Membrane transport proteins of the malaria parasite

The malaria parasite-infected erythrocyte is a multi-compartment structure, incorporating numerous different membrane systems. The movement of nutrients, metabolites and inorganic ions into and out of the intraerythrocytic parasite, as well as between subcellular compartments within the parasite, is mediated by transporters and channels – integral membrane proteins that facilitate the movement of solutes across the membrane bilayer. Proteins of this type also play a key role in antimalarial drug resistance. Genes encoding transporters and channels account for at least 2.5% of the parasite genome. However, ascribing functions and physiological roles to these proteins, and defining their roles in drug resistance, is not straightforward. For any given membrane transport protein, a full understanding of its role(s) in the parasitized erythrocyte requires a knowledge of its subcellular localization and substrate specificity, as well as some knowledge of the effects on the parasite of modifying the sequence and/or level of expression of the gene involved. Here we consider recent work in this area, describe a number of newly identified transport proteins, and summarize the likely subcellular localization and putative substrate specificity of all of the candidate membrane transport proteins identified to date.     

Caveat emptor: limitations of the automated reconstruction of metabolic pathways in Plasmodium

The functional reconstruction of metabolic pathways from an annotated genome is a tedious and demanding enterprise. Automation of this endeavor using bioinformatics algorithms could cope with the ever-increasing number of sequenced genomes and accelerate the process. Here, the manual reconstruction of metabolic pathways in the functional genomic database of Plasmodium falciparum--Malaria Parasite Metabolic Pathways--is described and compared with pathways generated automatically as they appear in PlasmoCyc, metaSHARK and the Kyoto Encyclopedia for Genes and Genomes. A critical evaluation of this comparison discloses that the automatic reconstruction of pathways generates manifold paths that need an expert manual verification to accept some and reject most others based on manually curated gene annotation.     

Membrane Potential Greatly Enhances Superoxide Generation by the Cytochrome bc1 Complex Reconstituted into Phospholipid Vesicles

The mitochondrial cytochrome bc₁ complex (ubiquinol/cytochrome c oxidoreductase) is generally thought to generate superoxide anion that participates in cell signaling and contributes to cellular damage in aging and degenerative disease. However, the isolated, detergent-solubilized bc₁ complex does not generate measurable amounts of superoxide except when inhibited by antimycin. In addition, indirect measurements of superoxide production by cells and isolated mitochondria have not clearly resolved the contribution of the bc₁ complex to the generation of superoxide by mitochondria in vivo, nor did they establish the effect, if any, of membrane potential on superoxide formation by this enzyme complex. In this study we show that the yeast cytochrome bc₁ complex does generate significant amounts of superoxide when reconstituted into phospholipid vesicles. The rate of superoxide generation by the reconstituted bc₁ complex increased exponentially with increased magnitude of the membrane potential, a finding that is compatible with the suggestion that membrane potential inhibits electron transfer from the cytochrome b_L to b_H hemes, thereby promoting the formation of a ubisemiquinone radical that interacts with oxygen to generate superoxide. When the membrane potential was further increased, by the addition of nigericin or by the imposition of a diffusion potential, the rate of generation of superoxide was further accelerated and approached the rate obtained with antimycin. These findings suggest that the bc₁ complex may contribute significantly to superoxide generation by mitochondria in vivo, and that the rate of superoxide generation can be controlled by modulation of the mitochondrial membrane potential.The mitochondrial oxidative phosphorylation system utilizes the energy derived from the oxidation of metabolic substrates to drive the synthesis of ATP. Electron transport through the NADH dehydrogenase complex, cytochrome bc₁ complex, and cytochrome c oxidase complex is coupled to proton translocation across the mitochondrial inner membrane, thus generating a protonmotive force (Δp) consisting of a membrane potential (ΔΨ) and a pH gradient (ΔpH) that drives the synthesis of ATP by the ATP synthase (reviewed in Ref. 1).Several of the mitochondrial electron transport complexes produce free radical intermediates that interact with oxygen to generate superoxide (reviewed in Refs. 2, 3). Superoxide is a highly reactive compound that can lead to the formation of other free radicals and reactive compounds and thus damage directly or indirectly cellular proteins, DNA, and phospholipids. It is also believed that free radical damage is a major cause of aging and contributes to many degenerative diseases (reviewed in Ref. 4).Studies with isolated mitochondria have attempted to evaluate the contributions of the different mitochondrial energy-transducing complexes to this process (5–9). An early study with isolated rat heart mitochondria suggested that the bc₁ complex produces large amounts of superoxide, but only when the mitochondrial membrane potential is high (10). This conclusion led to the suggestion that cells modulate the magnitude of the mitochondrial protonmotive force to protect the mitochondria from excess production of superoxide (11).However, it was shown later that with high concentrations of succinate as a substrate and without rotenone (as in Ref. 10), most of the superoxide is generated by reverse electron transport through complex I (8). Moreover, the rate of generation of superoxide by reverse electron transport through complex I was shown to be more strongly dependent on ΔpH than on Δψ (12). It was also suggested that the contribution of the bc₁ complex to superoxide generation by mitochondria is negligible compared with that produced by reverse electron transport through complex I (9), but it is not clear whether reverse electron transport is a significant process under most physiological conditions.Several groups have measured superoxide production by the detergent-solubilized bc₁ complex isolated from either yeast or beef heart. It was possible to observe superoxide production by the isolated, detergent-solubilized bc₁ complex that was mutated in key residues at the ubiquinol oxidation site (13). However, the native enzyme did not produce measurable amounts of superoxide except when inhibited by antimycin or other bc₁ complex inhibitors (14–18). The mechanism of the antimycin-induced generation of superoxide by the bc₁ complex is fairly well understood within the framework of the Q cycle mechanism, shown in Fig. 1. Following the oxidation of ubiquinol at center P, as electrons recycle through the b hemes, antimycin inhibits reduction of ubiquinone at center N and electrons back up in center P, resulting in the formation of a ubisemiquinone radical, which can interact with oxygen to form superoxide (15, 18). It also can be predicted that the membrane potential would inhibit electron transfer from heme b_L to b_H and stimulate the production of superoxide by the bc₁ complex. However, it is not known whether this prediction actually manifests and, if so, how strong is the dependence of superoxide production by the bc₁ complex on the magnitude of membrane potential.Open in a separate windowFIGURE 1.Mechanistic basis for production of superoxide by the reconstituted cytochrome bc₁ complex. The figure shows the protonmotive Q cycle mechanism and the leak of electrons to oxygen that is presumably the source of superoxide formation by the reconstituted enzyme. A shows the protonmotive Q cycle mechanism as it normally functions. Ubiquinol (QH₂) is oxidized at center P near the outer surface of the membrane or vesicle in a bifurcated reaction that transfers one electron to the Rieske iron-sulfur protein (ISP) and one electron to the b_L heme of cytochrome b. The electron on the iron-sulfur protein is then transferred to cytochrome c₁, and the electron on the b_L heme is transferred to the b_H heme, which then reduces ubiquinone (Q) to semiquinone at center N. When a second molecule of ubiquinol is oxidized, the electron that arrives on the b_H heme reduces semiquinone to ubiquinol. B shows the formation of superoxide anion that results when electron transfer from the b_L to b_H heme is inhibited, either by an opposing membrane potential or by antimycin, which blocks reoxidation of the b_H heme, causing electrons to accumulate in the b_L heme. Superoxide anion is formed by reaction of oxygen with ubisemiquinone, which is formed either by transfer of one electron from ubiquinol to the iron-sulfur protein or by reduction of ubiquinone by the reduced b_L heme. In both panels solid arrows indicate electron transfer reactions. Dashed arrows indicate movement of ubiquinone and ubiquinol between reaction centers in the bc₁ complex, release and uptake of protons at center P and center N, or changes in redox status of ubiquinone, ubiquinol, and oxygen. Solid bars in B show the opposition of electron transfer from the b_L to b_H heme by the membrane potential and inhibition of b_H reoxidation by antimycin.We have attempted to resolve this issue by reconstitution of the yeast cytochrome bc₁ complex into phospholipid vesicles, followed by measuring the rate of superoxide generation in parallel with the magnitude of the membrane potential that is generated by the reconstituted enzyme. Our findings indicate that superoxide anion formation by the bc₁ complex in situ depends strongly on membrane potential and can approach values similar to those promoted by antimycin.     

Glyphosate-induced anther indehiscence in cotton is partially temperature dependent and involves cytoskeleton and secondary wall modifications and auxin accumulation

Yield reduction caused by late application of glyphosate to glyphosate-resistant cotton (Gossypium hirsutum; GRC) expressing CP4 5-enol-pyruvylshikmate-3-P synthase under the cauliflower mosaic virus-35S promoter has been attributed to male sterility. This study was aimed to elucidate the factors and mechanisms involved in this phenomenon. Western and tissue-print blots demonstrated a reduced expression of the transgene in anthers of GRC compared to ovules of the same plants. Glyphosate application to GRC grown at a high temperature regime after the initiation of flower buds caused a complete loss of pollen viability and inhibition of anther dehiscence, while at a moderate temperature regime only 50% of the pollen grains were disrupted and anther dehiscence was normal. Glyphosate-damaged anthers exhibited a change in the deposition of the secondary cell wall thickenings (SWT) in the endothecium cells, from the normal longitudinal orientation to a transverse orientation, and hindered septum disintegration. These changes occurred only at the high temperature regime. The reorientation of SWT in GRC was accompanied by a similar change in microtubule orientation. A similar reorientation of microtubules was also observed in Arabidopsis (Arabidopsis thaliana) seedlings expressing green fluorescent protein tubulin (tubulin alpha 6) following glyphosate treatment. Glyphosate treatment induced the accumulation of high levels of indole-3-acetic acid in GRC anthers. Cotton plants treated with 2,4-dichlorophenoxyacetic acid had male sterile flowers, with SWT abnormalities in the endothecium layer similar to those observed in glyphosate-treated plants. Our data demonstrate that glyphosate inhibits anther dehiscence by inducing changes in the microtubule and cell wall organization in the endothecium cells, which are mediated by auxin.     

Autophagy in Yeast: Mechanistic Insights and Physiological Function

Unicellular eukaryotic organisms must be capable of rapid adaptation to changing environments. While such changes do not normally occur in the tissues of multicellular organisms, developmental and pathological changes in the environment of cells often require adaptation mechanisms not dissimilar from those found in simpler cells. Autophagy is a catabolic membrane-trafficking phenomenon that occurs in response to dramatic changes in the nutrients available to yeast cells, for example during starvation or after challenge with rapamycin, a macrolide antibiotic whose effects mimic starvation. Autophagy also occurs in animal cells that are serum starved or challenged with specific hormonal stimuli. In macroautophagy, the form of autophagy commonly observed, cytoplasmic material is sequestered in double-membrane vesicles called autophagosomes and is then delivered to a lytic compartment such as the yeast vacuole or mammalian lysosome. In this fashion, autophagy allows the degradation and recycling of a wide spectrum of biological macromolecules. While autophagy is induced only under specific conditions, salient mechanistic aspects of autophagy are functional in a constitutive fashion. In Saccharomyces cerevisiae, induction of autophagy subverts a constitutive membrane-trafficking mechanism called the cytoplasm-to-vacuole targeting pathway from a specific mode, in which it carries the resident vacuolar hydrolase, aminopeptidase I, to a nonspecific bulk mode in which significant amounts of cytoplasmic material are also sequestered and recycled in the vacuole. The general aim of this review is to focus on insights gained into the mechanism of autophagy in yeast and also to review our understanding of the physiological significance of autophagy in both yeast and higher organisms.     

Caffeine induces macroautophagy and confers a cytocidal effect on food spoilage yeast in combination with benzoic acid

Weak organic acids are an important class of food preservatives that are particularly efficacious towards yeast and fungal spoilage. While acids with small aliphatic chains appear to function by acidification of the cytosol and are required at high concentrations to inhibit growth, more hydrophobic organic acids such as sorbic and benzoic acid have been suggested to function by perturbing membrane dynamics and are growth-inhibitory at much lower concentrations. We previously demonstrated that benzoic acid has selective effects on membrane trafficking in Saccharomyces cerevisiae. Benzoic acid selectively blocks macroautophagy in S. cerevisiae while acetic acid does not, and sorbic acid does so to a lesser extent. Indeed, while both benzoic acid and nitrogen starvation are cytostatic when assayed separately, the combination of these treatments is cytocidal, because macroautophagy is essential for survival during nitrogen starvation. In this report, we demonstrate that Zygosaccharomyces bailii, a food spoilage yeast with relatively high resistance to weak acid stress, also exhibits a cytocidal response to the combination of benzoic acid and nitrogen starvation. In addition, we show that nitrogen starvation can be replaced by caffeine supplementation. Caffeine induces a starvation response that includes the induction of macroautophagy, and the combination of caffeine and benzoic acid is cytocidal, as predicted from the nitrogen starvation data.     

Oxidative stress in malaria parasite-infected erythrocytes: host-parasite interactions

Experimenta naturae, like the glucose-6-phosphate dehydrogenase deficiency, indicate that malaria parasites are highly susceptible to alterations in the redox equilibrium. This offers a great potential for the development of urgently required novel chemotherapeutic strategies. However, the relationship between the redox status of malarial parasites and that of their host is complex. In this review article we summarise the presently available knowledge on sources and detoxification pathways of reactive oxygen species in malaria parasite-infected red cells, on clinical aspects of redox metabolism and redox-related mechanisms of drug action as well as future prospects for drug development. As delineated below, alterations in redox status contribute to disease manifestation including sequestration, cerebral pathology, anaemia, respiratory distress, and placental malaria. Studying haemoglobinopathies, like thalassemias and sickle cell disease, and other red cell defects that provide protection against malaria allows insights into this fine balance of redox interactions. The host immune response to malaria involves phagocytosis as well as the production of nitric oxide and oxygen radicals that form part of the host defence system and also contribute to the pathology of the disease. Haemoglobin degradation by the malarial parasite produces the redox active by-products, free haem and H(2)O(2), conferring oxidative insult on the host cell. However, the parasite also supplies antioxidant moieties to the host and possesses an efficient enzymatic antioxidant defence system including glutathione- and thioredoxin-dependent proteins. Mechanistic and structural work on these enzymes might provide a basis for targeting the parasite. Indeed, a number of currently used drugs, especially the endoperoxide antimalarials, appear to act by increasing oxidant stress, and novel drugs such as peroxidic compounds and anthroquinones are being developed.     

How many functional transport pathways does Plasmodium falciparum induce in the membrane of its host erythrocyte?

Intraerythrocytic malaria parasites induce considerable change in the permeability of the membrane of their host cell. Using classical techniques of radiolabel uptake and iso-osmotic lysis, the permeability characteristics of the host-cell membrane have been determined. In a recent analysis of these results, we concluded that there are at least two types of channel that conform to the data: a low copy number (four channels per cell) type that mediates the transport of cations, anions and most other osmolytes that were tested, and a high copy number (300-400 channels per cell) type that is an anion channel that could also mediate the translocation of purine nucleosides. Patch-clamping experiments using cells infected with Plasmodium falciparum reveal 200-1000 anion channels of more than one type that are of host-cell endogenous provenance. Recent reports show that parasites can grow normally in erythrocytes that lack these endogenous agencies and in which the anion channels are not expressed, although their osmolyte permeability is present. We suggest that only the latter type of channel is important for normal development of the parasite.