Suc or its hydrolytic products, hexoses, from the apoplast of surrounding tissues in the style. Due to species-specific sugar requirements, reliance of pollen germination and tube growth on cell wall invertase and Suc or hexose transporters varies between species, but it is not known if plants have a sugar transporter that mediates the uptake of both hexose and Suc for pollen tube growth. Here, we show that a sugar transporter protein in apple (Malus domestica), MdSTP13a, takes up both hexose and Suc when expressed in yeast, and is essential for pollen tube growth on Glc and Suc but not on maltose. MdSTP13a-mediated direct uptake of Suc is primarily responsible for apple pollen tube growth on Suc medium. Sorbitol, a major photosynthate and transport carbohydrate in apple, modulates pollen tube growth via the MYB transcription factor MdMYB39L, which binds to the promoter of MdSTP13a to activate its expression. Antisense repression of MdSTP13a blocks sorbitol-modulated pollen tube growth. These findings demonstrate that MdSTP13a takes up both hexose and Suc for sorbitol-modulated pollen tube growth in apple, revealing a situation where acquisition of sugars for pollen tube growth is regulated by a sugar alcohol.