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At least 19 recordsLinked to original sources
PubMed2026

Phosphoproteomic Analysis of GAP50-Deficient Parasites Reveals the Inner Membrane Complex Is Involved in Ion Regulation in Toxoplasma gondii.

The Alveolata group of organisms is characterized by a vesicular structure located beneath the plasma membrane. In apicomplexa, this structure is known as the inner membrane complex (IMC). The IMC acts as a scaffold during the budding of the daughter parasite and is utilized as a rigid base for the glideosome. In this study, we discovered that the phosphatase activity of GAP50 is crucial for its function in the biogenesis of the IMC. Through further phosphoproteomic analysis of parasites lacking GAP50, we identified that an NIPA family transporter is localized in the IMC. Knocking out TgNIPA1 significantly impairs the robust growth of the parasites. Additionally, expressing TgNIPA1 in a Salmonella strain lacking magnesium transporters could rescue the growth phenotype of bacteria under low magnesium conditions, indicating that it functions as an active magnesium transporter. Our results strongly suggest that the flattened vesicles of the IMC may play important roles in ion regulation in T. gondii.

Toxoplasma↗
PubMed2025

Function and interactions of a protein bridge between the inner membrane complex and subpellicular microtubules in Toxoplasma gondii.

Toxoplasma gondii is an intracellular parasite that utilizes peripheral membrane and cytoskeletal structures for essential functions such as host cell invasion and replication. These include the inner membrane complex (IMC) and the underlying longitudinal subpellicular microtubules (SPMT) that provide support for the IMC and give the parasite its distinctive crescent shape. Although the IMC and SPMTs have been studied separately, the mechanisms linking these adjacent structures remain largely unknown. This study identifies a protein named IMT1 that localizes to the maternal IMC and SPMTs and appears to tether the IMC to the microtubules. We disrupt the IMT1 gene to assess function and then use deletion analyses and mutagenesis to reveal regions of the protein that are necessary for binding to the IMC cytoskeleton or SPMTs. Using proximity labeling, we identify candidate IMT1 interactors in the IMC or SPMTs. Exploration of these candidates reveals that the loss of IMT1 results in a dramatic reduction of the microtubule-associated protein TLAP2 and that IMT1 binds directly to the cytoskeletal IMC proteins IMC1, IMC18, and IMC24. Together, these interactions reveal a novel bridge that connects two key cytoskeletal structures and provides new insight into the organization of the structural backbone of T. gondii.

Toxoplasma↗
PubMed1977

[Freeze fracture study of Toxoplasma and Sarcocystis infective stages (author's transl)].

Infective stages of Toxoplasma and Sarcocystis have been studied by the freeze fracture technique. The outer membrane of the pellicle is continuous and shows an apical 8 + 1 particles rosette in the P fracture face. The inner membrane complex is made of rectangular flattened vesicles aligned in longitudinal rows and joined in a puzzle like fashion. Sarcocystis has 11 of these rows whereas their number varies in Toxoplasma. A posterior interruption is present. Anteriorly is one truncated conical plate apically opened by a vertical ridge. The membranes of the inner complex are characterized by parallel alignment of particles (in P faces) some of which are joined and continuous with double rows radiating in the apical cap. Those rows correspond in number and arrangement with the underlying microtubules. The rhoptries membranes show periodic circular arrays of particles.

Animals↗
PubMed1978

14-3-2 protein in rat brain.

The distribution of the 14-3-2 protein in rat brain was investigated by immuno-electron microscopy using antiserum to the protein conjugated with peroxidase. 14-3-2 was demonstrated in the nuclear membrane, the endoplasmic reticular membranes and in the plasma membrane of nerve cells. The protein was also localized to the presynaptic densities and to the pre- and postsynaptic membranes. It could not be demonstrated in the membranes of the Golgi complex, inner membrane of mitochondria or in the nucleoplasm of neurons. No 14-3-2 protein was found in astrocytes, oligodendrocytes or in non-neuroectodermal tissue elements.

Animals↗
PubMed1979

Ultrastructural study of schizogony of Eimeria bovis in cell cultures.

First-generation schizogony of Eimeria bovis in bovine cell culture was studied by electron microscopy. The intracellular sporozoite retained its structure for at least 6 days at which time it rounded up and lost its apical complex. Although the refractile body underwent certain morphologic changes, it was retained throughout the parasite's growth. The beginning of mitosis was marked by the formation of a cytoplasmic funnel which traversed the nucleus opening on each side toward a pair of centrioles. Subsequently, there developed an intranuclear spindle. Separation of the daughter nuclei was preceded by the formation of typical centrocones. Differentiation of merozoites was accomplished by exogenesis during the last mitotoc division. A dense fiber, interpreted as a link connecting the merozoite anlage with its nucleus, extended from the developing apical complex to the nearest division pole. In the anlage, the inner membrane complex was at first composed of patches associated with pairs of subpellicular microtubules. Rhoptries appeared early in merogenesis, whereas micronemes formed at the time the merozoites detached from the residuum. The level of amylopectin, low in schizonts, rose at the beginning of merozoite formation.

Animals↗
PubMed1975

The ultrastructure of macrogametes of Eimeria ferrisi Levine and Ivens 1965 in Mus musculus.

Macrogametes of Eimeria ferrisi occurred in epithelial cells of the cecum and colon of Mus musculus and were studied by electron microscopy. Young stages were identified as macrogamonts by the presence of wall-forming bodies. At first an outerlimiting membrane and remnants of the inner membrane complex of the former merozoite pellicle were present; the latter was later lost but in mature macrogametes 3 limiting membranes were observed. Type II wall-forming bodies appeared before type I; the former developed in expanded cisternae of the endoplasmic reticulum whereas the latter were smaller in size and appeared in the ground substance of the cytoplasm. After formation of the oocyst wall the bodies of the 2 types were no longer visible. The presenceodies of the 2 types were no longer visible. The persistence of micronemes in mature macrogametes and the presence of numerous layers of rough endoplasmic reticulum during wall formation have not been previously reported.

Animals↗
PubMed1978

Evidence for the specific association of the chromosomal origin with outer membrane fractions isolated from Escherichia coli.

DNA-envelope complexes isolated from osmotically lysed spheroplasts of Escherichia coli contained 0.2 to 1% of the total cellular DNA after labeling with [3H]thymidine. Molecular weight determinations indicated that the amount of bound DNA was equivalent in most cases to a maximum of three binding sites per chromosome. Bound DNA from E. coli B/r was distributed approximately equally between inner and outer membrane components when envelopes were fractionated on sucrose equilibrium gradients. Outer membrane-DNA complexes, in particular, fraction H1, with a density of 1.24 g/cm3, were quite stable against shearing and against Sarkosyl NL97. In the case of E. coli B/r, H1-DNA was also relatively resistant to deoxyribonuclease. Inner membrane-DNA complexes, in contrast, were quite labile and readily dissociated to release free DNA. The outer membrane fractions did not appear to contain replication fork DNA, but small amounts may have been present in the inner membrane complexes. A two- to eightfold enrichment for chromosomal origin DNA in the envelope was obtained when cultures of E. coli K-12, synchronized for DNA replication, were pulse labeled at different times in the replication cycle. This enrichment was found invariably in the outer membrane fractions. However, the data do not exclude the possibility that this DNA is bound to regions of adhesion between inner and outer membranes which sediment with a density indistinguishable from that of the outer membrane.

Binding Sites↗
PubMed1979

Labeling of cytochrome c oxidase with [35S]diazobenzenesulfonate. Orientation of this electron transfer complex in the inner mitochondrial membrane.

Isolated cytochrome c oxidase was fractionated by native-gel electrophoresis in Triton X-100, and a preparation of enzyme almost completely free of the usual impurities was recovered. This fraction was used to generate antibodies specific to cytochrome c oxidase. These antibodies inhibited cytochrome c oxidase activity rapidly and completely and immunoprecipitated an enzyme containing seven different subunits from detergent-solubilized mitochondria or submitochondrial particles. Reaction of detergent-solubilized cytochrome c oxidase with [35S]diazobenzenesulfonate labeled all seven subunits although I and VI were much less reactive than the other five components. When cytochrome c oxidase was immunoprecipitated from mitochondria which had been reacted with [35S]DABS, subunits II and III were the only components labeled. When the complex was immunoprecipitated from labeled submitochondrial particles, II, III, IV, V, and VII were all labeled. Polypeptides I and VI were not labeled from either side of the membrane. These results confirm earlier studies which showed that cytochrome c oxidase spans the mitochondrial inner membrane and is asymmetrically arranged across this permeability barrier.

Animals↗
PubMed1977

The development of oxidative enzymes in rat liver mitochondria.

During early postnatal development there was an increase in the specific activity of a number of oxidative enzymes localized on the outer and inner mitochondrial membrane. The succinic oxidase complex of the inner mitochondrial membrane, whose activity in 1-day-old rats was 50% of the value in adult animals, attained the maximum on about the 10th day after birth. Activity of the choline and the proline oxidase complex, both of which are also localized in the inner mitochondrial membrane, was minimal in 1-day-old rats and went on rising after the 10th day. Rotenone-insensitive NADH-cytochrome c reductase activity, which is localized on the outer mitochondrial membrane, remained stable up to the 10th day, and rose between the 10th and the 90th day. Developmental changes in monoaminooxidase activity, which is likewise localized on the outer mitochondrial membrane, followed a similar course to the choline and proline oxidase complexes. The amount of cytochromes a+alpha3 and cytochrome b in isolated mitochondria did not alter during development. The protein spectrum of the mitochondrial particles, determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate, likewise displayed no marked changes during postnatal development. The above findings show that the metabolic functions of the mitochondria mature during development and that changes in the different enzymes have their own characteristic time course.

Animals↗
PubMed1978

The mitochondrial localization of coproporphyrinogen III oxidase.

The location of coproporphyrinogen III oxidase in mitochondria was studied in rat liver by using the digitonin method or hypo-osmotic media for fractionation. The enzyme was found in the intermembrane space with a fraction loosely bound to the inner membrane. This fraction was released by washing the inner-membrane-matrix complex with alkaline solutions or solutions of high ionic strength. The enzyme in both fractions had the same Km (0.16 micrometer) for coproporphyrinogen III. When incubation was performed in a medium that avoided destruction of enzyme membrane binding, a dramatic increase in activity was observed after sonication of whole mitochondria or of the inner-membrane-matrix complex.

Animals↗
PubMed1978

Biosynthesis of mitochondrial membrane proteins: co-ordination with special reference to cytochrome c oxidase.

This paper reviews mechanisms by which the rate of synthesis of subunits of mitochondrial inner membrane protein complexes and the assembly of these subunits are co-ordinated. Current models are evaluated and critically discussed in the light of some recent evidences. The focus is on the incorporation of cytoplasmically-synthesized cytochrome c oxidase subunits in the development of a newer model, which introduces some twists into a combination of several current ideas. A mechanism which governs both organized assembly and the co-ordination of rates of polypeptide synthesis is illustrated and the principles of the model are applied to the elucidation of some odd features of certain mutants. The possibilities that mitochondrial ATPase and cytochrome c reductase may also be synthesized and assembled according to this model are discussed.

Cytoplasm↗
PubMed1979

[Fine-structure changes in Toxoplasma gondii trophozoites after deep-freezing with dimethyl sulphoxide (author's transl)].

The changes observed in trophozoites of Toxoplasma gondii after deep-freeze preservation were examined by electron microscopy. Toxoplasmas (strain BK) from peritoneal exudate of infected NMRI mice were supended in Ringer's solution, deep-frozen in liquid nitrogen with 5% dimethylsulphoxide (DMSO), and compared after thawing with control samples with and without the addition of DMSO. Slight structural changes such as widening of endoplasmic reticulum, formation of fissures in the cytoplasm, and loosening of chromatin were only observed in some of the free toxoplasmas of the DMSO control. Among the deep-frozen parasites, about 1/5 of the free stages showed no or only slight morphological changes. In contrast to this, almost all intracellular forms found in macrophages showed lesions. The most remarkable change was a partial destruction of the inner cell membrane complex. The outflow of ribosome-containing protoplasm with ballon-like swelling of the outer elementary membrane was observed as a consequence of this frequent lesion. The outflow of protoplasm induced a drastic decrease in the electronic density of the whole cytoplasm. Other characteristic degenerative signs were vacuolation of cytoplasm up to formation of great optically empty spaces, widening of the perinuclear space, swelling of mitochondria, disintegration of rhoptria, micronemata, and Golgi zone, coarse-plaque loosening, and displacement of electron-dense areas of the nucleus up to disintegration with maintenance of the karyoplasm. In some almost completely disintegrated trophozoites, enlarged mitochondria with remarkable electronic density were observed. Apart from the cell membrane, the conoid was the longest-persisting organelle. The alterations observed after deep-freezing permit the conclusion that the free cells, which were only slightly impaired or not at all, remained infective.

Animals↗
PubMed1979

Labeling of complex III, with [35S]diazobenzenesulfonate: orientation of this electron transfer segment in the mitochondrial inner membrane.

[34S]Diazobenzenesulfonate has been used to tag the surface-exposed polypeptides of isolated complex III. All nine different component polypeptides were labeled, indicating that each is at least partially exposed on the surface of the isolated, detergent-dispersed complex. Labeling studies were also conducted on the membrane-bound complex. Preparations of intact mitochondria and submitochondrial particles were separately labeled with [35S]diazobenzenesulfonate in order to determine the distribution of the polypeptides of complex III between the outer (cytoplasmic) and inner (matrix) surfaces of the mitochondrial inner membrane, respectively. Polypeptides II and III were the only components labeled in a significant amount in submitochondrial particles (i.e., from the matrix side). Polypeptides III, IV, and VI were heavily labeled in mitochondria (i.e., from the cytoplasmic side). Polypeptides I,II, V, and VII were also labeled in mitochondria but to a much lesser extent. Polypeptides VIII and IX were not significantly labeled from either side of the membrane. The labeling data and information obtained from previous crosslinking studies [Smith, R.J. & Capaldi, R.A. (1977) Biochemistry 16, 2629-2633] are used to derive a picture of the arrangement of complex III in the mitochondrial inner membrane.

Animals↗
PubMed1977

[Characterization of the membrane attached to the folded chromosome isolated from Escherichia coli].

Phospholipid analysis of the membranes associated with fast sedimenting folded chromosomes prepared by lysis of E. coli CR 34 shows that both inner and outer membranes are parts of the complex, in proportions not very different from that found in the whole bacteria. During the preparation of the folded chromosomes, the most recently synthesized molecules of phosphatidylglycerol and phosphatidylethanoamine are more sensitive to solubilisation, particularly those from the cytoplasmic membrane. Identification of a dominant fraction, the outer membrane, in some complexes, results from a preferential solubilization of the inner membrane. These results do not favor any specific association between the folded chromosome and the membranes.

Cell Fractionation↗
PubMed1977

The character of protein-nucleic interaction in relation to the mtDNA-membrane complex.

Specific sites that interact with structural proteins of the mitochondrial inner membrane were found in mitochondrial DNA (mtDNA) of rat liver. Analysis of the isolated DNA fragments revealed their capacity to form a complex with membrane proteins in vitro and allowed the detection of a protein with a molecular weight 40,000. The size of the fragments was found to be 12-18 nucleotide pairs with an average molecular weight 10,000 MtDNA sites recognized by membrane protein proved to be quite unique in having a secondary structure, a high content of AT sequences (82%) and oligopyrimidine blocks. It was shown that the light mtDNA strand, rich in adenine, is 60% more active in the binding with membrane mitochondria than the heavy one.

Binding Sites↗
PubMed1975

Iron-sulfur components of succinate dehydrogenase: stoichiometry and kinetic behavior in activated preparations.

Extensively or completely activated preparations of beef heart succinate dehydrogenase have been investigated by electron paramagnetic resonance (EPR) techniques at 6 to 97 K. Reductive titrations with dithionite and rapid kinetic studies were performed with various types of soluble and membrane-bound preparations of the enzyme. The following components were detected and their behavior analyzed: a free radical, presumably arising from the covalently bound flavin on reduction, two iron-sulfur centers of the ferredoxin type, the signals of which appear on reduction, and a highpotential iron-sulfur component, detectable in the oxidized state. The high-potential component was only detected in complex II and inner-membrane preparations. This component and one of the ferredoxin-type centers were present in amounts close to stoichiometric with the flavin and were reduced by substrate. The other ferredoxin-type center was present in amounts between 0.1 and 0.5 times that of the flavin and was reduced only by dithionite. Of the components reduced by succinate, however, only a fraction (up to 50% of the high-potential iron-sulfur center and 40-60% of the ferredoxin-type iron-sulfur center) was reduced within the turnover time of the enzymes; In complex II not more than about 10% of the flavin appeared in the semiquinone form at any time. Soluble, purified preparations behaved similarly except that the high-potential component was nearly or completely absent and extensive accumulation of the free radical occurred (up to 70 to 80% of the flavin) in titration and kinetic experiments. No significant difference was observed between the rates of semiquinone formation and the reduction of the ferredoxin-type or high-potential centers by the substrate. Also no qualitative differences in the properties studied in this work became apparent between prepatations containing 4 or 8 iron atoms, respectively.

Animals↗