Stephan Nussberger

Cloning and characterization of a mammalian proton-coupled metal-ion transporter

Metal ions are essential cofactors for a wealth of biological processes, including oxidative phosphorylation, gene regulation and free-radical homeostasis. Failure to maintain appropriate levels of metal ions in humans is a feature of hereditary haemochromatosis, disorders of metal-ion deficiency, and certain neurodegenerative diseases. Despite their pivotal physiological roles, however, there is no molecular information on how metal ions are actively absorbed by mammalian cells. We have now identified a new metal-ion transporter in the rat, DCT1, which has an unusually broad substrate range that includes Fe2+, Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Ni2+ and Pb2+. DCT1 mediates active transport that is proton-coupled and depends on the cell membrane potential. It is a 561-amino-acid protein with 12 putative membrane-spanning domains and is ubiquitously expressed, most notably in the proximal duodenum. DCT1 is upregulated by dietary iron deficiency, and may represent a key mediator of intestinal iron absorption. DCT1 is a member of the ‘natural-resistance-associated macrophage protein’ (Nramp) family and thus its properties provide insight into how these proteins confer resistance to pathogens.

The Preprotein Translocation Channel of the Outer Membrane of Mitochondria

The preprotein translocase of the outer membrane of mitochondria (TOM complex) facilitates the recognition, insertion, and translocation of nuclear-encoded mitochondrial preproteins. We have purified the TOM complex from Neurospora crassa and analyzed its composition and functional properties. The TOM complex contains a cation-selective high-conductance channel. Upon reconstitution into liposomes, it mediates integration of proteins into and translocation across the lipid bilayer. TOM complex particles have a diameter of about 138 A, as revealed by electron microscopy and image analysis; they contain two or three centers of stain-filled openings, which we interpret as pores with an apparent diameter of about 20 A. We conclude that the structure reported here represents the protein-conducting channel of the mitochondrial outer membrane.

Mammalian ion-coupled solute transporters.

Active transport of solutes into and out of cells proceeds via specialized transporters that utilize diverse energy‐coupling mechanisms. Ion‐coupled transporters link uphill solute transport to downhill electrochemical ion gradients. In mammals, these transporters are coupled to the co‐transport of H+, Na+, Cl‐ and/or to the countertransport of K+ or OH‐. By contrast, ATP‐dependent transporters are directly energized by the hydrolysis of ATP. The development of expression cloning approaches to select cDNA clones solely based on their capacity to induce transport function in Xenopus oocytes has led to the cloning of several ion‐coupled transporter cDNAs and revealed new insights into structural designs, energy‐coupling mechanisms and physiological relevance of the transporter proteins. Different types of mammalian ion‐coupled transporters are illustrated by discussing transporters isolated in our own laboratory such as the Na+/glucose co‐transporters SGLT1 and SGLT2, the H(+)‐coupled oligopeptide transporters PepT1 and PepT2, and the Na(+)‐ and K(+)‐dependent neuronal and epithelial high affinity glutamate transporter EAAC1. Most mammalian ion‐coupled organic solute transporters studied so far can be grouped into the following transporter families: (1) the predominantly Na(+)‐coupled transporter family which includes the Na+/glucose co‐transporters SGLT1, SGLT2, SGLT3 (SAAT‐pSGLT2) and the inositol transporter SMIT, (2) the Na(+)‐ and Cl(‐)‐coupled transporter family which includes the neurotransmitter transporters of gamma‐amino‐butyric acid (GABA), serotonin, dopamine, norepinephrine, glycine and proline as well as transporters of beta‐amino acids, (3) the Na(+)‐ and K(+)‐dependent glutamate/neurotransmitter family which includes the high affinity glutamate transporters EAAC1, GLT‐1, GLAST, EAAT4 and the neutral amino acid transporters ASCT1 and SATT1 reminiscent of system ASC and (4) the H(+)‐coupled oligopeptide transporter family which includes the intestinal H(+)‐dependent oligopeptide transporter PepT1.

Stoichiometry and pH dependence of the rabbit proton-dependent oligopeptide transporter PepT1.

1. The intestinal H(+)‐coupled peptide transporter PepT1, displays a broad substrate specificity and accepts most charged and neutral di‐ and tripeptides. To study the proton‐to‐peptide stoichiometry and the dependence of the kinetic parameters on extracellular pH (pHo), rabbit PepT1 was expressed in Xenopus laevis oocytes and used for uptake studies of radiolabelled neutral and charged dipeptides, voltage‐clamp analysis and intracellular pH measurements. 2. PepT1 did not display the substrate‐gated anion conductances that have been found to be characteristic of members of the Na(+)‐ and H(+)‐coupled high‐affinity glutamate transporter family. In conjunction with previous data on the ion dependence of PepT1, it can therefore be concluded that peptide‐evoked charge fluxes of PepT1 are entirely due to H+ movement. 3. Neutral, acidic and basic dipeptides induced intracellular acidification. The rate of acidification, the initial rates of the uptake of radiolabelled peptides and the associated charge fluxes gave proton‐substrate coupling ratios of 1:1, 2:1 and 1:1 for neutral, acidic and basic dipeptides, respectively. 4. Maximal transport of the neutral and charged dipeptides Gly‐Leu, Gly‐Glu, Gly‐Lys and Ala‐Lys occurred at pHo 5.5, 5.2, 6.2 and 5.8, respectively. The Imax values were relatively pHo independent but the apparent affinity (Km(app) values for these peptides were shown to be highly pHo dependent. 5. Our data show that at physiological pH (pHo 5.5‐6.0) PepT1 prefers neutral and acidic peptides. The shift in transport maximum for the acidic peptide Gly‐Glu to a lower pH value suggests that acidic dipeptides are transported in the protonated form. The shift in the transport maxima of the basic dipeptides to higher pH values may involve titration of a side‐chain on the transporter molecule (e.g. protonation of a histidine group). These considerations have led us to propose a model for coupled transport of neutral, acidic and basic dipeptides.

Recognition of preproteins by the isolated TOM complex of mitochondria

A multisubunit complex in the mitochondrial outer membrane, the TOM complex, mediates targeting and membrane translocation of nuclear‐encoded preproteins. We have isolated the TOM holo complex, containing the preprotein receptor components Tom70 and Tom20, and the TOM core complex, which lacks these receptors. The interaction of recombinant mitochondrial preproteins with both types of soluble TOM complex was analyzed. Preproteins bound efficiently in a specific manner to the isolated complexes in the absence of chaperones and lipids in a bilayer structure. Using fluorescence correlation spectroscopy, a dissociation constant in the nanomolar range was determined. The affinity was lower when the preprotein was stabilized in its folded conformation. Following the initial binding, the presequence was transferred into the translocation pore in a step that required unfolding of the mature part of the preprotein. This translocation step was also mediated by protease‐treated TOM holo complex, which contains almost exclusively Tom40. Thus, the TOM core complex, consisting of Tom40, Tom22, Tom6 and Tom7, is a molecular machine that can recognize and partially translocate mitochondrial precursor proteins.

Differential modulation of the uptake currents by redox interconversion of cysteine residues in the human neuronal glutamate transporter EAAC1

Control of extrasynaptic glutamate concentration in the central nervous system is an important determinant of neurotransmission and excitotoxicity. Mechanisms that modulate glutamate transporter function are therefore critical factors in these processes. The redox modulation of glutamate uptake was examined by measuring transporter‐mediated electrical currents and radiolabelled amino acid influx in voltage‐clamped Xenopus oocytes expressing the human neuronal glutamate transporter EAAC1. Up and down changes of the glutamate uptake currents in response to treatment with dithiothreitol and 5,5′‐dithio‐bis‐(2‐nitrobenzoic) acid (DTNB) were observed in oocytes clamped at ‐60 mV. The redox interconversion of cysteines induced by dithiothreitol/DTNB influenced the Vmax (Imax) of transport, while the apparent affinity for glutamate was not affected. Formation or breakdown of disulphide groups did not affect the pre‐steady‐state currents, suggesting that these manipulations do not interfere with the Na+ binding/unbinding and/or the charge distribution on the transporter molecule. The glutamate‐evoked net uptake current of EAAC1 was composed of the inward current from electrogenic glutamate transport and the current arising from the glutamate‐activated CI‐ conductance. The structural rearrangement produced by the formation or breakdown of disulphide groups only affected the current from electrogenic giutamate transport. The electrogenic currents of EAAC1 were significantly reduced by peroxynitrite, an endogenously occurring oxidant formed in certain pathological brain processes, and the mechanism of inhibition partially depended on the formation of disulphide groups.

Symmetry of H+ Binding to the Intra- and Extracellular Side of the H+-coupled Oligopeptide Cotransporter PepT1

Ion-coupled solute transporters exhibit pre-steady-tate currents that resemble those of voltage-dependent ion channels. These currents were assumed to be mostly due to binding and dissociation of the coupling ion near the extracellular transporter surface. Little attention was given to analogous events that may occur at the intracellular surface. To address this issue, we performed voltage clamp studies of Xenopus oocytes expressing the intestinal H+-coupled peptide cotransporter PepT1 and recorded the dependence of transient charge movements in the absence of peptide substrate on changing intra- (pHi) and extracellular pH (pHo). Rapid steps in membrane potential induced transient charge movements that showed a marked dependence on pHi and pHo. At a pHo of 7.0 and a holding potential (Vh) of −50 mV, the charge movements were mostly inwardly directed, whereas reduction of pHo to below 7.0 resulted in outwardly directed charge movements. When pHi was reduced, inwardly directed charge movements were observed. The data on the voltage dependence of the transient charge movements were fitted by the Boltzmann equation, yielding an apparent valence of 0.65 ± 0.03 (n = 7). The midpoint voltage (V0.5) of the charge distribution shifted linearly as a function of pHi and pHo. Our results indicate that, as a first approximation, the magnitude and polarity of the transient charge movements depend upon the prevailing H+ electrochemical gradient. We propose that PepT1 has a single proton binding site that is symmetrically accessible from both sides of the membrane and that decreasing the H+ chemical potential (ΔμH) or increasing the membrane potential (Vm) shifts this binding site from an outwardly to an inwardly facing occluded state. This concept constitutes an important extension of previous kinetic models of ion-coupled solute transporters by including a more detailed description of intracellular events.

Role of Tom5 in maintaining the structural stability of the TOM complex of mitochondria.

Transport of nuclear encoded proteins into mitochondria is mediated by multisubunit translocation machineries in the outer and inner membranes of mitochondria. The TOM complex contains receptor and pore components that facilitate the recognition of preproteins and their transfer through the outer membrane. In addition, the complex contains a set of small proteins. Tom7 and Tom6 have been found in Neurospora and yeast, Tom5 has been found so far only in the latter organism. In the present study, we identified Neurospora Tom5 and analyzed its function in comparison to yeast Tom5, which has been proposed to play a role as a receptor-like component. Neurospora Tom5 crosses the outer membrane with its carboxyl terminus facing the intermembrane space like the other small Tom components. The temperature-sensitive growth phenotype of the yeast TOM5 deletion was rescued by overexpression of Neurospora Tom5. On the other hand, Neurospora cells deficient in tom5 did not exhibit any defect in growth. The structural stability of TOM complexes from cells devoid of Tom5 was significantly altered in yeast but not in Neurospora. The efficiency of protein import in Neurospora mitochondria was not affected by deletion of tom5, whereas in yeast it was reduced as compared with wild type. We conclude that the main role of Tom5, rather than being a receptor, is maintaining the structural integrity of the TOM complex.

High-Level Expression, Refolding and Probing the Natural Fold of the Human Voltage-Dependent Anion Channel Isoforms I and II

The voltage-dependent anion channel (VDAC) is the major protein found in the outer membrane of mitochondria. The channel is responsible for the exchange of ATP/ADP and the translocation of ions and other small metabolites over the membrane. In order to obtain large amounts of pure and suitably folded human VDAC for functional and structural studies, the genes of the human isoforms I and II (HVDAC1 and HVDAC2) were cloned in Escherichia coli. High-level expression led to inclusion body formation. Both proteins could be refolded in vitro by adding denatured protein to a solution of zwitterionic or nonionic detergents. A highly efficient and fast protocol for refolding was developed that yielded more than 50 mg of pure human VDACs per liter of cell culture. The native and functional state of the refolded porins was probed by Fourier transform infrared spectroscopy to determine the secondary structure composition and by electrophysiological measurements, demonstrating the pore-forming activity of HVDAC1. Furthermore, binding of HVDAC1 to immobilized ATP was demonstrated. Limited proteolysis of HVDAC1 protein embedded in detergent micelles in combination with matrix-assisted laser desorption ionization mass spectrometric analysis was applied to identify micelle-exposed regions of the protein and to develop an improved topology model. Our analysis strongly suggests a 16-stranded, antiparallel β-barrel with one large and seven short loops and turns. Initial crystallization trials of the protein yielded crystals diffracting to 8 Å resolution.

PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha

BackgroundPoly(3-hydroxybutyrate) (PHB) granules are important storage compounds of carbon and energy in many prokaryotes which allow survival of the cells in the absence of suitable carbon sources. Formation and subcellular localization of PHB granules was previously assumed to occur randomly in the cytoplasm of PHB accumulating bacteria. However, contradictionary results on subcellular localization of PHB granules in Ralstonia eutropha were published, recently.ResultsHere, we provide evidence by transmission electron microscopy that PHB granules are localized in close contact to the nucleoid region in R. eutropha during growth on nutrient broth. Binding of PHB granules to the nucleoid is mediated by PhaM, a PHB granule associated protein with phasin-like properties that is also able to bind to DNA and to phasin PhaP5. Over-expression of PhaM resulted in formation of many small PHB granules that were always attached to the nucleoid region. In contrast, PHB granules of ∆phaM strains became very large and distribution of granules to daughter cells was impaired. Association of PHB granules to the nucleoid region was prevented by over-expression of PhaP5 and clusters of several PHB granules were mainly localized near the cell poles.ConclusionSubcellular localization of PHB granules is controlled in R. eutropha and depends on the presence and concentrations of at least two PHB granule associated proteins, PhaM and PhaP5.