Richard Wagner

Solute pores, ion channels, and metabolite transporters in the outer and inner envelope membranes of higher plant plastids.

All plant cells contain plastids. Various reactions are located exclusively within these unique organelles, requiring the controlled exchange of a wide range of solutes, ions, and metabolites. In recent years, several proteins involved in import and/or export of these compounds have been characterized using biochemical and electrophysiological approaches, and in addition have been identified at the molecular level. Several solute channels have been identified in the outer envelope membrane. These porin-like proteins in the outer envelope membrane were formerly thought to be quite unspecific, but have now been shown to exhibit significant substrate specificity and to be highly regulated. Therefore, the inter-envelope membrane space is not as freely accessible as previously thought. Transport proteins in the inner envelope membrane have been characterized in more detail. It has been proved unequivocally that a family of proteins (including triose phosphate-/phosphoenolpyruvate-, and glucose 6-phosphate-specific transporters) permit the exchange of inorganic phosphate and phosphorylated intermediates. A new type of plastidic 2-oxoglutarate/malate transporter has been identified and represents the first carrier with 12 putative transmembrane domains, to be located in the inner envelope membrane. The plastidic ATP/ADP transporter also contains 12 putative transmembrane domains and possesses striking structural similarity to ATP/ADP transporters found in intracellular, human pathogenic bacteria.

Antisense technology and prospects for therapy of viral infections and cancer.

Eighteen years ago, antisense oligonucleotide therapeutics that can selectively knock out disease-causing genes could easily have been viewed as science fiction. Yet today, through much persistence and focused investment, the technology has nearly evolved to the point of realization. A number of first-generation antisense compounds have entered human clinical trials. Some of these compounds appear to work by an antisense mechanism to inhibit the expression of disease-causing genes, while others probably work by unanticipated, yet clinically beneficial, mechanisms. In this review, the current status of antisense oligonucleotide development will be described as it relates to two areas of concentrated effort: antiviral and anticancer applications.

Bacterial Porin Disrupts Mitochondrial Membrane Potential and Sensitizes Host Cells to Apoptosis

The bacterial PorB porin, an ATP-binding β-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (ΔΨm). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of β-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of ΔΨm. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce ΔΨm loss and apoptosis, demonstrating that dissipation of ΔΨm is a requirement for cell death caused by neisserial infection.

Characterization of Horizontal Lipid Bilayers as a Model System to Study Lipid Phase Separation

Artificial lipid membranes are widely used as a model system to study single ion channel activity using electrophysiological techniques. In this study, we characterize the properties of the artificial bilayer system with respect to its dynamics of lipid phase separation using single-molecule fluorescence fluctuation and electrophysiological techniques. We determined the rotational motions of fluorescently labeled lipids on the nanosecond timescale using confocal time-resolved anisotropy to probe the microscopic viscosity of the membrane. Simultaneously, long-range mobility was investigated by the lateral diffusion of the lipids using fluorescence correlation spectroscopy. Depending on the solvent used for membrane preparation, lateral diffusion coefficients in the range D(lat) = 10-25 mum(2)/s and rotational diffusion coefficients ranging from D(rot) = 2.8 - 1.4 x 10(7) s(-1) were measured in pure liquid-disordered (L(d)) membranes. In ternary mixtures containing saturated and unsaturated phospholipids and cholesterol, liquid-ordered (L(o)) domains segregated from the L(d) phase at 23 degrees C. The lateral mobility of lipids in L(o) domains was around eightfold lower compared to those in the L(d) phase, whereas the rotational mobility decreased by a factor of 1.5. Burst-integrated steady-state anisotropy histograms, as well as anisotropy imaging, were used to visualize the rotational mobility of lipid probes in phase-separated bilayers. These experiments and fluorescence correlation spectroscopy measurements at different focal diameters indicated a heterogeneous microenvironment in the L(o) phase. Finally, we demonstrate the potential of the optoelectro setup to study the influence of lipid domains on the electrophysiological properties of ion channels. We found that the electrophysiological activity of gramicidin A (gA), a well-characterized ion-channel-forming peptide, was related to lipid-domain partitioning. During liquid-liquid phase separation, gA was largely excluded from L(o) domains. Simultaneously, the number of electrically active gA dimers increased due to the increased surface density of gA in the L(d) phase.

Potent and selective gene inhibition using antisense oligodeoxynucleotides

The development of antisense technology as a generally useful tool relies on the use of potent agents and the utilization of many controls in experiments. Here we describe our experience using oligodeoxynucleotides (ODNs) containing C-5 propynyl pyrimidine and phosphorothioate modifications as broadly applicable gene inhibition agents in cell culture. Methods include selection of antisense sequences, synthesis and purification of ODNs, choice of controls, delivery methods (microinjection, cationic lipid transfection, and electroporation), and analysis of gene inhibition.

Alternative Splicing-Mediated Targeting of the Arabidopsis GLUTAMATE RECEPTOR3.5 to Mitochondria Affects Organelle Morphology

A unique mitochondrial ion channel affects organelle physiology and its lack is associated with senescence in the model plant Arabidopsis. Since the discovery of 20 genes encoding for putative ionotropic glutamate receptors in the Arabidopsis (Arabidopsis thaliana) genome, there has been considerable interest in uncovering their physiological functions. For many of these receptors, neither their channel formation and/or physiological roles nor their localization within the plant cells is known. Here, we provide, to our knowledge, new information about in vivo protein localization and give insight into the biological roles of the so-far uncharacterized Arabidopsis GLUTAMATE RECEPTOR3.5 (AtGLR3.5), a member of subfamily 3 of plant glutamate receptors. Using the pGREAT vector designed for the expression of fusion proteins in plants, we show that a splicing variant of AtGLR3.5 targets the inner mitochondrial membrane, while the other variant localizes to chloroplasts. Mitochondria of knockout or silenced plants showed a strikingly altered ultrastructure, lack of cristae, and swelling. Furthermore, using a genetically encoded mitochondria-targeted calcium probe, we measured a slightly reduced mitochondrial calcium uptake capacity in the knockout mutant. These observations indicate a functional expression of AtGLR3.5 in this organelle. Furthermore, AtGLR3.5-less mutant plants undergo anticipated senescence. Our data thus represent, to our knowledge, the first evidence of splicing-regulated organellar targeting of a plant ion channel and identify the first cation channel in plant mitochondria from a molecular point of view.

Protein conducting nanopores

About 50% of the cellular proteins have to be transported into or across cellular membranes. This transport is an essential step in the protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Almost all proteins of the endosymbiotic organelles chloroplasts and mitochondria are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic, biochemical and biophysical approaches led to rather detailed knowledge on the composition of the translocon-complexes which catalyze the membrane transport of the preproteins. Comprehensive concepts on the targeting and membrane transport of polypeptides emerged, however little detail on the molecular nature and mechanisms of the protein translocation channels comprising nanopores has been achieved. In this paper we will highlight recent developments of the diverse protein translocation systems and focus particularly on the common biophysical properties and functions of the protein conducting nanopores. We also provide a first analysis of the interaction between the genuine protein conducting nanopore Tom40(SC) as well as a mutant Tom40(SC) (S(54 --> E) containing an additional negative charge at the channel vestibule and one of its native substrates, CoxIV, a mitochondrial targeting peptide. The polypeptide induced a voltage-dependent increase in the frequency of channel closure of Tom40(SC) corresponding to a voltage-dependent association rate, which was even more pronounced for the Tom40(SC) S54E mutant. The corresponding dwelltime reflecting association/transport of the peptide could be determined with t(off) approximately = 1.1 ms for the wildtype, whereas the mutant Tom40(SC) S54E displayed a biphasic dwelltime distribution (t(off)(-1) approximately = 0.4 ms; t(off)(-2) approximately = 4.6 ms).

Exploring protein import pores of cellular organelles at the single molecule level using the planar lipid bilayer technique.

Proteins of living cells carry out their specialized functions within various subcellular membranes or aqueous spaces. Approximately half of all the proteins of a typical cell are transported into or across membranes. Targeting and transport to their correct subcellular destinations are essential steps in protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Virtually all proteins of the endosymbiotic organelles, chloroplasts and mitochondria, are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic and biochemical techniques led to rather detailed knowledge on the subunit composition of the various protein transport complexes which carry out the membrane transport of the preproteins. Conclusive concepts on targeting and cytosolic transport of polypeptides emerged, while still few details on the molecular nature and mechanisms of the channel moieties of protein translocation complexes have been achieved. In this paper we will describe the history of how the individual subunits forming the channel pores of the chloroplast, mitochondrial and endoplasmic reticulum protein import machineries were identified and characterized by single channel electrophysiological techniques in planar bilayers. We will also highlight recent developments in the exploration of the molecular properties of protein translocating channels and the regulation of the diverse protein translocation systems using the planar bilayer technique.

Conformational changes of the isolated ferredoxin-NADP-oxidoreductase upon nucleotide binding as revealed by the triplet lifetime of bound eosin-SCN

The existence in chloroplasts of a flavoprotein with NADPH-speciflc diaphorase activity was first reported by Avron and Jagendorf [ 1 ] and later it was discovered that this enzyme is essential for the photoreduction of NADP + [2]. It is active when isolated in monomeric form at M r 35 000-40 000 [3]. In the thylakoid membrane it is accessible to impermeant antibodies and probably located at the outer side of the nonstacked parts of the membrane [4,5]. Recently, regulation of the reductase activity by pH and by light, via a transmemb rane pH-difference, was demonstrated [6,7]. In vitro the reductase forms an equimolar complex with NADP ÷ which differs from the uncomplexed form in its visible absorption spectrum [8], fluorescence yield of the flavin moeity [9] and in sensitivity to SH-reagents [ 10]. These alterations have been ascribed to conformational changes of the protein, without further specification of their nature and their physiological role. It is known, however, that the complex formation requires unmodified lysyl [ 11], arginyl [12] and carboxyl groups [7]. We attempted to further characterize the conformational changes with the probe eosin-isothiocyanate. We have applied eosin-SCN to study conformational changes of the membrane-bound [13] as well as of the isolated coupling factor of photophosphorylation [19,22]. When excited with a short laser flash eosin is efficiently transferred into a relatively stable triplet state. When iosin-SCN is covalently bound to an enzyme its triplet lifetime depends on the access of oxygen to the respective binding site. This can vary under conformational changes [13], shorter lifetime is a qualitative indicator for closer proximity of a site to the surface of a protein. Besides via its triplet lifetime we also used eosin-SCN for photoselection studies on the rotational diffusion of the coupling factor (CFI) in the thylakoid membrane. The rotational correlation time was also dependent, e.g., on the existence of a protonmotive force across the membrane and on ATP, served as another indicator of conformational changes [13]. In this study we labelled the isolated and purified ferredoxin-NADP reductase with eosin-isothiocyanate and measured the activity of the enzyme and the triplet lifetime of the probe. We found at least two binding sites which differ in their effect on the activity and in their triplet lifetime. Binding of eosin-SCN to site A inhibits the NADPH-diaphorase activity. The triplet lifetime is rather short, and therefore site A will be close to the surface of the protein. In contrast to this, binding of eosin-SCN to site B does not affect the diaphorase activity but rather the interaction with ferredoxin. The triplet lifetime of eosin-SCN at site B is longer than at A; however, it is shortened upon binding of NADPH to the (unmodified) site A. This may indicate that the environment of the ferredoxin binding site of the reductase is modified by the presence of NADPH at the other catalytic site.

Effects of CO2-depletion on proton uptake and release in thylakoid membranes

The ability of COz to stimulate the Hill reaction in thylakoid membrane is well established [ 11. When NaHCOs is added to COz-depleted thylakoids, a large (4-6-fold) increase in oxygen evolution is observed. Recent experiments [2-61 have demonstrated that a major site of inhibition of Hill reaction by CO2 depletion is between electron acceptor Q of photosystem II (PS II) and the plastoquinone (PQ) pool, and more specifically between the two electron acceptor R (or B) and PQ [4,5]. Although a major site of bicarbonate action has been clearly established, and recent experiments suggest that CO1 is the species that diffuses and may initially bind to the membrane [7], the mechanism of its action is not known. During illumination of thylakoids there is an uptake of protons from the external phase and a release of protons into the interior space [g-lo]. There are two proton uptake sites on the outer side of the membrane [ 1 I] and two sites of proton release on the inner side [ 121. One of the sites on the outer surface is attributable to the reduction of the electron acceptor used. The second site of proton uptake is associated with the reduction of plastoquinone (PQ + 2 H’ + 2 e--, PQH2) which acts as a shuttle such that its oxidation releases protons at the inner side of the membrane. The other site of proton release into the inner phase is attributed to the oxidation of water (1/2H,0~1/402+I-l++e-).Thesitesofprotonbinding from the outer phase and proton release into the inner aqueous volume have been studied with pH