Regulation Of Transport Activity Research Articles

Assessing the negative impact of chlorantraniliprole, isoxaflutole, and simazine pesticides on phospholipid membrane models and tilapia gill tissues

The indiscriminate and, very often, incorrect use of pesticides in Brazil, as well as in other countries, results in severe levels of environmental pollution and intoxication of human life. Herein, we studied plasma membrane models (monolayer and bilayer) of the phospholipid Dioleoyl-sn-glycerol-3-phosphocholine (DOPC) using Langmuir films, and large (LUVs) and giant (GUVs) unilamellar vesicles, to determine the effect of the pesticides chlorantraniliprole (CLTP), isoxaflutole (ISF), and simazine (SMZ), used in sugarcane. CLTP affects the lipid organization of the bioinspired models of DOPC π-A isotherms, while ISF and SMZ pesticides significantly affect the LUVs and GUVs. Furthermore, the in vivo study of the gill tissue in fish in the presence of pesticides (2.0 × 10−10 mol/L for CLTP, 8.3 × 10−9 mol/L for ISF, and SMZ at 9.9 × 10−9 mol/L) was performed using optical and fluorescence images. This investigation was motivated by the gill lipid membranes, which are vital for regulating transporter activity through transmembrane proteins, crucial for maintaining ionic balance in fish gills. In this way, the presence of phospholipids in gills offers a model for understanding their effects on fish health. Histological results show that exposure to CLTP, ISF, and SMZ may interfere with vital gill functions, leading to respiratory disorders and osmoregulation dysfunction. The results indicate that exposure to pesticides caused severe morphological alterations in fish, which could be correlated with their impact on the bioinspired membrane models. Moreover, the effect does not depend on the exposure period (24h and 96h), showing that animals exposed to pesticides for a short period suffer irreparable damage to gill tissue. In summary, we can conclude that the harm caused by pesticides, both in membrane models and in fish gills, occurs due to contamination of the aquatic system with pesticides. Therefore, water quality is vital for the preservation of ecosystems.

The thylakoid proton antiporter KEA3 regulates photosynthesis in response to the chloroplast energy status

Plant photosynthesis contains two functional modules, the light-driven reactions in the thylakoid membrane and the carbon-fixing reactions in the chloroplast stroma. In nature, light availability for photosynthesis often undergoes massive and rapid fluctuations. Efficient and productive use of such variable light supply requires an instant crosstalk and rapid synchronization of both functional modules. Here, we show that this communication involves the stromal exposed C-terminus of the thylakoid K+-exchange antiporter KEA3, which regulates the ΔpH across the thylakoid membrane and therefore pH-dependent photoprotection. By combining in silico, in vitro, and in vivo approaches, we demonstrate that the KEA3 C-terminus senses the energy state of the chloroplast in a pH-dependent manner and regulates transport activity in response. Together our data pinpoint a regulatory feedback loop by which the stromal energy state orchestrates light capture and photoprotection via multi-level regulation of KEA3.

Structural insights into the conformational changes of BTR1/SLC4A11 in complex with PIP2

BTR1 (SLC4A11) is a NH3 stimulated H+ (OH-) transporter belonging to the SLC4 family. Dysfunction of BTR1 leads to diseases such as congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy (FECD). However, the mechanistic basis of BTR1 activation by alkaline pH, transport activity regulation and pathogenic mutations remains elusive. Here, we present cryo-EM structures of human BTR1 in the outward-facing state in complex with its activating ligands PIP2 and the inward-facing state with the pathogenic R125H mutation. We reveal that PIP2 binds at the interface between the transmembrane domain and the N-terminal cytosolic domain of BTR1. Disruption of either the PIP2 binding site or protonation of PIP2 phosphate groups by acidic pH can transform BTR1 into an inward-facing conformation. Our results provide insights into the mechanisms of how the transport activity and conformation changes of BTR1 are regulated by PIP2 binding and interaction of TMD and NTD.

Arabidopsis COPT1 copper transporter uses a single histidine to regulate transport activity and protein stability

Copper acquisition and subsequent delivery to target proteins are essential for many biological processes. However, the cellular levels of this trace element must be controlled because of its potential toxicity. The COPT1 protein rich in potential metal-binding amino acids functions in high affinity copper uptake at the plasma membrane of Arabidopsis cells. The functional role of these putative metal-binding residues is largely unknown. Through truncations and site-directed mutagenesis, we identified His43, a single residue within the extracellular N-terminal domain as absolutely critical for copper uptake of COPT1. Substitution of this residue with leucine, methionine or cysteine almost inactivated transport function of COPT1, implying that His43 fails to serves as a copper ligand in the regulation of COPT1 activity. Deletion of all extracellular N-terminal metal-binding residues completely blocked copper-stimulated degradation but did not alter the subcellular distribution and multimerization of COPT1. Although mutation of His43 to alanine and serine retained the transporter activity in yeast cells, the mutant protein was unstable and degraded in the proteasome in Arabidopsis cells. Our results demonstrate a pivotal role for the extracellular residue His43 in high affinity copper transport activity, and suggest common molecular mechanisms for regulating both metal transport and protein stability of COPT1.

Investigation on the morphology and the permeability of biomimetic cellulose triacetate (CTA) impregnated membranes (IM): In-situ synchrotron imaging, experimental and computational studies

The goal of the present study is to ascertain the controlled permeability of the biomimetic cellulose triacetate (CTA) membranes at different temperatures and salt concentrations by investigating the electrical oscillatory behaviour across the impregnated membranes (IM). The novel biomimetic CTA membranes were infused with a capric fatty acid to induce the electrical oscillatory behavior, and hence, to ensure the controlled permeability. In-situ synchrotron-based X-ray tomography (SR-μCT) at BioMedical Imaging and Therapy (BMIT) Beamline at the Canadian Light Source (CLS) was used to evaluate the main impregnated membrane morphology compared to neat CTA to ensure the fatty acid penetration inside membrane pores along membrane matrices. This study has revealed changes in phase transition temperatures of the impregnated CTA membrane at the melting temperature of the fatty acid. The pulsations of measured voltages were observed to be related to changes in the KCl concentration in the vicinity of the Ag/AgCl electrode. In this study, amplitudes and frequencies of voltage pulsations were dependent on the temperature and concentration of the KCl solution to control the permeability of the biomimetic membrane membranes. Increasing the KCl concentration led to a higher frequency of oscillation which reflects a higher permeability. In addition, no mechanical stirring or disturbance of the system was required for the electrical oscillatory phenomenon to occur. Nevertheless, for the average waveforms, the peak-to-peak value obtained was doubled when stirring was performed, while for the higher amplitude waveforms, the amplitude increased by 5 times. In addition, interactions at the molecular level between water molecules and the fragments of infused membranes at different temperatures and their effect on controlling permeability was investigated using pair interaction energy decomposition analysis (PIEDA) as part of the fragment molecular orbital (FMO) method's framework. Those results opened new frontiers for facilitation and regulation of active transport and permeability through biomimetic smart membranes for a variety of biomedical and drug delivery applications.

Distinct Functions of the Atypical Terminal Hydrophilic Domain of the HKT Transporter in the Liverwort Marchantia polymorpha.

K+/Na+ homeostasis is important for land plants, particularly under salt stress. In this study, the structure and ion transport properties of the high-affinity K+ transporter (HKT) of the liverwort Marchantia polymorpha were investigated. Only one HKT gene, MpHKT1, was identified in the genome of M. polymorpha. Phylogenetic analysis of HKT proteins revealed that non-seed plants possess HKTs grouped into a clade independent of the other two clades including HKTs of angiosperms. A distinct long hydrophilic domain was found in the C-terminus of MpHKT1. Complementary DNA (cDNA) of truncated MpHKT1 (t-MpHKT1) encoding the MpHKT_Δ596-812 protein was used to examine the functions of the C-terminal domain. Both MpHKT1 transporters fused with enhanced green fluorescent protein at the N-terminus were localized to the plasma membrane when expressed in rice protoplasts. Two-electrode voltage clamp experiments using Xenopus laevis oocytes indicated that MpHKT1 mediated the transport of monovalent alkali cations with higher selectivity for Na+ and K+, but truncation of the C-terminal domain significantly reduced the transport activity with a decrease in the Na+ permeability. Overexpression of MpHKT1 or t-MpHKT1 in M. polymorpha conferred accumulation of higher Na+ levels and showed higher Na+ uptake rates, compared to those of wild-type plants; however, phenotypes with t-MpHKT1 were consistently weaker than those with MpHKT1. Together, these findings suggest that the hydrophilic C-terminal domain plays a unique role in the regulation of transport activity and ion selectivity of MpHKT1.

Biochemical and structural insight of regulation in yeast vacuolar ABC transporter Ycf1

Yeast Cadmium Factor-1 (Ycf1) is a vacuolar membrane localized ATP Binding Cassette (ABC) transporter belonging to the C-subfamily and plays a crucial role in cell survival during stress by maintaining the homeostasis of the heavy metals (Cd, Hg and Pb) and glutathione in cytoplasm by transporting them in vacuole. Many ABCC subfamily transporters adopts a fast switch on-and-off mechanisms to cope with stress conditions. One of such mechanism is regulation of transport activity by phosphorylation of intrinsically disordered domains, known as regulatory domains (R-domains).

RESEARCH OF THE GEOINFORMATION SYSTEM OF AUTOMATION AND ACCOUNTING AND CONTROL OF CAPACITIES OF HIGHWAYS OF THE REPUBLIC (ON THE EXAMPLE OF THE ALMATY-KAPSHAGAY-TALDYKORGAN HIGHWAY)

The work is aimed at studying the intensity of roads in the Republic of Kazakhstan (for example, Almaty-Kapshagai-Taldykorgan highway) and improving the traffic management system. The development of a single economic space of the country largely depends on the level of development of transport infrastructure in each region. In this regard, the need to strengthen the interaction of central and local executive bodies on issues of state regulation of transport activities, while maintaining the trend of balanced development and decentralization of the transport system of the regions is particularly relevant. The analysis also shows that several measures can be taken to improve traffic management. The issues under consideration are devoted to topical problems of the development of a network of highways, which ensures the integrity of the territory of the largest cities and their zones of influence. The issues of the complex interrelation of urban planning factors, social and functional requirements that affect the degree of the necessary development of the network of the main structure-forming highways in the development of master plans of cities, planning projects of agglomerations, schemes of territorial planning of interconnected systems of settlements are considered. A method is proposed for the approximate development of the main network of highways formed by streets and high-speed roads. Modified classification of streets and roads in large cities is proposed, as well as a model for the planning organization of a single network of highways of cities and interconnected settlement systems, considering the modern requirements of sustainable development of urbanized territories and the growing level of motorization. The effectiveness of the proposals is illustrated by specific examples of domestic cities. For employees of scientific and design organizations engaged in the design and prospective development of main streets and roads in large and large cities, in urban agglomerations and areas of their influence. The methodological basis of the work was the results of fundamental and applied research in the field of vehicle operation in order to determine the main directions for increasing energy efficiency by improving the traffic management system. The solution to these problems requires to a large extent not only scarce financial, material, labor and other resources, but also the development of information technologies in the road industry. There is an urgent need for qualitative changes at all stages of collection, processing and ordering of information, at all stages of the continuous spiral of life cycles of the development of both the road itself and its structures - surveys, design, construction, operation and again surveys for design, reconstruction, repair, etc.
 The article considers the problems of the Almaty-Kapshagai-Taldykorgan highway and ways to solve them in the geographic information system.

The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2

NKCC and KCC transporters mediate coupled transport of Na++K++Cl− and K++Cl− across the plasma membrane, thus regulating cell Cl− concentration and cell volume and playing critical roles in transepithelial salt and water transport and in neuronal excitability. The function of these transporters has been intensively studied, but a mechanistic understanding has awaited structural studies of the transporters. Here, we present the cryo-electron microscopy (cryo-EM) structures of the two neuronal cation-chloride cotransporters human NKCC1 (SLC12A2) and mouse KCC2 (SLC12A5), along with computational analysis and functional characterization. These structures highlight essential residues in ion transport and allow us to propose mechanisms by which phosphorylation regulates transport activity.

Problems of legal regulation of transport activities on inland waterways of Ukraine

The article examines the problems of legal regulation of transport activities on inland waterways of Ukraine. The current absence of legal framework governing the implementation of river transport and port infrastructure in Ukraine is indicated. The purpose of the article is to reveal the problematic aspects of legal regulation of transport activities on inland waterways of Ukraine, namely – the absence of the relevant Law of Ukraine «On Inland Water Transport» and the importance of its adoption for legal regulation of transport activities and development of shipping on the inland waterways of Ukraine. The state policy on river transport and the strategy for its implementation are currently only prescribed and approved in the bill on inland water transport, which has been underway since 2015. Currently, there is a situation in which the shortcomings in the legal regulation of legal relations arising in the field of inland navigation in Ukraine are clearly defined. The lack of a mechanism for resolving the problems of the industry is confirmed by the disputes faced by enterprises and institutions of the organization regarding the operation by inland water transport. The settlement of the above situation requires a clear definition of the legal status of the river port, delimitation of the functions of the river port in terms of economic activity and state control and supervision over shipping safety, determination of the water area status and river port territory, consolidation of legal relations with business entities of all ownership forms at the legislative level. Adoption of this bill will make it possible to regulate at the legislative level legal relations in the field of domestic navigation on inland waterways, i.e. activities related to the use of vessels for cargo, passengers, luggage, mail, operation of inland waterways, registration of vessels and river cargo terminals and the right to sail under the State flag of Ukraine, the legal status of the ship’s crew, its composition, the rights and responsibilities of the captain (commander) of the ship, the rules of navigation safety, regulation of ship traffic, etc.

Regarding certain historical and legal preconditions for the formation and development of transport law

The article examines some historical and legal prerequisites for the formation and development of transport law as a complexbranch of law. It is emphasized that the regulation of transport activities in the ancient and medieval world was carried out mainlythrough trade and maritime customs, and points to the application of legal customs in transport law today.Transport law is a complex branch of law, and depending on the prevailing legal relationship, it can be considered as part of civil,commercial or administrative law. There was no separate school of transport law in the former USSR and Ukraine. Today, research onthe problems of transport law and the development of proposals to overcome them are carried out by scientists belonging to the scientificschools of commercial, civil and administrative law. Within these schools, there are separate groups of scientists or research departmentsthat carry out research to improve transport legislation.In transport law, the regulation of shipping and maritime trade developed first of all. It was there that the first legal customsappeared. Among the legal customs used in transport law, port customs (customs of the port) stand out.In Russia, trade was conducted mainly by river and sea “from the Vikings to the Greeks”, which ran from the Scandinavian Peninsula,the Baltic Sea, the Dnieper and the Black Sea to Byzantium. Thanks to this trade route, the inhabitants of Kievan Rus built boatsand developed a transport system. In addition to sea and river routes, the territory of modern Ukraine was a land route from Asia toEurope, stretching from southeast to northwest of our country – the legendary “Aryan route”, which according to some historians thousandsof years ago from the Indian subcontinent to Western and Northern Europe came the Aryan tribe, which gave development to theEuropean peoples.
 The possibility of applying port customs is provided by Art. 78 of the Code of Merchant Shipping of Ukraine. In addition to ports,the customs of merchant shipping are widely used. For example, in accordance with Art. 6 (concerning the permission to include in theagreements provided by the Code of Merchant Shipping of Ukraine, conditions on application of foreign legislation and customs ofmerchant shipping in case the parties may deviate from its rules in accordance with the current Code); art. 71 (in emergency cases, whenthe vessel has to be on the high seas for a long time and the body of the deceased cannot be saved, the captain of the vessel has the rightto give the body to the sea according to maritime customs, about which the corresponding act is made and the corresponding record ismade; Art. 146 (cargo is placed on the ship at the discretion of the captain, but can not be placed on deck without the written consentof the sender, except for cargo, the carriage of which on deck is allowed in accordance with applicable rules and customs); art. 293(concerning the possibility of determining the type of accident, calculating the size of the general accident and compiling the dispatchby the dispatcher in conditions of incompleteness of the law) of the Merchant Shipping Code of Ukraine of May 23, 1995.

Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs.

Cation-chloride-cotransporters (CCCs) catalyze transport of Cl- with K+ and/or Na+across cellular membranes. CCCs play roles in cellular volume regulation, neural development and function, audition, regulation of blood pressure, and renal function. CCCs are targets of clinically important drugs including loop diuretics and their disruption has been implicated in pathophysiology including epilepsy, hearing loss, and the genetic disorders Andermann, Gitelman, and Bartter syndromes. Here we present the structure of a CCC, the Mus musculus K+-Cl- cotransporter (KCC) KCC4, in lipid nanodiscs determined by cryo-EM. The structure, captured in an inside-open conformation, reveals the architecture of KCCs including an extracellular domain poised to regulate transport activity through an outer gate. We identify binding sites for substrate K+ and Cl- ions, demonstrate the importance of key coordinating residues for transporter activity, and provide a structural explanation for varied substrate specificity and ion transport ratio among CCCs. These results provide mechanistic insight into the function and regulation of a physiologically important transporter family.

Changes in transport activity regulation in the context of the coronavirus pandemic

In the article, the authors consider the activities of transport companies in the context of the coronavirus pandemic. The main research concerns the organization of the process of replacing the time of service provision by contractors when they are in emergency situations or when it is impossible to fulfill transport contracts at the set time. For the first time in the history of mankind, legal states are faced with the need to immediately close borders, prohibit citizens from leaving not only their own state, but also their place of residence in whole. Digitalization of the processes of instant solution of issues related to the activities of transport organizations turned out to be inoperable in relation to the realities of the new time. The state, trying not to destroy the country’s economic system and preserve the transport system, was forced to immediately introduce online services and other technological resources. Analysis of changes in the accompanying documentation and regulations at the level of federal laws, departmental and local regulations shows that civil society and its institutions in the future will not be able to do without thoughtful innovations and regulatory support for the technical component of transport regulation.

Regulation of transport activities taking into account the requirements of European standards

Regulation of transport activities taking into account the requirements of European standards

PID/WAG-mediated phosphorylation of the Arabidopsis PIN3 auxin transporter mediates polarity switches during gravitropism

Intercellular distribution of the plant hormone auxin largely depends on the polar subcellular distribution of the plasma membrane PIN-FORMED (PIN) auxin transporters. PIN polarity switches in response to different developmental and environmental signals have been shown to redirect auxin fluxes mediating certain developmental responses. PIN phosphorylation at different sites and by different kinases is crucial for PIN function. Here we investigate the role of PIN phosphorylation during gravitropic response. Loss- and gain-of-function mutants in PINOID and related kinases but not in D6PK kinase as well as mutations mimicking constitutive dephosphorylated or phosphorylated status of two clusters of predicted phosphorylation sites partially disrupted PIN3 phosphorylation and caused defects in gravitropic bending in roots and hypocotyls. In particular, they impacted PIN3 polarity rearrangements in response to gravity and during feed-back regulation by auxin itself. Thus PIN phosphorylation, besides regulating transport activity and apical-basal targeting, is also important for the rapid polarity switches in response to environmental and endogenous signals.

Alteration of transporter activities in the epididymides of infertile initial segment-specific Pten knockout mice.

A fully functional initial segment, the most proximal region of the epididymis, is important for male fertility. Our previous study generated a mouse model to investigate the importance of initial segment function in male fertility. In that model, phosphatase and tensin homolog (Pten) was conditionally removed from the initial segment epithelium, which resulted in epithelial de-differentiation. When spermatozoa progressed through the de-differentiated epithelial duct, they developed angled flagella, suggesting compromised sperm maturation, which eventually resulted in male infertility. To understand the molecular mechanisms, by which PTEN regulates epididymal sperm maturation, we compared the transcriptome profile of the initial segment between controls and initial segment-specific Pten knockouts and revealed that water, ion, and organic solute transporter activities were one of the top molecular and cellular functions altered following loss of Pten. Alteration in protein levels and localization of several transporters following loss of Pten were also observed by immunofluorescence analysis. Epithelial cells of the initial segment from knockouts were more permeable to fluorescein isothiocyanate-dextran (4000 Da) compared to controls. Interestingly, conditional deletion of Pten from other organs also resulted in changes in transporter activity, suggesting a common role of PTEN in regulation of transporter activity. Taken together, our data support the hypothesis that loss of Pten from the initial segment epithelium results in changes in the transporting and permeability characteristics of the epithelium, which in turn altered the luminal fluid microenvironment that is so important for sperm maturation and male fertility.

A Magnesium Transporter OsMGT1 Plays a Critical Role in Salt Tolerance in Rice.

Salt stress is one of the major factors limiting rice (Oryza sativa) production globally. Although several transporters involved in salt tolerance have been identified in rice, the mechanisms regulating their transport activity are still poorly understood. Here, we show evidence that a rice Mg transporter OsMGT1 is required for salt tolerance probably by regulating transport activity of OsHKT1;5, a key transporter for the removal of Na+ from the xylem sap at the root mature zone. Knockout of OsMGT1 did not affect total Na uptake, but increased Na concentration in the shoots and xylem sap, resulting in a significant increase in salt sensitivity at low external Mg2+ concentration (20-200 μm). However, such differences were abolished at a higher Mg2+ concentration (2 mm), although the total Na uptake was not altered. OsMGT1 was expressed in both the roots and shoots, but only that in the roots was moderately up-regulated by salt stress. Spatial expression analysis revealed that OsMGT1 was expressed in all root cells of the root tips but was highly expressed in the pericycle of root mature zone. OsMGT1 was also expressed in the phloem region of basal node, leaf blade, and sheath. When expressed in Xenopus laevis oocytes, the transport activity of OsHKT1;5 was enhanced by elevating external Mg2+ concentration. Furthermore, knockout of OsHKT1;5 in osmgt1 mutant background did not further increase its salt sensitivity. Taken together, our results suggest that Mg2+ transported by OsMGT1 in the root mature zone is required for enhancing OsHKT1;5 activity, thereby restricting Na accumulation to the shoots.

Cell-Type-Specific H+-ATPase Activity in Root Tissues Enables K+ Retention and Mediates Acclimation of Barley (Hordeum vulgare) to Salinity Stress.

While the importance of cell type specificity in plant adaptive responses is widely accepted, only a limited number of studies have addressed this issue at the functional level. We have combined electrophysiological, imaging, and biochemical techniques to reveal the physiological mechanisms conferring higher sensitivity of apical root cells to salinity in barley (Hordeum vulgare). We show that salinity application to the root apex arrests root growth in a highly tissue- and treatment-specific manner. Although salinity-induced transient net Na+ uptake was about 4-fold higher in the root apex compared with the mature zone, mature root cells accumulated more cytosolic and vacuolar Na+, suggesting that the higher sensitivity of apical cells to salt is not related to either enhanced Na+ exclusion or sequestration inside the root. Rather, the above differential sensitivity between the two zones originates from a 10-fold difference in K+ efflux between the mature zone and the apical region (much poorer in the root apex) of the root. Major factors contributing to this poor K+ retention ability are (1) an intrinsically lower H+-ATPase activity in the root apex, (2) greater salt-induced membrane depolarization, and (3) a higher reactive oxygen species production under NaCl and a larger density of reactive oxygen species-activated cation currents in the apex. Salinity treatment increased (2- to 5-fold) the content of 10 (out of 25 detected) amino acids in the root apex but not in the mature zone and changed the organic acid and sugar contents. The causal link between the observed changes in the root metabolic profile and the regulation of transporter activity is discussed.

Lipid-Protein Interactions in the Regulated Betaine Symporter BetP Probed by Infrared Spectroscopy

The Na(+)-coupled betaine symporter BetP senses changes in the membrane state and increasing levels of cytoplasmic K(+) during hyperosmotic stress latter via its C-terminal domain and regulates transport activity according to both stimuli. This intriguing sensing and regulation behavior of BetP was intensively studied in the past. It was shown by several biochemical studies that activation and regulation depends crucially on the lipid composition of the surrounding membrane. In fact, BetP is active and regulated only when negatively charged lipids are present. Recent structural studies have revealed binding of phosphatidylglycerol lipids to functional important parts of BetP, suggesting a functional role of lipid interactions. However, a regulatory role of lipid interactions could only be speculated from the snapshot provided by the crystal structure. Here, we investigate the nature of lipid-protein interactions of BetP reconstituted in closely packed two-dimensional crystals of negatively charged lipids and probed at the molecular level with Fourier transform infrared (FTIR) spectroscopy. The FTIR data indicate that K(+) binding weakens the interaction of BetP especially with the anionic lipid head groups. We suggest a regulation mechanism in which lipid-protein interactions, especially with the C-terminal domain and the functional important gating helices transmembrane helice 3 (TMH3) and TMH12, confine BetP to its down-regulated transport state. As BetP is also activated by changes in the physical state of the membrane, our results point toward a more general mechanism of how active transport can be modified by dynamic lipid-protein interactions.

A new metal binding domain involved in cadmium, cobalt and zinc transport.

The P1B-ATPases, which couple cation transport across membranes to ATP hydrolysis, are central to metal homeostasis in all organisms. An important feature of P1B-ATPases is the presence of soluble metal binding domains that regulate transport activity. Only one type of MBD has been characterized extensively, but bioinformatics analyses indicate that a diversity of MBDs may exist in nature. Here we report the biochemical, structural, and functional characterization of a new MBD from the Cupriavidus metallidurans P1B-4-ATPase CzcP (CzcP MBD). The CzcP MBD binds two Cd2+, Co2+, or Zn2+ ions in distinct and unique sites, and adopts an unexpected fold consisting of two fused ferredoxin-like domains. Both in vitro and in vivo activity assays using full length CzcP, truncated CzcP, and several variants indicate a regulatory role for the MBD and distinct functions for the two metal binding sites. Taken together, these findings elucidate a previously unknown MBD and suggest new regulatory mechanisms for metal transport by P1B-ATPases.