EmrE is an
Phosphate is an indispensable metabolite in a wide variety of cells and is involved in nucleotide and lipid synthesis, signaling, and chemical energy storage. Proton-coupled phosphate transporters within the major facilitator family are crucial for phosphate uptake in plants and fungi. Similar proton-coupled phosphate transporters have been found in different protozoan parasites that cause human diseases, in breast cancer cells with elevated phosphate demand, in osteoclast-like cells during bone reabsorption, and in human intestinal Caco2BBE cells for phosphate homeostasis. However, the mechanism of proton-driven phosphate transport remains unclear. Here, we demonstrate in a eukaryotic, high-affinity phosphate transporter from
- Publication Date:
- NSF-PAR ID:
- Journal Name:
- Proceedings of the National Academy of Sciences
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- Article No. e2101932118
- Proceedings of the National Academy of Sciences
- Sponsoring Org:
- National Science Foundation
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EmrE is an
Escherichia colimultidrug efflux pump and member of the small multidrug resistance (SMR) family that transports drugs as a homodimer by harnessing energy from the proton motive force. SMR family transporters contain a conserved glutamate residue in transmembrane 1 (Glu14 in EmrE) that is required for binding protons and drugs. Yet the mechanism underlying proton-coupled transport by the two glutamate residues in the dimer remains unresolved. Here, we used NMR spectroscopy to determine acid dissociation constants (p K a) for wild-type EmrE and heterodimers containing one or two Glu14 residues in the dimer. For wild-type EmrE, we measured chemical shifts of the carboxyl side chain of Glu14 using solid-state NMR in lipid bilayers and obtained unambiguous evidence on the existence of asymmetric protonation states. Subsequent measurements of p K avalues for heterodimers with a single Glu14 residue showed no significant differences from heterodimers with two Glu14 residues, supporting a model where the two Glu14 residues have independent p K avalues and are not electrostatically coupled. These insights support a transport pathway with well-defined protonation states in each monomer of the dimer, including a preferred cytoplasmic-facing state where Glu14 is deprotonated in monomer A and protonated in monomer B under pH conditions in the cytoplasm of E.more ». Our findings also lead to a model, hop-free exchange, which proposes how exchangers with conformation-dependent p K avalues reduce proton leakage. This model is relevant to the SMR family and transporters comprised of inverted repeat domains.
Arabidopsis thaliana zinc accumulation in leaf trichomes is correlated with zinc concentration in leaves
Zinc (Zn) is a key micronutrient for plants and animals, and understanding Zn homeostasis in plants can improve both agriculture and human health. While root Zn transporters in plant model species have been characterized in detail, comparatively little is known about shoot processes controlling Zn concentrations and spatial distribution. Previous work showed that Zn hyperaccumulator species such as
Arabidopsis halleriaccumulate Zn and other metals in leaf trichomes. To date there is no systematic study regarding Zn accumulation in the trichomes of the non-accumulating, genetic model species A. thaliana. Here, we used Synchrotron X-Ray Fluorescence mapping to show that Zn accumulates at the base of trichomes of A. thaliana. Using transgenic and natural accessions of A thalianathat vary in bulk leaf Zn concentration, we demonstrate that higher leaf Zn increases total Zn found at the base of trichome cells. Our data indicates that Zn accumulation in trichomes is a function of the Zn status of the plant, and provides the basis for future studies on a genetically tractable plant species to understand the molecular steps involved in Zn spatial distribution in leaves.
BIN1locus contains the second-most significant genetic risk factor for late-onset Alzheimer’s disease. BIN1undergoes alternate splicing to generate tissue- and cell-type-specific BIN1 isoforms, which regulate membrane dynamics in a range of crucial cellular processes. Whilst the expression of BIN1 in the brain has been characterized in neurons and oligodendrocytes in detail, information regarding microglial BIN1 expression is mainly limited to large-scale transcriptomic and proteomic data. Notably, BIN1 protein expression and its functional roles in microglia, a cell type most relevant to Alzheimer’s disease, have not been examined in depth. Methods
Microglial BIN1 expression was analyzed by immunostaining mouse and human brain, as well as by immunoblot and RT-PCR assays of isolated microglia or human iPSC-derived microglial cells.
Bin1expression was ablated by siRNA knockdown in primary microglial cultures in vitro and Cre-lox mediated conditional deletion in adult mouse brain microglia in vivo. Regulation of neuroinflammatory microglial signatures by BIN1 in vitro and in vivo was characterized using NanoString gene panels and flow cytometry methods. The transcriptome data was explored by in silico pathway analysis and validated by complementary molecular approaches. Results
Here, we characterized microglial BIN1 expression in vitro and in vivo and ascertained microglia expressed BIN1 isoforms. By silencing
Bin1expression in primary microglial cultures, wemore » Conclusions
Our convergent findings provide novel insights into microglial BIN1 function and demonstrate an essential role of microglial BIN1 in regulating brain inflammatory response and microglial phenotypic changes. Moreover, for the first time, our study shows a regulatory relationship between
Bin1and Ifitm3, two Alzheimer’s disease-related genes in microglia. The requirement for BIN1 to regulate Ifitm3upregulation during inflammation has important implications for inflammatory responses during the pathogenesis and progression of many neurodegenerative diseases. Graphical Abstract
Double-strand break repair (DSBR) is a highly regulated process involving dozens of proteins acting in a defined order to repair a DNA lesion that is fatal for any living cell. Model organisms such as
Saccharomyces cerevisiaehave been used to study the mechanisms underlying DSBR, including factors influencing its efficiency such as the presence of distinct combinations of microsatellites and endonucleases, mainly by bulk analysis of millions of cells undergoing repair of a broken chromosome. Here, we use a microfluidic device to demonstrate in yeast that DSBR may be studied at a single-cell level in a time-resolved manner, on a large number of independent lineages undergoing repair. Results
We used engineered
S. cerevisiaecells in which GFP is expressed following the successful repair of a DSB induced by Cas9 or Cpf1 endonucleases, and different genetic backgrounds were screened to detect key events leading to the DSBR efficiency. Per condition, the progenies of 80–150 individual cells were analyzed over 24 h. The observed DSBR dynamics, which revealed heterogeneity of individual cell fates and their contributions to global repair efficacy, was confronted with a coupled differential equation model to obtain repair process rates. Good agreement was found between the mathematical model and experimental results at differentmore » Conclusions
Our analysis paves the way to a significant advance in understanding the complex molecular mechanism of DSB repair, with potential implications beyond yeast cell biology. This multiscale and multidisciplinary approach more generally allows unique insights into the relation between in vivo microscopic processes within each cell and their impact on the population dynamics, which were inaccessible by previous approaches using molecular genetics tools alone.
Potential-induced wetting and dewetting in pH-responsive block copolymer membranes for mass transport controlWetting and dewetting behavior in channel-confined hydrophobic volumes is used in biological membranes to effect selective ion/molecular transport. Artificial biomimetic hydrophobic nanopores have been devised utilizing wetting and dewetting, however, tunable mass transport control utilizing multiple transport modes is required for applications such as controllable release/transport, water separation/purification and energy conversion. Here, we investigate the potential-induced wetting and dewetting behavior in a pH-responsive membrane composed of a polystyrene- b -poly(4-vinylpyridine) (PS- b -P4VP) block copolymer (BCP) when fabricated as a hierarchically-organized sandwich structure on a nanopore electrode array (NEA), i.e. BCP@NEA. At pH < p K a (P4VP) (p K a ∼ 4.8), the BCP acts as an anion-exchange membrane due to the hydrophilic, protonated P4VP cylindrical nanodomains, but at pH > p K a (P4VP), the P4VP domains exhibit charge-neutral, hydrophobic and collapsed structures, blocking mass transport via the hydrophobic membrane. However, when originally prepared in a dewetted condition, mass transport in the BCP membrane may be switched on if sufficiently negative potentials are applied to the BCP@NEA architecture. When the hydrophobic BCP membrane is introduced on top of 2-electrode-embedded nanopore arrays, electrolyte solution in the nanopores is introduced, then isolated, by exploiting the potential-induced wetting and dewetting transitionsmore »