Adenylate Translocator Research Articles

Duchenne muscular dystrophy is associated with the inhibition of calcium uniport in mitochondria and an increased sensitivity of the organelles to the calcium-induced permeability transition

Duchenne muscular dystrophy (DMD) is characterized by a pronounced and progressive degradation of the structure of skeletal muscles, which decreases their strength and lowers endurance of the organism. At muscular dystrophy, mitochondria are known to undergo significant functional changes, which is manifested in a decreased efficiency of oxidative phosphorylation and impaired energy metabolism of the cell. It is believed that the DMD-induced functional changes of mitochondria are mainly associated with the dysregulation of Ca2+ homeostasis. This work examines the kinetic parameters of Ca2+ transport and the opening of the Ca2+-dependent MPT pore in the skeletal-muscle mitochondria of the dystrophin-deficient C57BL/10ScSn-mdx mice. As compared to the organelles of wild-type animals, skeletal-muscle mitochondria of mdx mice have been found to be much less efficient in respect to Ca2+ uniport, with the kinetics of Na+-dependent Ca2+ efflux not changing. The data obtained indicate that the decreased rate of Ca2+ uniport in the mitochondria of mdx mice may be associated with the increased level of the dominant negative subunit of Ca2+ uniporter (MCUb). The experiments have also shown that in mdx mice, skeletal-muscle mitochondria have low resistance to the induction of MPT, which may be related to a significantly increased expression of adenylate translocator (ANT2), a possible structural element of the MPT pore. The paper discusses how changes in the expression of calcium uniporter and putative components of the MPT pore caused by the development of DMD can affect Ca2+ homeostasis of skeletal-muscle mitochondria.

Identification and Genetic Analysis of a Novel Allelic Variation of Brittle-1 with Endosperm Mutant in Maize

Endosperm mutants are critical to the studies on both starch synthesis and metabolism and genetic improvement of starch quality in maize. In the present study, a novel maize endosperm mutant A0178 of natural variation was used as the experimental material and identified and then characterized. Through phenotypic identification, genetic analysis, main ingredients measurement and embryo rescue, development of genetic mapping population from A0178, the endosperm mutant gene was located. The results showed that the mutant exhibited extremely low germination ability as attributed to the inhibited embryo development, and amounts of sugars were accumulated in the mutant seeds and more sugars content was detected at 23 days after pollination (DAP) in A0178 than B73. Employing genetic linkage analysis, the mutant trait was mapped in the bin 5.04 on chromosome 5. Sequence analysis showed that two sites of base transversion and insertion presented in the protein coding region and non-coding region of the mutant brittle-1 (bt1), the adenylate translocator encoding gene involved in the starch synthesis. The single base insertion in the coding region cause frameshift mutation, early termination and lose of function of Brittle-1 (BT1). All results suggested that bt1 is a novel allelic gene and the causal gene of this endosperm mutant, providing insights on the mechanism of endosperm formation in maize.

Simultaneous boosting of source and sink capacities doubles tuber starch yield of potato plants

An important goal in biotechnological research is to improve the yield of crop plants. Here, we genetically modified simultaneously source and sink capacities in potato (Solanum tuberosum cv. Desirée) plants to improve starch yield. Source capacity was increased by mesophyll-specific overexpression of a pyrophosphatase or, alternatively, by antisense expression of the ADP-glucose pyrophosphorylase in leaves. Both approaches make use of re-routing photoassimilates to sink organs at the expense of leaf starch accumulation. Simultaneous increase in sink capacity was accomplished by overexpression of two plastidic metabolite translocators, that is, a glucose 6-phosphate/phosphate translocator and an adenylate translocator in tubers. Employing such a 'pull' approach, we have previously shown that potato starch content and yield can be increased when sink strength is elevated. In the current biotechnological approach, we successfully enhanced source and sink capacities by a combination of 'pull' and 'push' approaches using two different attempts. A doubling in tuber starch yield was achieved. This successful approach might be transferable to other crop plants in the future.

α-Synuclein overexpression impairs mitochondrial function by associating with adenylate translocator

α-Synuclein (α-syn), a protein involved in the pathogenesis of Parkinson's disease (PD), is known to accumulate in mitochondria, disrupt mitochondrial function. However, the molecular mechanisms that link these pathological responses have not been investigated. In rats overexpressing α-syn in the substantia nigra (SN) through adeno-associated virus (AAV) transduction, about 50% of tyrosine hydroxylase positive neurons were lost after 24 weeks. Overexpression of α-syn was also associated with morphological deformation of mitochondria and depolarization of the mitochondrial membrane potential (ΔΨm). Both co-immunoprecipitation and confocal microscopy demonstrated that mitochondrial α-syn associated with adenylate translocator (ANT), a component of the mitochondrial permeability transition pore (mPTP). The depolarization of ΔΨm was partially reversed in vitro by bongkrekic acid (BKA), an inhibitor of ANT, suggesting that the molecular association between α-syn and ANT facilitated ΔΨm depolarization. Concomitant with α-syn accumulation in mitochondria, abnormal mitochondrial morphology, ΔΨm depolarization, and loss of TH-positive neurons, there was a decrease in apoptosis-inducing factor (AIF) within the mitochondrial matrix, suggesting possible translocation to the cytosol. Our findings suggest that overexpression of α-syn may cause mitochondrial defects in dopaminergic neurons of the substantia nigra through an association with adenylate translocator and activation of mitochondria-dependent cell death pathways. Disruption of normal mitochondrial function may contribute to the loss of dopaminergic neurons in Parkinson's disease.

Elucidation of maize endosperm starch granule channel proteins and evidence for plastoskeletal structures in maize endosperm amyloplasts

Previously, starch granules of maize ( Zea mays), sorghum ( Sorghum bicolor), and millet ( Pennisetum americanum) were found to contain channels connecting the interior cavity with the outside surface, and the channels of maize starch channels were found to contain, presumably to be lined with, proteins. One objective of this study was to identify and characterize channel proteins of maize starch granules. A putative starch granule channel protein extract was subjected to 2D-PAGE. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used to identify the proteins. Data analysis indicated the presence of actin-like and tubulin-like (FtsZ) proteins, ADP-glucose pyrophosphorylase large and small subunits (Shrunken2 [Sh2] and Brittle2 [Bt2], respectively), granule-bound starch synthase (GBSS), and Brittle1 (Bt1, an adenylate translocator). A combination of Western blot analysis and immunolocalization confirmed the presence of an actin-like protein within the starch granule structure. Another objective was to determine the origin of maize starch granule channels. TEM examination of maize endosperm amyloplasts that did not contain a formed starch granule revealed structures that were connected to the amyloplast membrane and extended inward to the center of the plastid.

Overriding the co‐limiting import of carbon and energy into tuber amyloplasts increases the starch content and yield of transgenic potato plants

Transgenic potato (Solanum tuberosum) plants simultaneously over-expressing a pea (Pisum sativum) glucose-6-phosphate/phosphate translocator (GPT) and an Arabidopsis thaliana adenylate translocator (NTT1) in tubers were generated. Double transformants exhibited an enhanced tuber yield of up to 19%, concomitant with an additional increased starch content of up to 28%, compared with control plants. The total starch content produced in tubers per plant was calculated to be increased by up to 44% in double transformants relative to the wild-type. Single over-expression of either gene had no effect on tuber starch content or tuber yield, suggesting that starch formation within amyloplasts is co-limited by the import of energy and the supply of carbon skeletons. As total adenosine diphosphate-glucose pyrophosphorylase and starch synthase activities remained unchanged in double transformants relative to the wild-type, they cannot account for the increased starch content found in tubers of double transformants. Rather, an optimized supply of amyloplasts with adenosine triphosphate and glucose-6-phosphate seems to favour increased starch synthesis, resulting in plants with increased starch content and yield of tubers.

Metabolic control analysis and regulation of the conversion of sucrose to starch in growing potato tubers

ABSTRACTStarch is of great importance both as a carbon storage reserve in plants and as a biotechnologically important product. The potato tuber is an attractive model system for the study of starch metabolism, because it is a relatively homogenous tissue in which conversion of sucrose to starch represents the dominant metabolic flux. All the major genes of the potato tuber sucrose to starch pathway have been cloned in recent years, allowing the generation of a suite of antisense transgenic lines to be produced in which the activity of each individual enzyme in the pathway is progressively decreased. Investigations of these plants have provided a complete picture of the distribution of control in this important pathway. Sucrose synthase, UGPase, hexokinase, cytosolic phosphoglucomutase, plastidial phosphoglucomutase, the amyloplastidial adenylate translocator, AGPase, starch synthase and starch branching enzyme have flux control coefficients (FCCs) of 0.10, approximating 0.00, approximating 0.00, 0.15, 0.23, 0.98, 0.35, 0.12 and approximating 0.00 for starch accumulation. These results show that the majority of the control on starch accumulation in potato tubers resides in the transfer of adenylate between the cytosol and the amyloplast, with a minor contribution being made by the first two steps of the plastidial starch synthesis pathway (the reactions catalysed by plastidial phosphoglucomutase and AGPase). This contrasts with leaves, in which the majority of the control has been found to reside in the reactions catalysed by plastidial phosphoglucomutase and AGPase. In leaves, ATP for starch synthesis is generated within the plastid via photophosphorylation. Several studies have attempted to increase the rate of starch synthesis by overexpressing pathway enzymes in tubers. The results of these studies and the role of other ATP producers in the starch synthetic process are reviewed. In the same time period methods of non‐aqueous fractionation have been adapted to potato tuber tissue in order to ascertain subcellular metabolite levels. Results obtained from these studies allow the calculation of mass action ratios of the constitutive enzymes of the sucrose to starch transition. When taken together with the known regulatory properties of these enzymes the combination of broad control analysis studies and assessment of the mass action ratios of the respective enzymes allows a comprehensive description of this important metabolic network. Some illustrative examples of how this network responds to environmental change are presented. Finally implications of this whole pathway evaluation for more general studies of plant metabolic pathways and networks are discussed.

C-terminal lysines determine phospholipid interaction of sarcomeric mitochondrial creatine kinase.

High affinity interaction between octameric mitochondrial creatine kinase (MtCK) and the phospholipid cardiolipin in the inner mitochondrial membrane plays an important role in metabolite channeling between MtCK and inner membrane adenylate translocator, which itself is tightly bound to cardiolipin. Three C-terminal basic residues revealed as putative cardiolipin anchors in the x-ray structures of MtCK and corresponding to lysines in human sarcomeric MtCK (sMtCK) were exchanged by in vitro mutagenesis (K369A/E, K379Q/A/E, K380Q/A/E) to yield double and triple mutants. sMtCK proteins were bacterially expressed, purified to homogeneity, and verified for structural integrity by enzymatic activity, gel filtration chromatography, and CD spectroscopy. Interaction with cardiolipin and other acidic phospholipids was quantitatively analyzed by light scattering, surface plasmon resonance, and fluorescence spectroscopy. All mutant sMtCKs showed a strong decrease in vesicle cross-linking, membrane affinity, binding capacity, membrane ordering capability, and binding-induced changes in protein structure as compared with wild type. These effects did not depend on the nature of the replacing amino acid but on the number of exchanged lysines. They were moderate for Lys-379/Lys-380 double mutants but pronounced for triple mutants, with a 30-fold lower membrane affinity and an entire lack of alterations in protein structure compared with wild-type sMtCK. However, even triple mutants partially maintained an increased order of cardiolipin-containing membranes. Thus, the three C-terminal lysines determine high affinity sMtCK/cardiolipin interaction and its effects on MtCK structure, whereas low level binding and some effect on membrane fluidity depend on other structural components. These results are discussed in regard to MtCK microcompartments and evolution.

The frequent classes of expressed genes in wheat endosperm tissue as possible sources of genetic markers

Frequently expressed genes in wheat endosperm tissue provide a potentially useful source of genetic markers because genes that are relatively highly expressed in seed development may provide new candidate genes contributing to quality traits in wheat. Wheat endosperm tissue at 8–12 days post anthesis (DPA) was characterised with respect to the accumulation of the major classes of seed storage proteins. The accumulation of these proteins characterises the wheat endosperm and provides a framework within which the frequencies of different expressed sequence tags (ESTs) could be categorised. Major seed storage protein classes such as glutenins and gliadins comprised approximately 40% of the proteins in 8–12 DPA endosperm tissue, and the levels of transcripts from the respective genes provided a benchmark for assessing the frequency of transcripts from other genes. Based on the analysis of 4374 sequences, 2 categories were defined: frequent class, comprising ESTs occurring at a frequency of >4 copies, and a second category containing ESTs occurring at a frequency of <3. Comparison of frequent class sequences of wheat with sequences from a corn library of an equivalent category identified genes apparently characteristic of wheat and others that were conserved during endosperm development. A significant new class of seed storage protein genes present in wheat but absent from corn, and related to avenin-3 of oats, was identified. Other prominent wheat ESTs, including a glycine-rich cell wall protein, End-1 (originally identified in early barley endosperm), Brittle-1 (corn, possible adenylate translocator), and an endosperm specific protein (EST expressed specifically in endosperm), were mapped in wheat using single nucleotide polymorphisms. The analysis demonstrated the feasibility of identifying new genes by large-scale sequencing and targetting these as possible markers for quality attributes.

Transport in and out of plastids: does the outer envelope membrane control the flow?

This recent work on OEPs is undoubtedly a significant contribution to our changing view of the properties of plastidic porins. However, electrophysiological measurements of the properties of the different OEPs only describe the potential of these channels to regulate solute flow through the outer envelope membrane and do not tell us about the situation in vivo. Is OEP21 actually regulated by the ATP:TPT-substrates ratio, allowing the efflux of triose phosphates under photosynthetic conditions? If on the one hand, the outer envelope membrane is unspecifically permeable to solutes as previously thought, then the metabolite status in the intermembrane space should reflect that of the cytosol. On the other hand, assuming that the outer envelope membrane can act as a permeability barrier for solutes, the metabolic situation in the intermembrane space should reflect that in the stroma rather than that in the cytosol, although the actual metabolite concentrations in the intermembrane space are unknown. Because the TPT mediates a strict 1:1 exchange of metabolites, the sum of the TPT-substrates in both the cytosol and the stroma is kept almost constant. Under photosynthetic conditions, ATP levels are approximately doubled in the stroma, leading to a higher ATP:TPT-substrates ratio in light compared with dark conditions. On the one hand, this should lead to an inward rectification of OEP21 channels and thus to a restriction of triose phosphate export. On the other hand, the export of triose phosphates has to be balanced by the import of inorganic phosphate (which is used for ATP production). Consequently, a restriction of phosphate import by outwardly rectifying OEP21 channels would lead to phosphate-depletion of chloroplasts and inhibition of photosynthesis13xRole of orthophosphate and other factors in the regulation of starch formation in leaves and isolated chloroplasts. Heldt, H.W. et al. Plant Physiol. 1977; 59: 1146–1155Crossref | PubMedSee all References13. On this basis it would appear unlikely that the rectifying properties of OEP21 are modified by the ATP:TPT-substrates ratio in vivo.In conclusion, the potential for the regulation of metabolite fluxes is clearly demonstrated for OEP21, although their contribution to flux regulation in vivo, particularly during transition from light to dark remains speculative. In view of the fact that several OEP channels present in the outer envelope membrane have overlapping and broad specificities (and there are probably more OEP channels than those identified to date), one wonders whether the permeation of a single solute across the outer membrane can be so restricted. It has been shown that the regulation of channel opening and closing could be achieved by voltage-gating. All three OEPs have the highest open probability at membrane potentials about zero and respond to higher potentials by reducing their open probabilities. However, the existence of a potential across the outer membrane is rather unlikely (except for a Donnan potential) thus the observed voltage gating might not be of physiological importance. The regulation of porin function could also be exerted by physiologically relevant factors (i.e. solutes or interacting proteins). Such interactions have been observed for bacterial and mitochondrial porins (e.g. the interaction of the mitochondrial VDAC with kinases and the inner membrane adenylate translocator14xComplexes between porin, hexokinase, mitochondrial creatine kinase and adenylate translocator display properties of the permeability transition pore. Implication for regulation of the permeability transition by the kinases. Beutner, G. et al. Biochim. Biophys. Acta. 1998; 1368: 7–18Crossref | PubMed | Scopus (249)See all References14), but are as yet poorly understood and are currently under investigation.What can be done to assess the control that the outer envelope membrane might exert on metabolic fluxes between chloroplasts and the cytosol, and to bridge the gap between the described biophysical properties of OEPs and their actual role in plant metabolism? One approach could be to identify the putative interacting factors of the OEPs and to analyse whether complexes formed between these factors and the OEPs can regulate permeability properties of individual OEPs. Another approach would be to identify OEP-homologs in Arabidopsis and to screen and characterize knockout-mutants. If the loss of gene function cannot be substituted by other OEPs (or is only partially substituted for), then these mutants should show alterations in plant metabolism (e.g. a reduced export of triose phosphates should result in a perturbation of starch and sucrose metabolism). However, it is more likely that only the simultaneous elimination of several OEPs will lead to clear-cut phenotypes. This could then be the basis to elucidate the regulation that a particular OEP-channel might exert on solute transport across the outer envelope membrane at the biochemical and physiological levels.

Brittle-1, an adenylate translocator, facilitates transfer of extraplastidial synthesized ADP--glucose into amyloplasts of maize endosperms.

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.

Metabolite transporters in plastids

Communication between plastids and the surrounding cytosol occurs via the plastidic envelope membrane. Recent findings show that the outer membrane is not as freely permeable to low molecular weight solutes as previously thought, but contains different channel-like proteins that act as selectivity filters. The inner envelope membrane contains a variety of metabolite transporters that mediate the exchange of metabolites between both compartments. Two new classes of phosphate antiporters were recently described that are different in structure and function from the known triose phosphate/phosphate translocator from chloroplasts. In addition, a cDNA coding for an ATP/ADP antiporter from plastids was isolated that shows similarities to a bacterial adenylate translocator.

The molecular structure of mitochondrial contact sites. Their role in regulation of energy metabolism and permeability transition.

Contact sites between the outer and peripheral inner membrane of mitochondria are involved in protein precursor uptake and energy transfer. Hexokinase and mitochondrial creatine kinase could be attributed by different techniques to the energy transfer contacts. Kinetic analyses suggested a functional interaction between the kinases, outer membrane pore protein, and inner membrane adenylate translocator (ANT). This suggestion was strongly supported by isolation of hexokinase and creatine kinase complexes that were constituted of kinase oligomers, porin and ANT. Phospholipid vesicles carrying reconstituted kinase-porin-ANT complexes enclosed internal ATP in contrast to vesicles containing free porin only. This indicated that unspecific transport through porin was regulated by its interaction with a specific antiporter, ANT. A direct interaction between porin and ANT in the hexokinase complex conferred the reconstituted system with permeability properties reminiscent of the mitochondrial permeability transition (PT) pore. In the creatine kinase complex this interaction between porin and ANT was replaced by contact of both with the kinase octamer. Thus PT-pore-like functions were not observed unless the creatine kinase octamer was dissociated, suggesting that the ANT was locked in the antiporter state by interaction with the octamer. Indeed, reconstituted pure ANT showed PT-pore-like properties concerning Ca2+ sensitivity. However, as cyclophilin was missing, sensitivity against cyclosporin was not observed.

Complexes between porin, hexokinase, mitochondrial creatine kinase and adenylate translocator display properties of the permeability transition pore. Implication for regulation of permeability transition by the kinases

Complexes between hexokinase, outer membrane porin, and the adenylate translocator (ANT) were recently found to establish properties of the mitochondrial permeability transition pore in a reconstituted system. The complex was extracted by 0.5% Triton X-100 from rat brain membranes and separated by anion exchanger chromatography. The molecular weight was approximately 400 kDa suggesting tetramers of hexokinase (monomer 100 kDa). By the same method a porin, creatine kinase octamer, ANT complex was isolated and reconstituted in liposomes. Vesicles containing the reconstituted complexes both retained ATP that could be used by either kinase to phosphorylate external creatine or glucose. Atractyloside inhibited this activity indicating that the ANT was involved in this process and was functionally reconstituted [1]. Exclusively from the hexokinase complex containing liposome internal malate or ATP was released by addition of Ca 2+ in a N-methylVal-4-cyclosporin sensitive way, suggesting that the hexokinase porin ANT complex might include the permeability transition pore (PTP). The Ca 2+ dependent opening of the PTP-like structure was inhibited by ADP (apparent I 50, 8 μM) and ATP (apparent I 50, 84 μM). Also glucose inhibited the PTP-like activity, while glucose-6-phosphate abolished this effect. Although porin and ANT were functionally active in vesicles containing the creatine kinase octamer complex, Ca 2+ did not induce a release of internal substrates. However, after dissociation of the creatine kinase octamer, the complex exhibited PTP-like properties and the vesicles liberated internal metabolites upon addition of Ca 2+. The latter process was also inhibited by N-methylVal-4-cyclosporin. The activity of peptidyl-prolyl- cis- trans-isomerase (representing cyclophilin) was followed during complex isolation. Cyp D was co-purified with the hexokinase complex, while it was absent in the creatine kinase complex. The inhibitory effect of N-methylVal-4-cyclosporin on the creatine kinase complex may be explained by direct interaction with the creatine kinase dimer that appeared to support octamer formation.

BT1, a possible adenylate translocator, is developmentally expressed in maize endosperm but not detected in starchy tissues from several other species

BT1, a possible adenylate translocator, is developmentally expressed in maize endosperm but not detected in starchy tissues from several other species

BT1, a possible adenylate translocator, is developmentally expressed in maize endosperm but not detected in starchy tissues from several other species

Brittle 1 protein (BT1), the major protein in amyloplast envelope membranes of wild‐type maize endosperm, is required for normal levels of starch accumulation in maize kernels. To gain information about the Bt1 locus in control of starch synthesis in plants, we examined Bt1 expression in kernels from different ages and from different genotypes of maize as well as in starchy tissues from different species. Both BT1 protein and Bt1 transcripts were detected in kernels from about 10 days post‐pollination (DPP) and increased up to 14 DPP. BT1 protein level was similar in kernels after 14 DPP. Furthermore, membranes from amyloplasts containing larger starch granules contained more BT1 protein than those containing smaller starch granules from the same age of kernels. These results indicate that Bt1 gene expression was developmentally regulated and that BT1 protein level was correlated with starch accumulation in normal maize endosperm. Unexpectedly, except for a weak band at a higher molecular mass in microsomal proteins extracted from spring barley grain which reacted with maize BT1 antibody, no BT1 protein was observed in starchy tissues from several other species, including grains of sorghum, wheat, winter barley and oats, potato tubers, pea roots and cauliflower buds. Thus either the BT1 antibody raised against a fusion protein including the 56 amino acid residues in the carboxyl terminal sequence of maize endosperm BT1 protein does not recognize BT1 homologues in amyloplasts from other tissues or species or amyloplasts from different tissues or species have alternative membrane transporters.

Complexes between hexokinase, mitochondrial porin and adenylate translocator in brain: regulation of hexokinase, oxidative phosphorylation and permeability transition pore.

Complexes between hexokinase, mitochondrial porin and adenylate translocator in brain: regulation of hexokinase, oxidative phosphorylation and permeability transition pore.

Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore

In vitro incubation of isolated hexokinase isozyme I or isolated dimer of mitochondrial creatine kinase with the outer mitochondrial membrane pore led to high molecular weight complexes of enzyme oligomers. Similar complexes of hexokinase and mitochondrial creatine kinase could be extracted by 0.5% Triton X-100 from homogenates of rat brain. Hexokinase and creatine kinase complexes could be separated by subsequent chromatography on DEAE anion exchanger. The molecular weight, as determined by gel-permeation chromatography, was approximately 400 kDa for both complexes. The M r suggested tetramers of hexokinase (monomer 100 kDa) and creatine kinase (active enzyme is a dimer of 80 kDa). The composition of the complexes was further characterised by specific antibodies. Besides either hexokinase or creatine kinase molecules the complexes contained porin and adenylate translocator. It was possible to incorporate the complexes into artificial bilayer membranes and to measure conductance in 1 M KCl. The incorporating channels had a high conductance of 6 nS that was asymmetrically voltage dependent. The complexes were also reconstituted in phospholipid vesicles that were loaded with ATP. Complex containing vesicles retained ATP while vesicles reconstituted with pure porin were leaky. The internal ATP could be used by creatine kinase and hexokinase in the complex to phosphorylate external creatine or glucose. This process was inhibited by atractyloside. The hexokinase complex containing vesicles were furthermore loaded with malate or ATP that was gradually released by addition of Ca 2+ between 100 and 600 μM. The liberation of malate or ATP by Ca 2+ could be inhibited by N-methylVal-4-cyclosporin, suggesting that the porin translocator complex constitutes the permeability transition pore. The results show the physiological existence of kinase porin translocator complexes at the mitochondrial surface. It is assumed that such complexes between inner and outer membrane components are the molecular basis of contact sites observed by electron microscopy. Kinase complex formation may serve three regulatory functions, firstly regulation of the kinase activity, secondly stimulation of oxidative phosphorylation and thirdly regulation of the permeability transition pore.

Molecular characterization of an Arabidopsis thaliana cDNA encoding a novel putative adenylate translocator of higher plants

We have isolated an Arabidopsis thaliana cDNA encoding a highly hydrophobic membrane protein of 589 amino acids which contains 12 potential transmembrane helices and shows a high degree of similarity (43.5% identity, 66.2% similarity) to the ATP/ADP translocase of the Gram-negative bacterium Rickettsia prowazekii, an obligate intracellular parasite responsible for the epidemic typhus. This rickettsial translocator resides in the cytoplasmic membrane and allows the bacterium to exploit the host cytoplasmic ATP pool. We hypothesize that the A. haliana homolog of the R. prowazekii ATP/ADP translocase is the functional eukaryotic equivalent and resides in the plastid inner envelope membrane where it functions as an ATP importer.

Starch synthesis and carbohydrate oxidation in amyloplasts from developing wheat endosperm

The rates of incorporation of various metabolites into starch by isolated amyloplasts from developing endosperm of spring wheat (Triticum aestivum L. cv. Axona) were examined. Of the metabolites tested that were likely to be present in the cytosol at concentrations sufficient to sustain starch synthesis, only glucose 1-phosphate (Glc1P) supported physiologically relevant rates of starch synthesis. Incorporation of Glc1P into starch was both dependent on the presence of ATP and intact organelles. The rate of incorporation of hexose into starch became saturated at a Glc1P concentration of less than 1 mol·m-3 in the presence of 1 mol·m-3 ATP. Starch synthesis from 5 mol · m-3 ADP-glucose supplied to the organelles occurred at rates 15-fold higher than from similar concentrations of Glc1P, but it is argued that this is probably of little physiological relevance. The net incorporation of hexose units into starch from GlclP was inhibited 50% by 100 mmol.m-3 carboxyatractyloside. Carbohydrate oxidation in the amyloplast was stimulated by the addition of 2-oxoglutarate and glutamine, and in such circumstances incorporation of14C-labelled metabolites into starch was reduced. Glucose 6-phosphate proved to be a better substrate for oxidative pathways than Glc1P. Our results suggest that Glc1P is the primary substrate for starch synthesis in developing wheat endosperm, and that ATP required for starch synthesis is imported via an adenylate translocator.