Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria.

Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex). We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex. Tom40 is organized in a high molecular mass complex of approximately 350 kD. It forms a high conductance channel. Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner. The secondary structure of Tom40 comprises approximately 31% beta-sheet, 22% alpha-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy. Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of approximately 2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly.

The TOM core complex: the general protein import pore of the outer membrane of mitochondria.

Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of approximately 2.1 nm and a height of approximately 7 nm. Tom40 is the key structural element of the TOM core complex.

Lipid-protein interactions in crystals of plant light-harvesting complex.

Two different thylakoid lipids are specifically associated with the light-harvesting complex of photosystem II (LHC-II). Digalactosyl diacyl glycerol (DGDG) binds to the isolated complex but can be removed by mild detergent treatment and anion-exchange chromatography. Removal of this lipid renders the complex unable to form two-dimensional or three-dimensional crystals. The ability to crystallize is completely restored by addition of pure DGDG, at a ratio of about four molecules per polypeptide for three-dimensional crystals, suggesting several binding sites at the periphery of the trimeric complex. Two-dimensional crystals of purified protein grown in the presence of DGDG are more highly ordered than those obtained from the unfractionated complex. The other lipid, phosphatidyl glycerol (PG), binds more firmly and cannot be removed with non-ionic detergent. Complete delipidation of LHC-II can be achieved either with phospholipase or by proteolytic cleavage of 49 amino acid residues at the N terminus. Both treatments dissociate the native, trimeric complex into monomers. This indicates that PG is directly involved in the formation of trimers, which are a prerequisite for two-dimensional and three-dimensional crystallization. Both lipids are therefore present in two-dimensional and three-dimensional crystals and have distinct roles in the structure of the complex.

S-palmitoylation represents a novel mechanism regulating the mitochondrial targeting of BAX and initiation of apoptosis.

The intrinsic pathway of apoptotic cell death is mainly mediated by the BCL-2-associated X (BAX) protein through permeabilization of the mitochondrial outer membrane (MOM) and the concomitant release of cytochrome c into the cytosol. In healthy, non-apoptotic cells, BAX is predominantly localized in the cytosol and exhibits a dynamic shuttle cycle between the cytosol and the mitochondria. Thus, the initial association with mitochondria represents a critical regulatory step enabling BAX to insert into MOMs, promoting the release of cytochrome c and ultimately resulting in apoptosis. However, the molecular mode of how BAX associates with MOMs and whether a cellular regulatory mechanism governs this process is poorly understood. Here we show that in both primary tissues and cultured cells, the association with MOMs and the proapoptotic action of BAX is controlled by its S-palmitoylation at Cys-126. A lack of BAX palmitoylation reduced BAX mitochondrial translocation, BAX oligomerization, caspase activity and apoptosis. Furthermore, ectopic expression of specific palmitoyl transferases in cultured healthy cells increases BAX S-palmitoylation and accelerates apoptosis, whereas malignant tumor cells show reduced BAX S-palmitoylation consistent with their reduced BAX-mediated proapoptotic activity. Our findings suggest that S-palmitoylation of BAX at Cys126 is a key regulatory process of BAX-mediated apoptosis.

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.

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 Cl- conductance. The structural rearrangement produced by the formation or breakdown of disulphide groups only affected the current from electrogenic glutamate 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.

Nonradioactive monitoring of organic and inorganic solute transport into single Xenopus oocytes by capillary zone electrophoresis.

Transport of organic and inorganic solutes into and out of cells requires specialized transport proteins. Given a sufficiently sensitive analytical method for measuring cellular solute concentrations, it should be possible to monitor solute transport across the plasma membrane at the level of single cells. We report a capillary zone electrophoresis approach that is generally applicable to monitor solute transport into Xenopus laevis oocytes, requires only nanoliters of sample, and involves no radioactive materials. The sensitivity of capillary electrophoresis with UV detection is typically on the order of 10(-5)-10(-6) M, resulting in the mass detection limits in the low femtomole range. We show that capillary zone electrophoresis serves as a simple technique to measure solute transport into oocytes. Studies of the mammalian oligopeptide transporter PepT1 and the Na(+)- and K(+)-coupled epithelial and neuronal glutamate transporter EAAC1 expressed in oocytes demonstrate that transport of the dipeptide Trp-Gly via PepT1 and transport of Na+ and K+ via EAAC1 across the oocyte plasma membrane can be monitored by measuring intracellular tryptophan absorption and by indirect UV detection of inorganic ions, respectively. The CZE method allowed the simultaneous detection of changes of intracellular Na+ and K+ concentrations in response to EAAC1-mediated Na+ cotransport and K+ countertransport. This is the first report of a capillary zone electrophoresis-based quantitative analysis of intracellular components of a single cell in response to transport activity.

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.

Spectroscopic characterization of three different monomeric forms of the main chlorophyll a/b binding protein from chloroplast membranes.

A detailed comparison has been made between dichroic steady-state spectroscopic properties at 77 K of several trimeric and monomeric forms of the major chlorophyll a/b binding protein (LHC-II) from pea. Monomeric forms were obtained by applying high concentrations of nonionic detergents, by a lipase treatment, or by a chymotrypsin/trypsin treatment. The latter treatments removed phosphatidyl glycerol essential for trimer formation. The absorption and dichroism spectra indicate that for trimeric LHC-II the chlorophyll b absorption region is centered around 649 nm and is composed of at least five subbands near 640, 647, 649, 652, and 656 nm. The chlorophyll a absorption region is centered around 670 nm and is composed of at least five bands near 661, 668, 671, 673, and 676 nm. The chlorophyll b band near 647 and 652 nm and the chlorophyll a bands near 668 and 673 nm are absent in the circular dichroism spectrum after monomerization. A configuration in which pigments of the same nature located on different monomers become excitonically coupled in the trimer could explain these results. In monomers obtained in high concentrations of nonionic detergents, no additional bands have disappeared, but the absorption spectra of the other two types of monomers lack the bands at 640 and 661 nm. These monomers have lost some chlorophyll a and b according to the fluorescence emission spectra, which show contributions from free chlorophyll a and b. The results suggest that phosphatidyl glycerol not only is involved in trimer formation but also has a structural role within the monomers.

Electrogenic properties of the epithelial and neuronal high affinity glutamate transporter.

Active ion-coupled glutamate transport is of critical importance for excitatory synaptic transmission, normal cellular function, and epithelial amino acid metabolism. We previously reported the cloning of the rabbit intestinal high affinity glutamate transporter EAAC1 (Kanai, Y., and Hediger, M. A. (1992) Nature 360, 467-471), which is expressed in numerous tissues including intestine, kidney, liver, heart, and brain. Here, we report a detailed stoichiometric and kinetic analysis of EAAC1 expressed in Xenopus laevis oocytes. Uptake studies of 22Na+ and [14C]glutamate, in combination with measurements of intracellular pH with pH microelectrodes gave a glutamate to charge ratio of 1:1, a glutamate to Na+ ratio of 1:2, and a OH-/H+ to charge ratio of 1:1. Since transport is K+ dependent it can be concluded that EAAC1-mediated glutamate transport is coupled to the cotransport of 2 Na+ ions, the countertransport of one K+ ion and either the countertransport of one OH- ion or the cotransport of 1 H+ ion. We further demonstrate that under conditions where the electrochemical gradients for these ions are disrupted, EAAC1 runs in reverse, a transport mode which is of pathologic importance. 22Na+ uptake studies revealed that there is a low level of Na+ uptake in the absence of extracellular glutamate which appears to be analogous to the Na+ leak observed for the intestinal Na+/glucose cotransporter SGLT1. In voltage clamp studies, reducing extracellular Na+ from 100 to 10 mM strongly increased K0.5L-glutamate and decreased I(max). The data indicate that Na+ binding at the extracellular transporter surface becomes rate-limiting. Studies addressing the cooperativity of the substrate-binding sites indicate that there are two distinct Na(+)-binding sites with different affinities and that Na+ binding is modulated by extracellular glutamate. A hypothetical ordered kinetic transport model for EAAC1 is discussed.

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 (DeltamuH) 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.

The neuronal and epithelial human high affinity glutamate transporter. Insights into structure and mechanism of transport.

High affinity transport of glutamate across plasma membranes of brain neurons and epithelial is mediated by a Na(+)- and K(+)-coupled electrogenic transporter. Here we report the primary structure and functional characterization of the human high affinity glutamate transporter (HEAAC1). A unique characteristic of HEAAC1-mediated transport is that the affinity for glutamate and the maximal transport rate are strongly dependent on membrane potential. Our data provide new insights into individual steps of high affinity glutamate transport and show that the transport mechanism is distinct from that of the gamma-aminobutyric acid transporter GAT-1 and the Na+/glucose transporter SGLT1. Under voltage clamp condition, HEAAC1 mediated large substrate-evoked inward currents (up to 1 microA). The substrate specificity, stereospecificity, the Km value (30 +/- 3 microM at -60 mV) of the L-glutamate-evoked current, and Northern analysis all agree with previously reported characteristics of high affinity glutamate transport in brain. In contrast to SGLT1 and GAT-1, voltage jump studies of HEAAC1 yielded only minor relaxation currents. Classic inhibitors of brain glutamate uptake such as DL-threo-beta-hydroxyaspartate, L-trans-pyrrolidine 2,4,-dicarboxylic acid (PDC), and dihydrokainate were found to be either transport substrates or to have no significant effect on glutamate transport. We also found that the maximal transport rate for PDC was markedly reduced compared to that for L-glutamate. We propose that PDC most likely reduces the turnover rate of the transporter. A search of the sequence data bases revealed weak homology of HEAAC1 to the H(+)-coupled vesicular monoamine transporter, suggesting an evolutionary link between plasma membrane and vesicular transporters.

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.

Expression cloning of a mammalian proton-coupled oligopeptide transporter.

In mammals, active transport of organic solutes across plasma membranes was thought to be primarily driven by the Na+ gradient. Here we report the cloning and functional characterization of a H(+)-coupled transporter of oligopeptides and peptide-derived antibiotics from rabbit small intestine. This new protein, named PepT1, displays an unusually broad substrate specificity. PepT1-mediated uptake is electrogenic, independent of extracellular Na+, K+ and Cl-, and of membrane potential. PepT1 messenger RNA was found in intestine, kidney and liver and in small amounts in brain. In the intestine, the PepT1 pathway constitutes a major mechanism for absorption of the products of protein digestion. To our knowledge, the PepT1 primary structure is the first reported for a proton-coupled organic solute transporter in vertebrates and represents an interesting evolutionary link between prokaryotic H(+)-coupled and vertebrate Na(+)-coupled transporters of organic solutes.

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.