The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na(+)/Cl(-)-dependent transporters) ...comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.
In the inner mitochondrial membrane, the respiratory chain complexes generate an electrochemical proton gradient, which is utilized to synthesize most of the cellular ATP. According to an increasing ...number of biochemical studies, these complexes are assembled into supercomplexes. However, little is known about the architecture of the proposed multicomplex assemblies. Here, we report the electron microscopic characterization of the two respiratory chain supercomplexes I1III2 and I1III2IV1 in bovine heart mitochondria, which are also two major supercomplexes in human mitochondria. After purification and demonstration of enzymatic activity, their structures in projection were determined by single particle image analysis. A difference map between the supercomplexes I1III2 and I1III2IV1 closely fits the x-ray structure of monocomplex IV and shows its location in the assembly. By comparing different views of supercomplex I1III2IV1, the location and mutual arrangement of complex I and the complex III dimer are discussed. Detailed knowledge of the architecture of the active supercomplexes is a prerequisite for a deeper understanding of energy conversion by mitochondria in mammals.
This publication forms part of the work of the Sub-Saharan Africa Transport Policy Program (SSATP) on identifying and promoting good policies and practices in rural transport in Africa. It provides ...an overall framework for identifying, planning, and prioritizing rural transport infrastructure and services interventions. Inadequate rural transport is a major factor contributing to the poverty of the rural population of most developing countries. For large parts of rural Africa, walking and headloading are by far the most important means of transport, most of this effort being undertaken by women. A key element is to encourage a holistic understanding of rural transport. In the context of this paper, the term covers both transport at the village and farm levels, as well as the transport services and infrastructure involved with the movement of people and goods within the village area and between villages, rural markets, and urban areas. First and foremost, though, the role of planning and prioritization is emphasized; a process based on reliable data is introduced, along with the elements of clarity and transparency. The planning process includes clearly identified objectives, relevant data, resources and constraints, and alternative scenarios. The intended audience for this paper comprises of officials, planners, economists, and engineers who are concerned with improving the livelihoods of the rural populations of Africa. Since the majority of external funding goes into initiatives for building rural roads, it is argued that much greater attention needs to be given to the other components of rural transport systems. The methods for planning and prioritization of infrastructure and services are discussed, and suggested further research is articulated. Two appendices providing examples of road planning and district planning procedures are included, along with ten figures interspersed throughout the paper.
Divalent metal-ion transporter-1 (DMT1) is a H+-coupled metal-ion transporter that plays essential roles in iron homeostasis. DMT1 exhibits reactivity (based on evoked currents) with a broad range of ...metal ions; however, direct measurement of transport is lacking for many of its potential substrates. We performed a comprehensive substrate-profile analysis for human DMT1 expressed in RNA-injected Xenopus oocytes by using radiotracer assays and the continuous measurement of transport by fluorescence with the metal-sensitive PhenGreen SK fluorophore. We provide validation for the use of PhenGreen SK fluorescence quenching as a reporter of cellular metal-ion uptake. We determined metal-ion selectivity under fixed conditions using the voltage clamp. Radiotracer and continuous measurement of transport by fluorescence assays revealed that DMT1 mediates the transport of several metal ions that were ranked in selectivity by using the ratio Imax/K0.5 (determined from evoked currents at −70 mV): Cd2+ > Fe2+ > Co2+, Mn2+ ≫ Zn2+, Ni2+, VO2+. DMT1 expression did not stimulate the transport of Cr2+, Cr3+, Cu+, Cu2+, Fe3+, Ga3+, Hg2+, or VO+. 55Fe2+ transport was competitively inhibited by Co2+ and Mn2+. Zn2+ only weakly inhibited 55Fe2+ transport. Our data reveal that DMT1 selects Fe2+ over its other physiological substrates and provides a basis for predicting the contribution of DMT1 to intestinal, nasal, and pulmonary absorption of metal ions and their cellular uptake in other tissues. Whereas DMT1 is a likely route of entry for the toxic heavy metal cadmium, and may serve the metabolism of cobalt, manganese, and vanadium, we predict that DMT1 should contribute little if at all to the absorption or uptake of zinc. The conclusion in previous reports that copper is a substrate of DMT1 is not supported.
Background: DMT1 plays essential roles in iron homeostasis, but questions remain about which other metals this transporter serves.
Results: DMT1 exhibits substrate selectivity Cd2+ > Fe2+ > Co2+, Mn2+ ≫ Ni2+, VO2+, Zn2+.
Conclusion: DMT1 is an iron-preferring transporter that does not transport copper.
Significance: These findings will help in predicting the contribution of DMT1 to absorption and cellular uptake of metal ions.
Mitochondrial respiratory chain (MRC) enzymes associate in supercomplexes (SCs) that are structurally interdependent. This may explain why defects in a single component often produce combined enzyme ...deficiencies in patients. A case in point is the alleged destabilization of complex I in the absence of complex III. To clarify the structural and functional relationships between complexes, we have used comprehensive proteomic, functional, and biogenetical approaches to analyze a MT‐CYB‐deficient human cell line. We show that the absence of complex III blocks complex I biogenesis by preventing the incorporation of the NADH module rather than decreasing its stability. In addition, complex IV subunits appeared sequestered within complex III subassemblies, leading to defective complex IV assembly as well. Therefore, we propose that complex III is central for MRC maturation and SC formation. Our results challenge the notion that SC biogenesis requires the pre‐formation of fully assembled individual complexes. In contrast, they support a cooperative‐assembly model in which the main role of complex III in SCs is to provide a structural and functional platform for the completion of overall MRC biogenesis.
Synopsis
The mitochondrial respiratory chain (MRC), necessary for aerobic cellular energy transduction in eukaryotic cells, consists of five large enzyme complexes that can assemble into larger supramolecular structures called supercomplexes (SCs). Biogenesis of the human MRC requires the cooperative and interdependent action of respiratory SCs.
Complex III is a master regulator of MRC maturation and SC formation.
Lack of respiratory complex III halts the assembly of complex I by preventing the incorporation of the NADH‐module, but it does not induce the degradation of fully assembled complex I.
Coenzyme Q and oxidoreductase activity of complex III are required for the maturation of complex I.
Mis‐assembly of complex III affects the biogenesis of complex IV as it causes the sequestration of unassembled complex IV subunits into complex III preassemblies.
Complex I, III and IV assemble in a cooperative way, interacting with each other prior to the formation of the individual complexes.
Biogenesis of the human mitochondrial respiratory chain requires the cooperative and interdependent action of respiratory supercomplexes.
This study is part of the Africa Infrastructure Country Diagnostic (AICD), a project designed to expand the world's knowledge of physical infrastructure in Africa. The AICD will provide a baseline ...against which future improvements in infrastructure services can be measured, making it possible to monitor the results achieved from donor support. It should also provide a more solid empirical foundation for prioritizing investments and designing policy reforms in the infrastructure sectors in Africa. The AICD is based on an unprecedented effort to collect detailed economic and technical data on the infrastructure sectors in Africa. The project has produced a series of original reports on public expenditure, spending needs, and sector performance in each of the main infrastructure sectors, including energy, information and communication technologies, irrigation, transport, and water and sanitation. The first phase of the AICD focused on 24 countries that together account for 85 percent of the gross domestic product, population, and infrastructure aid flows of Sub- Saharan Africa. Under a second phase of the project, coverage is expanding to include as many of the additional African countries as possible.
The respirasome is a multisubunit supercomplex of the respiratory chain in mitochondria. Here we report the 3D reconstruction of the bovine heart respirasome, composed of dimeric complex III and ...single copies of complex I and IV, at about 2.2-nm resolution, determined by cryoelectron tomography and subvolume averaging. Fitting of X-ray structures of single complexes I, III2, and IV with high fidelity allows interpretation of the model at the level of secondary structures and shows how the individual complexes interact within the respirasome. Surprisingly, the distance between cytochrome c binding sites of complexes III2 and IV is about 10 nm. Modeling indicates a loose interaction between the three complexes and provides evidence that lipids are gluing them at the interfaces.
Mitochondrial electron transport chain complexes are organized into supercomplexes responsible for carrying out cellular respiration. Here we present three architectures of mammalian (ovine) ...supercomplexes determined by cryo-electron microscopy. We identify two distinct arrangements of supercomplex CICIII
CIV (the respirasome)-a major 'tight' form and a minor 'loose' form (resolved at the resolution of 5.8 Å and 6.7 Å, respectively), which may represent different stages in supercomplex assembly or disassembly. We have also determined an architecture of supercomplex CICIII
at 7.8 Å resolution. All observed density can be attributed to the known 80 subunits of the individual complexes, including 132 transmembrane helices. The individual complexes form tight interactions that vary between the architectures, with complex IV subunit COX7a switching contact from complex III to complex I. The arrangement of active sites within the supercomplex may help control reactive oxygen species production. To our knowledge, these are the first complete architectures of the dominant, physiologically relevant state of the electron transport chain.
Biogenic amine transporters for serotonin, norepinephrine and dopamine (SERT, NET and DAT respectively), are the key players terminating transmission of these amines in the central nervous system by ...their high-affinity uptake. They are also major targets for many antidepressant drugs. Interestingly however, drugs targeted to a specific transporter do not appear to be as clinically efficacious as those that block two or all three of these transporters. A growing body of literature, reviewed here, supports the idea that promiscuity among these transporters (the uptake of multiple amines in addition to their "native" transmitter) may account for improved therapeutic effects of dual and triple uptake blockers. However, even these drugs do not provide effective treatment outcomes for all individuals. An emerging literature suggests that "non-traditional" transporters such as organic cation transporters (OCT) and the plasma membrane monoamine transporter (PMAT) may contribute to the less than hoped for efficacy of currently prescribed uptake inhibitors. OCT and PMAT are capable of clearing biogenic amines from extracellular fluid and may serve to buffer the effects of frontline antidepressants, such as selective serotonin reuptake inhibitors. In addition, polymorphisms that occur in the genes encoding the transporters can lead to variation in transporter expression and function (e.g. the serotonin transporter linked polymorphic region; 5-HTTLPR) and can have profound effects on treatment outcome. This may be accounted for, in part, by compensatory adaptations in other transporters. This review synthesizes the existing literature, focusing on serotonin to illustrate and revive a model for the rationale design of improved antidepressants.
The transport of pyruvate, the end product of glycolysis, into mitochondria is an essential process that provides the organelle with a major oxidative fuel. Although the existence of a specific ...mitochondrial pyruvate carrier (MPC) has been anticipated, its molecular identity remained unknown. We report that MPC is a heterocomplex formed by two members of a family of previously uncharacterized membrane proteins that are conserved from yeast to mammals. Members of the MPC family were found in the inner mitochondrial membrane, and yeast mutants lacking MPC proteins showed severe defects in mitochondrial pyruvate uptake. Coexpression of mouse MPC1 and MPC2 in Lactococcus lactis promoted transport of pyruvate across the membrane. These observations firmly establish these proteins as essential components of the MPC.
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