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Coenzyme Q (CoQ, ubiquinone) is a central electron carrier in mitochondrial respiration. CoQ is synthesized through multiple steps involving a number of different enzymes. The prevailing view that the CoQ used in respiration exists as a free pool that diffuses throughout the mitochondrial inner membrane bilayer has recently been challenged. In the yeast Saccharomyces cerevisiae, deletion of the gene encoding Coq10p results in respiration deficiency without inhibiting the synthesis of CoQ, suggesting that the Coq10 protein is critical for the delivery of CoQ to the site(s) of respiration. The precise mechanism by which this is achieved remains unknown at present. We have identified a Plasmodium orthologue of Coq10 (PfCoq10), which is predominantly expressed in trophozoite-stage parasites, and localizes to the parasite mitochondrion. Expression of PfCoq10 in the S. cerevisiae coq10 deletion strain restored the capability of the yeast to grow on respiratory substrates, suggesting a remarkable functional conservation of this protein over a vast evolutionary distance, and despite a relatively low level of amino acid sequence identity. As the antimalarial drug atovaquone acts as a competitive inhibitor of CoQ, we assessed whether over-expression of PfCoq10 altered the atovaquone sensitivity in parasites and in yeast mitochondria, but found no alteration of its activity.

Malaria parasites replicating inside red blood cells (RBCs) export a large subset of proteins into the erythrocyte cytoplasm to facilitate parasite growth and survival. PTEX, the parasite-encoded translocon, mediates protein transport across the parasitophorous vacuolar membrane (PVM) in Plasmodium falciparum-infected erythrocytes. Proteins exported into the erythrocyte cytoplasm have been localized to membranous structures, such as Maurer's clefts, small vesicles, and a tubovesicular network. Comparable studies of protein trafficking in Plasmodium vivax-infected reticulocytes are limited. With Plasmodium yoelii-infected reticulocytes, we identified exported protein 2 (Exp2) in a proteomic screen of proteins putatively transported across the PVM. Immunofluorescence studies showed that P. yoelii Exp2 (PyExp2) was primarily localized to the PVM. Unexpectedly, PyExp2 was also associated with distinct, membrane-bound vesicles in the reticulocyte cytoplasm. This is in contrast to P. falciparum in mature RBCs, where P. falciparum Exp2 (PfExp2) is exclusively localized to the PVM. Two P. yoelii-exported proteins, PY04481 (encoded by a pyst-a gene) and PY06203 (PypAg-1), partially colocalized with these PyExp2-positive vesicles. Further analysis revealed that with P. yoelii, Plasmodium berghei, and P. falciparum, cytoplasmic Exp2-positive vesicles were primarily observed in CD71(+) reticulocytes versus mature RBCs. In transgenic P. yoelii 17X parasites, the association of hemagglutinin-tagged PyExp2 with the PVM and cytoplasmic vesicles was retained, but the pyexp2 gene was refractory to deletion. These data suggest that the localization of Exp2 in mouse and human RBCs can be influenced by the host cell environment. Exp2 may function at multiple points in the pathway by which parasites traffic proteins into and through the reticulocyte cytoplasm., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na(+) regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na(+) homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na(+) homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes.

Apicomplexan parasites enter host cells by many sophisticated steps including use of an ATP-powered invasion machinery. The machinery consists of multiple proteins, including a special myosin (MyoA) which moves along an actin fiber and which is connected to the myosin tail interaction protein (MTIP). Here we report a crystal structure of the major MyoA-binding domain (D3) of Plasmodium falciparum MTIP in complex with an anti-MTIP nanobody. In this complex, the MyoA-binding groove in MTIP-D3 is considerably less accessible than when occupied by the MyoA helix, due to a shift of two helices. The nanobody binds to an area slightly overlapping with the MyoA binding groove, covering a hydrophobic region next to the groove entrance. This provides a new avenue for arriving at compounds interfering with the invasion machinery since small molecules binding simultaneously to the nanobody binding site and the adjacent MyoA binding groove would prevent MyoA binding by MTIP., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The myosin motor of the malaria parasite's invasion machinery moves over actin fibers while it is making critical contacts with the myosin-tail interacting protein (MTIP). Previously, in a "compact" Plasmodium falciparum MTIP•MyoA complex, MTIP domains 2 (D2) and 3 (D3) make contacts with the MyoA helix, and the central helix is kinked, but in an "extended" Plasmodium knowlesi MTIP•MyoA complex only D3 interacts with the MyoA helix, and the central helix is fully extended. Here we report the crystal structure of the compact P. knowlesi MTIP•MyoA complex. It appears that, depending on the pH, P. knowlesi MTIP can adopt either the compact or the extended conformation to interact with MyoA. Only at pH values above ~7.0, can key hydrogen bonds can be formed by the imidazole group of MyoA His810 with an aspartate carboxylate from the hinge of MTIP and a lysine amino group of MyoA simultaneously., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Plasmodium falciparum pathogenesis is affected by various cell types in the blood, including platelets, which can kill intraerythrocytic malaria parasites. Platelets could mediate these antimalarial effects through human defense peptides (HDPs), which exert antimicrobial effects by permeabilizing membranes. Therefore, we screened a panel of HDPs and determined that human platelet factor 4 (hPF4) kills malaria parasites inside erythrocytes by selectively lysing the parasite digestive vacuole (DV). PF4 rapidly accumulates only within infected erythrocytes and is required for parasite killing in infected erythrocyte-platelet cocultures. To exploit this antimalarial mechanism, we tested a library of small, nonpeptidic mimics of HDPs (smHDPs) and identified compounds that kill P. falciparum by rapidly lysing the parasite DV while sparing the erythrocyte plasma membrane. Lead smHDPs also reduced parasitemia in a murine malaria model. Thus, identifying host molecules that control parasite growth can further the development of related molecules with therapeutic potential., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Microarray studies using in vitro cultures of synchronized, blood-stage Plasmodium falciparum malaria parasites have revealed a 'just-in-time' cascade of gene expression with some indication that these transcriptional patterns remain stable even in the presence of external stressors. However, direct analysis of transcription in P. falciparum blood-stage parasites obtained from the blood of infected patients suggests that parasite gene expression may be modulated by factors present in the in vivo environment of the host. The aim of this study was to examine changes in gene expression of the rodent malaria parasite, Plasmodium yoelii 17X, while varying the in vivo setting of replication., Methods: Using P. yoelii 17X parasites replicating in vivo, differential gene expression in parasites isolated from individual mice, from independent infections, during ascending, peak and descending parasitaemia and in the presence and absence of host antibody responses was examined using P. yoelii DNA microarrays. A genome-wide analysis to identify coordinated changes in groups of genes associated with specific biological pathways was a primary focus, although an analysis of the expression patterns of two multi-gene families in P. yoelii, the yir and pyst-a families, was also completed., Results: Across experimental conditions, transcription was surprisingly stable with little evidence for distinct transcriptional states or for consistent changes in specific pathways. Differential gene expression was greatest when comparing differences due to parasite load and/or host cell availability. However, the number of differentially expressed genes was generally low. Of genes that were differentially expressed, many involved biologically diverse pathways. There was little to no differential expression of members of the yir and pyst-a multigene families that encode polymorphic proteins associated with the membrane of infected erythrocytes. However, a relatively large number of these genes were expressed during blood-stage infection regardless of experimental condition., Conclusions: Taken together, these results indicate that 1) P. yoelii gene expression remains stable in the presence of a changing host environment, and 2) concurrent expression of a large number of the polymorphic yir and pyst-a genes, rather than differential expression in response to specific host factors, may in itself limit the effectiveness of host immune responses.

The glideosome associated protein GAP50 is an essential protein in apicomplexan parasites such as Plasmodium, Toxoplasma and Cryptosporidium, several species of which are important human pathogens. The 44.6kDa protein is part of a multi-protein complex known as the invasion machinery or glideosome, which is required for cell invasion and substrate gliding motility empowered by an actin-myosin motor. GAP50 is anchored through its C-terminal transmembrane helix into the inner membrane complex and interacts via a short six residue C-terminal tail with other proteins of the invasion machinery in the pellicle of the parasite. In this paper we describe the 1.7Å resolution crystal structure of the soluble GAP50 domain from the malaria parasite Plasmodium falciparum. The structure shows an αßßα fold with overall similarity to purple acid phosphatases with, however, little homology regarding the nature of the residues in the active site region of the latter enzyme. While purple acid phosphatases contain a phosphate bridged binuclear Fe-site coordinated by seven side chains with the Fe-ions 3.2Å apart, GAP50 in our crystals contains two cobalt ions each with one protein ligand and a distance between the Co(2+) ions of 18Å., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The excessive production of proinflammatory cytokines plays a significant role in the pathogenesis of severe malaria. Mammalian macrophage migration inhibitory factor (MIF) (mMIF) is an immune mediator that promotes a sustained proinflammatory response by inhibiting the glucocorticoid-mediated downregulation of inflammation. In addition, Plasmodium parasites also encode a homologue of mammalian MIF that is expressed in asexual-stage parasites. We used the Plasmodium yoelii murine model to study the potential role of parasite-encoded MIF in the pathogenesis of malaria. Antibodies raised against purified, non-epitope-tagged P. yoelii MIF (PyMIF) were used to localize expression in trophozoite- and schizont-stage parasites and demonstrate extracellular release. In vitro, recombinant PyMIF was shown to actively induce the chemotaxis of macrophages but did not induce or enhance tumor necrosis factor alpha (TNF-α) production from peritoneal macrophages. To examine the role of parasite-derived PyMIF in vivo, two transgenic parasite lines that constitutively overexpress PyMIF were generated, one in a nonlethal P. yoelii 17X background [Py17X-MIF(+)] and the other in a lethal P. yoelii 17XL background [Py17XL-MIF(+)]. Challenge studies with transgenic parasites in mice showed that the increased expression of PyMIF resulted in a reduction in disease severity. Mice infected with Py17X-MIF(+) developed lower peak parasitemia levels than controls, while malaria-associated anemia was unaltered. Infection with Py17XL-MIF(+) resulted in a prolonged course of infection and a reduction in the overall mortality rate. Combined, the data indicate that parasite-derived MIF does not contribute significantly to immunopathology but, through its chemotactic ability toward macrophages, may attenuate disease and prolong infection of highly virulent parasite isolates.

Malaria is endemic in most developing countries, with nearly 500 million cases estimated to occur each year. The need to design a new generation of antimalarial drugs that can combat the most drug-resistant forms of the malarial parasite is well recognized. In this study, we wanted to develop inhibitors of key proteins that form the invasion machinery of the malarial parasite. A critical feature of host-cell invasion by apicomplexan parasites is the interaction between the carboxy terminal tail of myosin A (MyoA) and the myosin tail interacting protein (MTIP). Using the cocrystal structure of the Plasmodium knowlesi MTIP and the MyoA tail peptide as input to the hybrid structure-based virtual screening approach, we identified a series of small molecules as having the potential to inhibit MTIP-MyoA interactions. Of the initial 15 compounds tested, a pyrazole-urea compound inhibited P. falciparum growth with an EC(50) value of 145 nM. We screened an additional 51 compounds belonging to the same chemical class and identified 8 compounds with EC(50) values less than 400 nM. Interestingly, the compounds appeared to act at several stages of the parasite's life cycle to block growth and development. The pyrazole-urea compounds identified in this study could be effective antimalarial agents because they competitively inhibit a key protein-protein interaction between MTIP and MyoA responsible for the gliding motility and the invasive features of the malarial parasite.

Soil contamination, such as heavy metals and benzene compounds, is a widespread problem on military installations. It is important to be able to determine the effects of soil contamination before any adverse effects appear in organisms in surrounding areas. We examined gene expression in Arabidopsis thaliana grown in soil from three sites at the Radford Army Ammunition Plant in Radford, Virginia, USA, using DNA microarrays. We analyzed soil, germination, and growth rate to compare with the microarray data. Soil contamination affected both external phenotype and gene expression. Plants grown in soil with high levels of contaminants were chloritic and were smaller than control plants grown in potting soil. Plants grown in soil with the highest copper concentration had the lowest growth rates and had genes up-regulated across several functional groups. Plants grown in soils with elevated lead had many genes down-regulated that were related to photosystem II, metabolism, cellular transport, and protein synthesis. Genes consistently up-regulated across most microarrays were genes related to photosystem I, genes related to water deprivation and oxidative stress response, heat shock proteins, and toxin catabolism genes such as glutathiones. DNA microarrays, in concert with a model genetic organism such as A. thaliana, were an effective assessment tool to determine the presence of toxic substances in soil at a site used for the production of military explosives.

The malaria parasite sporozoite transmission stage develops and differentiates within parasite oocysts on the Anopheles mosquito midgut. Successful inoculation of the parasite into a mammalian host is critically dependent on the sporozoite's ability to first infect the mosquito salivary glands. Remarkable changes in tissue infection competence are observed as the sporozoites transit from the midgut oocysts to the salivary glands. Our microarray analysis shows that compared to oocyst sporozoites, salivary gland sporozoites upregulate expression of at least 124 unique genes. Conversely, oocyst sporozoites show upregulation of at least 47 genes (upregulated in oocyst sporozoites [UOS genes]) before they infect the salivary glands. Targeted gene deletion of UOS3, encoding a putative transmembrane protein with a thrombospondin repeat that localizes to the sporozoite secretory organelles, rendered oocyst sporozoites unable to infect the mosquito salivary glands but maintained the parasites' liver infection competence. This phenotype demonstrates the significance of differential UOS expression. Thus, the UIS-UOS gene classification provides a framework to elucidate the infectivity and transmission success of Plasmodium sporozoites on a whole-genome scale. Genes identified herein might represent targets for vector-based transmission blocking strategies (UOS genes), as well as strategies that prevent mammalian host infection (UIS genes).

The sexual cycle of Plasmodium is required for transmission of malaria from mosquitoes to mammals, but how parasites induce the expression of genes required for the sexual stages is not known. We disrupted the Plasmodium yoelii gene encoding high mobility group nuclear factor hmgb2, which encodes a DNA-binding protein potentially implicated in transcriptional regulation of malaria gene expression. We investigated its function in vivo in the vertebrate and invertebrate hosts. Deltapyhmgb2 parasites develop into gametocytes but have drastic impairment of oocyst formation. A global transcriptome analysis of the Deltapyhmgb2 parasites identified approximately 30 genes whose expression is down-regulated in the Deltapyhmgb2 parasites. These genes are conserved in all malaria species, and more than 90% of these genes show a peak of mRNA expression at the gametocyte stage. Surprisingly, the transcripts coding for the Plasmodium berghei orthologues of those genes are stored and translated in the ookinete stage. Therefore, sexual stage protein expression appears to be both transcriptionally and translationally regulated with Plasmodium HMGB2 acting as an important regulator of malaria sexual stage gene expression.

Efficient and specific host cell entry is of exquisite importance for intracellular pathogens. Parasites of the phylum Apicomplexa are highly motile and actively enter host cells. These functions are mediated by type I transmembrane invasins of the TRAP family that link an extracellular recognition event to the parasite actin-myosin motor machinery. We systematically tested potential parasite invasins for binding to the actin bridging molecule aldolase and complementation of the vital cytoplasmic domain of the sporozoite invasin TRAP. We show that the ookinete invasin CTRP and a novel, structurally related protein, termed TRAP-like protein (TLP), are functional members of the TRAP family. Although TLP is expressed in invasive stages, targeted gene disruption revealed a nonvital role during life cycle progression. This is the first genetic analysis of TLP, encoding a redundant TRAP family invasin, in the malaria parasite.

For 50 years since their discovery, the malaria parasite liver stages (LS) have been difficult to analyze, impeding their utilization as a critical target for antiinfection vaccines and drugs. We have undertaken a comprehensive transcriptome analysis in combination with a proteomic survey of LS. Green fluorescent protein-tagged Plasmodium yoelii (PyGFP) was used to efficiently isolate LS-infected hepatocytes from the rodent host. Genome-wide LS gene expression was profiled and compared with other parasite life cycle stages. The analysis revealed approximately 2,000 genes active during LS development, and proteomic analysis identified 816 proteins. A subset of proteins appeared to be expressed in LS only. The data revealed exported parasite proteins and LS metabolic pathways including expression of FASII pathway enzymes. The FASII inhibitor hexachlorophene and the antibiotics, tetracycline and rifampicin, that target the apicoplast inhibited LS development, identifying FASII and other pathways localized in the apicoplast as potential drug targets to prevent malaria infection.

The Myosin A-tail interacting protein (MTIP) of the malaria parasite links the actomyosin motor of the host cell invasion machinery to its inner membrane complex. We report here that at neutral pH Plasmodium falciparum MTIP in complex with Myosin A adopts a compact conformation, with its two domains completely surrounding the Myosin A-tail helix, dramatically different from previously observed extended MTIP structures. Crystallographic and mutagenesis studies show that H810 and K813 of Myosin A are key players in the formation of the compact MTIP:Myosin A complex. Only the unprotonated state of Myosin A-H810 is compatible with the compact complex. Most surprisingly, every side-chain atom of Myosin A-K813 is engaged in contacts with MTIP. While this side-chain was previously considered to prevent a compact conformation of MTIP with Myosin A, it actually appears to be essential for the formation of the compact complex. The hydrophobic pockets and adaptability seen in the available series of MTIP structures bodes well for the discovery of inhibitors of cell invasion by malaria parasites.

The causative agents of malaria have developed a sophisticated machinery for entering multiple cell types in the human and insect hosts. In this machinery, a critical interaction occurs between the unusual myosin motor MyoA and the MyoA-tail Interacting Protein (MTIP). Here we present one crystal structure that shows three different conformations of Plasmodium MTIP, one of these in complex with the MyoA-tail, which reveal major conformational changes in the C-terminal domain of MTIP upon binding the MyoA-tail helix, thereby creating several hydrophobic pockets in MTIP that are the recipients of key hydrophobic side chains of MyoA. Because we also show that the MyoA helix is able to block parasite growth, this provides avenues for designing antimalarials.

Immunization with Plasmodium yoelii merozoite surface protein-8 (PyMSP-8) has been shown to protect mice against lethal P. yoelii 17XL malaria. Here we demonstrate that PyMSP-8-specific antibodies preferentially suppress P. yoelii 17XL growth in mature erythrocytes compared to growth in reticulocytes and do not suppress the growth of nonlethal P. yoelii 17X, a parasite that primarily replicates in reticulocytes. The protection against normocyte-associated P. yoelii malaria parasites is mediated by antibodies that recognize conformational epitopes of PyMSP-8 that are nonpolymorphic. We examined changes in gene expression in reticulocyte-restricted P. yoelii 17XL parasites that escaped neutralization by PyMSP-8-specific antibodies using P. yoelii DNA microarrays. Of interest, Pymsp-8 gene expression decreased, while the expression of msp-1, msp-7, and several rhoptry protein genes increased. Breakthrough parasites also exhibited increases in the expression of a subset of yir and Pyst-a genes that are predicted to encode polymorphic antigens expressed on the surface of infected erythrocytes. These data suggest that changes in the expression of parasite proteins expressed on the merozoite surface, as well as the surface of infected erythrocytes, may alter host cell tropism and contribute to the ability of malaria parasites to evade merozoite-specific, neutralizing antibodies.

The Pho85-Pho80 cyclin-CDK (cyclin-dependent protein kinase) complex of Saccharomyces cerevisiae functions as a key regulator of the phosphate-repressible acid phosphatase system. We have further characterized the Pho85-Pho80 kinase complex and identified the Pho80 cyclin subunit and the Pho81 CDK inhibitor as substrates of the Pho85 protein kinase. The phosphorylation sites within Pho80 have been identified at Ser234 and Ser267. Of the two sites, phosphorylation of Ser234 is required for Pho80 function, to form an active kinase complex and repress acid phosphatase expression. Evidence suggests that the activity of Pho81 is regulated by a post-translational modification and therefore that Pho85-mediated phosphorylation of Pho81 may alter its ability to function as a CDK inhibitor. Thus, the control of acid phosphatase expression involves the phosphorylation of several of the regulatory components of the system.

Cyclins, cyclin-dependent kinases (CDK) and their inhibitors play a critical role in many biological processes. In yeast, the ankyrin repeat protein Pho81p, by being an inhibitor of the Pho85p-Pho80p cyclin-dependent protein kinase complex, transcriptionally regulates the production of repressible acid phosphatase, encoded by the PHO5 gene. Recent studies in our laboratory showed that Pho81p is phosphorylated by the Pho80p-Pho85p CDK complex in vitro; and, to determine the significance of the phosphorylation, we used site-directed mutagenesis to alter the potential phosphorylation sites for this kinase complex. The resulting mutations were introduced into a yeast strain containing a deletion of PHO81 and the effect of the mutation on PHO5 expression was assayed. Results suggest that phosphorylation of particular residues within Pho81p is crucial for its activity as an inhibitor. Studies using a green fluorescent protein-Pho81p fusion and Western analysis indicate that the localization and half-life of the mutants are similar to wild-type Pho81 proteins. However, an in vivo binding assay indicates that the mutant Pho81p is deficient in binding to the Pho80p-Pho85p kinase complex. These findings support the observation that the mutant fails to inhibit kinase activity in low phosphate. These studies provide insight into the mechanism of regulation of CDK inhibitor activity.

In Plasmodium falciparum malaria, erythrocyte invasion by circulating merozoites may occur via two distinct pathways involving either a sialic acid-dependent or -independent mechanism. Earlier, we identified two nonglycosylated exofacial regions of erythrocyte band 3 termed 5ABC and 6A as an important host receptor in the sialic acid-independent invasion pathway. 5ABC, a major segment of this receptor, interacts with the 42-kDa processing product of merozoite surface protein 1 (MSP1(42)) through its 19-kDa C-terminal domain. Here, we show that two regions of merozoite surface protein 9 (MSP9), also known as acidic basic repeat antigen, interact directly with 5ABC during erythrocyte invasion by P. falciparum. Native MSP9 as well as recombinant polypeptides derived from two regions of MSP9 (MSP9/Delta1 and MSP9/Delta2) interacted with both 5ABC and intact erythrocytes. Soluble 5ABC added to the assay mixture drastically diminished the binding of MSP9 to erythrocytes. Recombinant MSP9/Delta1 and MSP9/Delta2 present in the culture medium blocked P. falciparum reinvasion into erythrocytes in vitro. Native MSP9 and MSP1(42), the two ligands binding to the 5ABC receptor, existed as a stable complex. Our results establish a novel concept wherein the merozoite exploits a specific complex of co-ligands on its surface to target a single erythrocyte receptor during invasion. This new paradigm poses a new challenge in the development of a vaccine for blood stage malaria.

Previously, we described the isolation of the Plasmodium yoelii sequence-related molecules P. yoelii MSP-7 (merozoite surface protein 7) and P. yoelii MSRP-2 (MSP-7-related protein 2) by their ability to interact with the amino-terminal end of P. yoelii MSP-1 in a yeast two-hybrid system. One of these molecules was the homologue of Plasmodium falciparum MSP-7, which was biochemically isolated as part of the shed MSP-1 complex. In the present study, with antibodies directed against recombinant proteins, immunoprecipitation analyses of the rodent system demonstrated that both P. yoelii MSP-7 and P. yoelii MSRP-2 could be isolated from parasite lysates and from parasite culture supernatants. Immunofluorescence studies colocalized P. yoelii MSP-7 and P. yoelii MSRP-2 with the amino-terminal portion of MSP-1 and with each other on the surface of schizonts. Immunization with P. yoelii MSRP-2 but not P. yoelii MSP-7 protected mice against a lethal infection with P. yoelii strain 17XL. These results establish that both P. yoelii MSP-7 and P. yoelii MSRP-2 are expressed on the surface of merozoites and released from the parasite and that P. yoelii MSRP-2 may be the target of a protective immune response.

Apicomplexan host cell invasion and gliding motility depend on the parasite's actomyosin system located beneath the plasma membrane of invasive stages. Myosin A (MyoA), a class XIV unconventional myosin, is the motor protein. A model has been proposed to explain how the actomyosin motor operates but little is known about the components, topology and connectivity of the motor complex. Using the MyoA neck and tail domain as bait in a yeast two-hybrid screen we identified MTIP, a novel 24 kDa protein that interacts with MyoA. Deletion analysis shows that the 15 amino-acid C-terminal tail domain of MyoA, rather than the neck domain, specifically interacts with MTIP. In Plasmodium sporozoites MTIP localizes to the inner membrane complex (IMC), where it is found clustered with MyoA. The data support a model for apicomplexan motility and invasion in which the MyoA motor protein is associated via its tail domain with MTIP, immobilizing it at the outer IMC membrane. The head domain of the immobilized MyoA moves actin filaments that, directly or via a bridging protein, connect to the cytoplasmic domain of a transmembrane protein of the TRAP family. The actin/TRAP complex is then redistributed by the stationary MyoA from the anterior to the posterior end of the zoite, leading to its forward movement on a substrate or to penetration of a host cell.

Merozoite surface protein 1 (MSP-1) is a high-molecular-weight protein expressed on the surface of the malaria merozoite in a noncovalent complex with other protein molecules. MSP-1 undergoes a series of proteolytic processing events, but no precise biological role for the various proteolytic fragments of MSP-1 or for the additional proteins present in the complex is known. Through the use of the yeast two-hybrid system, we have isolated genes encoding proteins that interact with a region of the amino-terminal proteolytic fragment of MSP-1 from the mouse parasite Plasmodium yoelii. This analysis has led to the isolation of two sequence-related molecules, one of which is the P. yoelii homologue of MSP-7 originally described in Plasmodium falciparum. BLAST analysis of the P. falciparum database has revealed that there are six related protein molecules present in this species encoded near each other on chromosome 13. In P. falciparum, we designated these molecules MSRP-1 to -5. Analysis of the P. yoelii database indicates a similar chromosomal organization for the two genes in the mouse parasite species. The three P. falciparum sequences with the highest degree of homology to the P. yoelii sequences isolated in the two-hybrid screen have been characterized at the molecular level (MSRP-1 to -3). Expression analysis indicated that the mRNAs are expressed at various levels in the different asexual stages. Immunofluorescence studies colocalized the expression of the MSRP molecules and the amino-terminal portion of MSP-1 to the surfaces of trophozoites. In vitro binding experiments confirmed the interaction between MSRP-1, MSRP-2, and the amino-terminal region of P. falciparum MSP-1.

Species of malaria parasite that infect rodents have long been used as models for malaria disease research. Here we report the whole-genome shotgun sequence of one species, Plasmodium yoelii yoelii, and comparative studies with the genome of the human malaria parasite Plasmodium falciparum clone 3D7. A synteny map of 2,212 P. y. yoelii contiguous DNA sequences (contigs) aligned to 14 P. falciparum chromosomes reveals marked conservation of gene synteny within the body of each chromosome. Of about 5,300 P. falciparum genes, more than 3,300 P. y. yoelii orthologues of predominantly metabolic function were identified. Over 800 copies of a variant antigen gene located in subtelomeric regions were found. This is the first genome sequence of a model eukaryotic parasite, and it provides insight into the use of such systems in the modelling of Plasmodium biology and disease.

Several model systems of plasmodia have demonstrated the potential of the merozoite surface protein, MSP-1, to induce protective immunity. However, little is known about the function of this protein or its interaction with other surface molecules that may also serve as immunological targets. To identify potentially significant inter- and intra-molecular interactions involving MSP-1, we have utilized the yeast two-hybrid system. A cDNA activation domain library was constructed from the erythrocytic stages of the murine malarial parasite Plasmodium yoelii yoelii 17XL. A 795 bp region of Py17XL MSP-1 (bait), homologous to the Plasmodium falciparum MSP1(33) fragment, was inserted into a Gal4p DNA binding domain vector and used to screen the activation domain library (target). Several randomly selected clones that demonstrated bait-target interaction were found to express overlapping regions of Py17XL MSP-1. Deletion constructs further localized the peptide fragments retaining interaction indicating that a region within the MSP-1(38) fragment interacts with the MSP-1 bait domain. Subsequent studies confirmed this interaction, as both peptides were co-precipitated from cell lysate by a peptide tag-specific antibody. It was observed that the interaction of these two fragments significantly increased the half-life of the MSP-1(38) within yeast cells. The specific interaction described here demonstrates the potential of this approach to elucidate additional inter- or intra-molecular interactions of Py17XL MSP1 and other malarial proteins.

The yeast transcription factor Pho4p is required for expression of the phosphate-repressible acid phosphatase encoded by the PHO5 gene. Functional studies have shown that the molecule is composed of an N-terminal acidic activation domain, a central region which is necessary for interaction with a negative regulatory factor (the cyclin Pho80) and a C-terminal basic helix-loop-helix domain, which mediates DNA binding and homodimerization. In this study the homodimerization domain maps specifically to helixII of this region and a cysteine residue within this region is essential for this function. Experiments support the role of an intermolecular disulfide bond in stabilization of homodimerization, which is critical for DNA binding.

The cyclin-dependent protein kinase Pho85 is a known negative regulatory factor for two stress response genes, PHO5 and GSY2, which encode the inducible form of acid phosphatase and glycogen synthase, respectively, in the yeast Saccharomyces cerevisiae. Cells carrying a disruption of the PHO85 gene inappropriately express both PHO5 and GSY2, resulting in the increase in phosphate scavenging and hyperaccumulation of glycogen in nutrient-rich conditions. Constitutive activation of PKA in a pho85 mutant suppresses the hyperaccumulation of glycogen. This work presents data to show that, at least in part, the suppression of glycogen biosynthesis upon activation of PKA in a pho85 mutant results from the suppression of GSY2 expression. In addition to GSY2, disruption of the PHO85 gene inappropriately triggers the derepression of two other stress response genes, HSP12 and UBI4. At least in the case of GSY2, regulation of transcription by Pho85 is not through the stress-responsive cis-promoter elements (STRE). Furthermore, Pho85 may associate with the known cyclin Pho80 in the transcriptional regulation of these genes.

In Saccharomyces cerevisiae, expression of acid phosphatase, encoded by the PHO5 gene, requires two positive regulatory factors, Pho4 and Pho2 (also called Bas2 or Grf10). Using GAL4-PHO4 fusions, we demonstrate that a functional interaction between these two proteins is necessary for transcriptional activation to occur. This functional interaction between Pho4 and Pho2 is independent of the presence of the negative regulatory factor, Pho80, which also interacts with Pho4. Interestingly, truncations of Pho4 missing amino acids 252-265, which encompass the basic region of the basic helix-loop-helix (bHLH) DNA binding motif, exhibit high transcriptional activation that is independent of the Pho2 molecule. Single amino acid mutations of highly conserved residues within this area all display this Pho2-independent phenotype. A region near the C-terminus of Pho2 appears to be critical for this interaction with Pho4. A model to account for the requirement for Pho2 in Pho4-dependent transcriptional activation is proposed.

Pho85, a protein kinase with significant homology to the cyclin-dependent kinase, Cdc28, has been shown to function in repression of transcription of acid phosphatase (APase, encoded by PHO5) in high phosphate (Pi) medium, as well as in regulation of the cell cycle at G1/S. We described several unique phenotypes associated with the deletion of the PHO85 gene including growth defects on a variety of carbon sources and hyperaccumulation of glycogen in rich medium high in Pi. Hyperaccumulation of glycogen in the pho85 strains is independent of other APase regulatory molecules and is not signaled through Snfl kinase. However, constitutive activation of cAPK suppresses the hyperaccumulation of glycogen in a pho85 mutant. Mutation of the type-1 protein phosphatase encoded by GLC7 only partially suppresses the glycogen phenotype of the pho85 mutant. Additionally, strains containing a deletion of the PHO85 gene show an increase in expression of GSY2. This work provides evidence that Pho85 has functions in addition to transcriptional regulation of APase and cell-cycle progression including the regulation of glycogen levels in the cell and may provide a link between the nutritional state of the cell and these growth related responses.

The PHO85 gene of Saccharomyces cerevisiae encodes a cyclin-dependent kinase involved in both transcriptional regulation and cell cycle progression. Although a great deal is known concerning the structure, function, and regulation of the highly homologous Cdc28 protein kinase, little is known concerning these relationships in regard to Pho85. In this study, we constructed a series of Pho85-Cdc28 chimeras to map the region(s) of the Pho85 molecule that is critical for function of Pho85 in repression of acid phosphatase (PHO5) expression. Using a combination of site-directed and ethyl methanesulfonate-induced mutagenesis, we have identified numerous residues critical for either activation of the Pho85 kinase, interaction of Pho85 with the cyclin-like molecule Pho80, or substrate recognition. Finally, analysis of mutations analogous to those previously identified in either Cdc28 or cdc2 of Schizosaccharomyces pombe suggested that the inhibition of Pho85-Pho80 activity in mechanistically different from that seen in the other cyclin-dependent kinases.

Total RNA from rat Schwann cells grown in culture and adult rat skeletal muscle was reverse transcribed, amplified for glial cell line-derived neurotrophic factor (GDNF) messenger RNA (mRNA) using the polymerase chain reaction (PCR), and the PCR products sequenced. Two forms of GDNF were detected in the PCR step, one of a predicted size (GDNF633) and a second smaller form missing a 78-base pair sequence (GDNF555). Sequence analysis demonstrated that GDNF633 is similar to the published sequence of GDNF differing only at three nucleotides. Southern and Northern blot analyses reveal that the two forms are probably derived from a single RNA species that is alternatively spliced. Interestingly, GDNF633 mRNA was found to be selectively upregulated in denervated rat skeletal muscle at 1-2 weeks following axotomy, providing evidence that the innervation status of the muscle may determine the expression profile of the two alternatively spliced forms. Given these findings, we suggest that GDNF may function as a target-derived trophic factor for neuronal populations innervating skeletal muscle, including sensory neurons and spinal cord motoneurons.

It has been suggested that several Trypanosoma cruzi antigens have possible protective epitopes which may be suitable vaccine candidates. We found previously that animals resistant to T. cruzi infection produced antibodies against the 75-77-kDa parasite antigen. To test the ability of the recombinant form of this antigen to protect animals from T. cruzi infection, the cDNA which encodes a portion of the 75-77-kDa antigen was cloned using a cDNA library constructed in an orientation-specific bacteriophage expression vector (lambda gt 11) from poly (A)+ RNA of Brazil strain epimastigotes. One clone, named SFS-40, was selected by screening the library using affinity purified antibodies specific for the 75-77-kDa parasite antigen as probe. The cDNA corresponding to the 1.7-kilobase insert of SFS-40 was subcloned into plasmid vectors and characterized. The cDNA sequence encodes a polypeptide of about 40 kDa. The putative product of the cDNA was homologous to members of the 70-kDa stress protein family. When epimastigotes were shifted from 29 degrees C to 37 degrees C, there was no change in the level of SFS-40 mRNA. In contrast, the 70-kDa heat shock protein mRNA of the parasite was increased about four fold by this treatment.

1. Site directed mutations were constructed in the yeast iso-1-cytochrome c gene adjacent to the lysine 77 (methylation site) codon. 2. These mutant genes were then cloned and transformed into the S. cerevisiae strain B-6642 which contains a deficiency in the iso-1-cytochrome c gene. 3. The resulting transformants were screened for cytochrome c production using gel electrophoresis. 4. Amino acid analysis of the mutated cytochromes c demonstrated varying levels of trimethyllysine formation, depending on the nature of the site directed mutation. 5. The resulting transformants were then used as tools in order to investigate the relationship between trimethyllysine formation and various aspects of cytochrome c metabolism including protein stability and heme conjugation.

The PHO81 gene product is a positive regulatory factor required for the synthesis of the phosphate repressible acid phosphatase (encoded by the PHO5 gene) in Saccharomyces cerevisiae. Genetic analysis has suggested that PHO81 may be the signal acceptor molecule; however, the biochemical function of the PHO81 gene product is not known. We have cloned the PHO81 gene and sequenced the promoter. A PHO81-LacZ fusion was shown to be a valid reporter since its expression is regulated by the level of inorganic phosphate and is controlled by the same regulatory factors that regulate PHO5 expression. To elucidate the mechanism by which PHO81 functions, we have isolated and cloned dominant mutations in the PHO81 gene which confer constitutive synthesis of acid phosphatase. We have demonstrated that overexpression of the negative regulatory factor, PHO80, but not the negative regulatory factor PHO85, partially blocks the constitutive acid phosphatase synthesis in a strain containing a dominant constitutive allele of PHO81. This suggests that PHO81 may function by interacting with PHO80 or that these molecules compete for the same target.

1. A mutant of the iso-1-cytochrome c gene from Saccharomyces cerevisiae has been constructed which contains an Arg codon, replacing the normal trimethylated Lys at position 77. 2. This mutated gene was cloned into a pGem 1 vector and used for the in vitro translation of yeast iso-1-cytochrome c. 3. Utilizing an in vitro mitochondria binding assay, it was found that the mutant cytochrome c could transverse the yeast mitochondrial membrane, however the amount of protein incorporated was 3-fold less that of the trimethylated wild type. 4. Omission of the protein methyltransferase from assays containing the wild type cytochrome c caused only a slight reduction (15%) in the amount of protein incorporated. 5. These results suggest while the lysine residue 77 of apocytochrome c is important for mitochondria uptake, the methylation of this residue seems to play a relatively minor role.

The PHO80 and PHO85 gene products encode proteins necessary for the repression of transcription from the major acid phosphatase gene (PHO5) of Saccharomyces cerevisiae. The deduced amino acid sequences of these genes have revealed that PHO85 is likely to encode a protein kinase, whereas no potential function has been revealed for PHO80. We undertook several approaches to aid in the elucidation of the PHO80 function, including deletion analysis, chemical mutagenesis, and expression analysis. DNA deletion analysis revealed that residues from both the carboxy- and amino-terminal regions of the protein, amounting to a total of 21% of the PHO80 protein, were not required for function with respect to repressor activity. Also, 10 independent single-amino-acid changes within PHO80 which resulted in the failure to repress PHO5 transcription were isolated. Nine of the 10 missense mutations resided in two subregions of the PHO80 molecule. In addition, expression analysis of the PHO80 and PHO85 genes suggested that the PHO85 gene product was not necessary for PHO80 expression and that the PHO85 gene was expressed at much higher levels in the cell than was the PHO80 gene. Furthermore, high levels of PHO80 were shown to suppress the effect of a PHO85 deletion at a level close to full repression. Implications for the function of the negative regulators in this system are discussed.

We have investigated the process of protein glycosylation in an attempt to answer the question of whether glucosamine and mannose are added to nascent chains prior to chain completion or only to completed chains after release from the ribosome. The MPC 11 mouse plasmacytoma cell line used in these studies synthesizes a glycosylated gamma2b heavy chain which accounts for 12% of the total protein synthesis. Nascent chains were separated from completed chains by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. Our results indicate that both glucosamine and mannose are incorporated into nascent heavy chains prior to chain completion and release from the ribosome. Gel analysis of specifically immunoprecipitated nascent chains indicates that the carbohydrate moiety can be added to the nascent heavy chains very soon after the presumptive asparaginyl glycosylation site (CH2 domain) is synthesized on the ribosome.

The initial glycosylation of MPC 11 gamma 2b heavy chains occurs quantitatively in vivo when the nascent heavy chains reach a size of approximately 38 000 daltons. Nonglycosylated, completed MPC 11 heavy chains cannot be glycosylated in these cells. Other classes of mouse heavy chains (i.e., mu, alpha, and gamma 1) also appear to be glycosylated as nascent chains; nonglycosylated, completed heavy chains cannot be glycosylated by the cell in any of these cases. In contrast, variant MPC 11 cells synthesizing a heavy chain with a carboxy-terminal deletion appear to glycosylate some heavy chains prior to chain completion and some heavy chains after chain completion and release from the polysomes. Similar to the variant MPC 11 cells, MOPC 46B cells (which synthesize a kappa light chain containing an oligosaccharide attached to an asparagine located 28 residues from the amino terminus) glycosylate the majority of light chains after prior to chain completion but also some light chains after chain completion and release from the polysomes. In addition, it appears that, although completed MOPC 46B light chains can be glycosylated if they are present in a monomeric form, they cannot be glycosylated if they are present in a covalent dimeric form.

We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG2b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG1), where a heavy chain dimer is the major initial disulfide linked intermediate.

The functional relationship of nucleosome positioning and gene expression is not known. Using high-copy plasmids, containing the yeast phosphate-repressible acid phosphatase gene (PHO5) and the TRP1/ARS1 vector system, I have determined the nucleosomal structure of the 5' region of the PHO5 gene and demonstrated that the nucleosomal positioning of this region is independent of orientation or position in the various plasmid constructions utilized. However, deletion of a 278-base pair BamHI-ClaI fragment from the 5'-flanking sequences of the PHO5 gene causes the nucleosome positioning to become dependent on orientation or position in the plasmids tested. Use of PHO5-CYC1-lACZ fusions have demonstrated that this DNA fragment contains the sequences responsible for the transcriptional regulation of the PHO5 gene in response to the level of phosphate in the growth media. The nucleosome positioning in the 5' region of PHO5 may be determined by an interaction with the sequences or machinery responsible for transcriptional regulation of the gene.

The expression of the PHO5 gene of Saccharomyces cerevisiae is transcriptionally regulated in response to the level of inorganic phosphate present in the growth medium. We have identified, by DNA deletion analysis, the sequences (upstream activator sequences) that mediate this response. The sequence 5' CTGCACAAATG 3' is present in two copies located within a 60-base-pair region. The presence of a single copy of the sequence is sufficient for the phosphate-mediated transcriptional response. In addition, a DNA fragment that contains two copies of this sequence will act to repress transcription of a CYC1-lacZ fusion when placed either upstream or downstream of the CYC1 activator sequence.

The initial step of intermolecular covalent assembly of immunoglobulins molecules involves formation of heavy chain-light chain or heavy chain-heavy chain disulfide bonds. Using QAE-Sephadex chromatography to isolate microsomal nascent polypeptides, we have shown that this initial step of intermolecular covalent assembly occurs, to a substantial extent, on nascent heavy chains, as well as on completed heavy chains as previously demonstrated by others. In MPC 11 mouse myeloma cells, completed light chains are assembled covalently to nascent heavy chains, whereas in MOPC 21 mouse myeloma cells, completed heavy chains are assembled covalently to nascent heavy chains. These results are consisted with the heavy-light half-molecule being the major initial intermediate in the assembly of MPC 11 IgG2b and heavy-heavy dimer being the major initial intermediate formed in assembly of MOPC 21 IgG1. The nascent MPC 11 heavy chain must be at least 38,000 daltons in size before assembly with the light chain occurs, even though the heavy chain cysteine involved in this disulfide bond is 131 residues (approximately 15,000 daltons) from the NH2 terminus. In addition, pulse-chase labeling studies of MPC 11 cells have shown that the assembly of completed light chains with the nascent heavy chain must occur within a few minutes of the synthesis of the light chain even though a large excess of unassembled MPC 11 light chains remain inside the cell for an average time of 2 h before being secreted.

Sea urchin sperm chromatin contains forms of H1, H2A, and H2B which differ from those present in adult tissues. We have delineated some effects of the variant H2A and H2B on chromatin by study of the structure of the core particle from Strongylocentrotus purpuratus sperm. The particle contains 145 base pairs of DNA and equal amounts of the four smaller histones. It sediments at 11 S and has a circular dichroism spectrum similar to that of particles containing more typical histones. The sperm core particle undergoes a shape change at low ionic strength, as observed for chicken erythrocyte particles. In contrast to these similarities, the melting profile of the sperm particle is quite different from that of erythrocyte; both the reversible transition and the irreversible denaturation of the core particle occur at higher temperatures. The sperm core particle is digested by DNase I more slowly than the core particle from chicken erythrocyte. Cutting site maps for the sperm core particle reveal the same basic organization of DNA by these histones; however, certain features of the map differ significantly from that for chicken erythrocyte, demonstrating a modulation of the canonical core particle structure by the unusual histones present in sea urchin sperm.

We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription. The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase. Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased. Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid. This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase. Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.

Analysis of nascent heavy chains isolated from MPC11 (gamma 2b heavy chains) and MOPC 21 (gamma 1 heavy chains) mouse myeloma cells demonstrates an accumulation of nascent heavy chains which are slightly smaller in mass (approximately 35,000 daltons) than nascent heavy chains which have just been glycosylated (approximately 38,000 daltons). The accumulation of 35,000-dalton nascent heavy chain appears to be a consequence of the glycosylation process since tunicamycin, an inhibitor of glycosylation, abolishes the apparent translational block manifested by the accumulation of 35,000-dalton nascent chains. Tunicamycin also causes a 15 to 25% increase n the relative rate of synthesis of heavy chain compared to the corresponding rate of synthesis of the nonglycosylated light chain synthesized by the same cell. These results suggest that the translation block, caused by the glycosylation process, of heavy chain synthesis contributes to the imbalance of heavy chain and light chain biosynthesis observed in malignant and normal lymphoid cells.